DE102004025988A1 - Use of vanadyl compounds for the treatment of tumor diseases - Google Patents

Abstract

The invention relates to the use of vanadyl compounds for the treatment of tumor diseases. DOLLAR A basis of the invention is a way of influencing the switch regions of certain proteins for the targeted regulation of signal transduction processes. In particular, the invention relates to the substitution of the normally nickel-tide-bound metal atom Mg · 2 + by VO · 2 + and the associated conformational changes in the switch regions of the superfamily of P-loop-containing mono-nucleotide binding proteins, including the important family guanosine-binding proteins. Such a substitution offers a variety of medical applications, u. a. the possibility of developing oncogene regulators for cancer therapy, but may also be applicable in a variety of other fields.

Description

Field of the invention

These This invention relates generally to the medical-pharmaceutical field inorganic biochemistry with decisive implications for de novo Therapy applications, in particular cancer therapy.

Quick Facts

The basis of the invention is a possibility of influencing the switch regions of specific proteins for the targeted regulation of signal transduction processes. In particular, the invention relates to the substitution of the normally nucleotide-bound metal atom Mg ²⁺ by VO ²⁺ and the associated conformational changes in the switch regions of the superfamily of "P-loop" -containing mononucleotide binding proteins including the important family of guanosine binding proteins. Such substitution offers a variety of medical applications, including the possibility of developing oncogene regulators for cancer therapy, but may also find application in a variety of other fields.

When so-called 'classic' mono-nucleotide binding proteins (MNBPs) are referred to below as regulatory hydrolases be characterized by the presence of quite characteristic structural features in the primary, secondary and tertiary structure of the nucleotide binding pocket or Distinguish folding (see for example the review article: Walker et al., 1982; Bourne et al., 1991; Kjeldgaard et al., 1996), which is also referred to as a common GTP / ATP binding topology can. The homology of the proteins is expressed by the presence a glycine-rich domain with an amino acid sequence highly conserved in all of these proteins of the form G × X × X × X × G × K × S / T (where X is any amino acid is and the rest Sign the usual one-letter Nomenclature of amino acids also known as Walker motif A (Walker et al., 1982), the part of a tertiary structure which is also known as "P-loop" (phosphate binding 'loops'), sometimes called G-1 box is called. This is a binding region that has all the classic 'MNBPs, and thus can said MNBPs also as a "P-loop" -owned superfamily of regulatory nucleotide triphosphate (NTP) hydrolases (Saraste et al., 1990). All these proteins are common, that she act as molecular switches. Overall, the superfamily, to which reference is hereby characterized by that she 1. a "P-loop" (Walker A) motif owns, 2. a switch I region and 3. a switch II region (Walker motif II) and thus a great homology of the "active-site" which also the functional similarity accounts.

A large and important subfamily of these MNBPs are the G proteins; these are proteins that bind guanosine 5'-triphosphate (GTP) and perform so-called regulatory switch functions, which are described in detail below. These include essentially the Ras-like proteins, such as the small monomeric GTPases such as Rab, Rap1, Rap2, TC21, Ran, Ral, Rheb, Arf, Arl, etc. and more recently probably the β-subunit of the recently identified signal recognition particles -Rezeptoren SRβ be counted; the Ras homologues (RHO) such as Rho, Rac, Cdc42 etc., but also other proteins such as the translation elongation factors EF-Tu, EF-G and EF-T or proteins of the family of heterotrimeric G proteins with the homologous G _α subunit. All these proteins have in common that they undergo critical GTP hydrolysis-induced conformational changes. The nifH gene product encoding the nitrogenase Fe protein is another protein that has a nucleotide-binding fold like the G proteins, but unlike the G proteins binds ATP and hydrolyzes to the same superfamily of "P-loop" -containing nucleotide triphosphate -Hydrolasen is counted.

A representative of the human ras proteins is the ras proto-oncogene product p21. In the following, p21 ^ras is described in more detail as a representative of 'classical' MNBPs, as this was the key protein whose switch function was first studied crystallographically by X-ray crystallography, ie in the GDP- and GTP-bound state and also as heteroprotein complexes with regulator or effector proteins Functional mechanisms were analyzed in detail structurally.

p21 ras: One of the identified proto-oncogenes is the ras gene. There are several ras genes (derived from rat sarcoma) whose most common representatives are termed about 85% sequence identity as H-ras, K-ras and N-ras, and which are grouped together as p21 ^ras gene products. Even in the subset of Ras and Ras-like proteins consisting of M-Ras, R-Ras, Rap and Ral, these still have an amino acid identity of 40-50%. The monomeric human ras gene product p21, which is referred to below as p21 ^ras or only as Ras, is a membrane-associated signal transduction protein. Said GTPase acts as a binary switch which switches between the active "on" (GTP bound) and inactive "off" (GDP bound) states sunk. This elementary switching mechanism does not only apply to Ras, but is based on all the protein families mentioned here. Only the GTP-bound active conformation allows Ras to bind and activate multiple downstream effectors. This active state initiates in eukaryotic cells by means of the effector molecules, inter alia, a cascade of "mitogen activated protein kinase" (MAPK) steps, which transmit a signal to the nucleus via various phosphorylation reactions by activating "extracellular signal-regulating kinases" ERKs. As such, p21 ras represents a central component in the signal transduction chain that is primarily responsible for processes such as cell growth and proliferation, differentiation and survival.

The p21 ^ras gene product has been examined by both X-ray and GTP-bound conditions by X-ray diffraction analysis (DeVos et al., 1988; Pai et al., 1989; Pai et al., 1990; Milburn et al., 1990 Tong et al., 1991), so that structural conformational changes are relatively well understood. The oncogenic mutant transformation has also been characterized crystallographically (Krengel, 1990, Tong et al., 1991, Franken et al., 1993). Despite the precise knowledge of p21 ^ras structures, conventional oncological research, with respect to inhibitor design, is largely limited to preceding or downstream steps in the Ras-dependent signal transduction chain (see, eg, Adjei, 2001). Examples include the farnesylation step of farnesyl transferase (Kohl et al., 1994, Park et al., 1997, Long et al., 2002) or MEK blockade by compounds such as 2- (2-chloro-4-iodo-phenylamino ) -N-cyclopropylmethoxy-3,4-difluoro-benzamide (Sebold-Leopold et al., 1999). However, the Ras-driven signal transduction pathway is not only involved in carcinogenic transformations but can control a variety of processes.

0 Without external activation, Ras is primarily in the inactive state since GDP is tightly bound and the dissociation rate corresponding is low. Activation of Ras happens when the GDP-bound Ras protein from so-called "guanine nucleotide exchange Factors "(GEFs) which is the dissociation or exchange of the nucleotide accelerate. Because there is much more GTP than GDP in the cell Ras is mainly GTP bind and thus put into the active state.

GEFs act as positive regulators of Ras (and quite generally for the entire class of NBPs described here), while "GTPase-activating proteins" (GAPs), which preferentially interact with the active (GTP-bound) form, are either the effectors of the Ras function or its negative regulator. The exchange of guanosine nucleotides is stimulated by GEFs, such as Sos or Cdc24, which accelerate the dissociation rate of the nucleotide and is inhibited by guanine-nucleotide dissociation inhibitors (GDIs). This means that Ras activation is modulated by the local balance of GEFs and GAPs. This is in 1 shown schematically. On the other hand, the interaction of Ras and GAP usually accelerates GTP hydrolysis by several orders of magnitude so that GAP ensures that Ras is predominantly in its GDP bound "off" conformation.

In the active GTP-bound conformation, Ras transmits a signal to an effector molecule, which can lead to cell growth and proliferation as well as apoptosis. The interaction of p21 ^ras with its effector Raf activates an entire kinase cascade (MAPK signaling pathway). In addition to the MAPK-dependent transmission pathway, there is also a MAPK-independent pathway that mediates Ras-induced cellular responses and ultimately controls diverse transcription factors. The Ets, c-Myc and c-Jun proteins are those Ras-dependent transcription factors necessary for cellular transformations in vitro and in vivo. The nuclear transcription factor κB (NF-κB) is activated in the case of oncogenic Ras, which is associated with protection against apoptosis. Phosphoinositide 3-kinase (PI3-K) is, in addition to Raf, another direct Ras effector whose γ unit in complex with Ras has also been thoroughly characterized by X-ray crystallography (Pacold et al., 2000). This effector activates the serine / threonine kinase Akt, also known as protein kinase B (PKB), which also plays a role in protection against apoptosis.

traditionally, it was assumed that the Ras protein is only active on the outer plasma membrane. Recently the evidence condenses that at least H-Ras not only as a plasma membrane-associated component acts, but also on intracellular membranes, such as the endoplasmic membrane Reticulum, the endosome membrane and the Golgiapparates, to the signal transduction processes contributes (Bivona et al., 2003). Through these new signal transduction pathways, Ras a surprise multifunctional role, giving this protein a far more important key function, as already accepted, assigns.

The (de) phosphorylation of the bound nucleotide leads to significant conformational changes of the protein as in 2 shown. These hydrolysis-induced conformational changes take place primarily in two regions, which are generally referred to as switch I and switch II regions. The Flexibility of certain polypeptide regions and the concomitant ability to control certain regulatory switch processes is a central feature of said GTPases and also of certain ATPases.

The nucleotide binding environment in these regions is characterized by highly conserved amino acid sequences that include both residues important for nucleotide binding and ligands of the nucleotide-coordinating metal atom. These regions are described in more detail below. A schematic representation of the Mg ²⁺ coordination structures for the GDP- and GTP-bound conformation of p21 ^ras is given in 4 shown. These illustrate that in the GTP-bound form Mg ² + coordinate two water molecules located in the two axial positions. Furthermore, the protein side-chain hydroxyl groups of serine-17 and threonine-35 coordinate the metal in the equatorial plane. Also important is the bidentate Nukleotidkoordinationsstruktur with contacts to oxygen atoms of two P _β - _γ P and phosphate groups in the two remaining metal equatorial positions. As a consequence of GTP hydrolysis, the Mg ²⁺ : γ phosphate contacts must be ^disrupted , including complex contacts with the protein backbone, leading to significant conformational changes. Thus, after the cleavage of the terminal phosphate group only a monodentate nucleotide coordination structure and the binding of the threonine-35 Residuums no longer exists which leads to the collapse of the switch I region. Furthermore, the bond between the γ-phosphate group and glycine-60 is abolished, which in part initiates changes in the switch II region. The liberated metal-ligand positions are filled by water molecules, so that a total of four water molecules in the GDP-bound form coordinate the Mg ²⁺ atom, two in the equatorial plane, the other two as axial ligands. In addition, the invariant serine-17 and an equatorial-bonded oxygen atom of the terminal nucleotide P _β phosphate group complete the octahedral inner coordination shell of the metal, in GTP and GDP bound form.

This shows that the divalent nucleotide-bound metal atom plays a central role in the Ras-induced signal transduction chain, since this contributes significantly to the coordination-related conformational changes. This interpretation is supported, inter alia, by the supposed mechanisms of action of GEFs, which accomplish the increased nucleotide exchange thereby; that the nucleotide Mg ²⁺ binding is blocked by the displacement of the residues that usually coordinate the metal atom and the terminal phosphate group (specifically, the switch I region) and the introduction of hydrophobic and acidic residues, which leads to the weakening of the nucleotide bond , Thus, the metal atom, which contributes significantly to the strength of nucleotide binding, has a key position in the regulatory mechanisms of G proteins.

The characteristics described above for p21 ^{ras of} the tertiary structure of the catalytic nucleus, the so-called nucleotide binding folding, can essentially be found in all G proteins. However, even slight steric changes can obviously lead to significant changes in factors such as the nucleotide dissociation rate.

Apart from the possibilities already mentioned above for influencing the Ras-dependent signal transduction chain, there are currently a number of further approaches for the direct modification of oncogenic p21 ^ras , which is (1) the proposal of the use of the non-steroidal anti-inflammatory agent (NSAID) sulindac sulfide (Herrmann et al., 1998), (2) the replacement of guanosine 5'-triphosphate with diaminobenzophenone-phosphoroamidate GTP (DABP-GTP) and similar compounds that modify the nucleotide binding pocket (Ahmadian et al., 1999) and (3 ) the development of peptides that, similar to the arginine finger in the GAP mechanism, contribute to an arginine-like residue for hydrolysis stimulation (Scheffzek et al., 1996, Scheffzek et al., 1997, Scheffzek et al., 1998a). X-ray diffraction analysis of the Ras · p120 ^GAP complex has shown that amino acids of both switch I and switch II regions of Ras make numerous interactions with corresponding amino acids of GAP. The most significant structural modifications are to be observed in the switch II region, eg this very mobile, hypervariable region in Ras is well defined in the Ras · RasGAP complex. It is also important to note at this point that the use of an arginine finger to stimulate the rate of hydrolysis is not limited to Ras but is the underlying mechanism of many of the protein families mentioned herein. On the other hand, the replacement of guanosine 5'-triphosphate has shown that the rate of hydrolysis stimulation can also be significantly increased by an additional functional group such as a 3,4-diaminobenzophenone phosphonoamidate group on the terminal phosphate of GTP. By contrast, sulindac sulfide binds Ras directly in a non-covalent fashion, with the result that the p21 ^ras binding domain appears to inhibit with Raf, which also allegedly interferes with nucleotide exchange with CDC25 and acceleration of hydrolysis by p120GAP. The molecular bonding position and geometry of sulindac sulfide, however, is unknown. Another approach, described in more detail below, is the replacement of the nucleotide-bound Mg ²⁺ ion by Mn ²⁺ (Schweins et al., 1997).

2. Summary the invention

Signal transduction processes that deviate from the norm are the cause of a variety of diseases. Key components of these processes are certain guanosine-binding (and sometimes adenosine-binding) proteins. The conformation of these proteins is largely determined by structural rearrangements in two flexible domains, the so-called switch I and II regions, which interact with regulatory and effector molecules regulated. Decisive for the conformation is the nucleotide that is bound in the presence of a divalent metal ion, usually Mg ²⁺ . The patent application relates to the use of the transition metal oxo-vanadium (IV) or vanadyl as a modulator of the switch family of the characteristic superfamily of 'classical' P-loop GTPases and ATPases contained in the catalytic core. These GTPases and ATPases are characterized by having a large homology of metal nucleotide binding fold. Substitution of the nucleotide-bound Mg ²⁺ cation by VO ²⁺ leads to significant changes in the ligand structure of these nucleotide-binding hydrolases, which decisively influences the switches I and switch II regions, which are essential for cellular signal transduction. Thus, VO ^{2+ is} able to modify the nucleotide binding fold such that it can modulate (control) the critical switch regions. This fact has important implications, in particular for the interactions of said GTPases and ATPases with effector proteins but also regulatory proteins, such as the guanosine exchange factors, which are very sensitive to rearrangements in the switch regions. In particular, such a modification of the "inner-sphere" metal coordination structure has a decisive influence on the modulation of the Ras oncogene product p21 ^ras and the associated signal transduction chain.

3. Detailed Description of the invention

The The invention relates to a method that directly relates to the switch regions certain GTPases and ATPases can influence. Such an influence the switch regions, which was previously not possible without changes the amino acid sequences of these regions, is the most straightforward approach to controlling downstream and partially GTPase / ATPase controlling protein processes et al directly influence the Ras-enabled signal transmission path can take.

Nitrogenase Fe protein: Like Ras, the nitrogenase Fe protein belongs to the same superfamily of "P-loop" -containing nucleotide triphosphate hydrolases. The amino acid sequence homology and similarity of tertiary structures between the ATP-binding Fe protein and other 'classical' MNBPs, and especially the Ras-like GTP-binding proteins, specifically in terms of the active-site topology, have been reported in various publications (Georgiadis et al., 1992; Howard, 1993; Rees et al., 1993; Howard & Rees, 1994; Howard & Rees, 1996; Burgess & Lowe, 1996; Rees & Howard, 1999; Vetter & Wittinghofer. 1999, Jang et al., 2000), ie, the Walker sequence motifs are also observed in the Fe protein, so that similar principles of operation also apply to the Fe protein. A sequence comparison of the key nucleotide binding regions for p2lras and the nitrogenase Fe proteins of two different organisms is presented in 5 shown. However, this similarity is not limited to the primary and tertiary structure, but also relates to a functional analogy that causes multiple conformational changes to regulate the functioning of the protein. The Fe protein can also be referred to as a switch protein because it enables the nitrogenase complex only in the ATP-bound form to reduce substrate. However, the ATP-bonded state is needed to initiate electron transfer steps, several of which are necessary for substrate reduction. Also striking is the generally very narrow MgADP binding in the two proteins, so that the free protein is present primarily in the diphosphate-bound nucleotide conformation.

The nitrogenase Fe protein is one of two proteins that represent the minimal functional unit of certain microorganisms to catalyze N ₂ into NH ₃ . The Fe protein serves as a specific electron donor for the nitrogenase MoFe protein harboring the catalytic center. The Fe protein is a γ ₂ -Homodimer, and acts as a natural reducing agent of the MoFe protein, necessary to fix dinitrogen. Each of the two Fe protein subunits binds an adenosine nucleotide molecule. The hydrolysis of MgATP into MgADP leads to considerable conformational changes, which until now have been observed only indirectly through changes in the properties of the [4Fe-4S] cluster contained in the Fe protein (see: Burgess & Lowe, 1996).

Another important parallel between the two said proteins is the aluminum fluoride (AlF _x -bonding behavior). AlF _x is usually considered to be a compound that represents the transition state in hydrolysis, with AlF _{x being} the terminal phosphate group of the pentacoordinate triphosphate-bound state mimict in inhibitory Interestingly, Ras (Mittal et al., 1996) and Ras-related proteins (Kahn, 1991) bind AlF _x only in the presence of GAP th is also known for nitrogenase. Here, even under "turn-over" conditions, the individual nitrogenase protein components do not form stable AlF _x nucleotide complexes (Renner & Howard, 1996, Duyvis et al., 1996). This is another indication that the two metal nucleotide binding environments behave almost identically because even the intermediate transition states show high homology.

Another similarity between the nitrogenase Fe protein and Ras is the stimulation of ATPase or GTPase activity by other proteins. In the case of the Fe protein this is the MoFe protein as for p21 ^ras the GAPs, with the difference that despite the binding of MgATP the Fe protein has no or negligible intrinsic ATPase activity in the absence of the MoFe protein as opposed to the not insignificant intrinsic activity of Ras without GAPs. This gave rise to the speculation that the MoFe protein either contributes directly to MgATP hydrolysis by providing a catalytic group (which would be similar to the arginine finger of GAP in the p21 ^ras · RasGAP complex) or that it requires a conformational change with such a catalytic group placed in the correct position or orientation to initiate the hydrolysis reaction (Burgess & Lowe, 1996).

However, there are a few points that need to be mentioned that are different and concern the nucleotide binding pocket, so one of the two "β-sheets" in the Fe protein is antiparallel to the direction it has for p21 ^ras ( 3 ). This results in the metal nucleotide ligands for the Fe protein being at the end of these "β-sheets" while they are at the beginning for p2lras. Furthermore, the Fe protein has an additional α-helix after the "β ₂ -sheet", which does not possess p21 ^ras .

The X-ray crystal structures for the Fe protein of Azotobacter vinelandii and those of the human p21 ^ras protein, which illustrate the similarity of the proteins, is in 6 shown. The equivalent amino acid to the switch II stabilizing glycine-60 residue present in the Ras protein is conserved glycine-128 in the Fe protein (see amino acid sequence in FIG 5 ) which in the triphosphate-bound state structurally also contributes to the correct orientation of the γ-phosphate group. All this suggests that the nucleotide binding structure of the nitrogenase Fe protein may serve as an analog for the G proteins and that findings obtained for the Fe protein may be transferred to G proteins to a high degree.

Members of the Ras protein family were the first switch proteins to be investigated not only by X-ray structure but also spectroscopically by magnetic resonance techniques in the ADP and ATP-bound states by substitution of the nucleotide-bound metal atom, with X-ray diffraction with both Mg ²⁺ and Mn ²⁺ at the Nucleotide binding site was analyzed (Schweins et al., 1997). On the other hand, the other method, in principle, can only be studied with paramagnetic metal centers. In connection with p21 ^ras , no other paramagnetic metal ions have yet been used for spectroscopic characterization except for Mn ²⁺ .

The direct coordination structure of the nucleotide-bound divalent metal ion (naturally, as for p21 ^ras, a Mg ²⁺ + atom) is essentially identical to the corresponding GDP for the ADP-bound conformation of the Fe protein, except for slight changes in the outer coordination shell. bound structure of p21 ^ras . The superposition of the "p-loop" region of p21 ^ras with that of one of the two Fe protein monomers, using the Cα atoms of the phosphate binding region, clearly shows that not only the positions and the orientation of the nucleotide but also partially position and orientation of the surrounding residuals are similar (Jang et al., 2000). The ATP-bound structure of the Fe protein is less thoroughly investigated. The only X-ray structural studies so far are for two Fe protein: MoFe protein complexes and these do not correspond to the conventional active state; these are the structure bound at 3.3 Å resolution according to the transition state with ADP and AlF _x (Schindelin et al., 1997) and a modified Fe protein at 3.0 Å resolution (Chiu et al., 2001) without, however, the electron densities of Oxygen atoms of the water molecules have been identified, so that the corresponding water ligand coordination for the Fe protein is still undetermined. The latter structure has an omission at position Leu127 (ΔL127), ie in the switch II region, and thus does not represent the native coordination structure. Interestingly, Leu127 is in the corresponding case for the Ras protein, close to a residue that is responsible for one of the malignant transformations (Q61). Also, this structure shows some homology to Ras by being fixed in the Mg ²⁺ triphosphate nucleotide-bound form and even binding with the MoFe protein (GAP) does not stimulate hydrolysis.

Although these two structures only partially reflect the natural ATP-activated structure, they nevertheless suggest that the Fe protein may serve as a good model between the various MNBPs also at the molecular level. The similarity of the X-ray crystal structures of the hydrolysis transition state for the respective diphosphate complex with AlF _x for the two proteins, p21ras and the nitrogenase Fe protein, is in 7 demonstrated.

Metal Nucleotide Coordination Structures: Of course, the nucleotide in said "classical" MNBPs binds in the presence of a divalent metal cation, usually Mg ²⁺ . In this case, the Mg ²⁺ ion is arranged in an octahedral coordination environment with the nucleotide phosphate group (s) in the equatorial plane. This is the case for this class of MNBPs for tri- and Diphosphatnukleotide, wherein a bidentate metal Nukleotidkoordination present for triphosphate nucleotides (P _β and P _γ), while after removal of the terminal phosphate group for Diphosphatnukleotide only a phosphate group (P _β) is bound. In each case one of the oxygen atoms of the nucleotide P _β and P _γ phosphate groups of GTP / ATP coordinate the metal in equatorial metal positions. Such a coordination structure can be observed for the cellular form of p2lras as well as in the wild-type Fe protein. The oncogenic transformation of p2lras assumes the same or at least a very similar metal coordination structure.

Substitution of the metal ion has recently been described for p2lras, and it has been suggested that such a possibility represents an interesting "target" for the development of an anti-cancer agent (Schweins et al., 1997). For this purpose, Mn ^{2+ was} used in this case, which showed that substitution with Mn ^{2+ is} able to significantly increase both the intrinsic k _cat rate of p2lras and the GAP-stimulated GTPase rate. This is possibly the result of an increase in the pK _a value of the terminal nucleotide phosphate group. Two more divalent metals have been tested, Zn ²⁺ and Cd ²⁺ , but both were unable to replace the strongly bound Mg ²⁺ ion. The importance of the nucleotide-bound divalent cation has been known for a long time; For example, the type of this cation is a crucial regulator in the GTP-binding tubulin protein (Jemiolo & Grisham, 1982).

Oxo-vanadium (IV) is a rather rare divalent metal ion with respect to biological systems, and has only recently gained more interest. Although it seldom leaves an active protein on its own, it has been shown to be capable of replacing the naturally-bound metal in a variety of metalloproteins (Chasteen, 1983). It has also recently been shown that the nucleotide-chelating metal ion, oxo-vanadium, very effectively competes with Mg ²⁺ for the binding site in 'classical' P-loop containing proteins (Petersen et al., 2002). The resulting IC ₅₀ value (value at which the activity has fallen by 50%) gave about 4.0 mM. This shows that VO ²⁺ indeed replaces the nucleotide-bound Mg ²⁺ ion.

Vanadium Chemistry and Molecular Structure: Vanadium is a transition metal that has recently gained more and more interest due to its unusual stereochemistry as well as its relevance to biological systems, as it forms the catalytic center in at least two enzymes; these are the bromo-chloro peroxidases and the vanadium-containing nitrogenase (but this is not to be confused with the use of vanadium in the present patent application, which is based on the more commonly used molybdenum-containing nitrogenase). Vanadium is a physiologically important trace element that is also present in foods in low concentrations (below 1ng / g). The daily vanadium intake of the US population is between 10 and 60 μg. Vanadium in conjunction with physiologically relevant systems exists in essentially two oxidation states, as tetravalent V (IV) and pentavalent V (V), and forms cationic (V = O) ²⁺ (vanadyl) or anionic (VO ₃ ) ^- (vanadate ) Oxo complexes.

In the following, the term vanadyl or vanadyl complex is to be understood as meaning a coordination compound having a central vanadium atom or ion, where vanadium has an oxidation state of +4 and forms a double bond to an oxygen atom (oxo vanadium) and may have different ligands, such as H ₂ O, substrate atoms or protein residues. It should also be expressly included such Oxo-vanadium compounds having one, maximum two, ligands that are not hydroxyl groups but may represent other single ions, such as Cl ^- , or 'small', not bulky compounds, but all have to meet the criterion in that they do not significantly affect the conformational ^geometry described here for VO ²⁺ .

In many cases, oxo vanadium (IV) binds in an octahedral coordination geometry with the vanadium oxo atom representing one of the axial ligands. The vanadium-oxo direction corresponds to the molecular z-axis. In axially symmetric systems this corresponds to the g ^∥ -bzw. A ^∥ axis, where g ^∥ and A ^{∥ is} the molecular g-factor or ⁵¹ V hyperfine (HF) component along the V = O connecting axis.

Vanadium (IV) in the form of oxo vanadium or vanadyl [V (IV) = O] ²⁺ is, in contrast to the commonly used vanadate, paramagnetic (electron spin S ≠ 0) and can therefore be used as a spin probe for the spectroscopic investigation of coordination environments serve. Such spectroscopic methods include "electron paramagnetic resonance" (EPR) and "electron nuclear double resonance" (ENDOR). Furthermore, the most commonly occurring isotope of vanadium, ⁵¹ V (99.75% has natuer frequency), a nuclear spin of I = 7/2, which in EPR spectra of aqueous solutions leads to 2I + 1 = 8 ia of well-resolved HF lines. In frozen solutions, these 8 lines are then split into powder-type parallel (g ^∥ , A ^∥ ) and perpendicular (g ^⊥ , A ^⊥ ) RF components for the usually axially symmetric spectrum of the vanadyl center.

This has recently been used to spectroscopically study the hydrolysis-induced conformational changes of 'classical' MNBPs with externally added vanadyl directly at the nucleotide binding site by means of EPR (Petersen et al., 2002). These studies showed that VO ²⁺ is bound in a distorted octahedral coordination geometry. It has also been shown that vanadyl inhibits nitrogenase substrate reduction in a manner unrelated to the inhibition mechanism of proteins, such as the inhibition of phosphatases by vanadate.

Methodology: The nucleotide binding environment of protein bound vanadyl has been characterized by high resolution ENDOR spectroscopy. The interaction of a paramagnetic center, in this case VO ²⁺ with unpaired electronic spin S = 1/2 (M _S = ± 1/2), with a nuclear magnetic core of nuclear spin I = 1/2, which in this case for the following two nuclei, ³¹ P and ¹ H's, holds, causes two ENDOR resonances, ν ⁺ and ν ^- , if the condition ν _n > | A / 2 | is satisfied, in first approximation at frequencies of ν ± = ν n (i) ± | A (i) / 2 | (1) where A is the superhyperfein interaction of the paramagnetic center with the nuclear magnetic species (i), where i in this case is either ³¹ P and ¹ H, and ν _n (i) is the corresponding magnetic field dependent nuclear Zeeman frequency of the corresponding nuclear species results from ν n = g n β n B 0 / h (2) where B _{0 is} the magnetic field, g _{n is} the core g-factor, β _{n is} the nuclear magneton and h is Planck's constant. For ν _n <| A / 2 | applies ν ± = | A (i) / 2 | ± ν n (i) (3), so that in this case the ENDOR signal pair is centered at half the superhyperfine (SHF) coupling constant A (i) and the two ENDOR lines are separated by 2ν _n (i). From the size of the SHF coupling constant important conclusions can be drawn on the nucleotide binding environment.

³¹ P Couplings: To characterize the vanadyl nucleotide coordination environment, angle-selective ENDOR spectra were recorded in different frequency regions. 8th shows the nucleotide phosphate coupling for ATP-bound vanadyl ions in the presence of the reduced nitrogenase Fe protein, recorded over a wide frequency range, to observe also larger metal-phosphate couplings, typical of covalently bonded nucleotides. Here, VOCl _{2 was} mixed with an excess of nucleotides as previously described (Petersen et al., 2002) to ensure that the entire VO ^{2+ is} nucleotide bound. An ENDOR signal pair with resonances at 7.0 MHz and 18.3 MHz is characteristic of SHF couplings to the equatorial, directly attached phosphate groups, for 'classical' MNBPs usually couplings of the P _β group for the diphosphate bound state and P _β and P _γ Represent couplings for the triphosphate-bound state. The _Pβ phosphate group does not normally interact directly with the metal ion. For these resonances the condition ν _n <| A | and the SHF coupling can thus be calculated using Equation (3). The determined SHF coupling | A | of 25.3 MHz is in good agreement with studies on inorganic VO ²⁺ nucleotides (Mustafi et al., 1992), a VO ²⁺ triphosphate complex (Dikanov et al., 2002), and nucleotide-bound 'classical' MNBPs such as F ₁ -ATPase in the presence of ATP and ADP (Buy et al., 1996). Thus, the large phosphate coupling can be attributed to directly bound equatorial phosphate groups of the nucleotide. These couplings are dominated by the isotropic SHF interaction, so that there is little difference between perpendicular and parallel magnetic field positioning.

Moreover, and the more important observation in the context of the patent application is that a second ENDOR "feature" is observed to have a "doublet" -like structure. This signal, by being symmetric about the nuclear Zeeman frequency of ³¹ P and its field dependence, can be unambiguously attributed to a coupling of vanadyl with a phosphate group. For this second set of resonances, the condition ν _n > | A | from which it follows that there is a further, much smaller phosphate coupling compared to the above. The coupling is shown in 8th through the horizontal bars, which are arranged symmetrically around ν _n with | A | = 0.31 MHz. Such a coupling has never before been observed with ENDOR spectroscopy. The only potentially comparable measurements have been recorded with electron-spike echo envelope modulation (ESEEM) spectroscopy. The first such measurements are in vivo studies of bone samples from rats after six weekly administration of bis (ethylmaltolato) oxovanadium (IV) (Dikanov et al., 1999) in drinking water or after four days of intraperitoneal injection of bis (picolinato) oxovanadium (IV) (Fukui et al., 1999). However, these signals were not assigned to any particular ³¹ P coordination structure. A second recent work by the former group investigates the coordination structure of VO ²⁺ triphosphate complexes (Dikanov et al., 2002). In this case, a similarly low ³¹ P coupling is observed, which in this case was attributed to a phosphate group which directly coordinates the metal in the single free axial position (the other axial position trans to the phosphate group is occupied by the fixed oxygen atom of oxovanadium ). It should be noted that this interpretation is based on the assumption that there is no axially coordinated water molecule or any other direct axial ligand. Such an interpretation certainly does not apply to the VO ²⁺ coordination structure here, as shown below.

The bottom track in 8A shows the corresponding ³¹ P-ENDOR spectrum recorded with the EPR field position centered on the M _I = -5/2 parallel RF resonance. With these two resonance positions one has the ³¹ P components of the SHF tensor in the direction of the molecular g main axis system. This coupling with a more than 80-fold weaker coupling constant is certainly attributable to no interaction of a directly coordinated equatorial nucleotide phosphate group. Thus, it can only be assigned to either a non-directly coordinated equatorial or an axial phosphate group. The former can be ruled out because the two spectra for vertical and parallel field positioning clearly have characteristics that point to axial coordination. This interpretation is supported by the fact that only when B _{0 is positioned} on a vertical EPR absorption line is a single ENDOR line pair observed corresponding to the A ^⊥ SHF coupling. In the case of equatorial coordination, both parallel and vertical SHF couplings should be observed with magnetic field positioning B ₀ on a vertical EPR absorption line (see, for example: Mustafi et al., 1992, Petersen et al., 1996). The characteristics of the observed small ³¹ P-ENDOR resonances confirm the presence of an axial phosphate group regardless of whether it is directly or indirectly coordinated to the central VO ²⁺ cation. It should be noted that, at a site where Mg ²⁺ naturally contains water molecules, there is an axially or quasi-axially coordinated nucleotide ^{phosphate group} in the presence of VO ²⁺ .

³¹ P-ENDOR spectra were also recorded for ADP as a coordinating nucleotide. The comparison of ³¹ P-ENDOR spectra for vanadyl in the presence of reduced Fe protein with bound ADP or ATP is in 9 shown. This comparison shows that the small phosphate coupling for ADP is completely absent, indicating that this coupling is a specific characteristic of the ATP-bonded form.

¹ H Couplings: To study the proton environment of the nucleotide-bound VO ²⁺ center in the presence of the reduced Fe protein, ENDOR spectra have also been recorded in the region around the Zeeman frequency of protons. The ¹ H ENDOR spectrum with the excitation field B ₀ centered on the M _I = -3/2 vertical HF line is in 10 shown. This spectrum clearly shows a pair of lines symmetrically arranged around ν _n ( ¹ H). The splitting of the line pair is 2.69 MHz. In the ¹ H-ENDOR spectrum after exchange of H ₂ O for D ₂ O in the buffer solution (mean spectrum in 10 ) this pair of lines is missing. This can also be clearly seen in the difference spectrum between preparations in H ₂ O and D ₂ O (lower spectrum in 10 ) meaning that this pair of lines must be attributed to an exchangeable proton (or protons). In the difference spectrum, even more exchangeable protons are observable, but with much lower SHF coupling constants. At this point it should also be pointed out that in particular the region for ENDOR splits of 2.5-3.1 MHz, even if this is 10 not immediately apparent, has a very complex structure. Presumably, this region represents a region in which different ENDOR components of different types of protons overlap. Since these are not relevant for the local patent application here will not be discussed in more detail here.

In addition, ¹ H-ENDOR spectra were also recorded with a field position on the M _I = -7/2 parallel RF line. These show an ENDOR line pair with a splitting of 5.53 MHz. This pair of lines, together with the other of 2.69 MHz for the vertical field position, can be attributed to the A ^∥ or A ^⊥ SHF component of protons of an axially coordinated water molecule. The characteristic of such protons is that A ^∥ ≈ 2A ^⊥ , which is sufficiently well-satisfied in the present case. From this it can be calculated, the isotropic portion of the SHF-coupling of the thus close to zero should be as vanadyl for the spin density of the unpaired electron of the metal ion is 3d ¹ d located in the _xy orbital. In fact, the isotropic component is calculated to A ^iso = +0.05 MHz. For such a low A ^iso value, it is allowed, from the purely dipolar SHF tensor components which can be calculated from this, the distance between the protons and the unpaired spinene to determine the The distance thus determined is 3.06 Å, which is in good agreement with literature values for axial hydroxyl protons. Thus, the ENDOR proton couplings can be clearly attributed to an axially coordinated water molecule. For Fe protein bound to VO ²⁺ -ATP, nearly identical ¹ H-ENDOR couplings are observed, from which a VO ²⁺ - ¹ H distance of 3.05 Å is calculated. This illustrates that such a water molecule is present in both ATP and ADP bound states. This also means that if at this metal ligand position a water molecule is bound, not an additional axial phosphate group can be bound.

It is important to note at this point that this water molecule can only be observed at a relatively low pH around pH 6.5. At neutral pH, corresponding ¹ H-ENDOR line pairs are absent so that no axial water molecule can be present at this pH. One might be tempted to assume that, at least for this case, a nucleotide phosphate group is directly coordinated in the axial position. However, this interpretation is almost excluded, since regardless of whether an axial water molecule is bound or not, the small observed ³¹ P coupling remains largely unchanged. Thus, it must be concluded that although this ³¹ P coupling has considerable axial character, and although a similarly small coupling was attributed to a directly axially coordinated phosphate group, this is inconsistent with the axial water molecule observed here. The lack of ³¹ P-ENDOR resonances with a low coupling constant for the protein with ADP shows that only in the presence of ATP is such an unusual axial coordination structure of the terminal phosphate group assumed. This means that for the inactive configuration with ADP no such dramatic changes occur as for ATP and thus presumably the signal transduction processes in the "off" state are not affected. Overall, it should be noted that the VO ²⁺ coordination environment shows a pronounced pH dependence.

The unusual nucleotide coordination structure is in 11 again shown schematically. Whether the axial nucleotide phosphate group VO ^{2+ is} indeed directly coordinated in a position trans to the position of the vanadyl oxo ligand or only has axial characteristics is difficult to definitively establish for a phosphate group because the interaction with such a ligand is generally weak (2.3 Å for an axially coordinated Water molecule) as well as the H ₂ O coordination for the low pH form (pH 5.0 to 6.8). Thus, it is almost impossible to say whether the trans position at neutral pH is occupied by a ligand or not, meaning that between 5-coordinated or 6-coordinated VO ²⁺ complexes neither by the EPR "spin Hamiltonian" parameters nor by the ENDOR characteristics can be clearly distinguished.

Regardless of whether the axial ligand is now a direct metal-phosphate contact or some other type of indirect axial phosphate coordination, most likely from the terminal nucleotide phosphate group in the triphosphate-bound state, the fact that such a coordination structure is capable of axial VO ²⁺ coordination position, which should normally be occupied by Mg ²⁺ of water molecules. This has a decisive influence on the switch regions of the protein since, as exemplified for p2lras, the bridging water molecules are normally responsible for critical contacts between the metal ion and, inter alia, two protein aspartate residues in the switch regions (see 4 ). Thus, the possibility opens up to modulate at least the switch II region of the large protein family, consisting of the above-classified GTPases and ATPases. Furthermore, such a coordination structure would twist the terminal nucleotide phosphate group from the position normally assumed to be Mg ²⁺ towards the axial coordination position, which also affects other phosphate ligands (such as Glu60 in p21 ras, which may be cleaved by this significant rearrangement). Consequently, at least the switch II region is decisively influenced by the substitution of Mg ²⁺ by VO ²⁺ .

It could give the impression that VO ²⁺ primarily modulates the switch II region. However, the two switch regions generally do not work independently, presumably for all the nucleotide-binding proteins described here. It has been suggested in numerous publications that a conformational change in the switch II region also interacts directly with the switch I region and vice versa, thus resulting in a change in one switch region also leading to a reorientation of the other (Kjeldgaard et al. , 1996). Thus, the coordination structure with VO ²⁺ , which specifically affects the axial ligand position by means of one switch region, would also have effects via various hydrogen bonds on the other switch region.

An axial nucleotide phosphate coordination position could be a specific feature of the nitrogenase Fe protein, but argues against such an interpretation that a similar small phosphate coupling was also observed for the only other nucleotide binding protein studied so far in the presence of VO ²⁺ (Buy et al., 1996). Although this was not considered in the publication in question or an axial phosphate coordination was not interpreted as such, but it seems to be clearly present in the local "hyperfine-sublevel correlation" (HYSCORE) spectra. This has already been mentioned in a study on isolated triphosphate complexes (Dikanov et al., 2002). Thus, such unusual phosphate coordination is not confined to the Fe protein, suggesting that it is specific for VO ²⁺ and is generally valid at least for proteins having the sequence motifs described above and a concomitant homologous nucleotide binding topology, and can therefore also be transferred to other 'classical' nucleotide binding proteins.

Furthermore, VO ²⁺ could only adopt such a coordination structure in vitro for the purified protein. However, there are indications that such a structure is also assumed in vivo. Thus, in vivo ESEEM measurements on rat bone samples after treatment with bis (ethylmaltolato) oxovanadium (IV) showed a similar low ³¹ P coupling constant (Dikanov et al., 1999). Here, the ³¹ P signals were attributed to interaction with the phosphate group (s) of the calcium phosphate polymer hydroxyapatite [Ca ₁₀ (PO ₄ ) ₆ (OH) 2] bone portion. This suggests that, at least for the proportion of VO ²⁺ that attaches to bone, such a structure is assumed, so that it can at least be assumed that this unusual coordination structure will also be assumed in vivo. In fact, a similar in vivo ESEEM study in which bone samples from rats were also investigated after treatment with bis (picolinato) VO ^{2+ shows} a phosphate interaction albeit without a resolvable splitting, so that no measurable coupling constant was detectable (Fukui et al., 1999). , Regarding the origin of the phosphate interaction in bone material, the authors come to a similar interpretation as Dikanov et al. (1999). However, it has been shown that the coordination structure of VO ²⁺ (pic) _{2 is} very similar to that of VO ²⁺ alone. In addition, ESEEM measurements were also performed on liver and kidney. These surprisingly show strikingly similar ESEEM spectra for rats treated with VO ²⁺ SO ₄ or the VO ²⁺ (pic) ₂ complex, suggesting that the VO ²⁺ complex in these organs is no more than VO ²⁺ (pic) ₂ , but at least partially decomposed into, for example, the monopicolinato complex, so that the ligand structure is more similar to that after ingestion of the vanadyl salt alone. The same is likely to apply to other oxovanadium complexes such as the bismaltolato-oxovanadium complex (BMOV) (Peters et al., 2003).

In contrast to the substitution with Mn ²⁺ (Schweins et al., 1997), a different strategy has been followed for VO ²⁺ , which is not so much aimed at influencing the intrinsic or stimulated reaction rate, but directly at the switch regions Influence can be controlled by the metal coordination structure and thus effector and / or regulator interactions. Metal coordination structures of 'classical' MNBPs are, apart from the structure of p2lras with Mn ²⁺ , so far only known for the naturally bound Mg ²⁺ atom. However, the structure with Mn ²⁺ shows an almost identical coordination environment as Mg ²⁺ . Since otherwise no other metal coordination structures are known, the unusual coordination geometry for VO ²⁺ is so far the only one that is capable of such an influence on the switch regions. In summary, the use of VO ^{2+ is} a novel method that is potentially able to directly influence cellular signal transduction processes by modifying the switch regions of particular GTPases.

Applying the VO ²⁺ nucleotide binding structure described above to other P-loop containing proteins that also have (1) the characteristic switch I and switch II regions and (2) the conserved Walker A and Walker B motifs, would be crucial Have an impact on their regulatory effects. This would consequently open up the possibility of being able to influence a large number of cellular processes which are controlled by the said GTPases (and ATPases). This in turn can potentially be used for new therapeutic approaches to treat certain diseases, especially in carcinogenic transformations. The signal transduction chain as a "target" for the therapy of diseases has often already been described in one way or another (see for example: Levitzki, 1994, Levitzki, 1996).

It has been suspected for some time that vanadium is capable to intervene in cellular regulatory processes (see the reviews: Ramasarma & Crane, 1981; Stern et al., 1993), often without, however, between the various oxidation states of vanadium or to elaborate on intracellular redox events. Generally, the literature refers almost exclusively to the use of vanadate and the effect of vanadate on special cellular components.

Although one of the earliest studies on the antineoplastic action of vanadium appeared as early as 1984 (Thompson et al., 1984), so far there are only a relatively few studies on vanadyl in the study of anticarcinogenic effects use. This original work found that orally administered vanadyl sulfate inhibits chemically-induced mammary carcinogenesis. Since then, there have been numerous studies investigating the antineoplastic effects of vanadium but almost exclusively in connection with the use of vanadate (V) and vanadate compounds (see review article by: Djordjevic, 1995; Evangelou, 2002). Recent studies have shown that vanadate can be associated with both the inhibition of liver carcinogenesis (Bishayee & Chatterjee, 1995) and the chemoprevention of mammary carcinogenesis (Bishayee et al., 2000). Antitumor activities for vanadium (IV) complexes and other metals with 2-methylaminopyridine have been reported (El-Naggar et al., 1998). However, this was attributed to increased superoxide dismutase-like activity in combination with a decrease in glutathione peroxidase and glutathione reductase activity, with concomitant reduction of reduced glutathione resulting in H ₂ O ₂ accumulation as a possible cause of the anti-carcinogenic effect. The only other work on oxo-vanadium concerns vanadyl compounds such as 1,10-phenanthroline-vanadyl in the presence of hydrogen peroxide (Sakurai et al., 1995), bis (maltolato) vanadyl (Schieven et al., 1995), and bis ( 4,7-dimethyl-1,10-phenanthroline) sulfatooxovanadium (IV) (Narla et al., 2000, Narla et al., 2001). Following on from the success of cisplatin in cancer therapy, other metallocene complexes have also been investigated, such as the cisplatin analogous vanadozene complex, bis (π-cyclopentadienyl) vanadium (IV) dichloride (Toney et al., 1986), and their apoptosis-inducing Effect on Human Testicular Cancer by Gosh et al. (2000) has been described in detail. Recently, another vanadozene complex, vanadonna (IV) acetylacetonate of the form [VCp ₂ (acac)] (CF ₃ SO ₃ ), exhibited antinoplastic effects with dual function, on the one hand with anti-angiogenic and on the other hand with anti-mitotic properties (Gosh et al., 2001). Apart from the few vanadyl complexes mentioned above, the reason that the effect of VO ^{2+ has} long been neglected for therapeutic purposes may possibly be the lack of a plausible approach to the physiological mode of action of VO ²⁺ .

A important observation in connection with the here described Invention is also that Vanadium seems to accumulate specifically in tumor cells. Like yourself by comparing carcinogenic human breast tissue with healthy tissues (Rizk & Sky-Peck, 1984), become specific Metals, u.a. Vanadium, propagated in neoplastic Tissue attached.

4. Examples

The examples presented below are intended to assist in the understanding of the invention and are not intended to limit the scope of, in any way, and should not be construed as such. The fact that only a few examples are described here is not intended to imply that the unusual coordination structure for VO ^{2+ is} limited to these examples only, but is intended to demonstrate the range of possible applications based on a few arbitrarily chosen examples. These examples are intended solely to illustrate the fact that VO ^{2+ can} affect carcinogenesis-causing transmission pathways in various ways (there are essentially five different possibilities presented in total); In addition, there are other medical applications. Even though the field of cellular signal transduction processes is a very young field and the understanding of extremely complex relationships is rapidly increasing but only slowly a clearer, more complete picture arises, the present patent application can not go into all the details and it must, as far as these processes already established, to be referred to the relevant specialist literature. Furthermore, in the present patent application, an emphasis is placed on cancer-causing processes, but this does not mean that a large number of other not so detailed medical conditions can not also be affected by the present patent application. In the following, only some of the most common diseases to which the invention might have an influence are mentioned, and more rarely occurring diseases are omitted. Furthermore, not human medical applications are conceivable because similar functional principles in various other organisms are effective. For example, applications are also possible in animal production and in other agricultural areas.

Example 1A:

Since the ras oncogene product is responsible for approximately 30% of all human cancers, especially for 95% of all carcinomas of the exocrine pancreas and 65% of adenocarcinomas of the colon, as well as endometrial carcinomas, neuroblastomas, lung carcinomas, bladder carcinomas, pancreatic carcinomas and other cancers pertaining to the ras gene, the ras gene product is a key protein in cancer therapy. Pancreatic carcinomas are known to be particularly aggressive and show a profound resistance to existing treatments. Colorectal cancer is the second leading cause of cancer death in the US Conventional treatments for many cancers, including cancer of the pancreas and colon, are based on tumor removal, chemotherapy and radiation therapy. Therefore, there is still an enormous need for effective and less invasive or toxic methods of treating cancer.

It it is assumed that the Ras protein not only in tumor initiation and progression is involved but also in cancer metastasis and thus in cell migration and invasion. H-Ras is usually activated in urinary tract tumors and Bladder carcinomas while K-Ras activation in about 50% of all colorectal and about 70 to 90% of all pancreatic tumors was observed and also in about 30% all adenocarcinomas of the lung; N-Ras activation is partial for epithelial cancers including colorectal cancer but also for Skin melanomas and acute non-lymphatic Leukemias. Furthermore, Ras mutations have been observed for colon adenocarcinomas and Adenomas, myeloid malfunctions such as myelodisplastic syndrome, acute myeloid leukemia and chronic myeloid leukemia and also for pediatric leukemia. In relation H-Ras mutations for rhabdomyosarcoma, especially on the soft tissue tumors, Leiomyosarcoma and peripheral nerve sheath tumors and K-Ras mutations for leiomyosarcoma and liposarcoma have been reported. In cases of malignant fibrous histiocytoma show H-Ras mutations changes of Codon 12 and K-Ras mutations on codon 13.

In contrast to cellular p21 ^ras , malignant transformation through interaction with GAP, Ras can no longer be converted into the inactive, GDP-bound conformation. Thus, Ras remains permanently in the GTP-bound, active conformation and the cell growth-stimulating signal is no longer inactivated and thus uninhibited cell growth is present, which can also be regarded as a "gain of function" mutation. The oncogenic form of p21 ^ras is essentially caused by point mutations of two residues, by a change of Gly12 and Gln61 (Adari et al., 1988), more rarely Gly13 or other residues. Furthermore, insertion mutations in the "P-loop" region can lead to a likewise oncogenic phenotype.

The X-ray structure analyzes of the oncogenic p21 ^ras transformation show that the X-ray structures are broadly similar to the wild-type structure. For example, the superposition of C shows _α chains of "wild-type" and the mutagenic altered p21 ^ras protein that either the mutation of Gly12 → Val or Gln61 → Leu houses, an almost complete identity. Deviations are observed only in the switch regions, but even there they are low. Only by looking very closely are there some slight changes in the nucleotide binding environment. Of particular importance to the invention are those in the immediate Mg ²⁺ nucleotide environment. Remarkable in this connection is that the ligands of the innermost coordination shell of Mg ²⁺ in the Gln61 → His mutant are less tightly bound compared to the wild-type protein. However, one of the axial water molecules is missing so that Mg ²⁺ only seems to be 5-fold coordinated. Whether this is just a lack of X-ray structure or of general relevance is unclear. For the Gly12 → Val mutation, the distances of equatorial Mg ²⁺ ligands of the innermost shell are comparable to the wild-type protein and somewhat shorter, but the deviations from the ideal octahedral coordination structure are appreciably larger than for the wild-type type "protein. Unfortunately, all water molecules are missing in this crystal structure analysis, so that the coordinates of the axial Mg ²⁺ water ligands are also unknown. Whether the equatorial changes in the coordination structures are sufficient for the oncogenic effect of the two mutations, in particular to influence the interaction with GAP, is still unclear. It has been speculated by comparative studies with oncogenic transformations that the reaction mechanism proceeds by means of a pentacoordinated γ-phosphate transition state and that the mutations of said amino acids possibly interfere with the formation of this state and thus cause oncogenic transformation (Privé et al., 1992). , Regardless, the structure of the p21 ^ras Leu61 mutation suggested that the 50-fold enhanced binding affinity with GAP resulted from changes in the switch II region (Krengel et al., 1990). Thus, especially this region should be sensitive to oncogenic changes.

In Regarding the oncogenic transformation of Ras, these could be doubly negative Impact, as, as mentioned above, Ras on the one hand via the MAPK transmission path for the Chronic cell growth is blamed and, on the other hand, over the Phosphoinositide 3-kinase (PI-3K) -induced pathway, the other Ras effector, is associated with apoptosis inhibition. The switch regions are key regions for the Interaction with the effectors. The crystal structure of Ras with PI3-K shows that both the switch I as well as switch II region in Ras protein crucial Makes contacts with PI-3Kγ (Pacold et al., 2000). This illustrates how important a targeted Control of Ras effector interactions is.

More generally, the critical signaling pathways involved in proliferation, differentiation, and apoptosis are tightly controlled Need to become. Ras plays a key role here. It should be noted that often multiple signal inputs are needed to initiate and maintain the signal responses required in cell proliferation.

Interestingly, there are also studies of a mutagenically modified nitrogenase Fe protein whose point mutation corresponds to that of the oncogenic Leu61 mutation in Ras. This is the Asp 129 → Glu Fe protein (Lanzilotta et al., 1995). It also binds MgADP and MgATP with slightly increased affinity for MgATP compared to the wild-type protein. It does, however, show that the function, in this case the relative substrate reduction activity, is completely inhibited by the change. The Asp129 → Glu Fe protein still forms a complex with the MoFe protein. Thus, the parallelism between Ras and the Fe protein does not seem to be confined to the cellular forms, but also applies to point mutations leading to neoplastic changes for the Ras protein. The reduction or exchange of Mg ²⁺ itself can also lead to a considerable change in the guanosine nucleotide exchange rates (Hall & Self 1986, Schweins et al., 1997).

One possible application of the coordination structure described above, wherein Mg ²⁺ is substituted by VO ²⁺ , is the Ras protein, for which it is possible that in the oncogenic transformation of p21 ^ras , portions of the switch regions are influenced such that regulator and effector interactions more similar to the cellular phenotype than the oncogenic phenotype. Thus, the signal transduction chain responsible for neoplastic transformations could be modulated with respect to the downstream regulatory mechanisms by at least partially back-transforming the switch region-dependent modifications and preventing effector association dependent on the GTP-linked conformation. Ultimately, this would lead to an abort of the growth-stimulating signal. Such modification of the switch regions is consistent with previous observations of the anticarcinogenic effect of vanadyl salts.

However, it should be remembered that the current generally accepted view is that more than one genetic defect is needed to transform a primary cell, corresponding to a multi-level nature of tumor development. It is believed that the Ras GTPase alone is unable to transform a primary cell but must go hand in hand with cooperating oncogenes or the loss of function of specific tumor suppressor genes. In this regard, cell cycle inhibition by cyclin-dependent kinase inhibitors (CDKIs) could play a role. Oncogenic changes that cooperate with Ras neutralize cell cycle inhibition through various mechanisms. This is believed to be associated with the induction of CDKI proteins, p16 and p21 ^Cipl, whose loss of activity results in an immortality or at least a very prolonged life span.

Very recently, another ras or ras-like gene product was discovered, which was called Eras (Takahashi et al., 2003). This occurs in embryonic stem cells (ES) and is associated with tumor-causing properties (teratomas). Eras contains all amino acid sequence motifs like the classic Ras protein, including the two Walker motifs A and B (see 5 ), so that it can be assumed that Eras also binds Mg ²⁺ GTP and, although this has not yet been confirmed experimentally, assumes similar switching functions as Ras. Interestingly, despite high sequence homology with Ras, Eras has some altered amino acids at sites responsible for oncogenic transformations in Ras, in particular the change of glycine-12 in Ras to serine in Eras. This observation therefore provides a plausible explanation for the tumor-causing properties of Eras in ES. On the other hand, unlike classical Ras, Eras does not seem to be able to stimulate the MAPK pathway because Eras does not interact with Raf. However, Eras specifically stimulates the other effector of Ras, PI3-K, indicating that this signaling pathway also exists in ES. These results indicate that the PI 3-K-directed signal transduction pathway can also lead to neoplastic changes, at least in ES. Substitution of Mg ²⁺ with VO ²⁺ would also allow modification of the metal-nucleotide coordination ^environment in this case, which could significantly influence the transformation potential of Eras.

With regard to oncogenic transformations of Ras, it should be noted that Ras is involved not only in actual tumor initiation and progression, but also in the neovascularization (angiogenesis) of hyperplasia, which is necessary for the further development of the tumors. Oncogenic Ras stimulates a number of effector transmission pathways involved in the transcriptional activation of genes that control angiogenesis. Perhaps the best known representative of this is the "vascular endothelial growth factor" (VEGF). VEGF is one of the main mediators of vascularization of solid tumors. There are at least two different Ras-activated routes to control VEGF expression, the classical MAPK pathway, and the PI-3K effector pathway with PI-3K>PDK> PKB, which are independent Control VEGF transcription from each other. The multitude of pathways and the corresponding diverse activation processes, in which Ras participates directly or indirectly, allows the use of VO ²⁺ substitution in Ras also for the regulation of angiogenic processes or as an anti-angiogenesis agent.

Furthermore, cellular Ras also appears to be involved in Na ⁺ channel activities. Experiments on aldosterone-induced Na ⁺ channel activity suggest such a relationship. Thus, Ras could also play some role in steroid hormone-controlled salt regulation and water homeostasis in the human body. Furthermore, it should be briefly mentioned at this point that oncogenic, activated Ras is also associated with an increased intrinsic radiation resistance. Ras inactivation should therefore also lead to radiation sensitization, as observed for post-translational modifications of Ras.

Not only p21 ^ras itself is an oncogene product, but there are also Ras GAPs and GEFs that lead to neoplastic transformations. For example, the guanosine exchange factor Vav, which is presumed to be a Ras-specific GEF, is an oncogene. Vav occurs in a wide variety of myeloid, macrophage, granulocyte, mast, erythroid, T cell and B cell-derived cell lines and has been observed in spleen, lung and fetal liver. Also, GAPs in the form of the neurofibromatosis type I (NF1) gene product, which is a negative regulator of p21 ras signaling, is associated with neoplastic changes when certain mutations occur in the tumor suppressor gene NF1. The exact molecular structure for the Ras-RasGAP complex and the NF1 "tumor suppressor" gene product neurofibromin have also been analyzed by X-ray diffraction analysis (Scheffzek et al., 1996, Scheffzek et al., 1998b). At least one amino acid, Lys1423, is mutated in Glu for neurofibromin and in Gln for neurofibromas and this is generally considered to be an important catalytic factor for the dramatically reduced GAP activity. Interestingly, activating mutations of Ras are associated with mutations that inactivate the tumor suppressor gene p53 in a number of transformation assays and models of tumorigenesis. Thus, several factors must come together for malignant transformations. In addition, typical of "tumor suppressor" genes, neurofibromin expression is absent in a number of sporadic tumors in subjects without NF1, including non-NF1 neurofibromas and malignant peripheral neurilemma, neuroblastomas, malignant melanomas, and pheochromocytomas.

Since both Ras regulatory processes are critically dependent on switch I and II conformations for both GAPs and GEFs, successful treatment of neurofibromatosis type I and Vav-induced tumors by said substitution with VO ^{2+ appears to be} a sub-example of Ras-related modifications of the nucleotide binding environment , which may take influence on the regulatory interactions with Ras and thus could be a regulator of misdirected signal transduction processes.

Example 1B:

diabetes is an epidemic malfunction resulting from i.a. the Insulin secretion of pancreatic β-cells is no longer capable is to keep the glucose level in a normal frame (glucose homeostasis). Insulin resistance is the main feature of type II diabetes mellitus ("non-insulin-inducing diabetes mellitus "or NIDDM), the most prevalent form of diabetes. Although defects Homeostasis in glucose has been known for decades is the molecular mechanism responsible for the diminished whole-body glucose intake responsible is still not complete Understood. The insulin signaling chain, as far as it is firmly established, i.a. the activation of the Ras GTPase. This is done by direct activation of the Ras Gunosina exchange factor, Sos in the complex with the "growth factor receptor bound protein-2 "Grb2 and Shc or SHP-2, either directly or through the insulin receptor of the downstream adapter protein of the family of the "insulin receptor substrates-1 "like IRS-1, which can then lead to the activation of Ras.

However, glucose homeostasis is much more complex. For example, the net rate of β-cell turn-over is an important factor in the regulation of glucose homeostasis. Interestingly, apart from the small GTPase Ras, which is directly involved in the IRS-1 signaling pathway, other GTPases could play at least indirectly in pancreatic β-cells. This is quite a number of members of herterotrimeren G proteins (G _i, G _s, G _q, etc.) (vide infra) and all three main representatives of the Rho family (vide infra). Functional studies have demonstrated that at least some of these GTPases are involved in the physiological control of insulin secretion (Kowluru et al., 1996, Kowluru et al., 1997). Although these signal transduction pathways are still poorly understood, they too may be related to the present patent application.

It has been known for some time that mainly vanadate and vanadate compounds but also vanadyl and vanadyl compounds are successful for diabetes mellitus type I and type II treatment can be used (see eg: Thompson et al., 1999, Shechter et al., 2003). The key advantage of vanadium treatment is the possibility of oral administration. This has led some vanadium complexes to enter the stage of initial clinical testing for their insulinomimetic effect, albeit with very limited numbers of subjects. The increased efficiency of these vanadium complexes over the simple vanadium salts, on the other hand, may possibly be related to more stable redox properties in body fluids or less favorable cellular transport properties, but not to the actual activity. The regulatory mechanisms described in the present patent application may also be of relevance for the homeostasis of the glucose balance, since in this case too, the Ras protein is decisively involved. While the effect of vanadate in the signal transduction chain is relatively secure, the effect of vanadyl is largely unclear. Thus, if Vanadyl's modification of Ras were indeed responsible for the insulin-mimetic effect of vanadyl, it would assign Ras a central role, much more central than previously thought.

Example 1C

With The effect of insulin is directly linked to another process. It has been known for some time that insulin also represents a crucial factor in lipogenesis. studies show that mature Adipocytes and myocytes emanate from a common mesenchymal precursor and the "insulin-like growth factor-1 "(IGF-1) plays a key role in cell differentiation. IGF-1 and Insulin activate a number of specific downstream Signal transduction pathways and one of these signaling pathways, which play a role here is the Ras-stimulated MAP kinase pathway already described above. The IGF-1 receptor is located upstream of Ras and will act as another Representatives regarded for the direct stimulation of Ras, so that a modulation the Ras activity also directly influence one of the IGF-1-dependent signal transmission paths would have.

Due to the insulino-mimetic effect of vanadium and vanadium complexes, it is obvious that vanadium can also directly influence cell differentiation. The VO ²⁺ -mediated Ras modulation thus creates a possibility to directly influence the adipogenesis.

A second possibility of influencing has recently been shown and one aspect should already be anticipated here, in the context of cell differentiation, although this GTPase will be described in more detail in Example 2 below. Apart from Ras, another GTP-binding protein, the GTPase Rho, which is normally thought to be responsible for the assembly of the cytoskeleton, has been shown to act as a switch between adipogenesis and myogenesis. It has been shown that myogenesis requires excessively sustained Rho activation, while adipogenesis is a result of Rho deactivation by the corresponding RhoGAP. The influence on the switch regions by the VO ²⁺ -substitution of Rho-proteins can thus also play a decisive role in the supposedly Rho-dependent cell differentiation.

Example 2:

The Ras homologous (RHO) subfamily of monomeric GTP-binding proteins, including Rho (with RhoA, RhoB and RhoC isoforms), Rac (with Rac1, Rac2 and Rac3 isoforms) and the cell division cycle protein, Cdc42 (with Cdc42Hs and G25K isoforms) provides another example of an application where substitution of nucleotide-bound Mg ²⁺ by VO ²⁺ may play a role. The following descriptions are limited to the best characterized members of this subfamily, Rho, Cdc42 and Rac. The functionality and efficiency of RhoGTPase signaling is the linchpin for a variety of biological processes. All three proteins of this subfamily are involved in one way or another in the actin polymerization and assembly of the cytoskeleton but also in gene expression and cell cycle progression.

These switch proteins, like p2lras, are activated by binding of GTP and inactivated by GDP, and activation is attenuated by the binding of GAPs, which accelerate the hydrolysis of GTP to GDP. Overall, the members of this subfamily have a very similar mechanism of action compared to Ras. In particular, they have a homologous nucleotide binding fold with Mg ²⁺ nucleotide-dependent switch I and switch II regions. Another noteworthy observation is the parallel in the AlF _x binding behavior for the Ras and the Rho G protein subfamily; all GTPases of these subfamilies bind AlF _x only in the simultaneous presence of their respective GAPs. Another parallelism is the involvement of a residue in the hydrolysis stimulation, which plays a direct role in the terminal phosphate group coordination by means of a GAP-contributed arginine finger, possibly by stabilizing the transition state of the GTPase reaction. In this example, however, it is also important to note that some may have important structural differences to the Ras subfamily exist and these specifically concern the switch I region. Although significant conformational changes have also been observed for RhoA in switch I and II regions between the GDP and a non-hydrolyzable GTP analog (GTPγS), the threonine of the switch I region through the Thr37 backbone oxygen atom already appears in the GDP-bound state To coordinate Mg ²⁺ atom, so that, in contrast to the four coordinated water molecules for Ras, Mg ²⁺ in the GDP-bound state has only three coordinated water molecules, apart from the monodentate GDP ligand and the invariable Thr (Ser) of the nucleotide-phosphate binding region , This means that the nucleotide binding folds for bound Mg ²⁺ GDP and Mg ²⁺ GTP analog are very similar. This also means that this region for bound GDP is much more rigid compared to the typical flexibility of this region in Ras. Similar behavior has also been observed for Cdc42 and it has been speculated for the complex with RhoGDI that stabilization of the switch I threonine might be a possible explanation for the nucleotide-inhibiting effect of RhoGDI. Overall, it can be said that despite the possible differences to Ras for Rho, the two switch regions are crucial for the interactions with regulatory and effector molecules.

Since it is believed that VO ^{2+ can} very effectively replace the guanosine nucleotide-bound Mg ²⁺ and modulate the switch regions, such substitution should also result in a possibility of modulating conformationally controlled effector and regulator interactions of all GTPases of this subfamily for this family of proteins. Interesting applications for VO ²⁺ -induced conformational changes could thus be the Rho family of proteins. However, unlike p21ras in the two examples presented below, not the nucleotide-binding protein itself is the potential trigger for the development of diseases, but the interactions of the Rho GTPases, especially Cdc42, with either the upstream or downstream molecules, ie in these cases the effectors or guanosine exchange factors (GEFs).

Cdc42 is a member of the Rho-related G protein subfamily. This regulates the cytoskeleton architecture through GTP-dependent binding of effector proteins possessing a Cdc42 / Rac interactive binding motif. One of the effector molecules is the product of the 'Wiskott-Aldrich syndrome' (WAS) gene, which is associated with the assembly of actin through the activation of Cdc42. WAS, a malfunction of hematopoietic cells characterized by disorders of the Aktingerüsts is caused by mutations in human WASP, which leads to the clinical WAS symptoms such as thrombocytopenia, eczema and immune system disorders. A number of mutations can cause classic WAS or XLT ("X-chromosomal linked thrombocytopenia"), which is somewhat milder in this disease limited to platelet abnormalities. This is confirmed by a recently published study of another hematopoietic disorder, the X-linked heavy congenital neutropenia (XLN). This apparently results from a single point mutation in WASP, namely the substitution of leucine at position 270 by proline. This residual is located in a region directly responsible for contacts between Cdc42 and WASP, and may interact directly with the Switch II region of Cdc42. This suggests that the interaction of Cdc42 with the WAS protein (WASP) is controlled by conformational changes in both switch regions I and II. It has been suggested that the WASP mutations act directly on the Cdc42 / WASP interactions and thereby influence the efficiency of WASP activation by the GTPase. With a VO ²⁺ -induced inactivation or modification of the switch regions of Cdc42 one would also be able to influence the signal transduction chain for defective WASP.

Originally it was assumed that the different Rho GTPases are only involved in the regulation of the actin cytoskeleton. More recently, however, Rho proteins have not only been implicated in cytoskeletal assembly, but are also involved in processes such as control of gene expression, membrane transport, cell growth regulation, transformation, and apoptosis, and this signal transduction pathway is in some cases remarkably similar to Ras -regulated MAPK activation path. It is sometimes claimed that the Rho family proteins are the key components for the transforming action of various oncoproteins, including Ras, since at least five Rho family proteins are believed to be critical regulators of oncogenic Ras transformations. Although convincing evidence for the involvement of Rho GTPases in human carcinogenesis was lacking for a long time, it is not surprising that Rho proteins have also been associated with carcinogenic transformations, but also with cardiovascular defects. Recent studies have shown that Rho GTPases are overexpressed in human tumors. It is generally believed that misdirected activation of Rho family proteins promotes uncontrolled proliferation and invasive and metastatic properties of tumor cells. Altered expression levels of Rho proteins have been observed in such genetically diverse cases as colon, breast, lung, and pancreatic cancers, meaning that Rho-driven processes are not restricted to actin-assembly-related processes such as cell motility, migration and cell-cell adhesion, but also with serum response factor regulated transcription, cytokinesis, apoptosis cell cycle progression, NFκB activation, cell transformation and NADPH oxidase regulation in Can be connected. Thus, Rho is thought to be a potent activator of Jun nuclear kinases (JNKs), linking Cdc42 to cellular processes that require JNK activation (eg, cell growth, differentiation and apoptosis). However, unlike Ras, all attempts at causing mutations in Rho himself (with the possible exception of RhoH) are responsible for the development of cancer. For example, a recent study looked at invasive human colorectal and breast cancer tumors for mutations and found no gene alterations for either RhoA, Rac1 or Cdc42, while those were found for both K-Ras and p53. On the other hand, intact Rho signaling has been shown to play an important role in the transformation of fibroblasts via oncogenic Ras, suggesting that inhibition of Rho function may be a therapeutic strategy for the treatment of Ras-transformed tumors. This could also be interesting in relation to the use of VO ²⁺ for the modification of Rho described here.

A Rho effector protein that plays an important role in this signal transduction pathway is the serine / threonine kinase PAK (p21 activating kinase). In contrast to WASP and ACK (activated Cdc42-associated tyrosine kinase), which have a high affinity for Cdc42, PAK binds either Cdc42 or Rac. PAK makes quite a few direct contacts with Cdc42 with both Switch I and Switch II region. Here, the binding of effectors, as the NMR structures show, stabilizes the switch regions, which can show a high degree of structural flexibility for the isolated Cdc42 protein, as already observed for Ras. However, the X-ray crystal structures for Rho proteins for bound Mg ²⁺ GDP have already assumed a much more stable configuration, especially of the switch regions, compared to Ras.

In addition, the rho-guanosine exchange factors (RhoGEFs) may also play a central role in carcinogenic effects, and are commonly believed to be crucial in promoting tumor cell proliferation. The Dbl family of oncogene products are RhoGEFs, formerly isolated from diffuse B-cell lymphoma (hence the name: Dbl), all of which contain a ~ 180 residue-long Dbl homology tandem domain (DH) and a ~ 100 residue pleckstrin homology domain (PH). have. The DH domain is the region essentially responsible for binding with Rho proteins. The accepted view is that DH represents the catalytic region of the protein and the PH domain regulates the DH region and is responsible for subcellular localization. However, the PH domain also makes contact with Rho proteins in a 'loop' region that spans a wide arc between the third and fourth 'β-sheet' (β ₃ / β ₄ -loop) towards the GTPase and thus contributes to an interaction with the switch II region of Rho proteins. Like all GEFs, the association of proteins displaces bound Mg ²⁺ and GDP; GEF thus preferentially binds GTPases in the absence of Mg ²⁺ and nucleotides. Dbs (Dbl's big sister ') is another Dbl-related oncogene product. The Dbs complex with Rho or Cdc42 is in 12 shown. It is clearly visible how both switch I and switch II regions influence the interactions with the RhoGEF regulator molecule. A Ras-like destabilization mechanism of the Mg ²⁺ cation is also likely to be responsible for the action of RhoGEF and the resulting nucleotide release. Thus, it suggests that an overall functionally similar GEF-induced activation process is also effective in Rho-Poteins.

The "T-lymphoma invasion and metastasis factor 1" (Tiam 1) or the "leukemia-associated Rho GEF" (LARG) are other Dbl-typical examples of medically relevant GEFs for Rho-typical proteins: the Tiam 1 GEF protein specific for Rac and Cdc42 but not for Rho, while LARG is specific for Rho but not an activator of Ras, Cdc42 or Rac The X-ray crystal structure of the eukaryotic Rac 1-TIAM 1 heteroprotein complex shows a similar overall structure to other RhoGEF complexes, in particular the Rho-DH domain topology, except for a substantial difference to the Dbl GEFs, is the orientation of the extended "β ₃ / β ₄ -loop" region of the PH domain, which in contrast to Dbl and a number of other GEFs, in In this case, assume an orientation that does not point in the direction of Cdc42 and thus can not make contacts that otherwise specifically affect the Switch II region, suggesting that the PH region is regulatory for others e mechanisms is responsible. However, since TIAM is a potent Rac activator, the Cdc42-DH domain contacts appear to be sufficient to achieve adequate destabilization of the GTPase Mg ²⁺ atom. As shown recently, TIAM appears to bind directly to c-Myc. The Myc protein plays a crucial role in apoptosis, and alterations of the Myc gene have been observed for human cancers.

Some other potentially interesting application examples, but not in detail here to be presented are the involvement of Rho family proteins in neurodegenerative disorders such as neuronal migration and polarization, axon conduction and dendritic formation, as well as synaptic organization and plasticity, which in one way or another may be associated with cytoskeletal assembly. These neural processes in many respects require the direct regulation and activity of Rho GTPases. One of these rather heterogeneous neuropathological disorders is the form of mental retardation (often referred to simply as MRX) associated with the X chromosome (as was previously responsible for disorders with WASP). Another Rho-related MRX gene is PAK3, which is a Rac (Cdc42) -specific effector and for which two different PAK3 mutations have been isolated in MRX. The MRX phenotype is relatively common and affects about one in every 500 men. Another case involving the RhoGTPase pathway with neuronal processes appears to be a mutation of a gene encoding a potential RhoGEF called alsin, for which amyotrophic lateral sclerosis (ALS) is co-responsible.

Pathogenic microbes have also developed a mechanism that affects the cellular cytoskeleton by secreting toxins that allow them to act directly on the Rho-driven signal transduction pathway. Covalent attachment of ADP-ribosyl moieties by the C-transferases of Clostridium botulinum, Staphylococcus aureus and other infectious bacteria, for example, blocks the ability of Rho to interact with the corresponding Rho-specific GEFs and thus prevent GEF-induced activation of Rho GTPases. An important class of bacterial toxins are the injected toxins that appear to mimic cellular GEFs and GAPs such that the invading toxin activates the Rho GTPases to alter the actin cytoskeleton such that the bacterium freely enters the corresponding cell via a type III secretion system can penetrate. It is striking that this family of GEFs for Rho GTPases lack DH and PH typical domains. The X-ray crystal structure of the enteropathogen Salmonella typhimurium SopE, which is one of the leading causes of gastroenteritis in developing countries, is a Rho-GEF unlike other injected toxins and has been shown to be Cdc42, despite any lack of sequence homology to Dbl in a Dbl-like Type can activate. Again, both switch I and switch II regions are involved, possibly being modified by interactions with SopE such that this leads to critical destabilization of the nucleotide-associated Mg ²⁺ atom. An important role is played by the catalytic element consisting of the GAGA sequence motif, which is unique to SopE. The most dramatic changes, however, are observable for the switch II region, as SopE presumably forces the Cdc42 side-chain methyl group of Ala59 to point in a direction that normally coordinates the nucleotide-bound Mg ²⁺ atom and thus leads to its repression. Modification by VO ²⁺ may play a crucial role in the connective tissue remodeling processes of pathogenic microbes, for example, to invade the host cell.

In general, for all of these cases, which are controlled by the switch regions of the Rho GTPases, it can be said that modulating them by the unusual coordination structure of VO ²⁺ would significantly affect the switching mechanism, which significantly affects the course of the signal transmission path and thus can directly regulate the actin cytoskeletal structure and other functions for Rho proteins.

In this context, it is also important to note that there are some interconnects between the Ras and the Rho signal transduction pathways. This means that the tumorigenesis process and tumor progression are likely to undergo a complex interplay between Ras and Rho-dependent factors and may not be attributed to a singular event. It has been speculated that Ras and other oncoproteins most likely require the Rho family proteins to propagate their transforming activities. Since the present method can influence both the Ras and Rho-dependent signal transduction mechanisms, a combined mechanism of action of the Ras and Rho-metal nucleotide coordination structures by VO ²⁺ substitution is also conceivable.

Example 3:

Another example of the application of the invention are the monomeric GTP-binding elongation factors (EF). The small G proteins, such as Ras, Rac and Ran, as well as the RHO subfamily, as well as the heterotrimeric G proteins occur only in eukaryotes, while GTPases, which play a role in protein synthesis, such as the elongation factors, in prokaryotes and Eukaryotes occur. Ribosomes synthesize proteins by means of translation factors for initiation, elongation and termination. During the bacterial elongation cycle, essentially two elongation GTPases are involved, EF-Tu and EF-G (EF-1α and EF-2 in other organisms). EF-Tu supports aminoacyl-tRNA binding to the codon-programmed ribosome. In contrast to that small p2lras protein, the elongation factors are significantly larger with a molecular weight of 45.3 kD for EF-Tu of Thermus aquaticus. Again, the key point for the activity of EF-Tu is the binding of Mg ²⁺ and GTP. Despite the differences in size, the nucleotide binding fold is homologous to that for p2lras. EF-Tu and EF-G are both hydrolysis-directed switch proteins, which, like Ras, can exist in two steric conformations, in the active GTP-bound state and in the inactive GDP-bound state. They have low intrinsic GTPase activity, which is increased by a factor of 10 ⁷ for EF-Tu when bound to the ribosome, the comparison suggesting that the ribosome is the natural equivalent of GAP. For EF-Tu, the interaction with EF-Ts (T stands for transfer) causes the Mg ^{2+ of} the nucleotide binding site to be destabilized, which then results in the release of the nucleotide. This mechanism shows some homology to the interaction with GEFs for Ras and demonstrates again, first, the key role of the divalent metal in the central mechanisms of switch protein activation, and second, the evolutionary homology of regulatory interactions.

Like p2lras and the nitrogenase Fe protein, the isolated GDP-bound EF-Tu protein does not appear to bind AlF _x as a phosphate analogue for the Pγ phosphate group (Hazlett et al., 1991), which changes significantly only after the addition of the ribosome. EF-Tu has been extensively studied by X-ray diffraction for both EF-Tu of Escherichia coli and T. thermophilus or T. aquaticus. This also applies to EF-G. These show that the elongation factors also have the characteristics of 'classical' MNBPs with the significant spatial regulatory effects of switch I and II regions. As in the other 'classical' MNBPs, the steric and electrostatic changes in nucleotide-binding folding due to the γ-phosphate group release are undoubtedly the driving force for switch rearrangements.

EF-Tu driven processes are in one way or another an attractive "target" for the use of existing and for the development of new antibiotics (see eg: Hogg et al., 2002). There are essentially two classes of antibiotics that interact with EF-Tu and inhibit function by affecting the elongation cycle in one or more stages. Cyclic thiazolyl peptide class antibiotics are among those that inhibit EF-Tu: GTP-aminoazyl-tRNA complex formation, whereas those of the Kirromycz family inhibit protein synthesis by preventing the release of EF-Tu from the ribosome after GTP hydrolysis. Other antibiotics that are thought to interact with EF-Tu are pulvomycin and enacyloxin IIa. It has been shown that pulvomycin and kirromycin bind with similar affinity, but they bind EF-Tu in completely different regions. It seems appropriate to compare the kryromycin binding with the effect of GEFs (or EF-Ts), as these stimulate the exchange of GDP by GTP. The main cause of the inhibitory effect of pulvomycin appears to be the disruption of EF-Tu: GTP binding with amino-azytylated tRNA. X-ray diffraction analysis of EF-Tu in the presence of Aurodox, an N-methyl-kirromycin derivative, shows that Aurodox binds in a cleft between the nucleotide binding region and the region primarily responsible for the interaction with the ribosome. The distance between the nucleotide-bound Mg ion and the Aurodox pyridone nitrogen is about 20.1 Å and the goldinoic acid content about 25 Å. Interestingly, the binding of Aurodox seems to induce a conformation in EF-Tu, despite bound GDP, as otherwise observed only for EF-Tu: GTP complexes. This is consistent with the assumption that Aurodox fixes the EF-Tu ribosome-bound state by freezing the ATP conformation. In particular, the Mg ²⁺ nucleotide-dependent switch II region extends to the Aurodoxbindungsstelle and is mainly in contact distance with the Goldinoik acid portion. Thus, there is a channel through which Aurodox can communicate with the nucleotide binding site and vice versa.

Another antibiotic, the thiazolyl peptide antibiotic GE2270A, binds EF-Tu at yet another, more distant site than the binding site for Aurodox. This binding site is at a distance from the nucleotide binding region with the shortest distance between the nucleotide-bound Mg ²⁺ ion and the peptide segment of ~ 35 Å and the farthest distance of ~ 52 Å. Other representatives of the cyclic thiazolyl antibiotics are GE37468 and Amythiamizine. Like GE2270A, all of these representatives seem to interact directly with EF-Tu. GE2270A appears to have high specificity for EF-Tu, which presumably does not exist for either EF-G or EF-1α. Here, GE2270A inhibits protein synthesis by blocking the conformational change between the GDP- and GTP-bound state and by competing with aminoazyl-tRNA for the same binding site on EF-Tu.

A possible mode of action of VO ²⁺ would be completely different from that of some of the antibiotics and nevertheless be able to inhibit the elongation cycle. While GE2270A EF-Tu interacts at sites that are not supposed to interact directly with the switch regions of the protein and still influence the conformational changes, VO ²⁺ substitution would directly affect these regions via modulation of the nucleotide binding site, which is also different Effect of cirromic-like antibiotics. This could open new ways of treatment.

Example 4:

Another example of sites where signal sorting is done is at the level of heterotrimeric G proteins. Vertebrate G-vertebrate protein, consisting of α, β and γ monomer subunits, usually attach to the cell surface of the plasma membrane and transmit signals from various external stimuli such as growth factors, vasoactive polypeptides, neurotransmitters, hormones, phospholipids, photons, odors and Taste ligands via sensory, transmembrane receptors ("G-protein coupled receptors" or GPCRs) to effector targets that relay them and regulate functions such as heart rate, smooth muscle contraction, synaptic transmission, endocrine secretion, odor and visual perception, and more. Numerous heterotrimeric G proteins are available for this wealth of regulatory functions. The completion of the human genome contributed to the identification of a total of 27 G _α proteins. These can be broadly classified according to their sequence homology into four different categories, namely G _s , G _i , G _q and G ₁₂ , which in general have 50% or higher identity within the categories. Slightly fewer variants are present for the G _β and G _γ subunits. Estimates showed that 1% of the mammalian genome alone encodes the GPCRs. In each of these cases, the receptor activates the G protein by enhancing the exchange of GTP for GDP bound in the G _α subunit made possible by the dissociation of the G _α : G _{β, γ} heterodimer from the receptor and also the two subunits of each other. In this case, the conformational changes in the switch I and II regions in the G _α -substance are the cause for the dissociation of the components, so that in the following the metastable GTP-bound G _α -protein (sometimes also G _{β, γ} or both component proteins together) may modulate downstream intracellular effectors such as adenylyl cyclase cAMP, phospholipase Cβ, K ⁺ and Ca ²⁺ channels, and cyclic GMP phosphodiesterases. Both switches I and switch II region are in place of the G _α subunit, which is closely linked to contacts to the β, γ subunits. However, the switch region probably does not make direct contact with some of the GPCRs and is likely to sense the binding of GPCRs only indirectly via the α ₂ β ₄ "loop" region of G _α . Recent studies have shown that the phosphorylation of G _α13 by the protein kinase A of a residue in the switch I region significantly destabilizes the coupling with the G _{β, γ} subunits and thus inhibits Rho activation. The G _{β, γ} subunits can _α as negative regulators of G subunit will be accepted in some way since they similarly stabilize the GAPs the inactive GDP-bound state, but they can not influence the hydrolysis take stimulation. G _{β, γ} appears to inhibit even the GAP activity as "single-turnover" assays showed possibly _β by direct competition for certain binding regions with the G _α substrate and CAP itself and thus the G _{can γ} subunits as GDIs for Nucleotide exchange can be viewed.

The G _α subunit of heterotrimeric G proteins has a striking similarity with Ras except for an additional helix domain. Nevertheless, G _{α has} an approximately 100-fold higher GTPase rate compared to Ras-like proteins. Another difference is that there appears to be an extra switch region (switch III region) in G _α compared to the two switch regions in Ras-like proteins. Nonetheless, the nucleotide-binding fold shows high homology with the Ras-like proteins.

The G _α subunits of the heterotrimeric G proteins have an important difference to all other G proteins described here, this is the AlF _x binding behavior. All the other G proteins bind AlF _x only in the presence of their GAPs or corresponding regulators, while the G _α subunit binds AlF _x without any regulator. This indicates that G _α should have an autoactivation process and means that the G _α subunits play a very special role in this respect. This was impressively confirmed by the X-ray crystal structures, which showed that in this case, unlike the other GTPases, the extra domain incorporated in G _{α represents} the arginine residue, which is the so-called 'arginine finger'. The crystal structure of the α-subunit of G _i1 in complex with GDP and AlF ₄ ^- in the presence of the "regulator of G-protein signaling" (or RGS protein) RGS4 showed that RGS4 does not have a catalytic residue either direct with GDP or AlF ₄ ^- interacts, contributes. Presumably, RGS4 exerts its GAP activity by primarily contributing to the stabilization of the transition state without, however, directly modifying the metal nucleotide binding site. Such behavior is also consistent with the fact that the heterotrimeric G-protein GAPs, unlike the Ras-like monomeric GTP-binding proteins, allosterically act on the G _α subunits and thus do not contribute to the direct chemistry of GTP hydrolysis , This property could also be of some importance for the effect on vanadyl substitution in the GTP-binding G _α subunit of heterotrimeric G proteins.

-Proteine involviert. Dies geschiet wahrscheinlich unter Beteiligung von Src. Damit eröffnet sich neben dem klassischen

→ cAMP → PKA → CREB Weg möglicherweise ein neuer Übertragungsweg, der bei der Zellproliferation und Transformation beteiligt ist und wichtig sein könnte für Tumoren von endokrinem Ursprung, wie beispielsweise Melanomas.It has been known for some time that a number of G _α subunits cross the MAPK transmission path, as here already for the Ras-dependent Ak described, can activate. Although many of the signal transduction pathways remain open to many questions and some of the components discussed are controversial, recent studies have shown that, in addition to the existing effector interactions, there may be another signal transduction pathway involving the activation of Stat3 by members of the

Proteins involved. This probably happened with the participation of Src. This opens up next to the classic

→ cAMP → PKA → CREB pathway may be a new pathway involved in cell proliferation and transformation that could be important for tumors of endocrine origin, such as melanomas.

GAPs for the heterotrimeric G proteins may also be simultaneously effectors in addition to the RGS-like proteins, ie those containing the RGS-typical conserved sequence domain of about 120 amino acids, such as the G _α -regulated effector protein PLC β1, p115RhoGEF, D -AKAP2 or GRK2.

G-Proteine. Somit scheinen interessanterweise einige der G_α Proteine auch eine wichtige Rolle in der Rho-GTPasen Aktivierung zu spielen. So sind wenigstens zwei heterotrimere G-Proteine,

und möglicherweise

mit der Rho-aktivierten Aktin-Filamentformation in Verbindung gebracht worden. Dies geschieht via der RhoGEFs wobei G_α mutmaßlich verschiedene RhoGEFs bevorzugt. Es scheint als ob

hauptsächlich p115RhoGEF aktiviert, während dies für

die Thyrosin phosphorylierte Leukämie-assoziierte RhoGEF (LARG) ist (Suzuki et al., 2003). Für

ist nur bekannt das es p115RhoGEF nicht stimuliert und somit andere Aktivierungswege vermutlich involviert sind. Darüberhinaus ist in jüngerer Zeit ebenfalls gezeigt worden, daß heterotrimere G-Proteine offenbar auch regulierend in den Ras- und den damit verbundenen MAPK-Signalübertragungsweg eingreifen können via der G_i-Proteine, d.h. Ras ist nicht nur mit der Rezeptor-Thyrosin-Kinase Signalübertragung assoziiert, sondern kann auch mit den rezeptorgesteuerten G-Proteinenübertragungswegen in Verbindung gebracht werden. "Lysophosphatidic acid" (LPA) und Thrombin waren die ersten G-Protein gekoppelten Rezeptoragonisten, die eine rapide stimulierte Ras:GTP Akkumulation in Säugetierzellen zeigten. LPA wird freigesetzt als Produkt der Blutkoagulationskaskade, sodaß daraus geschlossen wurde, daß LPA wahrscheinlich die Wundheilung fördert durch Stimulation der Zellmigration und Proliferation an der Stelle der Verwundung. Interessanterweise, ist die Akkumulation von LPA in Aszites-Flüssigkeit von Krebspatienten mutmaßlich verwickelt mit der Pathogenese von Intraperitonealkrebs.The latter group of RGSs is sometimes referred to as "RGS-like" (RGL) proteins. RGS proteins primarily bind to the family of

G-proteins. Interestingly, some of the G _α proteins also seem to play an important role in Rho GTPase activation. So at least two heterotrimeric G proteins,

and possibly

been associated with Rho-activated actin filament formation. This happens via the RhoGEFs where G _α presumably prefers different RhoGEFs. It seems as if

mainly p115RhoGEF enabled while this is for

the tyrosine phosphorylated leukemia-associated RhoGEF (LARG) is (Suzuki et al., 2003). For

it is only known that it does not stimulate p115RhoGEF and therefore other activation pathways are probably involved. In addition, it has also recently been shown that heterotrimeric G-proteins also appear to be able to regulate the Ras and the associated MAPK signaling pathway via the G _i proteins, ie Ras is not only involved in receptor tyrosine kinase signaling but may also be associated with the receptor-gated protein transfer pathways. Lysophosphatidic acid (LPA) and thrombin were the first G-protein coupled receptor agonists to show rapidly stimulated Ras: GTP accumulation in mammalian cells. LPA is released as a product of the blood coagulation cascade, thus suggesting that LPA is likely to promote wound healing by stimulating cell migration and proliferation at the site of wounding. Interestingly, the accumulation of LPA in ascites fluid from cancer patients is allegedly implicated in the pathogenesis of intraperitoneal cancer.

GTPase, kodiert durch das gsp Onkogen (hergeleitet aus G_s-Protein), sind in Hypophysentumoren, thyroiden Adenomas und thyroiden Karzinomas nachgewiesen worden. Eine Modifikation von Arg-201 oder Gln-227 in der

-Untereinheit ist als aktivierende Mutation für Hypophysenadenome identifiziert worden. Die für gewöhnlich häufigste Mutation in ca. 40% aller sporadisch auftretenden Wachstumshormon-ausschüttenden Hypophysenadenomen ist Arg-201→Cys. Beide aktivierenden Mutationen liegen erneut in konformations-sensitiven

-Regionen; Arg-201 befindet sich in der Schalter I Region nur zwei Residuen abwärts gelegen vom kritischen Threonin und Gln-227 befindet sich in der Schalter II Region (Walker Motiv B, siehe 5), sodaß es nicht verwunderlich ist, daß diese Mutationen zu kritischen physiologischen Veränderungen führen. Interessanterweise führt die kovalente Modifikation von Arg-201 durch das Cholera-Toxin ebenfalls zu einer

Aktivierung. Die schwere sekretorische Diarrhöe, die charakteristisch ist für die intestinale Cholera-Infektion resultiert aus erhöhten cAMP Werten.The oncogenic transformation activity of a number of heterotrimeric G proteins has been recently identified. All four categories of G _α -GTPases have been implicated, in one way or another, directly or indirectly, in carcinogenesis, development and / or progression. Some activating point mutations in the

GTPase, encoded by the gsp oncogene (derived from G _s protein), has been detected in pituitary tumors, thyroid adenomas, and thyroid carcinomas. A modification of Arg-201 or Gln-227 in the

Subunit has been identified as an activating mutation for pituitary adenomas. The most common mutation in approximately 40% of all sporadically occurring growth hormone-releasing pituitary adenomas is Arg-201 → Cys. Both activating mutations are again in conformation-sensitive

region; Arg-201 is located in the switch I region just two residuals downstream of critical threonine and Gln-227 is located in the switch II region (Walker motif B, see 5 ), so it is not surprising that these mutations lead to critical physiological changes. Interestingly, the covalent modification of Arg-201 by the cholera toxin also leads to a

Activation. The severe secretory diarrhea that is characteristic of intestinal cholera infection results from elevated cAMP levels.

kodiert durch das gip2 Onkogen (hergeleitet aus G_i-Protein-2), assoziiert worden mit Keimstrang-Stromatumoren des Ovars und Nebennierenrinden-Tumoren während Doppelmutationen in

mit einer Vielzahl von Tumoren in Verbindung gebracht werden. Obwohl Mutationen in

bisher noch in keinem Tumor gefunden wurden, erscheint es wahrscheinlich, daß auch

-Signalisierungsprozesse bei der Krebsentstehung und/oder Entwicklung eine Rolle spielen könnten, da

wie die meisten anderen heterotrimeren G-Proteine auch, in der Lage ist in der einen oder anderen Art mit typischen Ras-abhängigen Signaltransduktionsprozessen zu interferieren. Ferner scheint das aktivierte

-Protein auch p110α PI3-K und die stromabwärts gelegene Kinase Akt unter bestimmten Voraussetzungen zu inhibieren.On the other hand, single mutations are in

encoded by the gip2 oncogene (derived from G _i protein-2) associated with ovarian stromal stromal tumors and adrenocortical tumors during double mutations in

be associated with a variety of tumors. Although mutations in

so far it has not been found in any tumor, it seems probable that too

Signaling processes in cancer development and / or development could play a role, since

Like most other heterotrimeric G-proteins, it is capable of interfering in one way or another with typical Ras-dependent signal transduction processes. Furthermore, the activated one seems

Protein also inhibit p110Î ± PI3-K and the downstream kinase Akt under certain conditions.

sind in nahezu allen Zellarten vertreten und besitzen eine 67 %ige Aminosäureidentität untereinander aber nur eine 35-44%-ige Identität mit anderen α-Untereinheiten der heterotrimeren G-Proteine wie beispielsweise

oder

-Unterfamilie ist, ähnlich wie Ras, dirin Verbindung gebracht worden. Das

mit der Regulation desOnkogen ist als das entscheidende transformierende Onkogen in Weichteiltumoren identifiziert worden. Hierbei ist besonders die aktivierende

Gln-229→Leu hervorzuheben. Die beiden

Vertreter sind insbesondere involviert in der Regulation der Zellfunktion durch RHO- und Ras-abhängige Prozesse, d.h.

hat die kritischeAufgabe verschiedene Signaltransduktionswege zu koordinieren was sowohl Ras als auch Rap1, Rac und Rho-gesteuerte Übertragungswege betrifft. Von den heterotrimeren G-Proteinen gelten die aktivierenden Mutationen von

als die potentesten transformierenden α-Untereinheiten die bisher getestet.

werden mitunter auch als die gep-Familie der Onkogene bezeichnet. Vermutlich hängt die Fähigkeit so verschiedenartige wachstumsfördende Signalprozesse zu aktivieren mit den potenten onkogenen Eigenschaften zusammen, die einzig den

-Poteinen zugeschrieben werden.

are present in almost all cell types and have a 67% amino acid identity with each other but only a 35-44% identity with other α subunits of the heterotrimeric G proteins such as

or

Subfamily, like Ras, has been linked. The

with the regulation of oncogene is identified as the crucial transforming oncogene in soft tissue tumors been decorated. Here is especially the activating

Gln-229 → Leu. The two

Representatives are particularly involved in the regulation of cell function by RHO and Ras-dependent processes, ie

the critical task is to coordinate various signal transduction pathways involving both Ras and Rap1, Rac and Rho-driven transmission pathways. Of the heterotrimeric G proteins, the activating mutations of

as the most potent transforming α-subunits tested so far.

are sometimes referred to as the gep family of oncogenes. Presumably, the ability to activate such diverse growth-promoting signaling processes depends on the potent oncogenic properties that are unique to the patient

Be attributed to.

-abhängige Signalprozesse beinhalten könnte, und es ist spekuliert worden, daß dies zu einem Herzversagen in letzter Konsequenz führen könnte.Further interesting information about the function of G _α comes from experiments on mice lacking G _α . Among other things, these have led to the assumption that G-protein signaling pathways also play a role in the central nervous system, in certain developmental processes, the immune system, the sensory system and platelet aggregation in vascular injuries. In addition, G _α -controlled signaling processes also seem to play a role in certain cardiac disorders. Thus, G _α proteins are involved in cardiomyocyte growth and myocardial hypertrophy. Since adult cardiac myocytes can not divide, it is believed that G _α signaling also plays a role in apoptosis, both

-dependent signal processes, and it has been speculated that this could lead to heart failure in the final consequence.

durch spezifische "regulators of G-protein signaling" oder RGS-Proteine, die als natürliche äquivalente der GAPs in G-Proteingesteuerten Übertragungswegen angesehen werden. Dies legte den Schluß nahe, daß p53 möglicherweise Wachstum und/oder Überlebensfaktoren durch G-Protein gekoppelte Rezeptorübertragungswege reguliert.Interestingly, a link between the G _α subunits and the tumor suppressor gene product p53 also appears to exist. Recent studies have shown that during genotoxic stress, p53 inactivates both on

by specific "regulators of G-protein signaling" or RGS proteins, which are considered to be natural equivalents of GAPs in G-protein driven transmission pathways. This suggested that p53 may regulate growth and / or survival factors through G protein-coupled receptor transmission pathways.

It However, not only are the heterotrimeric G proteins themselves interesting "targets" for modifications but also the family of G-protein activating membrane receptors ( "G-protein-coupled receptors ": GPCRs). GPCRs are proteins that contain seven transmembrane helix domains and couple a cellular response to an external signal. These bind the inactive GDP-bound state of the G protein complex. Following agonist activation, the GPCRs catalyze nucleotide exchange and therefore act as natural equivalents the GEFs. Indeed GPCRs are probably the group of signal transduction components that can be found on the most successful as "targets" for new drug development to be used. GPCRs are also thought to be crucial carcinogens Factors are effective. The realization confirms that GPCRs themselves proliferative signal transduction pathways regulate, therefore, it seems likely that a chronic Stimulation or mutagenic activation of receptors to oncogenic Transformations can lead.

apart of the pituitary adenomas described above are a number of others Endocrine diseases have been attributed to mutations of G proteins as well as on GPCR mutations. Generally, the majority of all endocrine disorders can occur in two categories, either hyper- or hyposecretory act in relation to a particular hormone. Both effects can be through Mutations of either the G protein or the corresponding GPCR be caused. As examples for both GPCR-caused and caused by G protein endocrine diseases have been described in detail (see: Spiegel, 1996) should be waived here.

-Unterfamilie betreffen, legen den Schluß nahe, daß eine wirksame auf Signaltransduktionprozessen-basierende Krebstherapie auf eine Reihe von Signalproteinen Einfluß nehmen muß.The close involvement of G-protein-driven processes not only with Ras processes, where either the G _α or G _{β, γ} subunits may be the crucial triggers, but also, as already mentioned above, with RHO processes, where the latter especially the

Subfamily suggests that effective signal transduction-based cancer therapy must affect a variety of signaling proteins.

Again, by substitution of Mg ²⁺ by VO ^2+, the conformational changes in the VO ²⁺ coordination environment could significantly contribute to the modulation of the switch regions in the guanosine nucleotide-binding monomeric G _α subunit and thus control regulator and / or effector interactions. This might be particularly interesting, since the G _α subunit, in contrast to the other small GTPases, already has a "built-in" arginine finger and thus there is no possibility for modifications described above using an artificial extra arginine finger. The use of VO ²⁺ to modify the nucleotide binding site is independent thereof. Since G _α also interacts directly with GPCRs, which leads to the activation of G _α , such an interaction is conformation-dependent and should be influenced by the presence of VO ²⁺ . The close fusion of the Ras, RHO, and G-protein signal transduction processes, whose activation mechanism is described in the above subject to very similar principles in the corresponding GTPases, suggests that VO ^{2+ would} potentially be able to modulate several of these signal transduction pathways simultaneously, although this is likely to be different for the different GTPases.

A potential application of the invention in a completely different area concerns the disease resistance of plants. Since the early 1990s, it has become increasingly accepted that heterotrimeric G proteins also exist in plants. G _α subunit-encoding genes have been identified in tobacco, tomato, corn, peas, soybeans, etc. Arabidopsis. Although the plant G _α proteins show relatively low sequence homology with the α-subunits of mammals (only about 40% homology), but great homologies in regions GTP binding or are responsible for the interaction with the βγ subunits to observe. However, the G _α proteins appear to be as these are the case for the corresponding proteins in mammals a much more diverse class of proteins in plants. The analysis of mutations in a gene segment encoding the G _α subunit of rice, which is also referred to as dwarfl or dl, showed that G _α is involved in the formation of germ form Stielelongation and rice. Several studies suggest the involvement of heterotrimeric G proteins in defense signaling in numerous plant species. It is believed that the signal transduction pathway proceeds in a similar manner from the membrane receptor via the G _α subunit to an effector, in this case OsRac, which then gives rise, via other signal components, to specific disease resistance training.

Interesting In terms of plant GTPases, too, is that plant equivalent for many the other small GTPases in mammals exist. For some time, for example, GTPases have been identified that has been a herbal equivalent to the family of the mammalian rho GTPase described above. These GTPases are commonly referred to in plants as Rop proteins, which here supposedly similar Perform functions as in mammals, namely the regulation of the actin cytoskeleton.

Example 5:

Examples of adenosine-binding proteins with said nucleotide binding topology are the monomeric adenosine phosphate-binding protein RecA and possibly the family of multimeric DNA / RNA helicases. RecA is a ubiquitous protein whose structural and functional homologues can be found in all organisms from bacteria to humans. RecA is a highly conserved, multifunctional enzyme and plays a crucial role in homologous genetic DNA recombination and post-replication repair of damaged DNA. RecA forms nucleoprotein filaments for this purpose. Like the nitrogenase Fe protein, RecA possesses the characteristic conserved Walker A and B motifs responsible for the described Mg ²⁺ and nucleotide phosphate group interactions with the protein environment. The X-ray crystal structures for the bacterial RecA protein from Escherichia coli show that RecA belongs to the same superfamily of 'classical' P-loop containing proteins. The nucleotide-induced conformational changes are presumably responsible for the high affinity for DNA and catalysis of DNA strand exchange.

Possible RecA-related medical applications of the VO ²⁺ modification could be, for example, two known representatives of bacterial RecA's, RecA of Mycobacterium tuberculosis and of Mycobacterium leprae. Mycobacterium tuberculosis is the causative agent of tuberculosis and continues to claim more lives than any other infectious disease despite effective chemotherapeutic drugs and the 'Bacille Calmette Guerin' vaccine. The crystal structure for the RecA protein of Mycobacterium tuberculosis (mtRecA) has recently been analyzed and shows a very good overall agreement with the RecA structure of Escherichia coli (ecRecA) which is consistent with the high sequence homology between ecRecA and mtRecA of 62%. Nevertheless, differences are discernible, in particular the nucleotide binding cleft seems to be considerably extended in mtRecA compared to ecRecA, especially the "P-loop" region seems to be affected. This is consistent with biochemical differences in binding strength and hydrolysis and provides a plausible explanation for the more than half reduced affinity of mtRecA for ATP.

Another subexample is the eukaryotic RAD51 gene, which is a structural and functional homolog of the bacterial RecA gene. Like the RecA protein, in this case the human RAD51 protein promotes eukaryotic recombination / repair and plays an important role in the repair of DNA double strand breaks by homologous recombination. However, the ability to hydrolyze ATP and / or cause strand exchange is different. The ATP-binding RAD51 recombinase gene product also has the two Walker motifs (see 5 ), so that the Mg ²⁺ and ATP binding topology can be considered to be similar to that of Ras, although detailed X-ray diffraction analysis so far exists only for the apo protein (Pellegrini et al., 2002), but showing nucleotide-binding folding . which suggests a large homology between the molecular metal nucleotide binding structures of the proteins (see 13 ). The side-chain carboxyl group of the conserved aspartate of the switch II region, which probably interacts directly with the nucleotide-bound Mg ²⁺ ion in the ATP-bound state via a water molecule, is also involved in surface contacts between Rad51 and the breast cancer susceptibility protein BRCA2. The BRCA2 protein is a "tumor suppressor" and controls the function of RAD51. Germline mutations of BRCA2 are responsible for an increased autosomal dominant predisposition to breast cancer and are also considered to be more susceptible to ovarian, prostate, pancreatic and other cancers. Women with inherited BRCA1 and BRCA2 mutations and a family history of breast and ovarian cancers actually have a 50-85% chance of actually getting these forms of cancer. The X-ray structure of BRCA2 in the presence of single-stranded DNA (ssDNA) has also recently been resolved so that a detailed understanding of the relationships underlying recombination-induced double-strand break repair appears to be developing at the molecular level. The likely metal-conformation-induced modifications of RAD51 are another interesting application for the substitution of nucleotide-bound Mg ²⁺ by VO ²⁺ and the associated metal coordination changes. This is particularly interesting in relation to the requirement of RecA and RAD51 for the presence of Mg ^2+, especially since RecA and RAD51, despite the high homology, have significantly different behavior with respect to the requirement for Mg ²⁺ for DNA strand exchange. This suggests that the divalent metal ion is a critical factor in the differences between RecA and RAD51.

The important observation of Thompson et al. (1984), made as early as the early 1980's, that the presence of VO ²⁺ SO ₄ significantly reduces the breast cancer rate in mice could be directly related to this, as is the interaction with p2lras. Such an effect would be in accordance with the herein described mechanisms of Mg ²⁺ substitution by VO ²⁺ for said molecular switches proteins.

Claims (32)

Use of vanadyl or vanadyl compounds to change the conformation at least one switch region of the superfamily of the 'classical' P-loop containing mono-nucleotide binding proteins and associated physiological or pathophysiological Conditions. Use according to claim 1, characterized in that vanadyl or the vanadyl compound is present as oxo-vanadium (IV) (VO ²⁺ ) or oxo-vanadium (IV) compound. Use according to claim 1 or 2, characterized the metal coordination structure is a metal nucleotide coordination structure mono-nucleotide binding "classical" GTPases and ATPases is. Use according to one of claims 1 to 3 characterized characterized in that the mono-nucleotide binding proteins (MNPBs) selected are from the group comprising the proteins of the Ras family, the Rho family, the heterotrimeric G proteins, the elongation factors and the RecA family. Use according to one of Claims 1 to 4, characterized the metal coordination structure is a metal nucleotide coordination structure of the oncogene product p21Ras. Method according to one of claims 1 to 5, characterized that the physiological state stimulates growth, a Transformation a cell proliferation and / or differentiation of cells and / or tissue. Use according to one of Claims 1 to 6, characterized that the pathophysiological conditions include a cell growth disorder, a cell differentiation disorder and / or a cell division disorder is. Use according to claim 7, characterized that the cell growth disorder, Cell differentiation disorder and / or cell division disorder is tumor-like disease. Use according to the preceding claim characterized in that the tumor-like disease is selected from the group comprising a neoplastic change, an inflammatory Tumor and / or an abscess, effusion and / or edema. Use according to the preceding claim characterized in that the tumor is selected from the group comprising a pancreatic cancer, adenocarcinoma of the colon, a bladder carcinoma, a carcinoma of the colon Endometrium, a neuroblastoma, a rhabdomyosarcoma, a leiomyosarcoma, a liposarcoma, a malignant fibrous histiocytoma, peripheral Nerve sheath tumors, a mummy carcinoma, an ovarian cancer and / or a bronchial carcinoma. Use of oxo-vanadium (IV) for the manufacture of a medicament for the treatment of Tu Morphologies associated with conformational changes in the superfamily switch region of the 'classical' P-loop containing mono-nucleotide binding proteins. Use of oxo-vanadium (IV) according to claim 11 for the prevention of blood vessel education for the purpose of nutrient supply of tumors. Antitumor agent comprising oxo-vanadium (IV) thereby in that it is an anti-pancreatic cancer agent, an agent against adenocarcinomas of the colon, an anti-bladder cancer drug, a remedy against Carcinomas of the endometrium, an anti-neuroblastic agent and / or an anti-bronchial carcinoma agent. Anti-tumor agent according to claim 13, characterized that it's a tolerable one pharmaceutical carrier, an adjuvant and / or vehicle. Anti-tumor agent according to the preceding claim, characterized in that the carriers are selected from the group comprising fillers, Extenders, binders, humectants, disintegrants, dissolution inhibitors, absorption accelerators, Wetting agents, adsorbents and / or lubricants. Antitumor agent according to one of the preceding claims, characterized characterized in that the carriers Liposomes and / or nanocapsules are. Use of the anticancer agent according to claim 13 for the treatment of pancreatic cancer, adenocarcinomas of the Colon, bladder carcinoma, carcinoma of the endometrium, neuroblastroma and / or bronchial carcinomas. Use according to the preceding claim in that the anticancer agent according to claim 13 is used in a combination therapy is used. Use according to the preceding claim, characterized characterized in that the combination therapy is chemotherapy, a cytostatic treatment and / or radiotherapy. Use according to the preceding claim, characterized characterized in that the combination therapy is an adjuvant biological includes specified therapy form. Use according to the preceding claim, characterized characterized in that the form of therapy is an immunotherapy. Use according to one of the preceding claims increase the sensitivity of tumor cells Cytostatics and / or radiation. Use according to one of the preceding claims Inhibition of vitality, the rate of proliferation of cells, for the induction of apoptosis and / or a cell cycle arrest. Use according to one of the preceding claims, characterized in that the anticancer agent according to claim 13 is used as gel, powder, powder, Tablet, sustained-release tablet, premix, emulsion, infusion formulation, Drops, concentrate, granules, syrup, pellet, boluses, capsule, aerosol, Spray and / or inhalant is prepared and used. Use according to the preceding claim, characterized in that the anticancer agent according to claim 13 is in a concentration from 0.1 to 99.5, preferably from 0.5 to 95.0, more preferably from 20.0 to 80.0 percent by weight in a preparation. Use according to the preceding claim, characterized in that the preparation is administered orally, subcutaneously, intravenously, intramuscularly, intraperitoneally and / or used topically. Use according to one of the preceding claims, characterized characterized in that the anticancer agent according to claim 13 in total amounts from 0.05 to 500 mg per kilogram, preferably from 5 to 100 mg per kg body weight every 24 hours is used. Method of treating a tumor disease, characterized in that the anticancer agent according to claim 13 with an organism, preferably a human or an animal is brought into contact. Method according to the preceding claim, characterized characterized in that contacting oral, via injection, topically, vaginally, rectally and / or nasally. Process for the preparation of a pharmaceutical Agent for the treatment of a tumor disease, characterized that a compound according to claim 13 is reacted or used with a pharmaceutically acceptable carrier. Kit comprising compounds according to claim 13 optionally with information for combining the contents of the kit. Use of the kit according to the preceding claim for the prophylaxis or therapy of Tumor diseases.