MXPA98008005A - New tata-enlazante protein of candida albicans, nucleic acid sequences codificante de esta, and methods of selection for inhibitors of the development of candida albic - Google Patents

Abstract

The invention encompasses a novel transcription factor of Candida albicans, TBP, a nucleic acid sequence encoding TBP, and methods of protection by inhibitors of the development of Candida albicans for TBP.

Description

-, UK NUCLEIC ACID SEQUENCE ENCODING THIS, AND SELECTION METHODS FOR THE DEVELOPMENT INHIBITORS OF CANDIDA ALBICANS FIELD OF THE INVENTION The invention encompasses a novel transcription factor of candida albicans, TBP, a nucleic acid sequence encoding TBP, and methods of protection by inhibiting the development of candida albicans towards TBP as target.

The invention generally describes transcription factors and methods of protection by antifungal agents.

The invention was made in part using government funds, NIH grant No. GM46498, and therefore the United States government has certain rights in the invention.

BACKGROUND OF THE INVENTION The yeast Candida Albicans (C. albicans) is one of the most widespread pathogenic fungi in humans. It has the ability to opportunistically infect a diverse spectrum of exposed hosts, and invade very diverse tissues in the human body. You can, in many cases, avoid treatments with REF. : 28480 antibiotics and the immune system. However Candida Albicans is a member of the normal flora of respiratory mucosa, gastrointestinal, and genital tracts male and female, in such locations, may predominate and be associated with pathological conditions. Sometimes it produces progressive and systemic disease in weak or indepressant patients, particularly if the cell-mediated immunity is damaged. Sepsis could find it in patients with exposed cellular immunity, for example those under cancer chemotherapy or those with lymphoma, AIDS, or other conditions. Candida can cause invasion into the bloodstream, thrombophlebitis, endocarditis, or infection of the eyes and virtually any organ or tissue when it is introduced intravenously, for example, via tubing, needles, narcotic abuse, ... etc.

Candida Albicans has been shown to be diploid with lethal equilibrium, and therefore probably does not cross a sexual phase or meiotic cycle. This yeast appears to be capable of switching spontaneously and reversibly at high frequency between at least seven general phenotypes. It has been shown that this switching occurs not only in standard laboratory strains, but also in strains isolated from the mouths of healthy individuals.

Nystatin, etoconazole, and amphotericin B are medications that have been used for oral and systemic treatment of Candida infections. However, orally administered nystatin is limited to treatment in the intestine and is not applicable in systemic treatment. Some systemic infections are susceptible to treatment with ketoconazole or amphotericin B, but these medications may not be effective in such treatments unless combined with additional medications. Amphotericin B has a relatively narrow therapeutic index and numerous undesirable side effects and toxicity occurs at the level of therapeutic concentrations. While ketoconazole and other antifungal azoles exhibit significantly lower levels of toxicity, their mechanism of action, inactivation of the prosthetic cytochrome P450 group in certain enzymes, some of which are found in humans, is excluded for use in patients who are simultaneously receiving other medications that are metabolized by cytochrome P450 from the body's enzymes. In addition, resistance to these compounds has been demonstrated and may pose a serious problem in the future.

There is a need in practice for an effective treatment of opportunistic infections caused by Candida Albicans. Accordingly, an object of the invention is provide protection trials to identify potential inhibitors of the development of Candida Albicans. Another objective of the invention is to provide protection assays and identify potential inhibitors of the proliferation of Candida Albicans this is based on the inhibition of transcription in this organism.

The synthesis of mRNA in eukaryotes requires RNA polymerase II and transcription factors as accessories, some of which are in general virtually all, if not all, promoters, and others that confer specificity and control. In general five factors, a, b, d, e, and g, have been purified until homogeneity of yeast S. cerevisiae, and have been identified as duplicates of human or rat factors, TFIIE, TFIIH, TFIID, TFIIB and TFIIF, respectively. These factors bring together a promoter in a complex with Polymerase II RNA to initiate transcription. The ligaments studied have shown that the order of assembly of the initiation complex on the promoter DNA starts with the factor d (TFIID), followed by the factor e (TFIIB), and then by the polymerase and the remaining factors. The factors b (TFIIH), e (TFIIB) and g (TFIIF), however, bind directly to polymerase II, and in this way 4 of the 5 factors can be combined with the polymerase in a holoenzyme before binding to the promoter, the Functional significance of the interactions revealed by the ligaments studied is not clear in that only a small percentage of initiation complexes can get to give transcripts.

Many aspects of transcription by RNA polymerases II are conserved between yeasts and higher eukaryotes. For example, there is similarity between the extensive amino acid sequence and the larger subunits of the yeast, mammalian polymerases and Drosophila. Other components of the transcription apparatus, such as TATA-ligaments and binding factor enhancers, are in some cases interchangeable between yeasts and mammalian ligaments in vitro or transcription system. There are, however, significant differences between the two systems. TATA elements are located from the base pairs 40 to 120 or more upstream of the initiation site of a promoter S. cerevisiae, and where these elements are found, they are required by the gene expression. This is because the C. Albicans gene functions in S. cerevisiae suggest that it also uses the space 40 to 120 base pairs between the TATA elements and the place of initiation. In contrast, mammalian TATA elements (as well as S. Pombe and transcription initiation sites are only 5 to 30 bp apart), and the deletion of a TATA element is not always It reduces the frequency of transcription initiation, although it may alter the place of initiation. There are also several degrees of homology between sequences of transcription factors of origin, yeast and those of mammals. Some of the multi-subunit factors, such as RNA polymerase II, TFIIF, and TFIID, contain different numbers of subunits in humans and in yeast. The molecular weight of the corresponding polypeptides differ between humans and yeasts, with sequences that have been found in a given yeast factor, not having been found in their human duplicate and vice versa.

The TATA-binding protein (TBP) is the central initiation factor for the transcription of the three nuclear RNA polymerases, and is highly conserved throughout the eukaryotic kingdom. The carboxyterminal amino acid 180 in the center of the region is sufficient for the TATA binding element, for all essential functions in S. cerevisiae, and is 80% identical between S. cerevisiae and humans. In vitro, yeast and human TBPs can functionally replace each other in terms of transcription of polymerase II from basal RNA, and present almost identical requirements to DNA sequences for TATA elements. However TBP exhibits a specific behavior of the species in vivo. For example, human and yeast TBPs are not interchangeable species in support of cell growth. (Gilí and Tijan, Cell 65: 333-340, (1991); Cormack et al., Cell 65: 341-348 (1991)). Human and S. cerevisiae TFIIBs have 50-60% identical amino acid sequences, and neither are interchangeable species in support of cell growth.

The operative substitution of the same transcription factor in the transcription system of different yeast species is not predictable. This is true despite a high degree of identity of amino acid sequences between some transcription factors of different yeast species. For example, the ability of a given transcription factor to support efficient and safe transcription in heterogeneous species of yeast species is not predictable. Li et al. (1994), Science 263: 805) tested the interchangeability of the transcription factors S cßrevisiae and S Pombe in vitro, and reports that many components of S. cerevisiae can not be individually substituted for transcription factors a, e, or S. pnebe RNA polymerase II, but some combination of these components were effective. In one case, active transcription could not be reconstituted when the TFIIB derivative of S. cerevisiae was the single substitution in a TFIIB group depleted of S. Pombe factors. A combination of S. cerevisiae TFIIB RNA polymerase II, could be used, indicating that the functional interaction of these two components is not only important, but that the activity can be dependent on specific determinant species that can not be complemented by any of the two components derived from different organisms. The unpredictability of substitutions made from a given factor between different yeast species is also evident that such substitutions are not reciprocal. That is, substitutions of S. Pombe fractions in a S. cerevisiae transcription system are less effective than the opposite substitutions. (Li et al., Supra).

The yeast Candida albicans differs from most strains of yeast in that it does not use the same genetic code as most organisms, whether those used by mammals or yeast. Santos et al. (1995, Nucleic Acids Research, 23: 1481) report that the codon CDG, which in the universal code is read as a leucine, is decoded as a serine in Candida. Therefore, any CUG codon that is decoded in Candida albicans as a serine, could be decoded as a leucine in transformed 5. cervisiae. Any The gene containing a CUG codon could therefore be translated as different amino acid sequences in Candida albicans and S. cerevisiae. Such erroneous translation can produce an inactive protein, since the amino acids serine and leucine have markedly different chemical properties and serine is known to be an essential residue in the active sites of some enzymes. Replacements of leucine by serine in coded CUG residues are a serious problem in the use of many reporter systems (for example: β-galactosidase, chloramphenicol acetyltransferase, Flux) in Candida albicans. Previous experiments have shown that the Candida translation of CUG such as leucine serine is often the result of the production of inactive reporter proteins.

Another objective of the invention is to provide a test for protection by selective inhibition of the growth of Candida albicans and / or its viability.

Still another purpose of the invention is to provide a molecular target for the inhibition of transcription of Candida albicans or initiation of transcription.

DESCRIPTION SUCCESS OF THE INVENTION The invention encompasses a recombinant nucleic acid consisting of a nucleic acid sequence encoding TBP Candida albicans The invention also encompasses a vector consisting of a nucleic acid sequence encoding TBP from Candida albicans, and a transformed host cell containing a nucleic acid sequence encoding TBP from Candida albicans.

The invention also encompasses a method for the production of TBP from recombinant Candida albicans, consisting of cultures of host cells transformed with a nucleic acid encoding TBP of Candida albicans under conditions sufficient to allow the expression of the nucleic acid encoding TBP of Candida albicans, and isolate TBP from Candida Albicans.

The invention also encompasses a method of protection for the identification of a growth inhibitor of Candida albicans, consisting of detecting the inhibition of mRNA transcription in an in vitro transcription test including a standard DNA, RNA polymerase II, recombinant Candida albicans TBP, and an inhibitory candidate, wherein the production of a complementary mRNA transcript in the standard DNA takes place if the inhibitory candidate is absent.

The invention also encompasses a method of protection for identifying a growth inhibitor of Candida albicans, consisting of detection in the presence of an inhibitory candidate, inhibition of the formation of a complex including a DNA standard and TBP of recombinant Candida albicans, in the absence of an inhibiting candidate, complex formation takes place. The method can also be effected in the presence of additional factors, such as TFIIB, RNA polymerase II, and TFIIF.

The invention also encompasses a method of protection for identifying a growth inhibitor of Candida albicans, including detection of an inhibitory candidate, inhibition of the formation of a complex consisting of TFIIB of Candida albicans and TBP of Candida albicans, wherein absence of the inhibitory candidate complex formation takes place. Preferably the complex will include a standard DNA.

The invention also encompasses a protective method for identifying a Candida albug growth inhibitor which includes detecting in the presence of an inhibitory candidate the inhibition of complex formation including RNA polymerase II, Candida albicans TBP, and Candida TFIIB. albicans where in the absence of the inhibitory candidate the formation of the complex takes place. Preferably, the complex will include a DNA standard and RNA polymerase II from C. albicans.

In the protection methods described above, the detection can be carried out in the presence of a plurality of inhibitory candidates. In the protection methods of the invention which involve protection of a plurality of inhibitory candidates, the plurality of inhibitors can be protected together in a single test or individually using multiple and simultaneously individual detection steps.

The invention also encompasses a method of preventing growth growth in Candida albicans, which consists of contacting the culture with an inhibitor that selectively inhibits the biological activity of the TBP of Candida albícans.

The invention also encompasses a method of preventing the growth of Candida albicans, in a mammal, which consists in administering to a mammal a therapeutically effective amount of an inhibitor that inhibits the biological activity of the TBP of Candida albicans.

The term "inhibition" refers here to a reduction in the parameter that is being measured, whether the growth or viability of Candida albícans, the average transcription of the TBP of Candida albicans or the formation of the transcription complex of the Candida TBP. albicans The quantification of such reduction is a measure in relation to a standard (control). Because of the multiple interactions of the TBP of Candida albicans in a transcription initiation, the target product for detection varies with respect to the particular protector employed in the test. Three preferred detection products are presented in this discovery and are: a) mRNA again transcribed; b) a TBP-DNA complex, and c) a polymerase II complex of RNA-TFIIB-TBP. "Reduction" is defined herein as a decrease of at least 25% relative to a control, preferably at least 50%, and more preferably at least 75%.

"Growth", refers here to the normal growth pattern of Candida albícans, for example, in a cell doubling the time of 60-90 minutes. "Viability" refers to the ability of Candida albicans to survive in 48-hour cultures.

"Biological activity" refers to the ability of TBP to form a transcription complex with a standard DNA or another protein of the transcription complex, or to interact with other transcription components as permitted by the initiation of transcription.

"DNA standard" refers to DNA of double filament and, where indicated by the particular DNA binding test of a filament, at least 10 nucleotides in length, which could be negatively superspirated if it is double filament, has a promoter region , and contains a region of TATA consensus in the yeast. The DNA standards used herein will preferably contain a TATA sequence that is located from 40 to 120 or more base pairs up from the starting point (measured distance from the first T of the TATA element to 5'- most, from the starting point) . A particularly efficient DNA standard for use in the methods of the invention involving transcription is devoid of guanosine residues, and therefore a "G-minus" or "G-minus" cassette is preferred.

"Transcript mRNA" refers to a full-length transcript as well as a truncated transcript, oligonucleotide and dinucleotide RNA transcripts.

"Complex formation" refers to the ligament of TBP to other transcription factors (eg, protein-protein ligaments) in the same manner as the ligament of TBP to a standard DNA; such will bind, of course, being a non-covalent association.

Other features and advantages of the invention will be apparent from the description, preferred embodiments thereof, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 presents the nucleotide and the amino acid sequences of the transcription factor TBP of Candida albícans.

Fig. 2 presents the nucleotide and the amino acid sequence of the transcription factor TFIIB of Candida albicans.

DESCRIPTION The invention is based on the discovery of a new protein, TBP of Candida Albicans, and on the isolation of recombinant DNA that encodes the transcription factor TBP of Candida albicans. Because TBP is essential for cell viability, a compound that blocks the biological activity of the protein is expected to have fungicidal properties. Accordingly, the invention is also based on the development of tests for the protection of TBP inhibitors.

Isolation and Characterization of the TBP gene Candida Given the unpredictability with respect to operational substitutions of a given transcription factor between different strains of fungi, one can not guess which strategies for cloning the genes that encode a transcription factor since they are based on the function of the factor, such as genetic complementation, will work-Other cloning strategies, which do not require functional complementation, as well as that based on the level of nucleic acid, can be used in an attempt to avoid a requirement for the factor function. For example, Polarized hybridizations of specific sequences of the stock carrying E. coli and PCR amplification of highly potential homologous regions of a gene are two strategies that have been successfully used to clone homologous genes from different organisms.

The approach used to clone the Candida albicans homolog of TBP involves genetic complementation of the mutant strain S. cerevisiae. A stock of genomic sequences of Candida albicans was introduced into a strain of S. cerevisiae that contained a mutated TBP gene (spt 15). This mutant strain was able to develop at 30 ° C, but was not viable at 37 ° C, due to a temperature-sensitive mutation in the TBP gene. Following the transformation of the stock into the strain, the cells were cultured at 37 ° C, and the colonies that grew at this non-allowable temperature were ad- minally studied as potential vehicles of a Candida albicans homologue of the defective gene. This approach will only be worked on if the homolog of Candida albicans is capable of being functionally substituted in vivo by the defective gene.

After the candidate clones were isolated by culture at the non-allowed temperature, the plasmid DNA pool was recovered from the cell and tested again for confirm that the C. albicans sequences on the plasmid were replaced by the S. cerivisiae gene. Subclones of the C. aZLbicans sequences were constructed by standard cloning methods, and the minimal sequences of Candida DNA that they replaced were sequenced using standard methods.

The nucleotide sequence encoding the TBP of Candida albicans and the predicted amino acid sequence of the encoded protein is presented in FIG. 1 (SEQ ID NOS: 1 and 2). The nucleotide sequence encoding the ~ TFIIB of Candida albicans and the predicted amino acid sequence of the encoded protein is presented in Fig. 2 (SEQ ID NOS 3 and 4).

Methods to protect the potential inhibitors of the growth of Candida albicans and / or its viability.

Because the TBP initiation factor is essential for the initiation of transcription, the recombinant TBP Candida albicans gene and the recombinant protein encoded by this gene can be used in protection tests for Candida albicans growth inhibitors and viability. The protection tests of this invention detected inhibition of the TBP-media component of Candida albicans from the start of transcription, either by measuring the inhibition of transcription, the initiation of transcription, or the formation of initiation complexes, or by testing the formation of a protein / DNA or a protein / protein complex.

EXAMPLE 1 Protection for Transcription Inhibitors a) Components of the Transcription Test An in vitro transcription test consists of the same components necessary to synthesize an mRNA transcript; a standard DNA can be used as a protection for the inhibition of mRNA production. The elements of such tests consist of: a) a standard DNA, b) an RNA polymerase II, c) TBP of recombinant Candida albícans, and d) a TFIIB that is preferably TFIIB of Candida albicans. In order to increase the transcription efficiency additional components of the transcription complex can be included, as desired; for example TFIIE, TFIIF, TFIIH, ..., etc.

Pervin and Sharp (Cell 73, 533-540, 1993) has reconstituted the transcription of a gene in vitro with a minimal reaction containing a standard DNA, DNA polymerase II, TFIIB, and TBP. For efficient transcription under minimal conditions, the standard DNA (a) is spiraloid, and (b) it has a promoter region containing a consensus TATA region. Additionally, Lue et al. 8Sc en 246, 661-664, 1989) has determined that transcription can be detected more efficiently with a standard DNA devoid of guanosine residues (a G-minu or G-minus cassette). Promoter dependence was demonstrated by signal loss when a promoter sequence lacking a plasmid is used as a standard. The correct initiation is demonstrated by the production of a band with a mobility consistent with the size of the expected product by electrophoresis on denatured polyacrylamide gels.

As stated above, the TBP of Candida albicans forms a transcriptional initiation complex with RNA polymerase II. Accordingly, it is desirable that an in vitro transcription test according to the invention contains RNA polymerase II. Although it is possible to carry out an inhibitor protection test using the RNA polymerase II of a yeast strain different from Candida albicans, for example S. cerevisiae, it is more desirable to use a homologous test in which the components of the transcription complex are of Candida albícans.

A method for the purification of poIII RNA from cerevisiae is described in Edwards et al. (Proc.

Nati Acad. Sci. USA 87: 2122-2126 (1990)). Alternatively, highly purified RNA polymerase II from Candida albicans was obtained as follows.

The activity of RNA polymerase II was measured in reactions containing 50 mM Tris-Cl, pH 7.9 (4 ° C), 50nM (NH4) 2S04, 2.5 M MnC12, ol mM EDTA, 5 mM DTT, 100 μg / ml . BSA, 0.6 mM ATP, CTP and GTP, 25 μM UTP (2.5 μCi) (at E-32P) UTP and 100 μg / ml. of calf thymus DNA denatured to heat in a final volume of 50 μl. The reactions were incubated for 60 minutes at 30 ° C and terminated by the addition of 50 μl of 15% trichloroacetic acid (weight / volume). The acid-insoluble radioactivity was collected by filtration through glass fiber filters and quantified by liquid scintillation spectrophotometry. One unit of RNA polymerase activity catalyzes the incorporation of 1 pmol of UTP into the acid-insoluble material in 60 minutes under the conditions described above.

Candida albicans was obtained from "American Type Culture Collection" ATCC 10231) and cultured in YPD medium (Current Protocols in Molecular Biology, Vol. 2, 13, Suppl. 19 (1989)) at 30 ° C with vigorous stirring and aeration. All procedures were carried out at 4 ° C using 18 liters of cultures. Cells were harvested by centrifugation (5000rpm, 10 min., Sorvall H6000 rotor), washed once with apróx. 1 Lt. of ice made with deionized and polished water as explained above. The cell in lentils (200-300 g net weight) was resuspended through a volume of buffer A (50 mM Tris-HCl, pH 7.9, 4 ° C, 10% glycerol, 1 mM EDTA, mM of MgC12, and protease as inhibitor) containing 300 mM of (NH4) 2S04 equivalent to the volume of packed cells (determined by weight, assuming a density of 1 g./ml of cells). The resuspended cells were either processed immediately as described below or frozen by pipetting in liquid N2 and stored at -80 ° C. The frozen cells were thawed on the previous ice as a procedure. Following the addition of NP-40 to a final concentration of 0.1%, the cells were separated by trituration with 1 ml. of glass beads pre-washed with acid per ml. of cell suspension (Sigma, 400-625 μM) using 12 runs of 30 seconds each in a "Bead Beater" blender (bioSpec). The glass beads are allowed to settle and the supernatant is centrifuged at 30,000 x g. for 40 minutes. The solids (NH4) 2S04 were added slowly to a final supernatant concentration of 0.4 g / ml. and the resulting precipitate was transformed into lentils by centrifugation at 100,000 x g. for 30 minutes. The lentil was resuspended with a volume of buffer A sufficient for a conductivity field equivalent to a buffer containing 75 M (NH4) 2S04.

Following the centrifugation of the resuspension at 10,000 x g. for 10 minutes, this supernatant (approximately 1-1.5 mg protein / ml.) was loaded onto a 300 ml column. of Cellulose-DEAE DE52, equilibrated with buffer A containing 75 ir? M (NH4) S04, After washing with 5 column volumes of buffer solution A containing 0.4 M (NH4) 2S04, the RNA polymerase II was eluted with 5 ml. column volumes of buffer solution containing o.4 M of (NH4) 2S04. The fractions were collected containing the protein peak, determined by absorbance at 80 nm and combined. The whole was dialyzed against a regulatory solution containing 20% glycerol for 3 hours at 4"c. (0.4 M NH4J2S04 eluted from cellulose-DEAE (261 mg protein, 290 ml.) Was diluted with sufficient buffer solution A to lower the conductivity to the equivalent of buffer solution containing (NH4) 2S04 0.15 M, centrifuged at 10,000 x g. for 10 minutes, and the supernatant It was charged at an expense of 30 ml./hr. on a cellulose-DEAE column equilibrated with buffer solution A containing (NH4) 2S04 0.15 M. After rinsing with 3 column volumes of buffer solution A containing (NH4) 2S04 0.15 M, the column was developed with a linear gradient of 200 ml . of (NH4J2S04 0.15 M- 0.4 M in buffer A at a cost of 45 ml./hr Fractions of the single peak of RNA polymerase activity-amanitin, eluting around (NH4J2S04 0.22 M, was pooled (21.1 mg of protein, 45 ml) and loaded directly onto a 5 ml column of Heparin agarose equilibrated with buffer solution A containing 0.2 M NH4J2S04. The column was washed with three column volumes of buffer solution A containing (NH4J2S04 and developed with 80 ml of a linear gradient of (NH4J2S04 of 0.2-0.6 Men buffer A. Active fractions, which eluted were approximately (NH4) 2S04 0.42 M were pooled (2 mg of protein, 15 ml.), Frozen in aliquots of 300 μl in liquid N, and stored at -80 ° C, conditions in which the activity remains stable for at least 6 months.

The purification of the protein initiation factors used in the test is carried out by the standard methods known in practice (for example, phosphocellulose chromatography followed by gel filtration), as described in (Nature 346, 387-390 (1990)).

Protection by transcription inhibition of TBP medium from Candida albícans, a transcription test is reconstituted using TBP of recombinant Candida albicans. The spiroidal DNA plasmid containing the CYC1 promoter bound to the G-minus cassette described by Lue et al. (Science 246, 661-664 (1989)), is purified by standard methods for the purification of circular spirochete DNA (Currents Protocols in-Molecular Biology, Vol. 2, 13, Suppl 19 (1989)). 10-100 ng. of TFIIB of Candida albicans, 10-100 ng. of TBP of Candida albicans 10-100 ng. depolymerase II of Candida albicans RNA and 1 μg. of plasmid DNA are added to 50 μl of reaction mixture containing 50 mM HEPES, pH 7.5, 10% glycerol, 90 mM potassium glutamate, 0.75% polyethylene glycol (molecular weight 3350), 10 mM acetate magnesium, 5 mM EGTA, 5 mM DTT, 0.4 mM ATP, 0.4 mM CTP, 10 μM (E-32P) ÜTP, 0.2 mM 3 '- O-methyl-GTP, and containing or missing one molecule of candidate for inhibitor. The reactions are incubated at 30 ° C for 30-60 minutes and RNA synthesis is detected as described below. b) Detection of the transcribed RNA.

The detection of the newly transcribed RNA is achieved by standard methods (Current Protocols in Molecular Biology, Vol. 1, 4.10, Suppl 24 (1989)). As an example, RNA synthesis can be detected by incorporation of a fluorescently labeled nucleotide or radioactively into RNA products with higher molecular weight, determined by one of the following methods: 1) quantification by the appropriate method of labeled insoluble acid material (for example, scintillation counting for radioactive precursors, fluorometry, for fluorescent precursors); 2) labeled reaction products that hybridize to the complementary oligonucleotides in the correctly initiated transcript (eg, polarized separation analysis); 3) the presence of a marked band with the appropriate mobility is detected by autoradiography, by electrophoresis on denatured polyacrylamide gels: 4) Any other method that discriminates the mononucleotides of the polynucleotides, where the polynucleotides are the desired RNA products. Such methods may use one or more well-known techniques of molecular biology (Current Protocols in Molecular Biology, Vol. 2, 13, Suppl 19 (1989)), for example; UV analysis; affinity systems (for example: chromatographic affinity, nitrocellulose filtration, streptavidin / biotin systems, Immunoaffinity, Current Protocols in Molecular Biology, Vol. 2, 13, Suppl. 19 (1989)); and HPLC.

The inclusion of an inhibitory molecule that interferes with the biological activity of the TBP of Candida albícans inhibits transcription. In this test the inhibition is measured as a reduction in the amount of the mRNA transcript produced relative to the amount of mRNA transcript produced in the absence of the inhibitor (the positive control). A decrease in the amount of the mRNA transcript is indicative of an inhibitor. The determination of the effective levels of inhibition of the RNA transcript is described below.

EXAMPLE 2 Protection by Inhibition of the formation of the Pro-DNA complex A ligament test of the DNA-protein complex consists of the minimum components necessary to allow the binding of TBP of Candida albicans-RON to take place, can be protected by inhibition of the formation of the TBP complex of Candida albicans -DNA during the initiation of the transcript The essential elements in a test such consist of: a) a standard DNA, b) TBP of recombinant Candida albicans, and optionally c) a candidate for TBP inhibitor of Candida albicans.

The inclusion of an inhibitory molecule that interferes with the interaction between the TBP of Candida albicans and the standard DNA inhibits the initiation of transcription. The inhibitor can interact directly with the TBP protein of Candida albicans, and / or can interact with the standard DNA at the DNA site of the TBP binding of Candida albícans. In this test the inhibition is measured as a reduction in the amount of the TBP complex of Candida alhicans-AOH produced relative to the amount of the complex or 'TBP of Candida albícans DNA produced in the absence of the inhibitor (positive control). A decrease in the amount of the TBP complex of Candida al-icans-DNA is indicative of an inhibitor. The determination of the effective levels of inhibition of TBP of Candida albicans-DNA is described below.

A DNA link test is constructed as follows. 10-100 ng. of TBP of Candida albicans, expressed in a purified E. coli as described above, is incubated with 0.5 ng of labeled oligonucleotide (eg fluorescently or radioactively labeled) containing a TATA element as described by Buratowski et al. (Cell 56, 549 -561 (1989)) in reactions containing 10-20 mM HEPE? (or equivalent), p.H 7.5-8.0, 5 mM MgC12, 12% glycerol, 10 mM Dithiitritol (DTT), 100 μg / ml. of BSA, 5-20 μg / ml. of poly (dG-dC): (dG-dC) and an ihnibidor candidate for complex formation. The reactions are incubated at 30 ° C for 30-60 minutes.

The formation of a TBP-DNA complex can be detected as retention of labeled DNA (the label being detected by appropriate methodologies such as scintillation counting for radiolabeled DNA or fluorometry for fluorescently labeled DNA) using known affinity methods for immobilization of proteins ( example: biotin / streptavidin, nitrocellulose filtration, chromatographic affinity, immunoaffinity). The non-retention of labeled DNA due to the failure in the formation of the DNA-TBP complex of Candida albicans is an indicator of an effective inhibitor.

The formation of the complex can also be detected as TBP retention of labeled Candida albicans (eg radioactively or fluorescently) using known methods for DNA immobilization. The non-retention of TBP of Candida albicans marked due to the failure in the formation of the DNA-TBP complex of Candida albicans is indicator of an effective ihnibidor. These methods are suitable for protection of the stock of highly finished chemical compounds, applications such as those commonly used in drug discovery.

A third example of detection of protein / DNA complex formation involves detection of a change in the electrophoretic mobility of labeled DNA on 4% polyacrylamide gels containing 5% (v./v.) Glycerol, 25 mM Tris, 100 mM glycine, 1 mM EDTA, 5 mM MgC12, pH 8.3 in the presence of TBP of Candida albicans. The position of the labeled oligonucleotide is detected by appropriate methods (e.g., autoradiography for radioactive oligonucleotides). The absence or deviation of the changes in mobility expected due to the formation of the TBP complex of Candida albicans-AON is indicative of an effective inhibitor.

Finally, other methods for detection or separation of protein-DNA complexes can be used, including cross-linking analysis, HPLC, phage display technology (US Patent No. 5,403,484, Viruses Expressing Chimeric Binding Proteins), fluorescence polarization, and resonance. of plasmon surface (Biacore, Pharmacia Biosensor, North America) as described below EXAMPLE 3 Protection by Inhibition of Protein-DNA Complex Formation A test for the DNA-protein link consists of the minimum components necessary to allow the TBP association of Candida to Ibicans-DNA to be carried out. The protection by inhibition of the formation of the TFIIB complex of Candida a-bicar-5 can be used. TBP-DNA during the start of transcription. The components of such a test include: a) a standard DNA, b) TBP of recombinant Candida albicans, c) TFIIB, preferably of Candida albicans, and optionally d) a candidate inhibitor of TBP of Candida albicans.

The inclusion of an inhibitory molecule that interferes with the interaction between the TBP of Candida albicans and the standard DNA inhibits the initiation of transcription. The inhibitor can interact directly with the TBP protein of Candida albicans, and / or can interact with TFIIB and / or with the standard DNA at the TFIIB / TBP binding site. In this test the inhibition is measured as a reduction in the amount of the TFIIB-TBP-DNA complex produced in relation to the quantity of the TFIIB-TBP-DNA complex produced in the absence of the inhibitor (the positive control). A decrease in the amount of the TFII-TBP-DNA complex is an indicator of an inhibitor. The determination of the effective TFIIB-TBP-DNA inhibition levels is described below.

A DNA binding test is constructed as follows. 10-100 ng TBP of Candida albicans, expressed in a purified E. coli as described above, is incubated with 0.5ng of a labeled oligonucleotide (eg, radioactively or fluorescently labeled) containing a TATA element such as that described by Buratowski and collaborators (Cell 56, 549-561 (1989) 1 and 10-100 ng of TFIIB of Candida albícans in reactions containing 10-20 mM HEPES (or equivalent), pH 7.5-8.0 mM MgC12, 12% glycerol, 10 mM of Dithiothreitol (DTT), 100 μg / ml BSA, 5-20 μg / ml poly (dG-dC): (dG-dC) and a candidate inhibitor of complex formation The reactions are incubated at 30 ° C for 30-60 minutes.

The formation of the TFIIB-TBP-DNA complex can be detected as retention of labeled DNA (the label being detected by an appropriate methodology such as scintillation counting for radiolabeled DNA or fluorometry for fluorescently labeled DNA, using known affinity methods). for immobilization of proteins (for example, streptavma-biotin, filtration on nitrocellulose, chromatographic affinity, immunoaffinity). The non-retention of the labeled DNA due to faults in the formation of the DNA-TBP-TFIIB complex is an indicator of an effective inhibitor.

The formation of the complex can be detected as TBP retention of Candida albícans labeled (eg radioactively or fluorescently) using known methods for DNA immobilization. The non-retention of marked TBP of Candida albicans due to faults in the formation of the DNA-TBP-TFIIB complex of Candida albicans is an indicator of an effective inhibitor. The two preceding methods are suitable for collections of highly finished chemical compounds using protection in applications such as those commonly used in drug discovery.

A third example of detection of protein / DNA complex formation involves the detection of a change in the electrophoretic mobility of the labeled DNA in 4% polyacrylamide gels containing 5% (v./v glycerol, 25 mM TRIS, 100 mM of glycine, 1 mM EDTA, 5 mM MgC12, pH 8.3 in the presence of TBP and TFIIB of Candida albicans The position of the labeled oligonucleotide is detected by methods appropriate (for example, autoradiography for radioactive oligonucleotides). The absence or deviation of the expected change in mobility due to the formation of the TBP complex of Candida albicans-KDN is an indicator of an effective inhibitor.

Finally, other methods for detection or separation of protein-DNA complexes can be used, including analysis by UV cross-linking, HPLC, phage display technology (US Patent No. 5,403,484, Viruses Expressing Chi eric Binding Proteins), and plasmon surface resonance. (Biacore, Pharmacia Biosensor, North America) as described below.

EXAMPLE 4 Protection for Inhibition of Protelna-Protein Complex Formation A protein-protein binding test consists of the minimum components necessary to allow the TFIIB binding of Candida albicans-TBF of Candida albicans to be carried out. Protection can be used by inhibition of the formation of the TFIIB complex of Candida albicans-Candida TBP albicans during the start of transcription. The elements of such a test consist of: a) recombinant Candida albicans TBP, b) TFIIB, preferably a TFIIB of recombinant Candida albicans, and optionally c) a candidate for binding inhibitor.

The inclusion of an inhibitory molecule that interferes with the interaction between TBP of Candida albicans and TFIIB of Candida albicans inhibits the initiation of transcription. The inhibitor can interact with TBP of Candida albicans or with the TFIIB protein and thus induce a conformational change that prevents binding, or it can directly inhibit the TFIIB interaction of Candida albicans and TBP proteins. In this test, the inhibition is measured as a reduction in the amount of the TFIIB-TBP complex of Candida albicans produced in relation to the amount of the TFIIB-TBP complex of Candida albicans produced in the absence of the inhibitor (positive control). A decrease in the amount of the TBP-TFIIB complex is an indicator of an inhibitor. The determination of the effective levels of inhibition of the TFIIB-TBP binding of Candida albicans is described below.

A test for the formation of the TFIIB-TBP complex Candida albicans is provided below. 10-100 ng. from TFIIB of Candida albicans and 10-100 ng. of TBP of Candida albicans expressed in and purified from E. Coli as described above, and is added to reactions containing 10-20 mM HEPES (or equivalent), pH 7.5-8.0, 5 mM MgC12, 12% glycerol, 10 mM Dithiothreitol (DTT) 100 μg / ml. of BSA, and a candidate for inhibitor. The mixture is then incubated at 30 ° C for 30-60 minutes.

The formation of a complex consisting of TFIIB of Candida albícans and TBP of Candida albicans can be detected by a change in the electrophoretic mobility of labeled TBP or TFIIB (eg radioactive or fluorescent) on gels at 4% polyacrylamine containing 5% (v ./v.) of glycerol, 25 mM Tris, 10 mM glycine, 1 mM EDTA, 5 mM MgC12, pH 8.3 in the presence of an unlabeled partner. The position of the partner is detected by the appropriate methods (for example, autoradiography for radioactive oligonucleotides). The absence or deviation of expected changes in mobility due to the formation of the TBP-TFIIB complex of Candida albicans is an indicator of an effective inhibitor.

The formation of a complex consisting of TBP of Candida albicans and TFIIB of Candida albícans can be detected as retention of labeled TBP using known affinity methods for immobilization of the TFIIB protein of Candida albícans (for example, streptavidin / biotin, filtration by nltrolosam, chromatographic affinity, immunoaffinity). Failure in the formation of the TFIIB-TBP complex of Candida albicans is an indicator of inhibition, and is indicated by the non-retention of marked TBP. Alternatively, the immobilized element may be TBP of Candida albicans and the tagged partner TFIIB of Candida albicans.

In the example above, a strong signal can be picked up in the presence of both TBP and TFIIB and, in addition, a standard DNA containing a TATA element. The complex is then quantified by autoradiography, technology, "Phosphorimager" or scintillation counting for radioactive factors, fluoromotria for fluorescently labeled factors, luminometry for factors labeled with ligands are detected using chemoluminescence or phosphorescence test methodologies, or other similar detection methods or materials marked as described above that are standards in the area.

Other methods for detection or separation of protein-protein complexes can be used, including UV cross-linked analysis, HPLC, phage display technology, and plasmon surface resonance as described herein.

EXAMPLE 5 Tests for the formation of Complexes of TBP-TFIIB-Polymeres II of AKN-ADN The formation of a DNA complex, RNA polymerase II, TFIIB, TBP, is known to be markedly stimulated by the incorporation of another factor, TFIIF. Previous data indicate that TFIIF of S. cerevisiae can function in species such as those initially related to Schizosaccaharomices pombe and humans, strongly suggest that this factor can functionally replace its homologue C. albicans. Accordingly this factor is purified from S. cerevisiae by published methods (Sayre, 1992, J. Biol. Chem. 267: 23383) and used to reconstitute the formation of the complex containing RNA polymerase II, TFIIB, TBP of Candida albicans and the promoter. containing DNA such as that described for the reconstitution of the DNA-TBP-TFIIB complex.

The formation of the complex is carried out in reactions containing, for example, 10-100 ng of TBP of Candida albicans, 10-100 ng of TFIIB of Candida albicans, 10-100 ng. of polymerase II of Candida albicans, 10-100 ng. of TFIIF of S. cerevisiae, 0.5 ng. of oligonucleotides containing TATA double-filament elements, (same as that used for the TFIIB-TBP-DNA complex test) 10-20 mM HEPES (or equivalent), pH 7.5-8.0, 5 mM MgC12, 12% glycerol, 10 mM dithiothreitol (DTT) ), 100 μg / ml. BSA, 5-20 μg / ml. of poly (dG-dC); (dG-dC) and compounds to be tested for their inhibitory activity. Following incubation at 30 ° C for 30-60 minutes, the complexes are detected by one of the methods described above for the TBP-TFIIB-DNA complex. The TBP-TFIIB-polymerase II RNA-DNA complex has slower electrophoretic mobility than the TBP-TFIIB-DNA complex identified by using the electrophoretic method. In addition complex formation can be detected as TBP, TFIIB retention dependent on RNA polymerase II activity (measured by incorporation of labeled precursor nucleotides into soluble acid products using the test for RNA polymerase activity described in the purification protocol of RNA polymerase II set forth above) on a matrix with TATA-element-containing DNA linkage. The IC50 of inhibitory compounds can be determined by titration in reactions constituted as described above. The IC 50 of these compounds against reactions reconstituted with TBP, TFIIB and polymerase II of human RNA will also be determined by the same method. Polymerase II of human RNA and TFIIF are purified as previously described (Flores y collaborators, J. Biol. Chem. 262: 3310-3321). These compounds in which IC50 contrasts with reactions containing C. albicans factors is < o - - 1/5 of their IC50 in contrast to reconstituted reactions with human factors will be tested for their ability to inhibit the development of C. albicans as described below.

EXAMPLE 6 Selection of -------- bidor by phage display In addition to the standard techniques mentioned above in practice, other technologies for molecular identification can be employed in the identification of inhibitory molecules. One of these technologies is the phage display technology (U.S. Patent No. 5,403,484, Viruses Expressing Chimeric Binding Proteins). Phage exposure allows the identification of a binding protein in contrast to a chosen target. Phage exposure is a molecular protection protocol that uses recombinant bacteriophages. The technology involves the transforming bacteriophage with a gene that encodes an appropriate ligand (in this case, an inhibitory candidate) capable of binding to the target molecule of interest. For the purposes of this discovery, the target molecule can be Candida albicans TBP or a protein-DNA or protein-protein complex formed using Tbp and / or TFIIB, as described herein. The transformed bacteriophage (which is preferably fixed to a solid support) expresses the inhibitory candidate and presents it in its phage envelope. Cells or viruses related to the candidate inhibitor that recognizes the target molecule are isolated and amplified. This characterizes the success of the inhibitor.

The phage display technology has advantages over the standard affinity protection technologies of ligands. The surface of the phage exposes the ligand of the microprotein in a third-dimensional conformation, more closely resembling that its natural conformation takes place. This allows more specific and higher affinity links for protection purposes.

EXAMPLE 7 Analysis by Interaction B speci ca A second and relatively new protection technology that can be applied to protection tests by inhibitor of this invention is analysis by biospecific interaction (BIAcore, Pharmacia Biosensor AB, Uppsala, Sweden). This technology is described in detail by Jonsson et al. (Biotechniques 11: 5, 620-627 (1991)). Analysis by biospecific interaction uses surface plasmon resonance (SPR) to monitor the adsorption of biomolecular complexes on a sensor "chip". The SPR measures the changes in the refractive index of a polarized light directed at the surface of the "ch p "sensor.

Specific ligands (for example, candidate inhibitors) capable of binding to the target molecule of interest (for example TBP of Candida albicans or a complex containing TBP of protein-protein or protein-DNA) are immobilized by the sensor "chip". In the presence of a target molecule, the specific bonds for the immobilization of the ligand are carried out. The nascent immobilized ligand-target molecule complex causes a change in the refractive index of the polarized light and is detected on a diode array. The analysis by biospecific interaction provides the advantages of; 1) allow the study by free labeling of the formation of molecular complexes; 2) study of molecular interactions in real time as it happened in the tests on the "chip" sensor; 3) detection in the surface of concentrations less than 10pg / mm2; detecting interactions between two or more molecules; and 4) being fully automatic (Biotechniques 11: 5, 620-6S7 (1991)).

EXAMPLE 8 Multiple and Simultaneous Protection of Potential Inhibitors It is contemplated according to the invention that the protection methods discovered here encompass protection of multiple samples simultaneously, also referred to herein as "multiple and simultaneous" protection. For example, in "multiple and simultaneous protection", several hundred to several thousand candidates for inhibitors can be protected in a single test. Several examples of "multiple and simultaneous protection" tests used according to the invention being as follows.

A fusion protein A (pA) -BP protein of C. albicans is generated by inserting the TBP coding sequence down the backbone of the pA sequence coding for the plasmid pRIT2T (Pharmacia Biotech). The construction of the fusion is induced, and the resulting recombinant protein is extracted and purified according to the recommended conditions by the manufacturer. This procedure can also be carried out for the preparation of a pA-TFIIB protein fusion of Candida albicans except that in the downstream part of the coding sequence is that of the TFIIB protein. All the other steps could stay the same.

A "Dinatech Microlite" with two microtiter plates or equivalent plate capacity for high-protein binding is covered with 1 μg / well tube of human IgG by incubation of 300 μl 3.33 μg / ml. of human IgG (Sigma) in regulatory cover (0.2 M sodium carbonate, pH 9.4) in the well tube for 4-12 hrs. at 4 ° C. The regulating coating is then decanted and the well tube is washed five times with 300 μl of PBS. 300 μl of regulatory blocker (Super Block ™ regulatory blocker; Pierce) containing 3.33 μg / ml. of pA-TBP or pA-TFIIB is added and the plate incubated for 4 or more hours at 4 ° C. The plates can be stored in this form at 4 ° C until they are ready for use. When ready for use the plates are washed five times with 300 μl of PBS. Tests composed at a final concentration of 20-200 μM, labeled TBP or TFIIB (eg unfused proteins), which are not added during the coating step, and 10-1000 promoter-containing fmoles containing oligonucleotides are suspended in HEG buffer containing 200 μg / ml. of BSA in a total volume of 150μl and are added and the incubated reaction at room temperature with slight agitation for 60 minutes. The plate is then washed five times with PBS using a "Dynetech" plate washer or equivalent. The bound labeled proteins are quantified by adding 250 μl of Microscint (Packard) per well tube and counted on a microtiter plate compatible with the scintillation spectrophotometer.

As an alternative, the fusion of protein A and the second, unfused protein are incubated in the presence of test compounds in polypropylene microtiter plates under the same regulator and -incubation conditions described above. The reaction mixture is then transferred to the well tubes of a microtiter plate coated with human IgG (which is prepared as described above, and is stored in regulatory blocker and washed five times with 300 μl of PBS immediately before its use) and is incubated in a blocking blocker for 60 minutes at room temperature with slight agitation. The retention of the radioactive protein is quantified as described above.

The interaction of TBP and TFIIB, which is measured as retention of radioactivity on the plate, is dependent on the plate coated with human IgG and the TBP or TFIIB of Candida albicans type sivestre, one of which can be fused to pA. The candidate inhibitor or extracts that inhibit retention or radioactivity for more than 30% are identified and the inhibitory activity is further purified if necessary.

The inhibitors identified as described above are then tested for their ability to inhibit TBP from Candida albicans-dependent transcription in a viral transcription system as described herein, and can also be tested for their ability to inhibit the development of Candida. albícans.

Other protein modification or fusion systems that have been contemplated to include, but are not limited to, glutathione-S-transferase, maltose protein anlace, influenza virus hemagglutinin, FLAG ™ and exaistidine fusions for TBP of Candida albicans or TFIIB of Candida albicans that are prepared, expressed, and purified by published methods or TBP or TFIIB of biotinylated Candida albicans that are prepared using commercially available reactive biotome precursors. The purified or modified fusion protein is immobilized on a microtiter plate containing the appropriate ligand for each protein fusion (e.g. glutathione, amylose, CA157 antibody, ... etc. respectively) and the test is carried out and the result evaluated in the same way essentially described above.

EXAMPLE 9 Can i data inhibitors A "candidate inhibitor" as interpreted herein, is any compound with a potential to inhibit TBP from Candida aibicans-medium initiation of transcription or complex formation. An inhibitor candidate is tested in a concentration range that depends on the molecular weight of the molecule and the type of test. For example, for inhibition of protein-protein or protein-DNA complex formation or transcription initiation, small molecules (as defined below) can be tested in a concentration range of 1 pg-100 μg / ml, preferably at about 100 pg-10 ng./ml; Large molecules, for example, peptides, can be tested in the range of 10 ng.-100 μg / ml, preferably 100 ng-10 μg / ml.

Inhibitors of the development of Candida albicans or its viability may be targeted by the new transcription factor described here, TBP, or the possible target a protein or nucleic acid that interacts with the new transcription factor in the same way that it prevents the natural biological interaction that takes place in vivo directing the start of transcription in Candida. Thus, an inhibitor identified as described here will possess two properties: 1) some concentration will inhibit the development of Candida albicans or its viability; and 2) at the same concentration, will not significantly affect the development of mammalian cells, particularly humans.

Inhibitory candidates will include peptide inhibitors and polypeptides having an amino acid sequence based on the new TBP sequences described herein. For example, a fragment of TBP can act as a competitive inhibitor with respect to the TBP linkage of other proteins involved in the transcription of Candida, for example, RNA polymerase II, TFIIB, or with respect to transcription complex linkages of the standard DNA .

Compounds that are candidates for inhibitors of large stocks of natural or synthetic compounds can be protected. Numerous media are commonly used for orange blossom and compounds based on targeted synthesis of saccharides, on peptides, and nucleic acids. Synthetic compound stocks are commercially available in a number of company including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ), Brandon Associates (Merrimack, NH), and Microsource (New Milford, CT). A rare chemical pool is available from Aldrich (Milwaukee, Wl). Combinatorial pools are available and can be prepared, alternatively, collections of natural compounds in the form of bacteria, fungi, plant extracts and animal proteins are available from for example, Pan Laboratories (Bothell, WA) or MycoSearch (NC), or are easily producible. Additionally, natural and synthetically produced collections and compounds that are easily modified by conventional chemical, physical and biochemical means.

The compounds used can be found in numerous chemical classes, although typically they have organic compounds, and preferably small organic compounds. Small organic compounds have a molecular weight of more than 50 even less than about 2,500 daltons, preferably less than about 750, more preferably less than about 350 daltons. Jemplar classes include heterocyclics, peptides, saccharides, spheroids and the like, the compounds can be modified to improve their efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent can be used to identify, generate, or agents of additional protection. For example, where peptide agents are identified, they can be modified in a variety of ways to improve their stability, such as the use of an unnatural amino acid, such as a D-amino acid, particularly D-alanine, by amino acid or carboxylic functionality terminal, for example, for the amino group, acylation or alkylation, and for the carboxylic group, esterification or amidation, or the like. Other stabilization methods may include encapsulation, for example, in liposomes, etc.

EXAMPLE 10 Measurement of effectiveness The amount of inhibition by an inhibitory candidate is quantified using the following formula, which describes the reactions reconstituted with a radioactively labeled moiety.

The percentage of Inhibition is equal to the difference between (CPM (positive control) - CPM (Sample) x 100) between (CPM (positive control)), where CPM (positive control) is the average of cpm in complexes of molecules of RNA formed in reactions in which the inhibitor candidate is missing, and CPM (sample) is the cpm in complexes formed in the reaction containing the inhibitory candidate. Inhibitory candidates for which the percent inhibition is 50% are titrated in reactions containing either TBP of Candida albicans or human TBP (expressed in a purified E. coli using existing recombinant clones (Peterson et al., Science 248, 1625 -1630, 1990; Kao et al., Science 248, 1646-1650, 1990; Hoffmann, et al., Nature 346, 387-390, 1990, and tested as described above) and their ICso with respect to human and Candida TBP. Albicans determined from graphs of compound concentrations Vs.% inhibition The ICso is defined as the concentration that results in 50% inhibition The inhibitory candidates for which the ICso against reactions containing TBP of Candida albicans is less than or equal to 1/5 the ICso against reactions containing human TBP are further tested for their ability to inhibit the development of Candida albicans in culture as described below.

EXAMPLE 11 Measures of the inhibition of the development of Candida? Lb ± cans in culture Once an inhibitor is identified in one or more of the binding or transcription tests described herein, it may be desirable to determine the effect of the inhibitor on the development or viability of Candida albicans in culture. An inhibitory candidate is tested for its ability to inhibit the development of Candida albicans cells in culture as follows. Methods for performing tests on growth inhibition in culture are well known in the art. Since the procedure is based on the NCCLS method M27P (The National Committee for Clinical Standards, Reference Method for Broth dilution antifungal Susceptibility Testing of Yeast, proposed standard, 1992), as follows. Sennated dilutions (two or three times the start steps of a maximum concentration of 100-200 μg / ml) of the inhibitor candidate are prepared using RPMI-1640 medium as a diluent and a 100 μl aliquot of each dilution is added to the Well tube. of a 96-well polystyrene microtiter plate. Five colonies of Candida albicans, injected from a dish Agar-Dextrose-Sabouraud inoculated for 14-20 hours previously with the strain of Candida albicans tested. { Catalog No. 10231 of the American Type Culture Collection, Yeast Catalog), are resuspended in RPMI-1640 medium such that the cell density is 10,000-30,000 cells / ml. 100 μl of the cell suspension is added to each of the wells of the 96-well microtiter plate containing the diluted inhibitor candidate and control medium. The cultures are mixed by shaking and incubated at 35 ° C for 48 hours, without agitation, after which cell development is monitored by visual inspection by the formation of turbidity and / or mycelial colonies. The minimum concentration of inhibitory candidate in which no cell growth is detected by this method is defined as the minimum inhibitory concentration (MIC) for this compound. Examples of MCIs by knowledge of antifungal compounds obtained using this technique are 0.125-0.5 μg / ml. for fluconazole and 0.25-1.0 μg / ml. for amphotericin B (The National Committee for Clinical Laboratory Standards, Reference Method for Broth Dilution antifungal Susceptibility Testing of Yeast, proposed standards, 1992). An inhibitor identified by the method described herein will have MICs that is equivalent to or less than the MICs for fluconazole or amphotericin B.

EXAMPLE 12 Counterprotection to Inhibition of Transcription Using TBP Hnpi? No A compound identified as an inhibitor of Candida albicans according to one or more of the tests described herein can be further tested in order to determine its effect on the host organism. In the development of the use of antifungal compounds for human therapeutics, it is desirable that such compounds act as effective agents in inhibiting the viability of the pathogenic fungi while not significantly inhibiting the human cellular system.

Specifically, inhibitors of Candida albicans identified in any of the tests described above can be counter-protected for the inhibition of human TBP.

Human recombinant TBP can be obtained from existing sources and purified by published methods (for example, see Petersonn et al., Kao et al., And Hoffmann et al., Supra) and contacted the inhibitory candidate in tests such as those described above but using a human system. The effectiveness of a TBP inhibitor of Candida albicans as a human therapeutic is determined as one exhibits a low level of inhibition against human TBP in relation to the level of inhibition with respect to TBP of Candida albicans. For example, it is preferred that the amount of inhibition for a given inhibitor of human TBP in a human system is no longer % with respect to the amount of TBP inhibition of Candida albicans / TFIB in a Candida system when tested in any of the tests described above.

Dosage and Pharmaceutical Formulations For therapeutic uses, the inhibitors identified as disclosed herein may be administered in a pharmaceutically acceptable formulation / biologically compatible formulation, for example, in the form of a cream, ointment, lotion or aerosol, for local use, or in a physiological solution, such as salts in solution, for internal administration. The amount of inhibitor administered will be determined according to the degree of pathogenic infection whether the infection is systemic or localized, and will typically be in the range of about 1 μg-100 mg./Kg. of body weight. Where the inhibitor is a peptide or polypeptide, it will be administered in the range of about 100-500 μg / ml. per dose. A single dose of inhibitor or multiple doses, daily, weekly or intermittently, is contemplated according to the invention.

The route of administration will be selected by the doctor, and may be local, oral, transdermal, nasal, rectal, intravenous, intramuscular, or subcutaneous.

Budapest Deposit Treaty E. coli transformed with a plasmid containing the TBP coding gene of Candida albicans has been deposited in an international deposit, ATCC, Rockville, MD, under accession number 69900, on September 15, 1995. E. coli transformed with a plasmid containing the TFIIB coding gene of Candida albicans has been deposited in an international deposit, A. T. C.C., Rockville, MD, under accession number 69899, September 15, 1995. A.T.C.C. No. 69900 and 69899 will be available to the public on the granting of a patent that discovers the access numbers in conjunction with the invention described herein. The deposits were made under the Budapest Treaty, and will be available for a longer period of time after the patent for which the deposit was made, and may be maintained for a period of at least thirty years after the date of deposit and at least five years after the date of deposit. years after the most recent requirement to supply a sample of the deposit is received by the ATCC It is understood that the availability of this deposit does not constitute a license to practice the subject of the invention in derogation of patent rights guaranteed for the subject of the invention by government action.

OTHER MODALITIES The preceding examples demonstrate the experiments carried out and contemplated by the present inventors in the preparation and execution of the invention. It is believed that these examples include an exposition of techniques that serve both information of the subject in the practice of the invention and to demonstrate its use. It will be appreciated by those skilled in the art that the techniques and modalities set forth herein are preferred embodiments only that in general numerous equivalent methods and techniques can be employed to achieve the same result.

All references identified above are hereby expressly incorporated herein for reference to the extensiveness they describe, hereinafter providing a basis for or facilitating compositions and / or methods that may be important for the practice of one or more modalities of the present invention.

LIST OF THE SEQUENCES (1. GENERAL INFORMATION (i) APPLICANT: SCRIPTEN PHARMACEUTICALS, INC. (ii) TITLE OF THE INVENTION: NEW TATA PROTEIN - CANDIDA ALBICANS ATTACHMENT, NUCLEIC ACID SEQUENCE ENCODING THIS, AND SELECTION METHODS FOR INHIBITORS, OF THE DEVELOPMENT OF CANDIDA ALBICANS (iii) SEQUENCE NUMBER: 4 (iv) ADDRESS FOR CORRESPONDENCE (TO) ADDRESS: DARBY S DARBY PC (B) STREET: 805 Third Avenue (C) CITY: New York (D) STATUS: New York (E) COUNTRY: United States of America (F) ZIP : 10022-7513 (v) READING COMPUTER (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS (D) "SOFTWARE": FastSEQ Version 1.5 (vi) NORMAL APPLICATION DATE: (A) APPLICATION NUMBER: (B) REGISTRATION DATE, (c) CLASSIFICATION: (vile) DATE OF PRIORITY APPLICATION: (A) APPLICATION NUMBER: 08 / 626,309 (B) REGISTRATION DATE: 01-aBRIL-1996 (viii) OFFICER / INFORMATION AGENT: (A) NAME: S. PETER LUDWIG, ESQ. (B) REGISTRATION NUMBER: 25,351 (c) REFERENCE / ACT NUMBER: 0342 / 2C488- OR (IX) TELECOMMUNICATION FOR INFORMATION: (A) TELEPHONE: (212) 527-7700 (B) TELEFAX: (212) 753-6237 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 219 amino acids (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: Linear (li) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (IV) CONTRASTING: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Mac Lyß Ser He alu alu Asp alu Lys Asn Lys Ala alu Aßp Leu Asp i 5 10 is lie lia Lys alu Asp He Asp alu Pro Lys ola alu Aßp Thr Thr 25 30 Asp Ser Asn Gly sly aly o1y He aly He Val Pro thr Leu Gln Asn 40 45 lie Val Ala Thr Val Asn Leu Asp Cys Arg Leu Asp Lau Lys Thr He SO 55 SO Ala Leu Kis Ala Arg Aßn Ala olu Tyr Asn Pro Lys Arg Phß Ala Ala 6S 70 7S 80 Val He Met Arg He Arg Asp Pro Lys Thr Thr Ala Leu He Phß Ala ßS 90 95 Ser Oly Lye Met Val Val Thr Oly Ala Lys Ser alu Asp Aßp Ser Lyß 100 105 110 Lau Ala Ser Arg Lys Tyr Ala Arg He Ha Oln Lyß Leu aly Phß Aßn US 120 125 Ala Lyß Phß Cyß Aßp Phe Lyß He Oln Asn He Val Oly Ser Thr Aßp 11C 1-.S 140 Val Lya Phe Wing He Arg Leu Glu Oly Leu Wing Phe Wing His Oly Thr 145 150 15S 160 Phß Ser Ser Tyr alu Pro Glu Leu Pro Pro Gly Leu He Tyr Arg Met 165 170 175 at Lya Pro Lyß He val Leu Leu He Phe Vai Sar Oly Lyß He Val 1. 80 185 190 Lau Thr sly Ala Lys Lyß Arg alu alu Hß Tyr Aßp Ala Phß alu Ser 195 200 205 Xle Tyr P1.0 Val Leu Asn alu Phe Arg Lyß Aßn 210 215 (2) INFORMATION OF SEQ ID NO: 2 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 344 amino acids (B) TYPE: amino acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: NO (iv) CONTRASTING: NO (v) TYPE OF FRAGMENT: internal (vi) ORIGINAL SOURCE: (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Mßt Ser Pro Ser Thr Ser Thr Ala Val aln alu Tyr Ilr 1 5 10 Leu Asn Val Thr Leu Thr Cyß Pro Olu Cyß Lyß He Phß 20 25 Leu Val Olu Ar-3 Phe Ser Glu Gly Aßp He Val Cyß Gly 35 40 45 Leu Val Leu be J p Arg Val Val Aßp Thr Arg Ser alu ' 50 55 £ 0 Phe Ser have Aßp Asp Gln Asn aly Asp Aßp Pro Ser Arg Vt 65 70 75 Ala aly Asn Pro Leu Leu Asp Thr slu Aap Leu Ser Thr Mßv 85 90 Tyr Ala Pro Aßp Ser Thr Lys Ala oly Arg Olu Leu Ser Arg 100 IOS 110 Ser Lys Ser Leu Val Asp Lys Lys Asp Asn Ala Leu Ala Wing. 115 120 125 He Lys I am aln Met Cys Asp aly Tyr Oln Leu Pro Lys I 130 135 140 Ser Asp Gly Ala Lyß alu Val Tyr Lyß Mßt Val Tyr Aßp alu Ly 145 150 155 165 170 175 aly Cys Arg Lys Ala Asn Val Ala Arg Ser Phß Lys Olu He Trj 'Ala 180 185 190 Lys Thr Asn Val pro Arg Lys olu He Oly Lyß Val Phß Lye .'le Met 195 200 205 Asp Lys He lie Arg Glu Lys Asn Ala Wing Asn Pro Aßn A '..-. íl-i Tyr 210 215 220 Tyr Oly Gln Asp be Hß sln Thr Thr Oln Thr Ser Wing Olu Asp Leu 225 230 235 240 Ha Arg Arg Phß Cya Sar His J.eu Gly Val Asa Thr Oln Val Thr Aan 245 250 2S5 aly Ala slu Tyr Ha Ala Arg Arg Cys Lys alu Val aly Val Leu Ala 2S0 2SS 270 aly Arg Ser Pro Thr Thr He Ala Ala Thr val Hß Tyr Mßt Ala Ser 275 280 285 Leu Val Phe Gly Ph-? Sp Lau Pro Pro S «r Lyß Hß Sar? Sp Lys Thr 290 295 300 sly Val Ser Aap Gly Thr Ha Lyß Thr Ser Tyr Ly »Tyr Mßt Tyr Glu 305 310 315 320 Glu Lyß alu Oln Leu He Aßp Pro Ser Trp xle Glu Sar Gly Lys Val 32S 330 335 Lys Leu alu Lys Xle Pro Lys? ßn 340 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 657 base pairs (B) TYPE: nucleic acid (C) TYPE OF FILAMENT: one only (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) CONTRASTING: NO (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) DESCRI PTION OF SEQ ID NO: 3: ATOAAGTCAA TAGAOGAAGA TGAAAAAAAT AAAGCCGA-W3 ATTTOGATAT TATAAAAAAC ßO GAAGATATTG ATGAACCTAA ACAAGAAO? T ACCACTOATA GTAATGGTaa TGGAGGTATT 120 GGTATAGTaC CCACATTACA AA? TATTCTT GCTACGGTsA ATCTTGATTO TCGACTTG? T 180 AAAACAATTO CTTTACATGC TAGAAATGCC OAATATAATC CAAAACGTTT TGCTGCGGTG 240 ATTATGAGAA TTAOAGATCC AAAAACTA G OC? TTAATCT TTQCTTCGGG OA? A? TOGTT 3 0 GTOACTOGOa CTAAATCCOA AGACGATTCC AAOTTOOCTT CAAGA? AGTA TOCTAGAATC 350 ATTCAAAAGT TGGGGTTCAA TGCTAAATTT TGTGATTTT? AAATTCAAAA TATAGTGGGG 420 TCAACAGATQ TTAAQTTTGC TATTAGATTA GAAGGCTT-W3 CTTTTGCTCA TGGTACTTTT 480 TCTTCATATO AACCAGAATT ATTTCCTGas TTAATTTATA GAATsGTGAA ACCA? AAATT S40 aTTTTACTTA TATTTOTTTC TGGGAAAATT GTTTTG? CGa GTGCCAAAA? OACAGAAGAA 600 ATTTATGATO CATTTOA? CT GATTTATCCO GTTTTAAATO AATTTCGTSA AAATTGA 657 (2) INFORMATION FOR SEQ ID NO: 4: (1) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1095 base pairs (B) TYPE: nucleic acid (C) TI PO DE FILAMENTO: (D) TOPOLOGY: (ii) TYPE OF MOLECULE: (iii) HYPOTHETICAL: NO (iv) CONTRASTING: NO (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: ATTGOACCAA ACTTOAATGT TACATTAACA TGTCCTGAOT GTAAOATATT TCCACCTOAT 120 TTG TAaAGA G3TTCAQCOA AGGTOACATT GTCTOTGGCA oTTOTaaacT AOTATTOAOT 180 0Atcatotta TOOATACaAG ATCAGAATOO AGAACTTTCA GTAACGATOA CCAAAATGOT 240 GATGATCCTT CTCGTGTTGG TOATOCAGOT AACCCTTTAT TAGACAC? GA aa? CTTGTCc 300 ACAATOATTT CTTATGCTCC TGATACTACC AAAGCAOOAA QAOAGTTAAG CCGAOCCCAA 360 TCTAAATCTC TAGTCGATAA AAAAGACAAT GCATTOOCTO CAaCATATAT CAAGATTTCT 420 CAAATGTOCO ATOGTTATCA ATTGCCTAAA ATAGTTCTGG ATGGGGCC ?? GGAAOTCTAC 480 AAAATOOTTT? TOACGAOAA ACCATTGCQA GOAAAATCAC AAGAOASTAT CATOaCAGCT S40 TCTATCTTTA TTOGTTaCAß AAAGGCCAAT GTTaCTCGTT CATTCAAAOA AATA-raaacA 600 AAOACTAATO TACCTCGTAA OGAAATTGaT AAAGTaTTCA AO? TCATOOA CAAGATCATT 660 COTOAAAAGA ATOCAaCCAA CCCTAATOCT GCATATTACO OEWG-U-ACAO C? TTCAAACC 720 ACCCAA CIT COaCCGAGGA TTTOATAGA AaArrctott CTCACTTGOO TOTTAACACA 780 CAAOTTACAA ATOGTGCGGA ATACATAGCC AGAAOATGTA AGOAAGTCOG GGTTTTAGCA 840 G3TAGATC0C CAACTACAAT TGCTGCAACT OTAATTT? CA TOGCTTCACT AGTGTTTOOA 900 TTTGACTTAC CTCCATCCAA GATATCTOAT AAAACTOOTa TCAGTGATOO TACTATCAAA 960 ACTTCATACA AGTACATOTA CGAGOAGAAA SAGs? ATTGA TTGATCCATC TTOOATAOAA 1020 AsTOOTAAAG TAAAATTOGA AAAAATACCA AAAAACTAAT? C? GCGGAGT CGCCACTGTT 1080 AATCCTTTAC CCTCT 1095 It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, it is claimed as property in the following:

Claims (15)

1. A recombinant nucleic acid characterized by a nucleic acid sequence coding for TBP of Candida albicans.

2. A vector characterized by a nucleic acid sequence encoding TBP of Candida albicans.

3. A transformed host cell containing a nucleic acid sequence coding for TBP of Candida alb.

4. A recombinant polypeptide characterized by TBP of Candida alb cans.

5. A fragment of TBP of Candida albicans, said fragment is characterized in that it inhibits the biological activity of TBP of Candida albicans at the start of transcription.

6. A fragment of TBP of Candida albicans, said fragment is characterized in that it prevents the development of Candida albicans.

7. A method for producing recombinant Candida albicans TBP is characterized by culturing the host cell of claim 3 under conditions sufficient to allow expression of the TBP coding nucleic acid of Candida albicans, and isolating said TBP from Candida albicans.

8. One method of protection for identifying an inhibitor of the development of Candida albícans, consists of detecting the inhibition of the transcription of mRNA in an in vitro transcription test that considers a DNA standard, Polymerase II of RNA, TBP of Candida albícans recombinante, and a inhibitory candidate, wherein the production of a mRNA transcript of said standard DNA is carried out in the absence of said inhibitory candidate.

9. A method of protection by identification of an inhibitor of the development of Candida albícans, is characterized by the detection in the presence of a candidate inhibitor, the inhibition of the formation of a complex characterized by a DNA standard and TBP of recombinant Candida albícans, is characterized because the formation of said complex takes place in the absence of said inhibitory candidate.

10. A method of protection by identification of an inhibitor of the development of Candida albicans, is characterized by the detection in the presence of a candidate inhibitor, the inhibition of the formation of a complex consisting of TFIIB of Candida albicans and TBP of Candida albicans is characterized in that the formation of said complex takes place in the absence of said inhibitory candidate.

11. A protection method by identification of an inhibitor of the development of Candida albicans, is characterized by the detection in the presence of a candidate inhibitor, the inhibition of the formation of a complex consisting of polymerase II RNA, TBP of Candida albicans, and TFIIB of Candida albicans, characterized in that the formation of said complex takes place in the absence of said inhibitory candidate.

12. The protection methods of claims 8, 9, 10 or 11 are characterized in that said detection is carried out in the presence of a plurality of inhibitory candidates such that said inhibition is indicative of inhibition by said inhibitor candidate of said plurality.

13. The method of protection of claims 8, 9, or 11, are characterized in that multiple steps are performed simultaneously using a plurality of inhibitory candidates, detection of inhibition is characterized in that any one of the inhibitory candidates is independently detectable from said plurality.

14. A method of preventing the development of Candida albícans in culture consists in contacting said culture with an inhibitor that selectively inhibits the biological activity of the TBP of Candida albícans.

15. One method of preventing the development of Candida albicans in a mammal consists of administering to said mammal a therapeutically effective amount of an inhibitor that inhibits the biological activity of TBP of Candida albicans.