HK1114619B - A bacterial atp synthase binding domain - Google Patents

Description

Binding domain of bacterial ATP synthase

The present invention provides isolated mutant atpE proteins and identifies an atpase binding domain from the mutant atpE proteins. The invention also provides related nucleic acids, vectors, host cells, pharmaceutical compositions and products. The invention further provides a method of determining whether a test compound interacts with an atpE protein (i.e. with an atpase binding domain of the invention) and also provides pharmaceutical compositions comprising said test compound, in particular for use as an antimicrobial, more particularly as an antimycobacterial agent, especially for use in the treatment of patients suffering from tuberculosis.

Background

Tuberculosis (TB) ranks front (secondary to AIDS) among adult causes of death worldwide, causing 2-3 million adult deaths each year, and is a critical barrier to reduce global poverty and disease work (1). Factors contributing to the resurgence of the disease include difficulties encountered in the implementation of anti-tuberculosis programs in many countries, a rapid increase in the number of immunosuppressed individuals (primarily due to HIV infection), and a move of people into and out of areas with high tuberculosis incidence. TB and HIV are contributing to each other in double-infected people-currently 1100 million people-increasing both morbidity and mortality (2, 3). Also among the causes of death in HIV-infected individuals, TB is also ranked top (4).

Although first-line anti-TB drugs can achieve efficiencies above 90%, their complexity can lead to poor drug compliance, and thus resistance, in the absence of adequate medical measures and anti-TB programs (5). Multiple Drug Resistant (MDR) strains of tuberculosis bacteria can make treatment very complicated (6). The global alliance for TB Drug Development has indicated that any new treatment must have at least one of The following three advantages over existing treatments: shortening or simplifying effective TB therapy; increasing efficiency against MDR strains; improving the treatment of potential forms of TB infection. This new drug can greatly improve patient compliance, thereby reducing the cost of TB Treatment programs, such as the world health organization's modern tuberculosis control strategy (dots) (7).

The new anti-TB drug candidates currently in development and clinical use are likely to be from the existing drug family (e.g. moxifloxacin), or from first-line drug analogues such as MJH-98-1-81 (from isoniazid), oxazolidinones, and rifapentine, a substance very similar to rifampicin (8). Although these drugs may be effective, structural analogs only temporarily solve the resistance problem (9) because their mechanism of action is consistent with the existing drug family.

Antibiotics in general inhibit bacterial replication by generally inhibiting bacterial metabolism through specific mechanisms. For example, isoniazid interferes with the enzymatic mechanism of action of synthetic mycolic acids, which are essential components of the cell wall, while rifampicin interferes with the DNA transcription mechanism of bacteria. It is therefore of interest to explore new ways to identify novel anti-TB compounds that target specific aspects of mycobacterial cell growth and replication compared to known drugs.

Summary of The Invention

The present invention provides an isolated mutant atpE protein, the amino acid sequence of which is selected from the group consisting of (SEQ ID No.1), (SEQ ID No.2), (SEQ ID No.3), (SEQ ID No.4) and (SEQ ID No.5), and the nucleic acid sequence encoding the isolated mutant atpE protein, which is selected from the group consisting of (SEQ ID No.6), (SEQ ID No.7), (SEQ ID No.8), (SFQ ID No.9) and (SEQ ID No.10), and a vector comprising a transiently expressed nucleic acid. In a particular embodiment the amino acid sequence of the mutant atpE protein is SEQ ID No.2 and the nucleic acid sequence encoding it consists of SEQ ID No. 7.

The invention also provides host-vector systems, including cells with transient expression vectors.

The invention also provides isolated cells comprising a mutant atpE protein, wherein the protein confers antimicrobial resistance to the cells.

The present invention also provides a method of identifying an antimicrobial compound, comprising the steps of:

(a) contacting a test compound with a cell expressing an atpE protein under physiological conditions;

(b) determining whether the test compound interacts with the atpE protein.

The present invention also provides a method of assessing the potential of a test compound to interact with an atpE protein, comprising the steps of:

(a) molecular modeling (molecular modeling) techniques are used to build the three-dimensional structure of the atpE protein;

(b) using computational means to perform a fit between the test compound and the three-dimensional structure of the atpE protein; and

(c) the results of the fit procedures were analyzed to quantify the relationship between the test compound and the three-dimensional structure of the atpE protein.

The present invention is also directed to providing F of ATPase₀Part of a binding site, Ala comprising at least one C subunit²⁴、Gly²⁷、Phe⁵³、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴And Phe⁶⁵(ii) a Ser of one A subunit¹⁸²、Leu¹⁸³、Ser¹⁸⁴、Leu¹⁸⁵And Arg¹⁸⁶And all of said amino acids having the amino acid sequence set forth in any one of tables 3, 4 or 5Atomic coordinates.

Another object of the present invention is to provide a method for identifying F capable of binding to ATPase using the aforementioned binding domain₀Partially interacting compounds and methods for identifying their potential as antimicrobial compounds, in particular anti-mycobacterial compounds using the aforementioned binding domains.

It is therefore another object of the present invention to provide a method for treating a subject infected with a microorganism, which comprises administering to the infected subject F, which is reactive with ATPase₀Compounds which interact in part, in particular with the mutated sites in the atpE protein which confer resistance thereto or with the binding sites of the invention. The invention also provides a method of treating a patient suffering from tuberculosis comprising administering to the patient an agent which interacts with the atpE protein obtained by any of the screening methods described above. This method of treatment uses F, which is compatible with ATPase₀Compounds which interact partially, in particular with the atpE protein, but which are previously known to interact with the F of ATPase₀Compounds that interact partially, particularly with the atpE protein, are to be excluded. In particular, the use of DARQ J (11) compounds according to any of the disclosed methods of treatment is to be excluded.

The invention also provides a pharmaceutical composition comprising an agent that interacts with atpE protein in a cell, and a pharmaceutically acceptable carrier. Finally, the invention provides products, including packaging and a medicament, wherein (a) the medicament interacts with the atpE protein in the cell, and (b) the packaging includes a label directing how to use the medicament for treating a person with a bacterial infection. In a specific embodiment of the invention, it is described how DARQ J is used to prepare antimicrobial drugs.

Various aspects of the invention are described in greater detail below.

Brief Description of Drawings

TABLE 1 Minimum Inhibitory Concentration (MIC) of lead DARQ compound (J), which concentration of J compound inhibits growth of different mycobacteria by 90%. If not otherwise stated, n is 1 for the number of strains tested.

Table 2 amino acids around the binding site of DARQ J compounds.

Table 3 atomic coordinates of amino acids around the binding site of DARQ J compounds in mycobacterium tuberculosis (m.tuberculosis) wild-type and DARQ J-resistant mutants.

TABLE 4 atomic coordinates of binding site for DARQ J compound in wild-type M.tuberculosis.

TABLE 5 atomic coordinates of binding site for DARQ J compound in mutant M.tuberculosis.

TABLE 6 atomic coordinates of Mycobacterium tuberculosis mutant atpE protein (SEQ ID No. 2).

TABLE 7 atomic coordinates of the wild-type atpE protein of Mycobacterium tuberculosis (SEQ ID No. 1).

FIG. 1, R207910, also referred to herein as the absolute configuration of J or DARQ J.

FIG. 2 sequence alignment of Mycobacterium tuberculosis and mutant Mycobacterium smegmatis (M.smegmatis) atpE proteins. Mtb _ S: a drug-sensitive strain of Mycobacterium tuberculosis H37Rv, atpE (1-81). Listing number (Accession number) Swiss-ProtQ10598(SEQ ID No. 1). Mtb _ R: a drug-resistant strain of Mycobacterium tuberculosis BK12, atpE (1-81) (SEQ ID No. 2). Msm _ S: a drug sensitive strain of Mycobacterium smegmatis, atpE (1-86). Sequence obtained from Institute for genome research (SEQ ID No. 3). Msm _ R09(SEQ ID No.4) and R10(SEQ ID No. 5): a drug-resistant strain of Mycobacterium smegmatis, atpE (1-86). Internally derived sequences, human: human (Homo sapiens), ATP5G3 (66-142). The recording number is as follows: ensembl ENSP 00000284727. Top sequence number: mycobacterium tuberculosis and Mycobacterium smegmatis atpE. Bottom sequence number: human ATP5G3 (66-142). The shaded areas indicate the amino acid similarity obtained using the BLOSUM62 matrix (black is highly similar; grey is moderately similar). Arrows indicate the positions of point mutations found in the resistant mutants.

FIG. 3 Total cellular ATP measurements in M.tuberculosis in the presence of DARQ J, isoniazid and DCCD. Relative Luminescence Units (Relative Luminescence Units) of oxyluciferin measured at a wavelength of 526nm within the M.tuberculosis wild-type and DARQ J resistant mutants.

FIG. 4 shows the band-like structure of the three C subunits (A chain, K chain and L chain) and A subunit (M chain) which together form the binding site for a DARQ J compound.

Detailed Description

Definition of

Unless otherwise explicitly stated, the meanings of the following terms used in the present application are listed below.

"atpE protein" means the ATPase Complex F₀The C chain of a subunit (represented by SwissProt entryQ10598, Mycobacterium tuberculosis), or a protein having at least 70, 80, 90, 95 or 99% sequence identity to the Mycobacterium tuberculosis sequence.

“F₁F₀ATPases also known as ATPases, ATP synthases, or F₁F₀Atpase, which refers to a large multi-subunit complex that catalyzes ATP synthesis or hydrolysis. F₁F₀Atpases are composed of two domains: f₁A moiety, located on the exterior of the membrane, containing catalytic sites; f₀And a moiety which spans the bilayer membrane and contains a proton pore (proton pore). Atpases are found on bacterial plasma membranes, the chloroplast inner vesicle membrane, and the mitochondrial inner membrane, and use a proton electrochemical gradient to drive ATP synthesis.

"administration" refers to delivery in some manner, and may be carried out by any of a variety of methods and delivery systems known to those skilled in the art. Can be administered, for example, topically, intravenously, intrapericardially, orally, by transplantation, transmucosally, transdermally, intramuscularly, subcutaneously, intrapleurally, intrathecally, intralymphatically, by intralymphatic injection, or epidurally. Administration may also be performed, e.g., once, multiple times, and/or over one or more extended periods of time.

"host cells" include, but are not limited to, bacterial cells, yeast cells, fungal cells, insect cells and mammalian cells. Bacterial cells can be transfected by methods well known in the art such as calcium phosphate precipitation, electroporation, and microinjection.

An "isolated" atpE protein refers to a preparation of membrane fragments that comprises an atpE protein that retains its native function and does not contain some/all of the other proteins in its native environment, or other suitable preparation. This means including F₀F₁F of ATPase₀Partial membrane preparations, in particular F containing the mutant atpE proteins of the invention₀Part of the membrane preparation.

"bacterial cell" refers to any bacterial cell. Bacterial cells include, but are not limited to, normal, abnormal and transformed cells such as mycobacteria, particularly Mycobacterium tuberculosis and Mycobacterium smegmatis, corynebacteria, Nocardia, gram-positive bacteria such as streptococci, staphylococci and enterococci, gram-negative bacteria such as E.coli, Haemophilus influenzae (Haemophilus influenzae) and Helicobacter pylori (Helicobacter pylori).

"nucleic acid" and "polynucleotide" are used synonymously herein and refer to polymers of deoxyribonucleotides and/or ribonucleotides. Deoxyribonucleotides and ribonucleotides may refer to either natural products or synthetic analogs.

When referring to a "physiological condition" of a given cell, it is generally intended to refer to the biochemical environment in which the cell is located. The biochemical environment of a cell includes (but is not limited to) all/some of the proteases that a cell normally faces. Such conditions include, but are not limited to, in vivo conditions.

"polypeptide", "peptide" and "protein" are used synonymously herein and all refer to a polymer of amino acid residues. The amino acid residue may be a natural product or a chemical analogue thereof. Polypeptides, peptides and proteins also include modified products such as glycosylation, lipid attachment, sulfation, hydroxylation and ADP-ribosylation.

A "subject," also referred to as an "infected" or "patient," includes any animal, e.g., mammals or birds, including (but not limited to) cows, horses, sheep, goats, pigs, dogs, cats, rodents such as mice or rats, turkeys, chickens, and primates. In a preferred embodiment the subject is a human.

"treating" includes, but is not limited to, eliminating a disease, reversing the disease process, delaying the progression of a disease, alleviating a pain, or otherwise ameliorating the disease in a subject.

"vector" refers to any nucleic acid vector known in the art. Such vectors include, but are not limited to, plasmid vectors, cosmid vectors, and phage vectors.

"candidate" and "test compound" are used synonymously herein and refer to substances that are believed to interact with other groups (i.e., atpE proteins) as biological response modifiers. For example, it is believed that representative candidates may interact with atpE proteins and may modify atpase activity. Candidates that can be studied using the methods of the invention include, for example, but are not limited to, peptides, enzymes, enzyme substrates, cofactors, sugars, oligonucleotides, small molecular weight compounds, and monoclonal antibodies.

"modulation" means increasing, decreasing, or otherwise altering any or all of the chemical and biological activities/properties of either the wild-type atpE protein or the mutant atpE protein.

"interaction" refers to a detectable interaction between molecules, including an intermolecular "binding" interaction. The interaction may be, for example, a naturally occurring protein-protein or protein-nucleic acid interaction. These interactions can be detected using methods known in the art, such as using a yeast two-hybrid system, immunoprecipitation, scintillation proximity assay, or filter binding detection.

As used herein, "atomic coordinates" or "structural coordinates" refer to mathematical coordinates describing the location of atoms in the Protein Database (PDB) format (format), including each atomX, Y, Z and B coordinates. It will be understood by those skilled in the art that the set of structural coordinates determined by X-ray crystallography are not without a standard deviation. In the present invention, a set of structural coordinates of ATP synthase from any source, when superimposed on non-hydrogen atom positions corresponding to the atomic coordinates set forth in Table 3, 4, 5,6 or 7, has a root mean square deviation of the non-hydrogen atom positions less thanI.e., they are all considered to have substantial identity or homology. In a more preferred embodiment, any set of structural coordinates of any source of ATP synthase, when superimposed on non-hydrogen atom positions corresponding to the atomic coordinates set forth in Table 3, 4, 5,6, or 7, has a root mean square deviation of the non-hydrogen atom positions less thanI.e., they are all considered to have substantial identity or homology.

Embodiments of the invention

Mutant atpE proteins

The present invention provides isolated mutant atpE proteins, particularly bacterial atpE proteins, and more particularly mycobacterial atpE proteins, and more particularly mycobacterium tuberculosis and mycobacterium smegmatis atpE proteins. The mutation is selected from a single point mutation, insertion or deletion. In one embodiment of the invention, the mutation comprises the introduction of at least one point mutation at any position between amino acids 20-40, in particular between amino acids 30-40 (preferably at amino acid position 34), or at least one point mutation at any position between amino acids 60-75, in particular between amino acids 62-73 (preferably at amino acid position 69), as shown in the sequence alignment in FIG. 2. In another embodiment, the isolated mutant atpE protein is selected from the group consisting of Mtb _ R (SEQ ID No.2), Msm _ R09(SEQ ID No.4) and Msm _ R10(SEQ ID No.5), as shown in fig. 2, or a protein having at least 70, 80, 90, 95, 97 or 98% sequence identity to any of the amino acid sequences described above.

The invention also provides a separated braidNucleic acid encoding said mutant atpE protein. In one embodiment, the nucleic acid sequence comprises all of the encoding F₀Part of a gene (see e.g.J.biol.chem, 1994, Vol.269(10), p.7285-7289), wherein said gene is transcribed from a single promoter and comprises a nucleic acid sequence encoding a mutant atpE protein of the invention. The nucleic acid may be DNA or RNA, preferably DNA, in another embodiment the nucleic acid is selected from a nucleic acid sequence encoding Mtb _ R (SEQ ID No.7), Msm _ R09(SEQ ID No.9) or Msm _ R10(SEQ ID No.10), or a nucleic acid sequence having at least 70, 80, 90, 95, 97 or 98% sequence identity to any of the nucleic acid sequences described above.

Commercial computational programs can be used to calculate percent identity of nucleic acid and polypeptide sequences, and these tools compare query sequences to reference sequences. The following programs (provided by the National Center for Biotechnology Information) can be used to determine homology/identity: BLAST, gapped BLAST, BLASTN, and PSI-BLAST (all with default parameters).

The algorithm GAP (Genetics Computer Group, Madison, Wis.) compares two complete sequences using Needleman and Wunsch algorithm, which maximizes the number of fits and minimizes the number of GAPs (GAP). Default parameters are typically used, and a gap deduction (gap creation dependency) is 12 and a gap extension dependency is 4.

The FASTDB software is based on the algorithm reported by Brutlag et al (Comp. App. biosci., 6; 237-. The software enables a comprehensive alignment of sequences. The results of the full alignment are in percent identity. Suitable parameters for searching DNA using FASTDB to calculate percent identity are: matrix equals unity, k-tuple equals 4, mismatch equals 1, Joining Penalty equals 30, Randomization Group Length equals 0, cutoff score equals 1, Gap Penalty equals 5, Gap Size Penalty equals 0.05, Window Size equals 500, or equals the base Length of the query sequence (the shorter of the two). Suitable parameters for calculating percent amino acid sequence identity and similarity are: matrix equals PAM 150, k-tuple equals 2, Mismatch Penalty equals 1, Joining Penalty equals 20, Randomization group Length equals 0, Cutoff Score equals 1, Gap Penalty equals 5, Gap Size Penalty equals 0.05, Window Size equals 500, or equals the base length of the query sequence (the shorter of the two).

The invention also provides vectors comprising the transient nucleic acids. In one embodiment the vector is a plasmid vector.

The invention also provides host-vector systems, including host cells with transient plasmid vectors. The host cell may be a prokaryotic or eukaryotic cell, in one embodiment the host cell is a bacterial cell, in particular a mycobacterial cell such as Mycobacterium tuberculosis or Mycobacterium smegmatis.

The invention also provides an isolated cell comprising a mutant atpE protein that confers antimicrobial resistance in the cell. In one embodiment, the isolated cell is a Mycobacterium smegmatis cell transformed with a mutant Mycobacterium atpE protein, in particular a mutant protein in which at least one point mutation is introduced at any position from amino acids 20 to 40, in particular from amino acids 30 to 40 (preferably at amino acid position 34), or at least one point mutation is introduced at any position from amino acids 60 to 75, in particular from amino acid position 62 to 73 (preferably at amino acid position 69), as shown in the sequence alignment of FIG. 2.

Screening method

The present invention also provides a method of identifying an antimicrobial compound, the steps of the method comprising:

(a) contacting a test compound with a cell expressing an atpE protein under physiological conditions;

(b) determining whether the test compound interacts with the atpE protein.

In one embodiment, the atpE protein used in the above method is a bacterial atpE protein (particularly a mycobacterial protein), including wild-type proteins and mutant proteins as described above. In a further embodiment of the present invention,the mycobacterial atpE protein used in the above method is a mutant mycobacterial atpE protein of the present invention. In a particular embodiment of the above method, a host cell transformed with a mutant atpE protein of the invention is used and is identified for F₁The possible inhibition of the enzymatic activity of the Fo-ATPase (comprising the mutant atpE protein) is used to measure the interaction between the test compound and the atpE protein. Determination of F pairs using methods known in the art₁Inhibition of Fo-ATPase activity, for example, by adding the substance to a system comprising ATPase and ATP as a substrate, and detecting the enzyme activity by coupling the production of ADP with the oxidation of NADPH (via pyruvate kinase and lactate hydrogenase reactions).

In one embodiment of this method, the atpE protein may be used in a binding assay. The binding assay may be competitive or non-competitive. Such assays allow for the rapid screening of a large number of compounds to identify any compound that is capable of binding to the polypeptide.

The invention herein provides a method for identifying whether a test compound binds to an isolated atpE protein of the invention, thereby identifying a potential antimicrobial compound, said method steps comprising:

a) contacting a cell expressing an atpE protein, in the presence or absence of a compound known to bind to the protein, with a test compound, wherein the cell does not normally express the atpE protein.

b) The binding of the test compound to the atpE protein was determined using as a reference a compound known to bind to the atpE protein.

The binding of the test compound or a compound known to bind to the atpE protein (hereinafter also referred to as the reference compound) is measured using methods known in the art for studying protein-ligand interactions. Such binding may be measured, for example, using a labeled substance or reference compound. The test compound or reference compound, particularly compound J (fig. 1), may be labeled using any convenient method known in the art, such as radiolabelling, fluorescent labeling or enzymatic labeling. In one embodiment of the above method, a compound known to bind to the atpE protein (i.e., the reference compound) is detectably labeled, and the label is used to determine the binding of the test compound to the atpE protein. The reference compound is labeled with a radioactive, fluorescent or enzymatic label, more preferably with a radioactive label,

in another alternative embodiment of the invention, the above binding assay is carried out on a cellular composition, i.e.in a cellular extract, cellular debris or organelles containing the atpE protein as defined above. More specifically, the above binding assay is carried out in a cellular composition, i.e.in a membrane preparation comprising an atpE protein fragment as defined above, wherein said cellular composition (i.e.membrane preparation) is obtained from a Mycobacterium smegmatis cell transformed with a mutant Mycobacterium atpE protein, in particular a mutant protein in which at least one point mutation is introduced at any position between amino acids 20-40, in particular between amino acids 30-40, preferably at amino acid 34, or at any position between amino acids 60-75, in particular between amino acids 62-73, preferably at amino acid 69, as shown in the sequence alignment in FIG. 2. With reference to the sequence numbers of Mtb _ S (SEQ ID No.1) or Mtb _ R (SEQ ID No.2), the above-mentioned region corresponds to amino acids 14 to 34, in particular amino acids 24 to 34, preferably to amino acid 28, or to amino acids 54 to 69, in particular amino acids 56 to 67, preferably to amino acid 63.

In one embodiment, the binding assay is performed using a membrane preparation. Such membrane preparations can be used in conventional filter-binding assays (e.g., using Brandel filtration assay equipment) or high throughput scintillation proximity assays (SPA and Cytostar-T flash plate technology; Amersham pharmacia Biotech) to detect atpE ligands with radiolabel(s) (including³H-labeled DARQs), and replacement of these radiolabels by binding site competitors. Packard Topcount or similar device capable of rapid measurement on 96-, 384-, 1536-microtiter plates may be usedIs placed to measure radioactivity. The SPA/Cytostar-T technique is particularly suitable for high throughput screening, and is therefore suitable for screening compounds that can replace standard ligands.

Other methods for studying ligand binding to atpE under near-natural conditions utilize the surface plasmon resonance effect developed by the biacore (biacore) device. The atpE protein in the membrane preparation or in the whole cell can be attached to a biosensor chip of Biacore and the binding of the ligand detected in the presence and absence of the compound to identify competitors of the binding site.

Molecular modeling

The present invention also provides a method of assessing the potential of a test compound to interact with an atpE protein, comprising the steps of:

(a) molecular modeling techniques were used to elucidate the three-dimensional structure of atpE proteins;

(b) performing a fit between the test compound and the three-dimensional structure of the atpE protein using computer methods;

(c) the results of the fit procedure were analysed to quantify the association between the test compound and the three-dimensional structure of the atpE protein.

Molecular modeling techniques known in the art, including hardware and software, are suitable for creating and utilizing receptor models and enzyme conformations.

There are many computer programs available that are suitable for performing computer modeling, modeling operations, and identifying, selecting, and measuring the atpE that interacts with a compound in the methods described herein. These programs include, for example, GRID (from Oxford University, UK), MCSS (from Accelrys, Inc., San Diego, CA), AUTODOCK from Oxford Molecular Group), FLEX X (from Tripos, St.Louis. MO), DOCK (from University of California, San Francisco, CA), CAVEAT (from University of California, Berkeley), HOOK (from Accelrys, Inc., San Diego, CA), and 3D database Systems such as MACCS-3D (from MDLInformation Systems, San Leandro, CA), UNITY (from Tripos, St.Louis. MO) and CATALYST (from Accelrys Diego, Inc., CA). Potential candidates can also be designed in silico from scratch ("de novo") using software packages such as LUDI (from Biosym Technologies, San Diego, CA), LEGEND (from Accelrys, Inc, San Diego, CA) and LEAPFROG (from Tripos, st. The deformation energy (deformation energy) and electrostatic repulsion of the compounds can be analyzed using programs such as GAUSSIAN 92, AMBER, QUANTA/CHARMM and INSIGHT II/DISCOVER. These computer measurement and modeling operations may be performed on any suitable hardware, such as Silicon Graphics, Sun Microsystems workstations, and the like. These modeling techniques, methods, hardware and software packages are representative only and are not intended to be limiting. Other Modeling techniques known in the art can also be used in accordance with the present invention, see, e.g., N.C. Cohen, Molecular Modeling in Drug Design, academic Press (1996).

In one embodiment of the invention, the three-dimensional structure of the atpE Protein is determined by the atomic coordinates of Ile28, Glu61 and Ile63 (E. coli (Protein Database 1Q01)) +/-the root mean square deviation of the amino acid backbone atoms (not more than that of the amino acid backbone atoms)Preferably not more than) Thus obtaining the product.

As illustrated in the examples below, it is an object of the present invention to provide a three-dimensional structure for the atpE protein. Tables 6 and 7 provide the atomic coordinates of the mutant atpE protein (SEQ ID No.2) and the wild-type atpE protein (SEQ ID No. 1). Thus in one embodiment the atomic coordinates of tables 6 or 7 are used to derive the three dimensional structure of the atpE protein. In particular embodiments, the atomic coordinates of Table 7 are used to derive the three-dimensional structure of the atpE protein. DARQ J Compounds inhibit Arg of A subunit¹⁸⁶Deprotonated form of Glu with the C subunit⁶¹The interaction between them. It is therefore an object of the present invention to provide a method for evaluating a test compound and a using the atomic coordinates in Table 6 or 7tpE protein.

Binding sites

In another embodiment of the invention, ATPase F is identified₀And (4) partial. The binding site was identified as a site capable of binding to DARQ J compounds and was found to be consistent with the region of the resistance conferring mutation site in the atpE proteins (17) of mycobacterium tuberculosis and mycobacterium smegmatis identified above. The invention thus provides F of an ATPase₀A binding site in the portion characterized as comprising a resistance conferring mutation site in the atpE protein. As used herein, resistance conferring mutation sites refer to amino acids 14-34, particularly amino acids 24-34, and amino acids 53-69, particularly amino acids 56-67 of the atpE protein, using the sequence numbers of Mtb _ S (SEQ ID No.1) or Mtb _ R (SEQ ID No.2) as a reference.

In another embodiment, the binding site comprises at least one amino acid Ala of the C subunit²⁴、Gly²⁷、Phe⁵³、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴And Phe⁶⁵And amino acid Ser of an A subunit¹⁸²、Leu¹⁸³、Leu¹⁸⁵And Arg¹⁸⁶(the A subunit is Ser 206-Leu 207-Leu 209 and Arg210 in tables 3, 4, 5), where the amino acids have the atomic coordinates set forth in any one of tables 3, 4, 5, or have homologous structural coordinates that, when superimposed on a non-hydrogen atom position corresponding to the atomic coordinates set forth in tables 3, 4, or 5, have a root mean square deviation of the non-hydrogen atom less thanPreferably less thanIn a particular embodiment, the binding site comprises the amino acid Ala of the first C subunit²¹、Gly²⁵(ii) a Amino acid Ala of the second C subunit²⁴、Gly²⁷、Phe⁵³、Phe⁵⁴、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴、Phe⁶⁵(ii) a Amino acid Met of the third C subunit¹⁷、Gly¹⁹、Gly²⁰、Ala²¹、Ile²²、Gly²³、Ala²⁴、Gly²⁵、Ile²⁶、Gly²⁷、Asp²⁸、Gly²⁹、Ala³¹、Phe⁵³、Thr⁵⁶、Val⁵⁷、Gly⁵⁸、Leu⁵⁹、Val⁶⁰、Glu⁶¹、Ala⁶²、Ala⁶³/Pro⁶³、Tyr⁶⁴And Phe⁶⁵(ii) a And the amino acid Leu of one A subunit¹⁸³、Leu¹⁸⁵And Arg¹⁸⁶Wherein said amino acid has the atomic coordinates set forth in any one of tables 3, 4, and 5, or has homologous structure coordinates wherein the root mean square deviation of a non-hydrogen atom is less than the root mean square deviation of a non-hydrogen atom when the homologous structure coordinates are superimposed on a non-hydrogen atom position corresponding to the atomic coordinates set forth in Table 3, 4, or 5Preferably less thanIn a more particular embodiment, the binding site consists of: ala of the first C subunit²¹、Gly²⁵(ii) a Ala of the second C subunit²⁴、Gly²⁷、Phe⁵³、Phe⁵⁴、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴、Phe⁶⁵(ii) a Met of the third C subunit¹⁷、Gly¹⁹、Gly²⁰、Ala²¹、Ile²²、Gly²³、Ala²⁴、Gly²⁵、Ile²⁶、Gly²⁷、Asp²⁸、Gly²⁹、Ala³¹、Phe⁵³、Thr⁵⁶、Val⁵⁷、Gly⁵⁸、Leu⁵⁹、Val⁶⁰、Glu⁶¹、Ala⁶²、Ala⁶³/Pro⁶³、Tyr⁶⁴、Phe⁶⁵And Leu of one A subunit¹⁸³、Leu¹⁸⁵And Arg¹⁸⁶Which isWherein said amino acid has the atomic coordinates set forth in any one of tables 3, 4, and 5. In a most particular embodiment, the binding site consists of: ala of the first C subunit²¹、Gly²⁵(ii) a Ala of the second C subunit²⁴、Gly²⁷、Phe⁵³、Phe⁵⁴、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴、Phe⁶⁵(ii) a Met of the third C subunit¹⁷、Gly¹⁹、Gly²⁰、Ala²¹、Ile²²、Gly²³、Ala²⁴、Gly²⁵、Ile²⁶、Gly²⁷、Asp²⁸、Gly²⁹、Ala³¹、Phe⁵³、Thr⁵⁶、Val⁵⁷、Gly⁵⁸、Leu⁵⁹、Val⁶⁰、Glu⁶¹、Ala⁶²、Ala⁶³/Pro⁶³、Tyr⁶⁴、Phe⁶⁵And Leu of one A subunit¹⁸³、Leu¹⁸⁵And Arg¹⁸⁶Where the amino acids have the atomic coordinates set forth in Table 3.

It is therefore an object of the present invention to use the above atomic coordinates in a computer screening program to evaluate the potential of a test compound to interact with an atpE protein. In one embodiment, the present invention provides a method of evaluating the potential of a test compound for interaction with an atpE protein, comprising: generation of ATPase F Using molecular modeling techniques₀The three-dimensional structure of the partial binding site; performing a fitting operation between the test compound and the three-dimensional structure of the binding site using a computer method; the results of the fitting procedure are analyzed to quantify the interrelationship between the test compound and the three-dimensional structure of the binding site. In another embodiment of the invention, the three-dimensional structure of the binding site is generated using the atomic coordinates set forth in Table 3, 4 or 5, or is generated using the coordinates of a homologous structure, wherein the homologous structure when superimposed on a non-hydrogen atom at a position corresponding to the atomic coordinates set forth in Table 3, 4 or 5, has a root mean square deviation of the non-hydrogen atom less than the root mean square deviationPreferably less thanIn a particular embodiment, the three-dimensional structure is derived from the atomic coordinates of Ala of the A chain of any one of tables 3, 4 or 5²¹、Gly²⁵(ii) a Ala of K chain of any one of tables 3, 4 or 5²⁴、Gly²⁷、Phe⁵³、Phe⁵⁴、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴、Phe⁶⁵(ii) a Met of L chain of any of tables 3, 4 or 5¹⁷、Gly¹⁹、Gly²⁰、Ala²¹、Ile²²、Gly²³、Ala²⁴、Gly²⁵、Ile²⁶、Gly²⁷、Asp²⁸、Gly²⁹、Ala³¹、Phe⁵³、Thr⁵⁶、Val⁵⁷、Gly⁵⁸、Leu⁵⁹、Val⁶⁰、Glu⁶¹、Ala⁶²、Ala⁶³/Pro⁶³、Tyr⁶⁴、Phe⁶⁵(ii) a Ser of M chain of any one of tables 3, 4 or 5²⁰⁶、Leu²⁰⁷、Leu²⁰⁷And Arg²¹⁰。

In this screening method, the degree of engagement of these compounds with the binding site is judged either by shape complementarity or by estimated interaction energy (Meng, E.C.et al, J.Coma. chem 13: 505-524 (1992)).

Use of binding sites

Two factors are generally considered in designing compounds that bind to the atpE proteins of the invention and enhance or inhibit their activity. First, the compound must be capable of physical, structural association with the atpE protein. In the interaction between the atpE protein and the compound, non-covalent molecular interactions play a significant role, including hydrogen bonding, van der waals forces, and hydrophobic interactions. Second, the compound must be able to assume a conformation that allows it to associate with atpE. Although some portions of the compound are not directly involved in its interaction with atpE, these portions may still affect the overall conformation of the molecule, thereby having a significant impact on binding affinity, therapeutic efficacy, drug-like (drug-like) properties and drug efficacy. The conformational requirements include the overall three-dimensional conformation and orientation of the chemical entity or compound involved in all or part of the atpE active site, or other region, or in the spacing of functional groups in the compound (containing multiple chemical entities that directly interact with atpE).

The potential, predicted, inhibitory agonist, antagonist or binding of the ligand or other compound to atpE may be analyzed using computer modeling techniques prior to the actual synthesis and testing of the ligand or other compound pair. If the theoretical structure of a given compound is not sufficient to associate and interact with atpE, then the compound may not be synthesized and tested. However, if computer modeling indicates that a compound has a strong effect on atpE, the molecule can be synthesized and tested for its ability to interact with atpE. This avoids the synthesis of compounds which are not functional. In some cases, compounds whose inactivation was predicted by modeling were synthesized and tested to investigate the SAP (structure-activity relationship) of compounds that interact with specific regions of atpE.

One skilled in the art can select from a variety of methods to screen chemical entity fragments, compounds or drugs for their ability to associate with atpE, particularly with a single binding pocket (pocket) or active site of atpE. When using this method, the site of activity, for example, can first be visualized on a computer (visual) based on the atomic coordinates of atpE or atpE-ligand complex. The selected chemical entities, compounds or drugs can then be placed in a number of different orientations, or they can be packed (dock) into a single binding pocket of atpE. The loading operation can be performed using software such as Quanta and Sybyl, and then molecular energy can be minimized using standard molecular force field (mechanics) software such as CHARMM and AMBER, and molecular dynamics analysis can be performed.

Specialized computer programs may be used to aid in the selection of chemical entities. This includes, but is not limited to, GRID (Goodford, P.J., "a method for determining energetically Favorable Binding Sites for important biological macromolecules", "A Computational Procedure for determining a defined energy efficient Binding Sites on biological macromolecules," J.Med.Chem.28: 849-; MCSS (Miranker, A.and M.Karplus, "functional map of binding Sites: A Multiple Copy Search method" "functional Maps of binding Sites: A Multiple Copy Simultaneous Search method," Proteins: Structure, Function and Genetics 11: 29-34(1991), from molecular Simulinations, Burlington, Mass.); AUTODOCK (Goodsel, D.S. and A.J.Olsen, "automated docking of substrate channel Proteins by Simulated Annealing" "automated docking of Substrates to Proteins by systematic assessment" Proteins: Structure. function, and Genetics 8: 195-; DOCK (Kuntz, I.D.et al, "genomic method for analysis of macromolecule-Ligand Interactions" "A Geometric Approach to macromolecules-Ligand Interactions," J. -mol.biol.161: 269-288(1982), from University of California, San Francisco, Calif.)

The surface sites can be analyzed using software to determine the structure of similar inhibitory proteins or compounds, such as GRID software, which can determine the possible sites of interaction between the probe and different characteristic functional groups and with the surface of macromolecules. With appropriate inhibitory groups on the molecule (e.g., protonated primary amines) as probes, the GRID tool can be used to identify potential hot spots around accessible sites at the appropriate energy contour (energy contour) level. The DOCK program can be used to analyze the active site or ligand binding site and to find ligands with complementary steric properties.

Once chemical entities, compounds or drugs are selected, they can be assembled into individual ligands, compounds, inhibitors or activators. The assembly can be performed by visually observing the interrelationship between the segments on the three-dimensional image. Then manually modeled using software such as Quanta or Sybyl.

Computer software programs that may facilitate the linking of individual Chemical entities, compounds or drugs include, but are not limited to, CAVEAT (Bartlett, P.A. et al, "CAVEAT: a tool for the Structure-origin Design of Biologically active molecules" ", CAVEAT: A Programm to facility the Structure-Derived Design of biological activities," In Molecular registration In Chemical and biological profiles, Special pub., Royal chem.Soc. 78, pp.82-196 (1989)); 3D database Systems such as MACCS-3D (MDL In Information Systems, San Leandro, Calif. and Martin, Y.C., "3D database In Drug Design" "3D database for database search In Drug Design", J.Med.chem.35: 2145-.

There are several methods available to search the three-dimensional database to test the pharmacophore hypothesis and select compounds for further screening. These methods include CAVEAT software (Bacon et al, J.mol.biol.225: 849-. For example, CAVEAT uses a database of cyclic compounds that can serve as spacers to link any number of chemical fragments that have been localized to the active site. The software allows one skilled in the art to quickly arrive at hundreds of possible methods for joining fragments that are known (or suspected) to be necessary for tight joining. Rather than stepwise constructing the inhibitory activators, agonists or antagonists of atpE, one chemical entity at a time as described above, the compounds can be designed entirely or de novo using empty active sites or any portion of known molecules. These methods include: LUDI (Bohm, H.J., LUDI software: a novel approach to De novo design of Enzyme Inhibitors, "The Computer Program LUDI: A New Method for The De Novodesign of Enzyme Inhibitors", J.Comm.aid.Molec.design, 6, pp.61-78(1992), Biosym Technologies, San Diego, Calif.); LEGEND (Nishibata, Y.and A.Itai, Tetrahedron 47: 8985(1991), molecular simulations, Burlington, Mass.) and LeapFrog (Tripos Associates, St.Louis, Mo.).

For example, LUDI software can identify a series of interaction sites to place hydrogen bonds and hydrophobic segments in. LUDI then uses a library of adaptors to join up to four different interaction sites to form a fragment. Smaller "bridging" molecules such as- -CH 2-and- -COO-are then used to link these fragments together. For example, in the case of the DHFR enzyme, LUDI replicates the placement of key functional groups within the putative inhibitor methotrexate. See also, Rotstein and Murcko, j.med.chem.36: 1700-1710(1992).

Other molecular modeling techniques may also be used in accordance with the present invention. See, e.g., Cohen, n.c. et., "Molecular Modeling Methods for Medicinal Chemistry, j.med.chem.33: 883-894 (1990); navia, M.A. and M.A. Murcko, "Use of Structural Information in Drug Design" "The Use of Structural Information in Drug Design," Current actions in Structural Biology, 2, pp.202-210 (1992).

Once a compound has been designed and selected by the methods described above, the compound can be tested and optimized for affinity for binding or association with atpE by in silico methods and/or by testing for biological activity after synthesis of the compound. The inhibitor or compound may interact with more than one conformation of atpE with similar overall binding energy. In these cases, the deformation energy imparted by binding is the difference between the energy of the free compound and the average energy of conformation observed when the compound binds to atpE.

The compound designed or selected to bind or associate with atpE may be further optimized by computational methods so that it and atpE preferably do not have electrostatic repulsive forces in their bound state. Such non-complementary (e.g., electrostatic) interactions include repulsive charge-charge, dipole (dipole) -dipole, and charge-dipole interactions. In particular, when the inhibitor binds, the sum of all electrostatic interactions between the inhibitor and atpE preferably acts to counteract or favour the enthalpy of binding. Weak binding compounds were also designed by these methods to determine SAR, see e.g. u.s.appl.nos.60/275,629; 60/331,235, respectively; 60/379,617, respectively; and, 10/097,249.

There is well-established computer software in the art to evaluate the deformation energy and electrostatic interaction of compounds. Software designed for this application such as Gaussian 92, version C (m.j. frisch, Gaussian, inc., Pittsburgh, Pa., COPYRGT 1992); AMBER, version 4.0 (P.A. Kollman, University of California at San Francisco, COPYRRGT 1994); QUANTA/CHARMM (Molecular diagnostics, Inc., Burlington, Mass. COPYRRGT 1994); insight II/Discover (biosystems technologies Inc., San Diego, Calif. COPYRGT 1994). Other hardware systems and software packages are known to those skilled in the art.

Once the compound associated with atpE has been optimally selected or designed as described above, some of its atoms or side groups may be replaced to improve or modulate its binding properties. Typically, substitutions are initially made with conservative substitutions, i.e., the new group has a similar size, shape, hydrophobicity, and charge as the original group. Of course, the use of components known in the art to alter conformation should be avoided. The validity of the fit of these chemically substituted compounds with atpE was then analyzed, again using the computer methods detailed above.

The present invention also provides systems, particularly computer systems, that incorporate the sequence and/or structure coordinates described herein. These systems were designed for atpE or ATPase F₀The binding sites in the moiety determine the structure and rational drug design. Computer system refers to the hardware, software and databases used to analyze the sequence and/or structural coordinates of the present invention in any of the computer methods described above. The hardware in the computer-based system of the present invention should include at least a CPU, an output device, an input device, and a data storage device. The skilled artisan will readily recognize that commercial computer systems are presently suitable for use with the present invention.

It is therefore an object of the present invention to provide a computer readable data storage device containing the structural coordinates described herein. As used herein, "computer-readable data storage device" refers to any device that is readable or directly accessible by a computer. Such devices include, but are not limited to, magnetic storage devices such as floppy disks, hard disks, and magnetic tape; optical storage devices such as compact disk CD-ROMs; electrical storage devices such as RAM and ROM; and combinations of these devices such as magnetic/optical storage devices.

Method of treatment

As indicated above, it is also an object of the present invention to provide a therapeutic method for treating a subject infected with a microorganism using a compound obtained from any of the screening methods described above. Bacterial pathogens are generally classified into gram-positive and gram-negative bacteria. Antimicrobial compounds that are both gram positive and gram negative are generally considered to have broad spectrum antimicrobial activity. The compounds of the present invention have activity against gram-positive bacteria and/or activity against gram-negative bacteria. In particular, the compounds of the present invention are resistant to at least one gram-positive bacterium, preferably to several gram-positive bacteria, more preferably to one or more gram-positive and/or one or more gram-negative bacteria.

Gram positive and gram negative aerobic and anaerobic bacteria include staphylococci such as staphylococcus aureus (s.aureus); enterococci such as enterococcus faecalis (e.faecalis); streptococci such as streptococcus pneumoniae (s.pneumoniae), streptococcus mutans (s.mutans), streptococcus pyogenes (s.pyogens); bacilli such as Bacillus (Bacillus subtilis); listeria such as Listeria monocytogenes (Listeria monocytogenes); haemophilus such as haemophilus influenzae (h.influenza); moraxella such as moraxella catarrhalis (m.catarrhalis); pseudomonas such as Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Escherichia such as Escherichia coli (E.coli).

Gram-positive pathogens such as staphylococci, enterococci and streptococci are of particular importance because resistant strains of these species, once established, are difficult to treat and difficult to eradicate (e.g. from hospital settings). Examples of such strains are methicillin-resistant staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative staphylococcus (MRCNS), penicillin-resistant streptococcus pneumoniae and multi-resistant enterococcus faecalis.

The compounds of the invention also show activity against resistant strains.

The compounds of the invention are dependent on F for those survival₁F₀Bacteria that normally function as ATP synthase are particularly effective. Unlike any existing theory (within the subject bound to animal), it is believed that the activity of the compounds of the invention is dependent on the activity on F₁F₀Inhibition of ATP synthase, in particular F₁F₀ATP synthase F₀Inhibition of complexes, more specifically F₁F₀ATP synthase F₀Arg from A subunit in the complex¹⁸⁶To C subunit Glu⁶¹Thereby killing the bacteria by reducing the ATP level of the bacterial cells. The compounds identified using any of the above screening methods are particularly effective against gram-positive bacteria, particularly mycobacteria, and are most particularly effective against infections caused by mycobacterium africanum (m.africanum), mycobacterium avium (m.avium), mycobacterium bovis (m.bovis), mycobacterium bovis-BCG (m.bovis-BCG), mycobacterium tortoise (m.chelonae), mycobacterium fortuitum (m.fortuitum), mycobacterium gordonae (m.gordonae), mycobacterium intracellulare (m.intracell), mycobacterium kansasii (m.kansasiii), mycobacterium microti (m.micorti), mycobacterium scrofulaceum (m.scorofulacum), mycobacterium paratuberculosis (m.paratuberculosis), mycobacterium leprea (m.tuberculosis), mycobacterium tuberculosis (m.tutulosis), mycobacterium ulcerosus (m.aereus) and mycobacterium phlei (m.ranium).

In both the above and the following, the ability of a compound to treat a bacterial infection means that the compound is able to treat an infection caused by one or more strains of bacteria. However, when DARQ J is used as an antimicrobial compound, "antimicrobial" herein means that the compound is capable of treating an infection caused by one or more strains, provided that the strain is not a mycobacterium.

Infections that can be treated with the compounds of the present invention include, for example, but are not limited to, central nervous system infections, external ear infections, middle ear infections such as acute secretory otitis media, sinus dura mater (craniallinuses) infections, eye infections, oral infections such as tooth, gum and mucosal infections, upper respiratory tract infections, lower respiratory tract infections, genitourinary infections, gastrointestinal infections, gynecological infections, septicemia, bone and joint infections, skin and skin structure infections, bacterial endocarditis, burns, post-surgical infection prophylaxis and immunosuppression prophylaxis in patients such as cancer patients and organ transplant patients.

The invention also provides a method of treating a patient suffering from tuberculosis comprising administering to the patient an agent capable of interacting with atpE protein.

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising an agent capable of interacting with atpE protein in a cell and a pharmaceutically acceptable carrier.

Such medicaments may be formulated as a composition comprising the medicament and a pharmaceutically acceptable carrier or diluent. The drug may be in the form of a functional derivative under physiological conditions, for example an ester or salt, such as an acid addition or alkali metal salt, or an N or S oxide. The compositions may be prepared according to any suitable route and method of administration. Pharmaceutically acceptable carriers or diluents include those used in formulations administered by: oral, rectal, nasal, inhalation, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intravenous, intramuscular, intradermal, intrathecal, epidural). Of course, the choice of carrier or diluent depends on the intended route of administration, which may depend on the drug and the therapeutic purpose. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The steps of these methods include bringing into association a carrier consisting of one or more additional ingredients and the active ingredient. In general, the active ingredient is uniformly and intimately associated with a liquid carrier (either finely divided solid carrier or both) to prepare a formulation which is then, if necessary, shaped.

For solid compositions, non-toxic solid carriers which are commonly used include, for example, pharmaceutical grades of mannitol, lactose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, sucrose, glucose, magnesium carbonate, and the like. The active compounds defined above may be formulated as suppositories using carriers such as polyalkylene glycols, acetyl triglycerides and the like. For example, liquid pharmaceutical compositions can be prepared by dissolving or dispersing the active compound as defined above and optional pharmaceutical excipients in a carrier, such as water, physiological saline dextran solution, glycerol, ethanol, and the like, to form a solution or suspension. If desired, the pharmaceutical compositions to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and the like. The actual methods for preparing these dosage forms are known (or readily understood) to those skilled in the art, see, e.g., Gennaroe et al, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 18th Edition, 1990.

In any event, the composition or formulation to be administered contains a dose of the active compound that is sufficient to effectively alleviate the symptoms of the subject being treated.

As is well known to those skilled in the art, the precise dosage and frequency of administration of a compound of the invention will be determined by the particular compound employed, the particular condition being treated, the severity of the condition being treated (severity), the age, body weight, sex, diet, time of treatment and the overall physiological condition of the patient, the mode of treatment, and other medications the patient may be taking. In addition, the effective daily dose can obviously be reduced or increased based on the response of the subject being treated and/or the evaluation of the physician prescribing the compounds of the instant invention.

The dosage form or composition may be prepared to contain the active ingredient in an amount ranging from 0.25 to 95% with the remainder being a non-toxic carrier. Depending on the mode of administration, the active ingredient is preferably present in the pharmaceutical composition in an amount of 0.05 to 99% by weight, more preferably 0.1 to 70% by weight, while the pharmaceutically acceptable carrier is present in an amount of 1 to 99.95% by weight, more preferably 30 to 99.9% by weight, all percentages being based on the total weight of the composition.

Pharmaceutically acceptable non-toxic compositions for oral administration may be prepared using any of the usual excipients, such as pharmaceutical grades of mannitol, lactose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, fossils, sucrose, glucose, magnesium carbonate and the like. The composition can be made into solution, suspension, tablet, pill, capsule, powder, sustained release preparation, etc. These compositions may contain from 1 to 95% of active ingredient, preferably from 2 to 50%, most preferably from 5 to 8%.

Parenteral administration is generally characterized by administration by injection, either subcutaneously, intramuscularly or intravenously. Injectables can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like. The pharmaceutical compositions to be administered may additionally, if desired, also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.

The amount of active ingredient in these parenteral compositions is highly dependent on the particular characteristics of the active ingredient, the activity of the compound and the needs of the subject. However, the active ingredient is suitably present in the solution in an amount of from 0.1% to 10% and may be present in higher amounts if the composition is a solid and subsequently diluted to the above-mentioned concentration. Preferably, the composition comprises 0.2-2% of the active agent in solution.

Finally, the invention provides products, including packaging and medicaments, wherein (a) the medicament interacts with the atpE protein in the cell and (b) the packaging contains a label indicating how to use the medicament for the treatment of a bacterial infected person, particularly for use as an anti-mycobacterial medicament.

All references herein to "standard methods", "standard Protocols" and "standard procedures", when used in the context of Molecular Biology techniques, refer to Protocols and procedures in conventional laboratory guidelines, such as Current Protocols in Molecular Biology, editors F.Ausubel et al, John Wiley and Sons, Inc.1994, or Sambrook, J.J., Fritsch, E.F.and Manitias, T.a., Molecular Cloning: a Laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989.

The present invention may be better understood by reference to the following experimental details, which, however, will be apparent to those skilled in the art for the purpose of illustration only, and the invention will be more fully defined in the claims that follow. Further, throughout this disclosure, various publications are referenced, the disclosures of which are incorporated herein by reference, to more fully describe the art to which this invention pertains.

Description of the experiments

Using M.smegmatis as an alternative, we found a series of DARQ with in vitro activity against several mycobacteria (11). Currently, 20 molecules in the DARQ series have a Minimum Inhibitory Concentration (MIC) of less than 0.5 μ g/ml against Mycobacterium tuberculosis (Mycobacterium tuberculosis) H37Rv, of which three molecules are confirmed to have anti-mycobacterial activity by in vivo mouse models.

DARQ differs significantly structurally and mechanistically from fluoroquinolones (including methoxyquinolones), and from other quinolines, including mefloquine and its analogs, 4-methylquinoline and 4-quinolinylhydrazone (12-16). One major structural difference between DARQ and other quinolones or quinolines is the specificity of the functional side chain (3') carried by DARQ. In addition, there was no cross-resistance to mycobacteria with existing chemicals, suggesting that the mechanism of action is also different.

A precursor compound of DARQ, hereinReferred to as J or DARQ J (fig. 1), we found that it has a unique potent and selective spectrum of anti-mycobacterial activity in vitro (table 1). It had a median MIC of 0.060. mu.g/ml for laboratory strain H37Rv and six fully sensitive isolates, and 1.00. mu.g/ml for rifampicin. The J compound also showed similar effects in vitro on clinically isolated strains of mycobacterium tuberculosis (m.tuberculosis) resistant to first-line and second-line TB drugs, which were rifampicin, streptomycin, ethambutol, pyrazinamide, and second-line TB drug resistant to moxifloxacin. The MIC of compound J against eight clinical isolates with isoniazid resistance was 0.010 μ g/ml. The J compound has no cross-resistance to currently available anti-TB drugs, suggesting that J may retain activity against MDR-TB strains. In fact, BACTEC was used^TMCulture systems, exposing MCR-TB strain to a fixed concentration of J-compound, clearly seen on bacterial growth of dose dependent inhibition. Of the 30 isolates of MDR-TB, 13 (43%) were sensitive to 0.100. mu.g/ml of J compound, and 17 (57%) were sensitive to 0.010. mu.g/ml of J compound. When BACTEC is used^TMWhen tested systematically, only one of the other 10 fully drug-sensitive strains showed similarly high sensitivity to J (MIC less than 0.010 μ g/ml), while all strains were sensitive to 0.100 μ g/ml of the J compound.

Potent activity has also been demonstrated in other mycobacteria, including M.bovis and M.kansasii, as well as in many bacteria that are naturally resistant to TB drugs and participate in opportunistic infections, such as M.avium complex (MAC), M.abscessus (Mycobacterium abscesses), M.fortuitum, and M.marinum.

Surprisingly, the activity of J shows specificity for mycobacteria. J has little activity against closely related species of mycobacteria such as Corynebacterium (Corynebacterium) (MIC 4.00. mu.g/ml) and Nocardia (Nocardia) (MIC > 4.00. mu.g/ml) and is inactive against other organisms including the gram-positive bacteria Streptococcus pneumoniae, Staphylococcus aureus (including methicillin-resistant strains) (MIC > 32. mu.g/ml) and enterococcus faecalis, as well as the gram-negative bacteria Escherichia coli, Haemophilus influenzae and helicobacter pylori.

Mycobacterium tuberculosis in logarithmic growth phase is treated with J compound with 100xMIC concentration, and the bacterial count is reduced by 3 orders of magnitude after 12 days (10)³log reduction), indicating that J has bactericidal activity in vitro. The effect of J on stationary stage tubercle bacilli is currently not studied.

Mutant screening, cross-resistance and putative drug targets

We have sought to identify molecular drug targets and to propose mechanisms of action by examining mycobacterium resistance. Resistant mutants of mycobacterium tuberculosis and mycobacterium smegmatis were selected in vitro using inhibitory concentrations of the J compound with the aim of:

determination of the proportion of resistant mutants in mycobacteria (rifampicin as control)

Evaluation of the resistance patterns of the resistant mutants (including cross/non-cross resistance to quinolones)

- - -examination of the mechanism of action

In the screening experiment, the proportion of the mutant strains with reduced J sensitivity in the mycobacterium tuberculosis and the mycobacterium smegmatis is respectively 5x10 under the MICx4 concentration^-7And 2x10^-8MICx8 concentration 5x10^-8And 1x10^-8(supporting online text)). The proportion of mutants in Mycobacterium tuberculosis compared to those resistant to rifampicin (10)^-7-10^-8) Rather, this indicates that there is little natural resistance to J. In addition, the sensitivity of the J-resistant mycobacterium tuberculosis strains to the anti-TB drugs isoniazid, rifampin, streptomycin, amikacin, ethambutol and moxifloxacin was not changed. Further studies of mutants with reduced J sensitivity in Mycobacterium tuberculosis and Mycobacterium smegmatis showed no mutations in the gyrA and gyrB regions of DNA gyrase, nor in sequences that typically produce quinolone resistance. This demonstrates the molecular targeting of the J compound with fluoroquineThe norketon targets differ.

One way to determine the molecular target of J and infer mechanism of action is to identify and compare resistance-conferring mutations in sensitive and resistant strains of Mycobacterium tuberculosis and Mycobacterium smegmatis. Genome sequencing of mycobacterium tuberculosis resistant strain BK12, two mycobacterium smegmatis resistant strains R09 and R10, and parent mycobacterium smegmatis is nearly complete. By comparative analysis of the genomic sequences of sensitive and resistant strains of mycobacterium tuberculosis and mycobacterium smegmatis, we identified resistance conferring mutations (fig. 2). Our results show that the only gene that functions in all three independent mutants compared to the corresponding parental wild-type encodes atpE (part of the F0 subunit of ATP synthase). This suggests that resistance of the mutant to J is caused by atpE, suggesting that J inhibits the proton pump of ATP synthase, a novel target of Mycobacterium tuberculosis.

Complementation studies were performed to show that the mutant atpE caused resistance to J, and it was directly concluded that the atpE gene product is the target for the action of J on mycobacteria. Given that all genes of the ATP synthase operon must be expressed in a coordinated manner, i.e. all genes encoding part F0 must be expressed from the same position, we amplified part F0 of the mycobacterium smegmatis resistant mutant (D32V) operon and selected clones without additional mutations by PCR. The wild type M.smegmatis was transformed with the plasmid containing the thus selected mutant F0 fragment. The resulting cells obtained resistance to J with MIC almost identical to that of M.smegmatis resistant mutant R09 (D32V). In addition, when the plasmid was re-isolated from this transformant and the atpE gene was sequenced, it was found to have a retained mutant allele (D32V).

The effect of DARQ J on ATP production in mycobacterium tuberculosis was further confirmed by measuring the effect of J on the amount of total cellular ATP present in the mycobacterium using the ATP Bioluminescence Luciferase Assay Kit HSII Kit from Roche. The basis of this experiment was that the ATP-driven conversion of D-luciferin to oxidized luciferin was measured at a wavelength of 526 nm.

Briefly, the effect of DARQ J on total ATP was detected in wild mycobacterium tuberculosis and mutant strains. DCCD (which is known to be an inhibitor of ATP synthase) was used as a positive control, and isoniazid (which inhibits the biosynthesis of certain components of the cell wall but has no effect on ATP production) was used as a negative control.

As shown in fig. 3, treatment of wild-type mycobacterium tuberculosis with DARQ J caused a dose-dependent decrease in ATP production in these bacteria. In contrast, isoniazid has no effect on ATP production. As described above, exposure of these bacteria to high concentrations of DARQJ resulted in diarylquinoline resistant mutants of Mycobacterium tuberculosis. Even when these M.tuberculosis mutants were treated with J at 100-fold MIC concentration, their ATP production was not reduced at all. In contrast, DCCD was able to repress ATP production in these mutants, suggesting that DARQ J and DCCD have distinct binding pockets at the ATP synthase.

Computer modeling and identification of DARQ J-binding regions

To further explore the different mechanisms of action of DCCD and DARQ J, a computer 3D model of ATP synthase from wild Mycobacterium tuberculosis and DARQ J resistant mutants, respectively, was established. The atomic coordinates in tables 4 and 5 were calculated by building 3D models disclosing the amino acid sequences P63691 and AJ 865377. The actual binding site for DARQ J was found to be located in the contact region of the a and C subunits, more specifically around "Arg 210" of the a subunit and "Glu 61" of the C subunit, as shown in tables 3, 4 or 5. This is in full agreement with the results of the resistance conferring mutation screen described above for sensitive and resistant mutants of Mycobacterium tuberculosis and Mycobacterium smegmatis.

The basis of this model is the result of a computational optimization of the relative positions of the A-and C-helices in the ATPase structure, the amino acid backbone and the orientation of the side chains with minimal intrinsic stress. Starting from the conventional spiral geometry of different organisms previously disclosed [ E-Coli PDB entry code 1C17-V.K.Rastogi and M.E.Girvin, Nature, 402, 263-268(1999) ], the geometry was obtained by several molecular dynamics simulation cycles and molecular mechanics relaxation methods. Molecular dynamics and relaxation geometry analysis were also performed using force field parameters (forcefield parameters) determined according to MMFF94s [ Halgren, T.A. (1996), J.Compout.chem., 17, 490-519 ]. Virtually any molecular dynamics analysis software can be used [ Berendsen, h.j.c., van der Spoel, d.and van Drunen, r., comp.phys.comm.91(1995), 43-56; lindahl, e, Hess, b.and van der Spoel, d, j.mol.mod.7(2001) 306-.

The calculated coordinates in tables 3, 4 and 5 include, based on the proposed inhibition pattern and the resistance-inducing mutation sites present in the biological assays, the partial predicted structure (radius 30. ANG. range) that is believed to be the structure of the region of these enzymes involved in the inhibition of MTB ATPase activity.

Discussion of the related Art

DARQ J is a member of a new class of anti-TB chemicals that has the same or lower MIC as the reference compound. The unique feature of its antibacterial spectrum is that it is specific to mycobacteria, including atypical mycobacteria, which are important among human pathogens; MAC, mycobacterium kansasii (m.kansasii) and fast growing mycobacterium fortuitum and mycobacterium abscessus (m.absessus). This specific antimycobacterial profile is different from isoniazid (which has no effect on MAC). The clinical use of the J compounds will be primarily directed to the treatment of TB and mycobacterial infections. The inability of J to inhibit non-mycobacterial microorganisms results in less selective pressure than antibiotics with broad spectrum antimicrobial capabilities, and also reduces the risk of resistance development by other species of bacteria (9).

The target and mechanism of action of the J-compound are different from those of other anti-TB drugs. The alignment between the sequences of different bacterial and eukaryotic ATP synthases, in particular the alignment of the ATP enzyme complex F0 subunit C chain sequence, and the 3D model of ATP synthases of wild-type and mutant M.tuberculosis, together provide the rationale for the specificity of the J antibiogram and to a lesser extent safety.

Kinetic analysis of the established mycobacterial atpase model revealed that a cavity (according to the atomic coordinates of table 3, i.e. the binding site) was present in the contact region between the a and C subunits in these structures (around "Arg 210" for the a subunit and "Glu 61" for the C subunit). Tables 4 and 5 provide the atomic coordinates around this site in the two studied variants, as well as their average position. By prohibiting these two amino acids from interacting, DARQ J inhibitors can interfere with the normal proton transfer steps involving these two amino acids. The stereospecificity of DARQ J can be understood from the predicted asymmetry of the binding site; the active chiral enantiomer is also ideally contained in this cavity, while other forms of the compound have a reduced degree of engagement with the variant ATPase.

We have derived here a binding site that is completely different on the atpase system than on the DCCD. According to bovine atpase crystal "1E 79" (see c.gibbons, m.g.montgomery, a.g.w.leslie, j.e.walker, nat.struct.biol., 7, 1055(2000)), the DARQ binding site is located in the membrane portion of the enzyme, while the DCCD binding site is about 90 angstroms intracellularly. The atoms which can participate in the inhibition of the DCCD-type MTB atpase are therefore not within the area listed in the atomic co-ordinate tables of the binding site herein (which spans only the membrane part of the enzyme), whereas the part of the enzyme involved in the DARQ J inhibition pattern is not present in the reported "1E 79" structure (which occurs only in the intracellular part). This difference in binding sites may explain the different responses of M.tuberculosis in vitro ATP production assays.

Earlier studies on the role of DCCD on mitochondrial atpase speculated that another binding site was present around an acidic amino acid in the lipophilic (lipophenyllic) environment of the F0 region of the enzyme, such as Sebald W, Machleidt W, waterer e., Proc natl acad Sci U S a.1980 feb; 77(2): 785-789. This binding site on mitochondrial ATPase can be considered similar to the mycobacterial site described herein.

At the same time, the new mechanism of action also ensures that the TB strains currently prevalent with resistance mutations to existing therapeutic measures are not cross-resistant to J compounds. It is clear from our in vitro studies that the J compound has a high antibacterial effect at least against MDR-TB isolates, and even against strains resistant to all four drugs, to the same extent as the pan-susceptable strains of M.tuberculosis. This finding is of great significance since it clearly demonstrates no cross-resistance between J and existing anti-TB drugs. If the binding pocket in the ATPase membrane moiety is further identified, the results of this study will help to further develop novel antibacterial compounds, particularly antimycobacterial compounds that target ATP synthesis in these organisms.

Reference to the literature

1.Global Alliance for TB Drug Development，Developing a fasterTB cure(2004；http://www.tballiance.org)(global consortium for TB drug development, development of rapid TB therapy).

2.E.L.Corbett et al.，Arch.Intern.Med.163，1009(2003).

3.UNAIDS，AIDS epidemic update 2003(2003；www.unaids.org/Unaids/EN/Resources).

4.World Health Organization，Tuberculosis(2004；http://www.who.int/health_topics/tuberculosis/en/)(world health organization, tuberculosis).

5.R.J.O′Brien，P.P.Nunn，Am.J.Respir.Crit.Care Med.163，1055(2001).

6.World Health Organization，Tuberculosis Fact Sheet No 104(2004；http://www.who.int/mediacentre/factsheets/fsl04/en/)(world health organization, tuberculosis fact report No. 104).

7.A.J.Claxton，J.Cramer，C.Pierce，Clin.Ther.23，1296(2001).

8.N.Lounis et al.，Antimicrob.Agents Chemother.45，3482(2001).

9.A.S.Ginsburg，J.H.Grosset，W.R.Bishai，Lancet Infect.Dis.3，432(2003).

10.C.K.Stover et al.，Nature 405，962(2000).

Guillemont J, Emile G, Patent (International publication No. WO 2004/011436, International publication No. 2004, 2/5).

Barbachyn MR, Brickner SJ, Patent (International publication No.: WO 93/09103, International publication No.: 5/13/1993, International application No.: PCT/US92/08267, International publication No.: 1992).

13.R.Jain，B.Vaitilingam，A.Nayyar，P.B.Palde，Bioorg.Med.Chem.Lett.13，1051(2003).

14.C.M.Kunin，W.Y.Ellis，Antimicrob.Agents Chemother.44，848(2000).

15.L.Savini，L.Chiasserini，A.Gaeta，C.Pellerano，Bioorg.MedChem 10，2193(2002).

16.S.Vangapandu，M.Jain，R.Jain，S.Kaur，P.P.Singh，Bioorg.Med.Chem.12，2501(2004).

17.K.Andries et al.，Science 307，223(2005).

Table 1.

TABLE 2

Sequence P63691 ═ SEQ ID No.1 ═ mycuba C-subunit, wt

Sequence AJ 865377-MYCTUB C-subunit, mutant

Sequence P63654 SEQ ID No.11 myctabb a-subunit, wt and mutant

Amino acids around the binding site based on the atomic coordinates of Table 3

# C-subunit 1 # # -A chain

Ala²¹、Gly²⁵

The # C-subunit 2 # # -K chain

Ala²⁴、Gly²⁷、Phe⁵³、Phe⁵⁴、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴、Phe⁶⁵

The # C-subunit # 3 # # -L chain

Met¹⁷、Gly¹⁹、Gly²⁰、Ala²¹、Ile²²、Gly²³、Ala²⁴、Gly²⁵、Ile²⁶、Gly²⁷、Asp²⁸、Gly²⁹、Ala³¹、Phe⁵³、Thr⁵⁶、Val⁵⁷、Gly⁵⁸、Leu⁵⁹、Val⁶⁰、Glu⁶¹、Ala⁶²、Ala⁶³/Pro⁶³、Tyr⁶⁴、Phe⁶⁵.

# # A subunit # # -M chain

Ser¹⁸²、Leu¹⁸³、Leu¹⁸⁵、Arg¹⁸⁶(Ser 206-Leu 207-Leu 209 and Arg210 in Table 3)

TABLE 3

TABLE 3 continuation

TABLE 3 continuation

TABLE 3 continuation

TABLE 3 continuation

TABLE 4

TABLE 4 continuation

TABLE 4 continuation

TABLE 4 continuation

TABLE 4 continuation

TABLE 4 continuation

TABLE 4 continuation

TABLE 4 continuation

TABLE 4 continuation

TABLE 4 continuation

TABLE 4 continuation

TABLE 5

TABLE 5 continuation

TABLE 5 continuation

TABLE 5 continuation

TABLE 5 continuation

TABLE 5 continuation

TABLE 5 continuation

TABLE 5 continuation

TABLE 5 continuation

TABLE 5 continuation

TABLE 6

TABLE 6 continuation

TABLE 6 continuation

TABLE 6 continuation

TABLE 6 continuation

TABLE 6 continuation

TABLE 6 continuation

TABLE 6 continuation

TABLE 6 continuation

TABLE 7

TABLE 7 continuation

TABLE 7 continuation

TABLE 7 continuation

TABLE 7 continuation

TABLE 7 continuation

TABLE 7 continuation

TABLE 7 continuation

TABLE 7 continuation

Sequence listing

<110>Janssen Pharmaceutica N.V.

<120>Mutant atpE proteins

<130>PRD2323p

<150>EP 04104720.0

<151>2004-09-28

<150>US 60/620500

<151>2004-10-20

<160>12

<170>PatentIn version 3.1

<210>1

<211>81

<212>PRT

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400>1

Met Asp Pro Thr Ile Ala Ala Gly Ala Leu Ile Gly Gly Gly Leu Ile

1 5 10 15

Met Ala Gly Gly Ala Ile Gly Ala Gly Ile Gly Asp Gly Val Ala Gly

20 25 30

Asn Ala Leu Ile Ser Gly Val Ala Arg Gln Pro Glu Ala Gln Gly Arg

35 40 45

Leu Phe Thr Pro Phe Phe Ile Thr Val Gly Leu Val Glu Ala Ala Tyr

50 55 60

Phe Ile Asn Leu Ala Phe Met Ala Leu Phe Val Phe Ala Thr Pro Val

65 70 75 80

Lys

<210>2

<211>81

<212>PRT

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400>2

Met Asp Pro Thr Ile Ala Ala Gly Ala Leu Ile Gly Gly Gly Leu Ile

1 5 10 15

Met Ala Gly Gly Ala Ile Gly Ala Gly Ile Gly Asp Gly Val Ala Gly

20 25 30

Asn Ala Leu Ile Ser Gly Val Ala Arg Gln Pro Glu Ala Gln Gly Arg

35 40 45

Leu Phe Thr Pro Phe Phe Ile Thr Val Gly Leu Val Glu Ala Pro Tyr

50 55 60

Phe Ile Asn Leu Ala Phe Met Ala Leu Phe Val Phe Ala Thr Pro Val

65 70 75 80

Lys

<210>3

<211>86

<212>PRT

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400>3

Met Asp Leu Asp Pro Asn Ala Ile Ile Thr Ala Gly Ala Leu Ile Gly

1 5 10 15

Gly Gly Leu Ile Met Gly Gly Gly Ala Ile Gly Ala Gly Ile Gly Asp

20 25 30

Gly Ile Ala Gly Asn Ala Leu Ile Ser Gly Ile Ala Arg Gln Pro Glu

35 40 45

Ala Gln Gly Arg Leu Phe Thr Pro Phe Phe Ile Thr Val Gly Leu Val

50 55 60

Glu Ala Ala Tyr Phe Ile Asn Leu Ala Phe Met Ala Leu Phe Val Phe

65 70 75 80

Ala Thr Pro Gly Leu Gln

85

<210>4

<211>86

<212>PRT

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400>4

Met Asp Leu Asp Pro Asn Ala Ile Ile Thr Ala Gly Ala Leu Ile Gly

1 5 10 15

Gly Gly Leu Ile Met Gly Gly Gly Ala Ile Gly Ala Gly Ile Gly Val

20 25 30

Gly Ile Ala Gly Asn Ala Leu Ile Ser Gly Ile Ala Arg Gln Pro Glu

35 40 45

Ala Gln Gly Arg Leu Phe Thr Pro Phe Phe Ile Thr Val Gly Leu Val

50 55 60

Glu Ala Ala Tyr Phe Ile Asn Leu Ala Phe Met Ala Leu Phe Val Phe

65 70 75 80

Ala Thr Pro Gly Leu Gln

85

<210>5

<211>86

<212>PRT

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400>5

Met Asp Leu Asp Pro Asn Ala Ile Ile Thr Ala Gly Ala Leu Ile Gly

1 5 10 15

Gly Gly Leu Ile Met Gly Gly Gly Ala Ile Gly Ala Gly Ile Gly Val

20 25 30

Gly Ile Ala Gly Asn Ala Leu Ile Ser Gly Ile Ala Arg Gln Pro Glu

35 40 45

Ala Gln Gly Arg Leu Phe Thr Pro Phe Phe Ile Thr Val Gly Leu Val

50 55 60

Glu Ala Ala Tyr Phe Ile Asn Leu Ala Phe Met Ala Leu Phe Val Phe

65 70 75 80

Ala Thr Pro Gly Leu Gln

85

<210>6

<211>246

<212>DNA

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400>6

atggacccca ctatcgctgc cggcgccctc atcggcggtg gactgatcat ggccggtggc 60

gccatcggcg ccggtatcgg tgacggtgtc gccggtaacg cgcttatctc cggtgtcgcc 120

cggcaacccg aggcgcaagg gcggctgttc acaccgttct tcatcaccgt cggtttggtt 180

gaggcggcat acttcatcaa cctggcgttt atggcgctgt tcgtcttcgc tacacccgtc 240

aagtaa 246

<210>7

<211>246

<212>DNA

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400>7

atggacccca ctatcgctgc cggcgccctc atcggcggtg gactgatcat ggccggtggc 60

gccatcggcg ccggtatcgg tgacggtgtc gccggtaacg cgcttatctc cggtgtcgcc 120

cggcaacccg aggcgcaagg gcggctgttc acaccgttct tcatcaccgt cggtttggtt 180

gaggcgccat acttcatcaa cctggcgttt atggcgctgt tcgtcttcgc tacacccgtc 240

aagtaa 246

<210>8

<211>261

<212>DNA

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400>8

atggatctcg atcccaacgc catcatcacg gccggcgccc tgatcggcgg tggattgatc 60

atgggtggcg gcgccatcgg tgccggtatc ggcgacggta tcgcgggtaa cgcgctgatc 120

tcgggtatcg cccgtcagcc cgaggcccag ggccggctgt tcaccccgtt cttcatcacc 180

gtcggtctgg tggaagccgc gtacttcatc aacctggcct tcatggcgct gttcgtcttc 240

gccactcctg gccttcagta a 261

<210>9

<211>261

<212>DNA

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400>9

atggatctcg atcccaacgc catcatcacg gccggcgccc tgatcggcgg tggattgatc 60

atgggtggcg gcgccatcgg tgccggtatc ggcgtcggta tcgcgggtaa cgcgctgatc 120

tcgggtatcg cccgtcagcc cgaggcccag ggccggctgt tcaccccgtt cttcatcacc 180

gtcggtctgg tggaagccgc gtacttcatc aacctggcct tcatggcgct gttcgtcttc 240

gccactcctg gccttcagta a 261

<210>10

<211>261

<212>DNA

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400>10

atggatctcg atcccaacgc catcatcacg gccggcgccc tgatcggcgg tggattgatc 60

atgggtggcg gcgccatcgg tgccggtatc ggcgtcggta tcgcgggtaa cgcgctgatc 120

tcgggtatcg cccgtcagcc cgaggcccag ggccggctgt tcaccccgtt cttcatcacc 180

gtcggtctgg tggaagccgc gtacttcatc aacctggcct tcatggcgct gttcgtcttc 240

gccactcctg gccttcagta a 261

<210>11

<211>753

<212>DNA

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400>11

atgactgaga ccatcctggc cgcccaaatc gaggtcggcg agcaccacac ggccacctgg 60

ctcggtatga cggtcaacac cgacaccgtg ttgtcgacgg cgatcgccgg gttgatcgtg 120

atcgcgttgg ccttttacct gcgcgccaaa gtgacttcga cggatgtgcc aggcggggtg 180

cagttgtttt ttgaggcgat caccattcag atgcgcaatc aggtcgaaag cgccatcggg 240

atgcggatcg cacccttcgt gctgccgctg gcggtgacca tcttcgtgtt catcctgatc 300

tccaactggc tggcagtcct cccggtgcag tacaccgata aacacgggca caccaccgag 360

ttgctcaaat cggcagcagc ggacatcaat tacgtgctgg cgctggcgct tttcgtgttc 420

gtctgctacc acacggccgg tatttggcgg cgcggtattg tcggacaccc gatcaagttg 480

ctgaaagggc acgtgacgct cctcgcgccg atcaaccttg tcgaagaagt cgccaagcca 540

atctcgttgt cgctccgact tttcggcaac attttcgccg gcggcattct ggtcgcactg 600

atcgcgctct ttccccccta catcatgtgg gcgcccaatg cgatctggaa agcatttgac 660

ctgttcgtcg gcgcaatcca ggccttcatt tttgcgctgc tgacaatttt gtacttcagc 720

caagcgatgg agctcgaaga ggaacaccac tag 753

<210>12

<211>250

<212>PRT

<213> Mycobacterium tuberculosis (Mycobacterium tuberculosis)

<400>12

Met Thr Glu Thr Ile Leu Ala Ala Gln Ile Glu Val Gly Glu His His

1 5 10 15

Thr Ala Thr Trp Leu Gly Met Thr Val Asn Thr Asp Thr Val Leu Ser

20 25 30

Thr Ala Ile Ala Gly Leu Ile Val Ile Ala Leu Ala Phe Tyr Leu Arg

35 40 45

Ala Lys Val Thr Ser Thr Asp Val Pro Gly Gly Val Gln Leu Phe Phe

50 55 60

Glu Ala Ile Thr Ile Gln Met Arg Asn Gln Val Glu Ser Ala Ile Gly

65 70 75 80

Met Arg Ile Ala Pro Phe Val Leu Pro Leu Ala Val Thr Ile Phe Val

85 90 95

Phe Ile Leu Ile Ser Asn Trp Leu Ala Val Leu Pro Val Gln Tyr Thr

100 105 110

Asp Lys His Gly His Thr Thr Glu Leu Leu Lys Ser Ala Ala Ala Asp

115 120 125

Ile Asn Tyr Val Leu Ala Leu Ala Leu Phe Val Phe Val Cys Tyr His

130 135 140

Thr Ala Gly Ile Trp Arg Arg Gly Ile Val Gly His Pro Ile Lys Leu

145 150 155 160

Leu Lys Gly His Val Thr Leu Leu Ala Pro Ile Asn Leu Val Glu Glu

165 170 175

Val Ala Lys Pro Ile Ser Leu Ser Leu Arg Leu Phe Gly Asn Ile Phe

180 185 190

Ala Gly Gly Ile Leu Val Ala Leu Ile Ala Leu Phe Pro Pro Tyr Ile

195 200 205

Met Trp Ala Pro Asn Ala Ile Trp Lys Ala Phe Asp Leu Phe Val Gly

210 215 220

Ala Ile Gln Ala Phe Ile Phe Ala Leu Leu Thr Ile Leu Tyr Phe Ser

225 230 235 240

Gln Ala Met Glu Leu Glu Glu Glu His His

245 250

Claims (12)

1. An isolated mutant atpE protein selected from the group consisting of SEQ ID nos: 2, Mtb _ R, SEQ ID No: msm _ R09 shown in FIG. 4 and SEQ ID No: msm _ R10 shown in FIG. 5.

2. Use of the isolated mutant atpE protein of claim 1 for the preparation of a reagent for identifying ATPase F₀Partially interacting compounds, and evaluating their potential as antimicrobial compounds.

3. A method of evaluating the potential of a test compound to interact with an atpE protein, said method comprising the steps of:

-using molecular modelling techniques to obtain the three-dimensional structure of the binding site of the protein of claim 1;

-performing a fitting operation between said test compound and the three-dimensional structure of said binding site using computer methods; and

-analysing the results of said engagement operation to quantify the association between said test compound and the three-dimensional structure of said binding site.

4. The method of claim 3, wherein the three-dimensional structure results from at least the following amino acids: amino acid Ala of one C subunit²⁴、Gly²⁷、Phe⁵³、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴And Phe⁶⁵And the amino acid Ser of one A subunit¹⁸²、Leu¹⁸³、Leu¹⁸⁵And Arg¹⁸⁶Derived using the atomic coordinates of any of tables 3, 4, or 5, or using homologous structure coordinates wherein the root mean square deviation of non-hydrogen atoms in the homologous structure coordinates is less than about 1.5 a.

5. The method of claim 3, wherein the three-dimensional structure is derived using the atomic coordinates of the following amino acids: ala of chain A of any one of tables 3, 4 or 5²¹、Gly²⁵(ii) a Ala of K chain of any one of tables 3, 4 or 5²⁴、Gly²⁷、Phe⁵³、Phe⁵⁴、Val⁵⁷、Gly⁵⁸、Glu⁶¹、Tyr⁶⁴、Phe⁶⁵(ii) a Met of L chain of any of tables 3, 4 or 5¹⁷、Gly¹⁹、Gly²⁰、Ala²¹、Ile²²、Gly²³、Ala²⁴、Gly²⁵、Ile²⁶、Gly²⁷、Asp²⁸、Gly²⁹、Ala³¹、Phe⁵³、Thr⁵⁶、Val⁵⁷、Gly⁵⁸、Leu⁵⁹、Val⁶⁰、Glu⁶¹、Ala⁶²、Ala⁶³/Pro⁶³、Tyr⁶⁴、Phe⁶⁵(ii) a And Ser of the M chain of any one of tables 3, 4 or 5¹⁸²、Leu¹⁸³、Leu¹⁸⁵、Arg¹⁸⁶。

6. An isolated nucleic acid sequence encoding the isolated mutant atpE protein of claim 1.

7. A vector comprising the nucleic acid sequence of claim 6.

8. A host cell carrying the vector of claim 7.

9. A method of identifying an antimicrobial compound in vitro, the method comprising:

a) contacting a test compound with a cell expressing atpE protein under physiological conditions, and

b) determining whether said test compound interacts with an atpE protein, wherein said atpE protein is a mutant atpE protein of claim 1.

10. A method of evaluating the potential of a test compound to interact with the mutant atpE protein of claim 1, which method comprises:

a) establishing the three-dimensional structure of the mutant atpE protein of claim 1 using molecular modeling techniques,

b) performing a fit between said test compound and the three-dimensional structure of the mutant atpE protein of claim 1 using in silico methods, and

c) analyzing the results of the fitting procedure to quantify the association between the test compound and the three-dimensional structure of the mutant atpE protein of claim 1.

11. The method of claim 10 wherein the three-dimensional structure of said atpE Protein is obtained using the root mean square deviation below 10 a of the Ile28, Glu61, and Ile63 atomic coordinates Protein Database 1Q01 +/-amino acid backbone atoms of e.

12. The method of claim 10 wherein the three-dimensional structure of the atpE protein is obtained using the root mean square deviation below 1.5 a of the atomic coordinates +/-amino acid backbone atoms of table 6 or 7.