EP2686687A2 - A method of screening anti-tubercular compounds - Google Patents

A method of screening anti-tubercular compounds

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Publication number
EP2686687A2
EP2686687A2 EP12716669.2A EP12716669A EP2686687A2 EP 2686687 A2 EP2686687 A2 EP 2686687A2 EP 12716669 A EP12716669 A EP 12716669A EP 2686687 A2 EP2686687 A2 EP 2686687A2
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EP
European Patent Office
Prior art keywords
protein
screening
biotin
tubercular
groel2
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EP12716669.2A
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German (de)
French (fr)
Inventor
Dhiman Sarkar
Sampa Sarkar
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Council of Scientific and Industrial Research CSIR
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Council of Scientific and Industrial Research CSIR
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the present invention relates to a method of screening anti-tubercular compounds. More particularly, the present invention provides novel target for screening of antitubercular agents. In particular, this invention discloses GroEL2 as an antitubercular target for screening of drugs having antitubercular activity.
  • Nl substituted 1, 2, 4-triazole compounds which are ergosterol biosynthesis inhibitors, while antitubercular activity is associated with the derivatives of 1, 2, 4- triazolethiols in which N4 is substituted.
  • the main objective of the present invention is thus to provide a process for the identification and isolation of new drug targets in M. tuberculosis.
  • Another objective of the invention is to provide a method for screening of novel drug candidates for treatment of tuberculosis.
  • the present invention provides only those molecules which have 1, 2, 4-triazole-3- thiols in their core structure (antimycobacterial activity) and substitution on Nl or N2 nitrogens (for sterol biosynthesis inhibition).
  • In vitro antimycobacterial activity of these propargylated triazole derivatives has been examined against actively replicating as well as non-replicating dormant phase of Mycobacterium bovis BCG and M. tuberculosis H37Ra.
  • Three molecules from these 1,2,4-triazole derivatives were found to be most effective against M. bovis BCG and M. tuberculosis with MIC values 2, 0.2 and 2 ⁇ g/ml respectively.
  • M.tuberculosis and M.bovis BCG there are about 23 copies of these genes but interestingly, in M.smegmatis 51 copies of the same is present. This indicates the importance of the role of this class of proteins in M.smegmatis, also where these triazolethiols identified from our screening programs are ineffective. This led to the search of possible new targets of these novel inhibitors in M. tuberculosis.
  • Fig-1 Binding reaction between biotin linker and 1, 2, 4, triazole compound
  • Fig- 2 Mass confirmation by MALDI-MS analysis.
  • MALDI mass spectra were obtained using the dried-drop method or the slow crystallization method of only inhibitor (a), only biotin -linker (b) and biotin-inhibitor conjugate (c). Spectra were obtained from a matrix solution consisting of 4HCCA-water/acetonitrile (2:1 v/v).
  • Fig- 3 HPLC profile of the product (biotin-inhibitor conjugate). Chromatogram of solvent (A), biotin at lmg/ml (B), 2d at MIC (C) and reaction mixture (D) were analyzed by preparative HPLC. The chromatograms are the representatives of result obtained from three identical set of each experiments. The rest of the details about the chromatography are provided in "Materials and Methods" section.
  • Fig- 4 Separation of identified proteins by SDS PAGE on the basis of the molecular weight Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the protein isolated from streptavidin beads. The gel picture represents the protein samples obtained from the whole cell extract previously treated with (Lane 2) and without (Lane 1) the inhibitor and Lane 3 is represented by high molecular weight protein markers.
  • Fig- 5 Pictorial diagrams by PLGS soft wear of MALDI- ESI-MS analysis. This data is analyses the single band taken out of SDS page representing molecular weight of -60 da.
  • the present invention describes a novel target for screening of antitubercular agents.
  • the instant invention discloses the identification of targets of one of the hits of propargylated triazole derivatives within M. tuberculosis. More particularly, the present invention discloses GroEL2 as an antitubercular target for screening of drug candidates.
  • the drug candidates are selected as propargylated 1, 2, 4, triazole compounds.
  • Identification of the intracellular target of a lead inhibition is an essential criterion for pursuing the scaffold in lead optimization program.
  • affinity pooling could be a straight and simple procedure to hit a protein target.
  • Mts-Atf-Biotin Label Transfer Reagents supplied by Thermo Scientific for labeling the inhibitor is selected for the purpose of the present invention.
  • the invention utilizes Mts-Atf-Biotin Label Transfer Reagents.
  • reagents are crosslinkers that enable purified test proteins to be labeled at sulfhydryl sites for later covalent attachment and transfer of biotinylation to an interacting protein.
  • Mts-Atf-Biotin Label Transfer Reagents Biotin Linker
  • Mts sulfhydryl-reactive methanethiosulfonate
  • Atf efficient photoactivatable tetrafluorophenyl azide
  • the product is purified from the rest of the reagents by preparative HPLC.
  • 100ml of M.tuberculosis culture is taken at, log phase in which 5ml of spheroplast solution (0.0006%) is added.
  • the culture is then allowed to incubate for 24hrs.
  • the biotin-linker modified with and without the hit compound is added in the culture and incubated for 30mins.
  • the cultures are then sonicated to release the intracellular proteins in to the medium.
  • the whole suspension is then centrifuged at 14,000rpm for lhr to get the supernatant where the target protein is expected to be present and bound with the inhibitor molecule.
  • the supernatant is dialysed against PBS to remove the excess Biotin-linker or inhibitor molecules.
  • MagnaBind Streptavidin Beads are added in the supernatant for pulling down the target protein already bound with biotin-labeled molecules by using an external magnetic field.
  • the beads are washed with PBS for 2-3 times to remove the unwanted proteins.
  • an excess of free biotin molecule is added before dialysis against water. Dialyzed sample is collected and precipitated using Chloroform-Methanol precipitation. Then, the individual protein in the sample is separated by carrying out SDS-PAGE.
  • the protein bands found in the gel is digested by trypsin and used for MALDI-ESI-MS analysis.
  • the protein identified according to the process of the present invention is a 60 kDa chaperonin 2 OS Mycobacterium tuberculosis GN groL2 PE 1 , S V 2.
  • the invention in its preferred embodiment provides a method of screening for anti- tubercular compounds that affect the binding between GroEL2 protein or a fragment thereof comprising;
  • the trifunctional cross linker according to invention is Mts-Atf-Biotin Label Transfer Reagents.
  • test compounds selected for the purpose of the invention are propargylated 1, 2, 4, triazole compounds as shown below: Compound 1 3-(allylthio)-5-(4-nitrophenyl)-lH-l,2,4-triazole
  • the cultures selected for the purpose of the invention are M. tuberculosis M. smegmatis and M. bovis.
  • the conjugate as referred above for the purpose of the invention is a conjugate of test compound with Mts-Atf-Biotin Label Transfer Reagents.
  • the conjugate of the present invention is purified using HPLC techniques.
  • the invention discloses an isolated GroEL 2 protein from M.tuberculosis having seq ID NO 1.
  • the invention provides a kit for screening of drug candidates comprising; an isolated gro EL 2 protein; Mts-Atf-Biotin Label Transfer Reagents and a culture medium comprising M. bovis BCG (ATCC 35745), M. smegmatis (ATCC 607). Bacterial strains, media and inoculum preparation M. bovis BCG (ATCC 35745), M. smegmatis (ATCC 607) was obtained from Astra Zeneca, Bangalore, India and M.
  • tuberculosis H37Ra (ATCC 25177) was obtained from Microbial Type Culture Collection, Chandigarh, India.
  • E. coli strain DH5a was obtained from National Collection of Industrial Microorganisms (NCIM), Pune, India. Sub culturing of all mycobacterial strains was routinely done in Dubos albumin agar slants or plates. Liquid inoculum was prepared in Dubos tween albumin broth, incubated in a shaker incubator rotating at a speed of 150 rpm at 370C. 1% of 1.0 O.D at 620nm of the culture was used as standard inoculum size for all the experiments, yielding final inoculum of approximately 105 CFU/ml. Thp-1 human monocyte and HL-60 cell line was obtained from, National Centre for Cell Science (NCCS), India. Cells were maintained routinely in RPMI 1640 cell culture medium supplemented with 10% FBS.
  • Identification of intracellular target of Triazolethiols within Mycobacterium tuberculosis is an essential criterion for pursuing the scaffold in lead optimization program.
  • affinity pooling is a straight and simple procedure to hit a protein target.
  • Mts-Atf-Biotin Label Transfer Reagents supplied by Thermo Scientific were selected for labeling the inhibitor.
  • Mts-Atf-Biotin Label Transfer Reagent has a trifunctional cross-linker that contain a biotin, a sulfhydryl-reactive methanethiosulfonate (Mts) moiety and an efficient photoactivatable tetrafluorophenyl azide (Atf) moiety for making covalent linkage with a chemical moiety of interest.
  • This biotin cross linker was allowed to react with the inhibitor (2d) and then analyzed the synthesis of the conjugate.
  • the product was then purified from the rest of the reagents by carrying out preparative HPLC.
  • the retention time of the solvent, biotin-linker and inhibitor were identified as 2.46, 6.01 and 11.6 minutes respectively.
  • Biotin Linker-Inhibitor conjugate was supposed to bind protein/s in the whole cell extract because of the higher affinity of the inhibitor remained for the target protein.
  • This whole complex could then be pooled down with the help of MagnaBind Streptavidin beads to the bottom by applying magnetic field. At this stage, the beads were washed with PBS for 2-3 times to remove the unwanted proteins. SDS-PAGE analysis of the target proteins pooled by streptavidin beads clearly indicated presence of a major protein band at ⁇ 62kd. This band was insignificantly present in the control sample where the extract was pre-treated with the inhibitor alone [figure 4].
  • the protein bands found in the gel was digested by trypsin and used for mass determination.
  • the protein mass was found to be 60 kDa which was further identified as chaperonin 20S Mycobacterium tuberculosis GN groL2 PE 1 SV 2 by using PLGS score.
  • GroEL 1 and 2 are functionally very similar because both belong to the class of protein representing 60Kd HSP family of chaperonines. Both of these two proteins are reported to. have ATPase activity and found to protect citrate synthase agglutination during heat shock.
  • groEL 1 was also taken in the experiments as control. The results clearly indicates that all the three compounds inhibited ATPase activity by -86% at their respective MIC when applied to these purified groEL2 but did not exhibit any significant effect on groELl (Table 4.3).
  • a further investigation on the effect of these inhibitors on citrate synthase agglutination was carried out and found to have no effect on it.
  • Mts-Atf-Biotin Label Transfer Reagents are tri-functional cross-linkers that contain a biotin, a sulfhydryl-reactive methanethiosulfonate (Mts) moiety and an efficient photoactivatable tetrafluorophenyl azide (Atf) moiety obtained from Thermo Scientific (Cat.No.33093).
  • ⁇ of the Biotin linker (5mg/ml) was mixed with 5 ⁇ 1 (lmg/ml) of triazole inhibitor (2d) and kept under Ultra Violet (UV) radiation for 72 hrs. The reaction mixture was then analysed for product formation.
  • MALDI-MS Matrix-Assisted Laser Desorption/ Ionization-Mass
  • All of the matrix solutions were saturated with 4HCCA and were prepared by adding 4HCCA (solid) to the organic solvent, followed by the addition of water and acid (as required). Each mixture was thoroughly vortexed and centrifuged, leaving a clear working matrix solution.
  • the solubility of 4HCCA were dependent on the solvent composition, ranging from a low of 5 raM (water/methanol, 2:1 v/v), to an intermediate value of 29 mM (formic acid/water/2- propanol, 1 :3:2 v/v/v), to a high value of 74 mM (water/acetonitrile, 1 :1 v/v) [53].
  • sample-matrix solution an aliquot (0.5 ⁇ ) of sample was combined with 15 ⁇ . of matrix solution in a small microcentrifuge tube (Tarson, India), Sample-matrix cocrystals were obtained by the following techniques.
  • sample-matrix solution An aliquot (0.5-1 ⁇ ,) of the sample-matrix solution was deposited onto an aluminum 10- sample MALDI probe tip and allowed to air-dry (several minutes) at room temperatures, resulting in a uniform layer of fine granular matrix crystals. Cold water was placed over the crystals for 10 s to help remove in volatile salts. The water was subsequently removed with vacuum suction.
  • Biotin-Inhibitor conjugate was allowed to keep for solvent evaporation in speed vac for 30 min, then dissolved in ⁇ DMSO and then added to the whole cell extract.
  • 5ml spheroplast solution (0.0006%) was added in 100ml log phase Mtb culture and allowed to incubate for 24hrs.
  • This Biotin linker-inhibitor conjugate was also added in the control culture, which already treated with 2d inhibitor, both control and test samples were kept for 30min. in incubator shaker at 37°C for binding to the target protein. Both the culture were sonicated in presence of protease cocktail and centrifuge the cell extract at 14,000 rpm for 1 hr. to releasing the intracellular protein.
  • the supernatant contained the target protein with Biotin-Inhibitor conjugate. Supernatant was taken out and dialysed against PBS to remove the excess Biotin-Inhibitor conjugate and inhibitor molecules.
  • MagnaBind Streptavidin Beads are convenient for affinity purification or separation methods involving of biotin-labelled molecules obtained from Thermo Scientific (Cat no 21344). MagnaBind Streptavidin Beads were added in the supernatant for pulling down the target protein already bound with Biotin-Inhibitor conjugate by using an external magnetic field, the beads were washed with PBS for 2-3 times to remove the unwanted proteins. To release the target protein attached with Biotin-Inhibitor conjugate, we added an excess of free biotin molecule before dialysis against water. Dialyzed sample was collected and processed for protein precipitation.
  • chloroform-methanol method was followed. ⁇ sample was added to 400 ⁇ 1 methanol, vortexed well, then ⁇ chloroform was added and vortexed. 300 ⁇ 1 mili Q water was added and again vortexed for 2min at the rate of 14,000g for 1 min. Removed the top aqueous layer (protein is between layers) then 400 ⁇ 1 methanol was added and vortexed for 2min @ 14,000g and removed the MeOH without disturbing the pellet. Sample was dried in speed- Vac. IX sample buffer was added for SDS PAGE.
  • Protein bands seen within the gel were cut properly and subjected to trypsin digestion, followed by peptide extraction and proteomic analysis in MALDI-ESI-MS.
  • the gel picture represents the protein samples obtained from the whole cell extract previously treated with (Lane 2) and without (Lane 1) the inhibitor and Lane 3 is represented by high molecular weight protein markers. The prominent band with Mol.Wt ⁇ 60kd was seen in the lane (Lane 1) where the sample was not previously treated with the inhibitor.
  • the ATPase activity of the purified GroEL 1 and groEL 2 was quantified with a colorimetric assay performed as described previously. Briefly, 25 ⁇ of the reaction buffer containing 50mM Tris-HCL (pH 8.0), lOMm KCL, lOMm MgCl 2 and 2.5 ⁇ of each GroEL was incubated with ImM ATP at 37°C for 20min. Enzymatic reactions were terminated by the addition of 100 ⁇ of an acidic solution of malachite green. The amount of inorganic phosphate liberated was measured at 655nm. In control, reaction was performed in absence of ATP and GroEL proteins. The estimation of liberated phosphate was done from the standard curve generated by using monobasic potassium phosphate with each experiment. Inhibitory effect of these leads on ATPase activity of groEL 2 conclusively proved that the potential anti-tubercular action of these triazolethiols was achieved through their binding with chaperonine 2 proteins.
  • Pig heart citrate synthase aggregation was performed as reported previously. Briefly, 0.15 ⁇ g/ml citrate synthase was incubated at 43 °C in the presence or absence of equimolar oligomer ratios of different GroEL variants in 40 mM HEPES-KOH buffer (pH 7.5). The ability of the said chaperones to prevent the aggregation of citrate synthase was monitored for 20 min on a LS55 spectro-fiuorimeter- with emission and excitation wavelengths set at 465 nm and corresponding band passes set at 3.0 nm. The temperature of the sample was maintained with a Julabo circulating water bath and was monitored by using a Physitemp type T microcouple. We did not observe any effect of the inhibitor on the aggregation of citrate synthase.
  • SEQ ID No. 1 Amino Acid sequence of groEL-2 MSKLIEYDETARRAMEVGMDKLADTVRVTLGPRGRHVVLAKAFGGPTVTNDGVTV AREIE

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Abstract

Novel targets for screening anti tubercular targets and methods to use those targets to screen potential drugs for anti tubercular therapy are addressed through this invention. A method of screening anti-tubercular compounds that affect the binding between GroEL2 protein or a fragment thereof and a kit for screening of antitubercular drug candidates having an isolated gro EL 2 protein; Mts- Atf-Biotin Label Transfer Reagents and a culture medium comprising M. bovis BCG (ATCC 35745), M. smegmatis (ATCC 607) are disclosed.

Description

A METHOD OF SCREENING ANTI-TUBERCULAR COMPOUNDS
FIELD OF THE INVENTION
The present invention relates to a method of screening anti-tubercular compounds. More particularly, the present invention provides novel target for screening of antitubercular agents. In particular, this invention discloses GroEL2 as an antitubercular target for screening of drugs having antitubercular activity.
BACKGROUND OF INVENTION AND DESCRIPTION OF PRIOR ART
In spite of an enormous amount of work done in understanding the genome sequence of mycobacteria, no new antituberculotic drug has been discovered in over 40 years. Hence, there is a pressing need to develop novel chemotherapeutic agents and to change current drug regimens in order to shorten the lengthy treatment, to minimize the resistance problem in mycobacterial strains, and/or to improve the treatment of latent TB infection. Various heterocyclic rings were taken in earlier attempts as a ground to constitute large series of compounds, e.g. imidazoles, tetrazoles, benzimidazoles, pyrazine, quinoxaline, and quinazoline for antitubercular activity. Special attention was given in recent past to synthesize triazole derivatives due to their potent antimycobacterial activity and known target of sterol synthesis. Commercially important antifungal derivatives such as fluconazole and hexacoazole are Nl substituted 1, 2, 4-triazole compounds, which are ergosterol biosynthesis inhibitors, while antitubercular activity is associated with the derivatives of 1, 2, 4- triazolethiols in which N4 is substituted.
Reference may be drawn to Jungblut et al in "Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens" in Molecular Microbiology Volume 33, Issue 6, pages 1103-1117, September 1999, wherein identified Twenty-five protein spots were identified as putative heat shock proteins, including Hsp60 (groEL2; Rv0440), Hsp70 (DnaK; Rv0350), HsplO (GroES; Rv3418) and ClpB (Rv0384c).
Reference may be drawn to Dahl et al in an article titled "The role of RelMtb-mediated adaptation to stationary phase in long-term persistence of Mycobacterium tuberculosis in mice" in Proceedings of the National Academy of Sciences, 10026-10031 _ PNAS _ August 19, 2003 _ vol. 100 _ no. 17 which disclosed that there was also a large set of known mycobacterial antigens differentially expressed between the two strains such as groELl and groES.
Reference may be drawn to Ojha et al in Cell . 123, 861-873, December 2, 2005 ttiled "GroELl : A Dedicated Chaperone Involved in Mycolic Acid Biosynthesis during Biofilm Formation in Mycobacteria which, stated that Mycobacteria are unusual in encoding two GroEL paralogs, GroELl and GroEL2. GroEL2 is essential— presumably providing the housekeeping chaperone functions. But there are no prior arts till date that identify a novel target such as groEL2 for screening of antitubercular agents.
OBJECTIVES OF THE INVENTION
The main objective of the present invention is thus to provide a process for the identification and isolation of new drug targets in M. tuberculosis.
Another objective of the invention is to provide a method for screening of novel drug candidates for treatment of tuberculosis.
SUMMARY OF THE INVENTION
The present invention provides only those molecules which have 1, 2, 4-triazole-3- thiols in their core structure (antimycobacterial activity) and substitution on Nl or N2 nitrogens (for sterol biosynthesis inhibition). In vitro antimycobacterial activity of these propargylated triazole derivatives has been examined against actively replicating as well as non-replicating dormant phase of Mycobacterium bovis BCG and M. tuberculosis H37Ra. Three molecules from these 1,2,4-triazole derivatives were found to be most effective against M. bovis BCG and M. tuberculosis with MIC values 2, 0.2 and 2μg/ml respectively. At MIC concentrations, these compounds yielded 0.82, 1.432 and 0.88 log reduction of CFU/ml against non- replicating M. tuberculosis. Insignificant effect of these inhibitors on growth of M. smegmatis, Escherichia coli and THP-1 macrophages indicated their selectivity and non-toxic character. Most significantly, effective killing of intracellular bacilli in THP-1 macrophages has identified these triazolethiol derivatives as potential antitubercular leads for further explorations. It is well known that 14-demethylase belongs to the Cytochrome P-450 class of proteins. In M.tuberculosis and M.bovis BCG, there are about 23 copies of these genes but interestingly, in M.smegmatis 51 copies of the same is present. This indicates the importance of the role of this class of proteins in M.smegmatis, also where these triazolethiols identified from our screening programs are ineffective. This led to the search of possible new targets of these novel inhibitors in M. tuberculosis.
BRIEF DESCRIPTION OF THE FIGURES
Fig-1 : Binding reaction between biotin linker and 1, 2, 4, triazole compound
Fig- 2: Mass confirmation by MALDI-MS analysis. MALDI mass spectra were obtained using the dried-drop method or the slow crystallization method of only inhibitor (a), only biotin -linker (b) and biotin-inhibitor conjugate (c). Spectra were obtained from a matrix solution consisting of 4HCCA-water/acetonitrile (2:1 v/v).
Fig- 3: HPLC profile of the product (biotin-inhibitor conjugate). Chromatogram of solvent (A), biotin at lmg/ml (B), 2d at MIC (C) and reaction mixture (D) were analyzed by preparative HPLC. The chromatograms are the representatives of result obtained from three identical set of each experiments. The rest of the details about the chromatography are provided in "Materials and Methods" section. Fig- 4: Separation of identified proteins by SDS PAGE on the basis of the molecular weight Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the protein isolated from streptavidin beads. The gel picture represents the protein samples obtained from the whole cell extract previously treated with (Lane 2) and without (Lane 1) the inhibitor and Lane 3 is represented by high molecular weight protein markers.
Fig- 5: Pictorial diagrams by PLGS soft wear of MALDI- ESI-MS analysis. This data is analyses the single band taken out of SDS page representing molecular weight of -60 da.
Fig- 6: Sequence of groELl and groEL 2 and their Multiple Sequence Alignments by CLUSTAL 2.1 DETAILED DESCRIPTION OF THE INVENTION
The subject application claims priority from Indian Patent Application No. 0737/DEL/201 1 dated March 16, 2012 whose disclosures are hereby incorporated by reference in their entirety in this application.
In accordance with the above objective, the present invention describes a novel target for screening of antitubercular agents. The instant invention discloses the identification of targets of one of the hits of propargylated triazole derivatives within M. tuberculosis. More particularly, the present invention discloses GroEL2 as an antitubercular target for screening of drug candidates.
For the purpose of the invention, the drug candidates are selected as propargylated 1, 2, 4, triazole compounds. Identification of the intracellular target of a lead inhibition is an essential criterion for pursuing the scaffold in lead optimization program. Among the different approaches known for identifying target of a novel inhibitor, affinity pooling could be a straight and simple procedure to hit a protein target. As the pharmacophor of this scaffold is still unknown, Mts-Atf-Biotin Label Transfer Reagents supplied by Thermo Scientific for labeling the inhibitor is selected for the purpose of the present invention. . Accordingly, in a preferred embodiment, the invention utilizes Mts-Atf-Biotin Label Transfer Reagents. These reagents are crosslinkers that enable purified test proteins to be labeled at sulfhydryl sites for later covalent attachment and transfer of biotinylation to an interacting protein. Mts-Atf-Biotin Label Transfer Reagents (Biotin Linker), a trifunctional cross-linker that contain a biotin, a sulfhydryl-reactive methanethiosulfonate (Mts) moiety and an efficient photoactivatable tetrafluorophenyl azide (Atf) moiety, from Thermo Scientific are taken and the linker is allowed to bind the hit compound/ inhibitor (1, 2, 4, triazole) under UV light. Confirmation of product formation is done by MALDI analysis. The product is purified from the rest of the reagents by preparative HPLC. For preparing the whole cell extract, 100ml of M.tuberculosis culture is taken at, log phase in which 5ml of spheroplast solution (0.0006%) is added. The culture is then allowed to incubate for 24hrs. The biotin-linker modified with and without the hit compound is added in the culture and incubated for 30mins. The cultures are then sonicated to release the intracellular proteins in to the medium. The whole suspension is then centrifuged at 14,000rpm for lhr to get the supernatant where the target protein is expected to be present and bound with the inhibitor molecule. At this stage, the supernatant is dialysed against PBS to remove the excess Biotin-linker or inhibitor molecules. Further, MagnaBind Streptavidin Beads are added in the supernatant for pulling down the target protein already bound with biotin-labeled molecules by using an external magnetic field. At this stage, the beads are washed with PBS for 2-3 times to remove the unwanted proteins. To release the target molecule attached with biotin linker-inhibitor conjugate, an excess of free biotin molecule is added before dialysis against water. Dialyzed sample is collected and precipitated using Chloroform-Methanol precipitation. Then, the individual protein in the sample is separated by carrying out SDS-PAGE. The protein bands found in the gel is digested by trypsin and used for MALDI-ESI-MS analysis. The protein identified according to the process of the present invention is a 60 kDa chaperonin 2 OS Mycobacterium tuberculosis GN groL2 PE 1 , S V 2.
Thus, the invention in its preferred embodiment provides a method of screening for anti- tubercular compounds that affect the binding between GroEL2 protein or a fragment thereof comprising;
preparing a conjugate of test compound with a trifunctional cross linker; contacting the GroEL2 protein or the fragment thereof in a culture having spheroplast solution medium with the conjugate ; determining the effect of the test compound on the binding of the GroEL2 protein or the fragment thereof, and
By measuring the difference in the binding between the GroEL2 protein or fragment in the presence of the test compound and the binding between the GroEL2 protein or fragment in the absence of the test compound (control).
The trifunctional cross linker according to invention is Mts-Atf-Biotin Label Transfer Reagents.
The test compounds selected for the purpose of the invention are propargylated 1, 2, 4, triazole compounds as shown below: Compound 1 3-(allylthio)-5-(4-nitrophenyl)-lH-l,2,4-triazole
Compound 3 3-(4-nitrophenyl)-5-(prop-2-ynylsulfonyl)-lH-l,2,4-triazole
The cultures selected for the purpose of the invention are M. tuberculosis M. smegmatis and M. bovis.
The conjugate as referred above for the purpose of the invention is a conjugate of test compound with Mts-Atf-Biotin Label Transfer Reagents.
The conjugate of the present invention is purified using HPLC techniques.
The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.
In another preferred embodiment, the invention discloses an isolated GroEL 2 protein from M.tuberculosis having seq ID NO 1. In another preferred embodiment, the invention provides a kit for screening of drug candidates comprising; an isolated gro EL 2 protein; Mts-Atf-Biotin Label Transfer Reagents and a culture medium comprising M. bovis BCG (ATCC 35745), M. smegmatis (ATCC 607). Bacterial strains, media and inoculum preparation M. bovis BCG (ATCC 35745), M. smegmatis (ATCC 607) was obtained from Astra Zeneca, Bangalore, India and M. tuberculosis H37Ra (ATCC 25177) was obtained from Microbial Type Culture Collection, Chandigarh, India. E. coli strain DH5a was obtained from National Collection of Industrial Microorganisms (NCIM), Pune, India. Sub culturing of all mycobacterial strains was routinely done in Dubos albumin agar slants or plates. Liquid inoculum was prepared in Dubos tween albumin broth, incubated in a shaker incubator rotating at a speed of 150 rpm at 370C. 1% of 1.0 O.D at 620nm of the culture was used as standard inoculum size for all the experiments, yielding final inoculum of approximately 105 CFU/ml. Thp-1 human monocyte and HL-60 cell line was obtained from, National Centre for Cell Science (NCCS), Pune, India. Cells were maintained routinely in RPMI 1640 cell culture medium supplemented with 10% FBS.
EXAMPLES
The following examples are given by way of illustration and therefor should not be construed to limit the scope of the present invention.
Example !
Identification of intracellular target of Triazolethiols within Mycobacterium tuberculosis Identification of the intracellular target of a lead inhibition is an essential criterion for pursuing the scaffold in lead optimization program. Among the different approaches known for identifying.target of a novel inhibitor, affinity pooling is a straight and simple procedure to hit a protein target. As the pharmacophor of this scaffold is still unknown, Mts-Atf-Biotin Label Transfer Reagents supplied by Thermo Scientific were selected for labeling the inhibitor. Mts-Atf-Biotin Label Transfer Reagent has a trifunctional cross-linker that contain a biotin, a sulfhydryl-reactive methanethiosulfonate (Mts) moiety and an efficient photoactivatable tetrafluorophenyl azide (Atf) moiety for making covalent linkage with a chemical moiety of interest. This biotin cross linker was allowed to react with the inhibitor (2d) and then analyzed the synthesis of the conjugate. Example 2
Purification of product (Inhibitor-biotin linker) by preparative HPLC and MALDI-MS analysis First, the linker was allowed to bind hit compound (2d) under UV light for 72hrs at room temperature. Then, the reaction mixture was subjected to MALDI-MS analysis. In this analysis the inhibitor, biotin-linker and one of the products possibly biotin linker- inhibitor conjugate in the reaction mixture were showing their masses 261.09, 840.79 and 1101.3kd respectively. The mass of the product (1101 kd, fig 4) clearly indicated the formation of biotirt linker-inhibitor conjugate.
The product was then purified from the rest of the reagents by carrying out preparative HPLC. The retention time of the solvent, biotin-linker and inhibitor were identified as 2.46, 6.01 and 11.6 minutes respectively.
One foreign peak was identified with 10.292 minute retention time when the reaction mixture was run through the HPLC column using similar conditions. This new peak was collected and subjected to MALDI-MS analysis and found to have Mol. Wt of 1101 kd confirming separation of the Biotin linker-inhibitor conjugate from the mixture. Then, the product was collected and pooled for further experiment.
Example 3
Identification of target protein by using Biotin linker-Inhibitor conjugate from Mycobacterium tuberculosis
Addition of Biotin Linker-Inhibitor conjugate was supposed to bind protein/s in the whole cell extract because of the higher affinity of the inhibitor remained for the target protein. This whole complex could then be pooled down with the help of MagnaBind Streptavidin beads to the bottom by applying magnetic field. At this stage, the beads were washed with PBS for 2-3 times to remove the unwanted proteins. SDS-PAGE analysis of the target proteins pooled by streptavidin beads clearly indicated presence of a major protein band at ~62kd. This band was insignificantly present in the control sample where the extract was pre-treated with the inhibitor alone [figure 4].
The protein bands found in the gel was digested by trypsin and used for mass determination. The protein mass was found to be 60 kDa which was further identified as chaperonin 20S Mycobacterium tuberculosis GN groL2 PE 1 SV 2 by using PLGS score.
Example 4 Validation of Triazolethiols, inhibiting Mycobacterium tuberculosis groEL 2 as target
GroEL 1 and 2 are functionally very similar because both belong to the class of protein representing 60Kd HSP family of chaperonines. Both of these two proteins are reported to. have ATPase activity and found to protect citrate synthase agglutination during heat shock. In order to validate the specific action of the three selected inhibitors (2d, 8f, 1 Id) on groEL 2; groEL 1 was also taken in the experiments as control. The results clearly indicates that all the three compounds inhibited ATPase activity by -86% at their respective MIC when applied to these purified groEL2 but did not exhibit any significant effect on groELl (Table 4.3). A further investigation on the effect of these inhibitors on citrate synthase agglutination was carried out and found to have no effect on it.
Table: 1 ATPase activity on purified groEL-1 and gro-EL-2
Example 5
Conjugation of Mts-Atf-Biotin linker with the Inhibitor
Mts-Atf-Biotin Label Transfer Reagents are tri-functional cross-linkers that contain a biotin, a sulfhydryl-reactive methanethiosulfonate (Mts) moiety and an efficient photoactivatable tetrafluorophenyl azide (Atf) moiety obtained from Thermo Scientific (Cat.No.33093). ΙΟμΙ of the Biotin linker (5mg/ml) was mixed with 5μ1 (lmg/ml) of triazole inhibitor (2d) and kept under Ultra Violet (UV) radiation for 72 hrs. The reaction mixture was then analysed for product formation.
Example 6
Matrix-Assisted Laser Desorption/ Ionization-Mass (MALDI-MS) analysis of Biotin- Inhibitor conjugate
Matrix Solution Preparation
All of the matrix solutions were saturated with 4HCCA and were prepared by adding 4HCCA (solid) to the organic solvent, followed by the addition of water and acid (as required). Each mixture was thoroughly vortexed and centrifuged, leaving a clear working matrix solution. The solubility of 4HCCA were dependent on the solvent composition, ranging from a low of 5 raM (water/methanol, 2:1 v/v), to an intermediate value of 29 mM (formic acid/water/2- propanol, 1 :3:2 v/v/v), to a high value of 74 mM (water/acetonitrile, 1 :1 v/v) [53].
Sample-Matrix Crystallization Procedures
To prepare the sample-matrix solution, an aliquot (0.5 μί) of sample was combined with 15 μΐ. of matrix solution in a small microcentrifuge tube (Tarson, India), Sample-matrix cocrystals were obtained by the following techniques.
Dried-Drop Method
An aliquot (0.5-1 μΐ,) of the sample-matrix solution was deposited onto an aluminum 10- sample MALDI probe tip and allowed to air-dry (several minutes) at room temperatures, resulting in a uniform layer of fine granular matrix crystals. Cold water was placed over the crystals for 10 s to help remove in volatile salts. The water was subsequently removed with vacuum suction.
Example 7
Preparative HPLC purification of Biotin-Inhibitor conjugate
Chromatographic separation and analysis of product and each component of the reaction was done by using reversed- phase X bridge C18 (5μπι, 46x250mm) column in preparative high- performance liquid chromatography (HPLC, SCL-lOvp-SHIMADZU system controller). The mobile phase composition used was in, 'A' solution - water (60, 60, 45, 40) % and 'B' solution - acetonitrile (40, 40, 55, 60) %. The analysis was carried out in gradient mode as flow rate lml/min with column effluent being monitored at 254 nm wave length.
Example 8
Binding of Biotin-Inhibitor conjugate with crude whole cell extract of Mycobacterium tuberculosis
The Biotin-Inhibitor conjugate was allowed to keep for solvent evaporation in speed vac for 30 min, then dissolved in ΙΟΟμΙ DMSO and then added to the whole cell extract. For whole cell extract preparation, 5ml spheroplast solution (0.0006%) was added in 100ml log phase Mtb culture and allowed to incubate for 24hrs. This Biotin linker-inhibitor conjugate was also added in the control culture, which already treated with 2d inhibitor, both control and test samples were kept for 30min. in incubator shaker at 37°C for binding to the target protein. Both the culture were sonicated in presence of protease cocktail and centrifuge the cell extract at 14,000 rpm for 1 hr. to releasing the intracellular protein. The supernatant contained the target protein with Biotin-Inhibitor conjugate. Supernatant was taken out and dialysed against PBS to remove the excess Biotin-Inhibitor conjugate and inhibitor molecules. Example 9
Purification of Biotin-Inhibitor conjugate tagged target Proteins by MagnaBind™ Streptavidin Beads
MagnaBind Streptavidin Beads are convenient for affinity purification or separation methods involving of biotin-labelled molecules obtained from Thermo Scientific (Cat no 21344). MagnaBind Streptavidin Beads were added in the supernatant for pulling down the target protein already bound with Biotin-Inhibitor conjugate by using an external magnetic field, the beads were washed with PBS for 2-3 times to remove the unwanted proteins. To release the target protein attached with Biotin-Inhibitor conjugate, we added an excess of free biotin molecule before dialysis against water. Dialyzed sample was collected and processed for protein precipitation.
Example 10
Chloroform-Methanol Precipitation of Target proteins
For removal of salt and detergents chloroform-methanol method was followed. ΙΟΟμΙ sample was added to 400μ1 methanol, vortexed well, then ΙΟΟμΙ chloroform was added and vortexed. 300μ1 mili Q water was added and again vortexed for 2min at the rate of 14,000g for 1 min. Removed the top aqueous layer (protein is between layers) then 400μ1 methanol was added and vortexed for 2min @ 14,000g and removed the MeOH without disturbing the pellet. Sample was dried in speed- Vac. IX sample buffer was added for SDS PAGE.
Example 11
SDS-PAGE and Proteomic Analysis of Captured Proteins Individual proteins in sample were separated by carrying out SDS-PAGE. Briefly, protein samples were first mixed with IX loading (sample) buffer containing 5% β-mercaptoethanol (Sigma, MO, USA). The samples were then incubated for 10 min at 80°C. 30 μΐ sample was then loaded onto 12.5% Bis-Tris pre-cast polyacrylamide gel and the SDS-PAGE was carried out using mini-cell system (Invitrogen, CA, USA). After electrophoresis, the gel was subjected to Comassie staining for overnight. Protein bands seen within the gel were cut properly and subjected to trypsin digestion, followed by peptide extraction and proteomic analysis in MALDI-ESI-MS. With reference to Figure 4, the gel picture represents the protein samples obtained from the whole cell extract previously treated with (Lane 2) and without (Lane 1) the inhibitor and Lane 3 is represented by high molecular weight protein markers.The prominent band with Mol.Wt ~60kd was seen in the lane (Lane 1) where the sample was not previously treated with the inhibitor.
Example 12
LC-MSE analysis
Two micro-liter digested peptides with final concentration of 100 ng/ μΐ, was analyzed by nano LC-MSE using nanoACQUITY online coupled to SYNAPT HDMS system (Waters Corporation, MA, USA) equipped with a nanolockspray ion source with flow rate of 300 nl/ • min (external lockmass standard: Glu-fibrinopeptide). Peptide samples were injected online onto a 5 μιτι Symmetry CI 8 trapping column (180 μηι.χ 2 cm length) at a flow rate of 15 μΐ/ min. Peptides were separated by in-line gradient elution onto BEH (Bridged Ethyl Hybrid) 130 C18 1.7 μΜ x 75 μΜ x 150 mm nanoACQUITY analytical column, at a flow rate of 300 nl/ min using a linear gradient from 5 to 40% B over 35 min (A. 0.1% formic acid in water, B. 0.1%) formic acid in acetonitrile) shown in Figure 2a and 2b. Acquisition was performed in positive V mode in a mass range of 50-1990 m/z with a scan time of 1 second with alternating low (5 eV) and high (15-40 eV) collision energy. MSE data were processed with ProteinLynx Global Server (PLGS version 2.4. Waters Corporation, MA, USA). The processed data were allowed to search against human subset of UniProt database containing all 44,987 protein entries for protein identification.
Example 13
ATPase activity assay
The ATPase activity of the purified GroEL 1 and groEL 2 was quantified with a colorimetric assay performed as described previously. Briefly, 25 μΐ of the reaction buffer containing 50mM Tris-HCL (pH 8.0), lOMm KCL, lOMm MgCl2 and 2.5μΜ of each GroEL was incubated with ImM ATP at 37°C for 20min. Enzymatic reactions were terminated by the addition of 100 μΐ of an acidic solution of malachite green. The amount of inorganic phosphate liberated was measured at 655nm. In control, reaction was performed in absence of ATP and GroEL proteins. The estimation of liberated phosphate was done from the standard curve generated by using monobasic potassium phosphate with each experiment. Inhibitory effect of these leads on ATPase activity of groEL 2 conclusively proved that the potential anti-tubercular action of these triazolethiols was achieved through their binding with chaperonine 2 proteins.
Example 1
Prevention of the aggregation of citrate synthase by chaperonins
Pig heart citrate synthase aggregation was performed as reported previously. Briefly, 0.15 μg/ml citrate synthase was incubated at 43 °C in the presence or absence of equimolar oligomer ratios of different GroEL variants in 40 mM HEPES-KOH buffer (pH 7.5). The ability of the said chaperones to prevent the aggregation of citrate synthase was monitored for 20 min on a LS55 spectro-fiuorimeter- with emission and excitation wavelengths set at 465 nm and corresponding band passes set at 3.0 nm. The temperature of the sample was maintained with a Julabo circulating water bath and was monitored by using a Physitemp type T microcouple. We did not observe any effect of the inhibitor on the aggregation of citrate synthase.
SEQ ID No. 1 = Amino Acid sequence of groEL-2 MSKLIEYDETARRAMEVGMDKLADTVRVTLGPRGRHVVLAKAFGGPTVTNDGVTV AREIE
LEDPFEDLGAQLVKSVATKTNDVAGDGTTTATILAQALIKGGLRLVAAGVNPIALGV GIG
KAADAVSEALLASATPVSGKTGIAQVATVSSRDEQIGDLVGEAMSKVGHDGVVSVE ESST
LGTELEFTEGIGFDKGFLSAYFVTDFDNQQAVLEDALILLHQDKISSLPDLLPLLEKVA G
TGKPLLIVAEDVEGEALATLVVNAIRKTLKAVAVKGPYFGDRRKAFLEDLAVVTGG QVVN
PDAGMVLREVGLEVLGSARRVVVSKDDTVIVDGGGTAEAVANRAKHLRAEIDKSDS DWDR
EKLGERLAKLAGGVAVIKVGAATETALKERKESVEDAVAAAKAAVEEGIVPGGGAS LIHQ
ARKALTELRASLTGDEVLGVDVFSEALAAPLFWIAANAGLDGSVVVNKVSELPAGH GLNV
NTLSYGDLAADGVIDPVKVTRSAVLNASSVARMVLTTETVVVDKPAKAEDHDHHH GHAH

Claims

We Claim:
1. A method of screening anti-tubercular compounds that affect the binding between GroEL2 protein or a fragment thereof comprising;
a) preparing a conjugate of test compound with a trifunctional cross linker; b) contacting the GroEL2 protein or the fragment thereof in a culture having spheroplast solution medium with the conjugate ; and c) determining the effect of the test compound on the binding of the GroEL2 protein or the fragment thereof, by measuring a difference in the binding between the GroEL2 protein or fragment in the presence of the test compound and the binding between the GroEL2 protein or fragment in the absence of the test compound (control).
2. The method of screening for anti-tubercular compounds according to claim 1, wherein said trifunctional cross linker is Mts-Atf-Biotin Label Transfer Reagents.
3. The method of screening for anti-tubercular compounds according to claim 1, wherein the test compound is selected from propargylated 1, 2, 4, triazole compounds selected from:
Compound 3
4. The method of screening anti-tubercular compounds as claimed in claim 1, wherein the culture is selected from M.tuberculosis M. smegm tis and M. Bovis.
5. The method of screening anti-tubercular compounds as claimed in claim 1, wherein the said conjugate is a conjugate of test compound with Mts-Atf-Biotin Label Transfer Reagents.
6. The method of screening anti-tubercular compounds as claimed in claim 1, wherein the conjugate is purified using HPLC techniques.
7. The method of screening anti-tubercular compounds as claimed in claim 1, wherein the GroEL 2 protein is represented by Seq ID NO 1.
8. A kit for screening of antitubercular drug candidates comprising; an isolated gro EL 2 protein; Mts-Atf-Biotin Label Transfer Reagents and a culture medium comprising M. bovis BCG (ATCC 35745), M. smegmatis (ATCC 607).
EP12716669.2A 2011-03-16 2012-03-16 A method of screening anti-tubercular compounds Withdrawn EP2686687A2 (en)

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