WO2019162964A1 - Anti-tubercular composition, and combinatorial implementations thereof - Google Patents

Anti-tubercular composition, and combinatorial implementations thereof Download PDF

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Publication number
WO2019162964A1
WO2019162964A1 PCT/IN2019/050138 IN2019050138W WO2019162964A1 WO 2019162964 A1 WO2019162964 A1 WO 2019162964A1 IN 2019050138 W IN2019050138 W IN 2019050138W WO 2019162964 A1 WO2019162964 A1 WO 2019162964A1
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mycobacterium
binding
argj
composition
enzyme
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PCT/IN2019/050138
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French (fr)
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Avadhesha Surolia
Archita MISHRA
Ashalatha Sreshty MAMIDI
Raju Shiva RAJMANI
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Indian Institute Of Science
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/133—Amines having hydroxy groups, e.g. sphingosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
    • G16B35/20—Screening of libraries
    • 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
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91051—Acyltransferases other than aminoacyltransferases (general) (2.3.1)
    • G01N2333/91057—Acyltransferases other than aminoacyltransferases (general) (2.3.1) with definite EC number (2.3.1.-)
    • 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
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure broadly relates to a composition for inhibiting growth of pathogens, and particularly relates to a composition comprising panlukast or sorafenib for inhibiting growth of Mycobacterium species.
  • Tuberculosis is an airborne infectious disease caused by organisms of the Mycobacterium tuberculosis complex. Although primarily a pulmonary pathogen, M. tuberculosis can cause disease in almost any part of the body.
  • the existing treatment regime to tackle tuberculosis is not adequate and novel therapeutic interventions are required to target Mycobacterium tuberculosis pathogenesis.
  • Most of the targets identified in the pathogen have significant similarity to the human counterparts, thus adding to the woes. Therefore, there is a dearth of a novel target which lacks a human counterpart and also not many compositions are available which binds to such targets to intervene with the growth of M. tuberculosis.
  • US5439891A discloses a new pharmaceutical composition for the treatment of tuberculosis and leprosy, said composition comprising piperine in combination with known antituberculosis or antileprosy drugs or the mixtures thereof.
  • W02014170820A3 discloses a pharmaceutical combination comprising therapeutically effective amounts of nano curcumin and isoniazide, for treating tuberculosis in a subject, is provided herein.
  • Nano curcumin and isoniazide of the pharmaceutical combination of the present invention may be administered simultaneously, separately or sequentially or as a single fixed dose combination.
  • Pharmaceutical compositions comprising nano curcumin and isoniazide are also described herein.
  • a composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species.
  • the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • a composition comprising: (a) pranlukast; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species.
  • the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • a composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species.
  • the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • a composition comprising: (a) sorafenib; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species.
  • the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • composition comprising pranlukast, for use in inhibiting growth of Mycobacterium species.
  • the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • composition comprising sorafenib, for use in inhibiting growth of Mycobacterium species.
  • the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ; and (b) predicting the binding of the candidate molecules to ArgJ to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen.
  • the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
  • a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ ; (b) predicting the binding of the candidate molecules to identify selected candidate molecules; and (c) performing in-vitro validation of the selected candidate molecules, to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen.
  • the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
  • a method for identifying a candidate molecule capable of binding to ArgJ enzyme of Mycobacterium species comprising:
  • TLC Thin layer chromatography
  • TSA Thermal Shift Assay
  • Figure 1 illustrates arginine biosynthesis in Mycobacterium tuberculosis (a) Arginine biosynthesis pathway in M. tuberculosis in detail; (b) Reaction catalysed by MtA rg J (. Mycobacterium tuberculosis ArgJ), in accordance with an embodiment of the present disclosure.
  • FIG. 2 illustrates characterization of allosteric site on M/ArgJ surface and its role in enzyme activity
  • (b) Fluorescence based assay; The graph shows increase in fluorescence upon ANS binding to M/ArgJ, in a concentration dependent manner (ANS 10 mM - 10 mM);
  • ANS 10 mM - 10 mM);
  • Figure 3 illustrates in-vitro kinetics and binding studies of inhibitory compounds (PRK and SRB) M/ArgJ.
  • Chemical structures of (a) Pranlukast (PRK) and (b) Sorafenib (SRB). Saturation curve fit using Michaelis-Menten plot for MtA rg J activity at two different inhibitor concentrations for (c) PRK and (d) SRB, quantified 26 by TLC based assay.
  • Dixon plot analysis performed by plotting l/v against varying inhibitor concentration, at three different substrate concentrations for (e) PRK and (f) SRB.
  • TSA Shift Assay
  • Figure 4 illustrates binding interactions of PRK and SRB with the allosteric pocket of M/ArgJ.
  • PRK range
  • Gln305 of B chain Ser3l0 of B and D chains in the allosteric pocket of the M/ArgJ
  • LIGPLOT for the interaction of PRK with the allosteric pocket of M/ArgJ
  • SRB magenta
  • LIGPLOT for the interaction of SRB with the allosteric pocket of MtA rg J
  • Figure 5 illustrates effect of PRK and SRB on Mtb H37Rv survival
  • a Representative image of Alamar Blue assay employed for MIC determination
  • b, c Mtb H37Rv cells were treated with varying concentration of inhibitors and cell viability was determined using Alamar Blue assay.
  • MIC90 was calculated by plotting cell viability (%) against increasing concentration of inhibitors PRK and SRB.
  • d, e Hill’s plot analysis of decline in Alamar Blue fluorescence with increasing 51 concentration of PRK and SRB, for IC50 determination. Effect of PRK and SRB on the Multi drug resistant strains of Mtb.
  • Mycobacterium tuberculosis MDR strains Jal 2261 and Jal 2287 were treated with varying concentration of inhibitors and cell viability was determined using Alamar blue assay. MIC was calculated by plotting cell viability (%) against increasing concentration of inhibitors: (f). Effect of PRK on Jal226l (g). Effect of PRK on Jal2287.
  • FIG. 6 illustrates effect of PRK and SRB on the macrophage internalized Mtb.
  • THP1 human monocytic cell-line
  • Raw264.7 mouse macrophage cell line
  • PRK 5 pM and 25 pM
  • SRB 10 pM
  • Figure 7 illustrates PRK treatment reduces the lung-associated granuloma from Mtb infected mice
  • the white spot (cyst like) depicted with an arrow corresponds to tubercular granulomas; (d) Lungs of mice treated with Rif alone and (e) Rif + PRK post 12 and 24 days of infection; (f) CFU analysis of Mtb from lungs of infected mice treated with PBS or PRK at 0 day, 15 day and 24 days of treatment; (g) CFU analysis of Mtb from lungs of infected mice treated with Rif and Rif + PRK at 0 day, 15 day and 24 days of treatment. Appropriate negative and positive controls taken as PBS and Rif treated mice, respectively.
  • L Lymphocytes
  • FC Foamy macrophages
  • PMNs Polymorphonuclear cells
  • Figure 8 illustrates the targeting of arginine biosynthesis by PRK in Mtb
  • SEQ ID NO: 1 depicts the nucleic acid sequence of the gene encoding ArgJ enzyme (ornithine acetyltransferase) of Mycobacterium tuberculosis.
  • SEQ ID NO: 2 depicts the amino acid sequence of ArgJ enzyme of M. tuberculosis.
  • MTDLAGTTRLLRAQGVTAPAGFRAAGVAAGIKASGALDLALVFNEG PDYAAAGVFTRNQVKAAPVLWTQQVLTTGRLRAVILNSGGANACTGPAGFA DTHATAEAVAAALSDWGTETGAIEVAVCSTGLIGDRLPMDKLLAGVAHVVH EMHGGLV GGDEA AH AIMTTDN VPKQ V ALHHHDNWT V GGM AKG AGMLAPS LATMLCVLTTDAAAEPAALERALRRAAAATFDRLDIDGSCSTNDTVLLLSSG ASEIPPAQADLDEAVLRVCDDLCAQLQADAEGVTKRVTVTVTGAATEDDAL VAARQIARDSLVKTALFGSDPNWGRVLAAVGMAPITLDPDRISVSFNGAA VCVHGVGAPGAREVDLSDADIDITVDLGVGDGQARIRTTDLSHAYVE
  • the articles“a”,“an” and“the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • Standard of care drugs for treatment of tuberculosis or standard of care anti- TB drugs refers to drugs which are recommended by World Health Organisation (WHO) for the treatment of tuberculosis.
  • WHO World Health Organization
  • the list is constantly updated by the WHO; thus, it is contemplated that the term‘standard of care drugs’ is referred to cover all the drugs listed out timely by the WHO.
  • pranlukast as used in the compositions disclosed herein is intended to cover all the possible analogs of pranlukast.
  • sorafenib as used in the compositions disclosed herein is intended to cover all the possible analogs of sorafenib.
  • in-silico screening refers to screening for candidate molecules using computer or computer simulation.
  • the expression is construed to cover all the simulation programmes known in the art to cover such screening using computer simulation.
  • in-vitro screening refers to screening for candidate molecules, wherein such screening takes place outside living biological organisms, i.e., in an environment that is distinct from their living environment. In-vitro screening can take place in a test-tube, petri-dish, cell lines, or microorganisms in a lab environment. It is intended to cover all the techniques known in the art to perform in-vitro studies.
  • in-vivo screening refers to screening for candidate molecules in which the effect is studied on whole living organisms, usually animals such as, mice, rat, rabbit, guinea pig, monkey and other lab animal models.
  • candidate molecules are referred to any drugs, small molecules, analogs, novel structures that are used for testing their possible effects on controlling a diseased condition by performing specific biological function.
  • “therapeutically effective amount” refers to an amount of a respective composition which would be required to decrease the infection caused by the causative microorganism.
  • the amount refers to any amount which is required to treat a subject suffering from infection caused by the causative microorganism.
  • “modulates” refer to either an increase or decrease in activity of a concerned molecule in presence of any other molecule. The increase or decrease is with respect to the activity exhibited by the concerned molecule in the absence of any other molecule.
  • Mycobacterial infection refers to any infection caused by a species belonging to the genus Mycobacterium. It is intended to cover an infection caused by one or more of species belonging to Mycobacterium.
  • the term“subject” refers to any vertebrate, including human being.
  • excipient include substances that serve as mechanisms to improve the delivery and the effectiveness of drugs.
  • a diluent also referred to as filler, dilutant, or thinner
  • An excipient is an inactive substance that serve as the vehicle or medium for a drug or other active substance.
  • Excipients include colouring agents, humectants, preservatives, emollients, and combinations thereof.
  • Mycobacterium genus belongs to Phylum Actinobacteria, Order Actinomycetales, and Family Mycobacteriaceae.
  • the present document depicts Mycobacterium species which is intended to cover all the species belonging to the genus Mycobacterium.
  • the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
  • the present disclosure provides a composition to inhibit growth of Mycobacterium species by targeting ArgJ enzyme of the pathogen.
  • the ArgJ enzyme is essential for survival, growth, and pathogenesis of M. tuberculosis.
  • the present disclosure exploits the use of ArgJ enzyme in identifying a candidate drug capable of binding to a novel allosteric binding pocket of the enzyme, thereby inhibiting growth of the pathogen.
  • Two such candidate drugs have been identified as part of the present disclosure namely, pranlukast and sorafenib.
  • the composition comprising a combination of pranlukast or sorafenib with standard care of drugs for tuberculosis treatment has been shown to inhibit growth of M.
  • tuberculosis in an enhanced manner as compared to the standard care of drugs used alone for tuberculosis treatment.
  • drugs used alone for tuberculosis treatment Following combinations like: pranukast and rifampicin; pranlukast, rifampicin, and isoniazid; sorafenib and rifampicin; sorafenib, rifampicin, and isoniazid have been shown to inhibit growth of M. tuberculosis in an enhanced manner as compared to the combination of standard care drugs (rifampicin, isoniazid, and ethambutol).
  • the present disclosure also discloses a screening method for identifying candidate molecules for allosterically binding to a novel pocket in ArgJ enzyme.
  • the screening method involves a combination of in-silico, in-vitro, and in-vivo methods to select the candidate molecules capable of binding to the ArgJ enzyme.
  • the identified novel allosteric binding pocket can be used for identifying candidate molecules for binding to the ArgJ enzyme, thereby inhibiting growth of pathogen harbouring said enzyme.
  • composition comprising: (a) pranlukast or sorafenib; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species.
  • a composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • a method for preparing a composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid, said method comprising: (i) obtaining pranlukast; (ii) obtaining rifampicin; (iii) obtaining isoniazid; and (iv) contacting pranlukast, rifampicin, and isoniazid to obtain the composition.
  • composition comprising: (a) pranlukast, and analogs thereof; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium,
  • Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
  • a composition comprising: (a) pranlukast; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • a method for preparing a composition comprising: (a) pranlukast; and (b) rifampicin, said method comprising: (i) obtaining pranlukast; (ii) obtaining rifampicin; and (iii) contacting pranlukast, and rifampicin to obtain the composition.
  • a composition comprising: (a) pranlukast, and analogs thereof; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • a composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • a method for preparing a composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid, said method comprising: (i) obtaining sorafenib; (ii) obtaining rifampicin; (iii) obtaining isoniazid; and (iv) contacting sorafenib, rifampicin, and isoniazid to obtain the composition.
  • a composition comprising: (a) sorafenib, and analogs thereof; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • a composition comprising: (a) sorafenib; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • a method for preparing a composition comprising: (a) sorafenib; and (b) rifampicin, said method comprising: (i) obtaining sorafenib; (ii) obtaining rifampicin; and (iii) contacting sorafenib, and rifampicin, to obtain the composition.
  • a composition comprising: (a) sorafenib, and analogs thereof; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • a composition comprising pranlukast, for use in inhibiting growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • a composition comprising sorafenib, for use in inhibiting growth of Mycobacterium species.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • composition as described herein, wherein the composition further comprises ethambutol.
  • compositions as described herein wherein the composition further comprises a standard of care drug for treatment of tuberculosis.
  • a composition as described herein wherein the composition further comprises a drug selected from the group consisting of pyrazinamide, bedaquiline (TMC-207), PA-824, AZD5847, linezolid, moxifloxacin, rifapentine, and BTZ043.
  • composition as described herein, wherein the composition further comprises: at least one excipient; at least one carrier; and at least one diluent.
  • the preparation method includes obtaining of relevant components and contacting the components to obtain respective composition.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
  • Mycobacterium species is selected from group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • ArgJ ornithine acetyl transferase enzyme of a microorganism as a target for identifying candidate molecules for binding to ArgJ enzyme, wherein the binding modulates function of ArgJ enzyme thereby inhibiting growth of the microorganism, and wherein the microorganism is selected from group consisting of Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
  • a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ; and (b) predicting the binding of the candidate molecules to ArgJ to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen.
  • the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
  • a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ ; (b) predicting the binding of the candidate molecules to identify selected candidate molecules; and (c) performing in-vitro validation of the selected candidate molecules, to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen, and wherein the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseri
  • the pathogen is Mycobacterium species, wherein the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • the binding of the candidate molecule comprises allosteric binding to the ArgJ enzyme, wherein the allosteric binding inhibits growth of the pathogen.
  • a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ ; (b) predicting the binding of the candidate molecules to identify selected candidate molecules; (c) performing in-vitro validation of the selected candidate molecules, to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen; and (d) performing an in-vivo validation of the selected candidate molecules, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen, and wherein the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis
  • the pathogen is Mycobacterium species, wherein the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • the binding of the candidate molecule comprises allosteric binding to the ArgJ enzyme, wherein the allosteric binding leads to inhibition of growth of Mycobacterium species.
  • Mycobacterium species comprising: (a) obtaining a computerized representation of FDA (Food and Drug Administration) approved small molecule drug library comprising candidate molecules; (b) obtaining a computerized representation of a reference molecule, wherein the reference molecule is ANS (8-anilinonaphthalene sulfonate); (c) performing in-silico screening with a computerized representation of ArgJ enzyme involving multiple steps comprising: i. flexible docking and filtering the candidate molecules of library to obtain first phase candidate molecules; ii.
  • FDA Food and Drug Administration
  • the five parameters are (1) Violation of Lipinski’s rule of five, (2) Binding free energies, (3) Ligand strain, (4) Change in Solvent accessible surface area (ASASA), and (5) Gap index, and (d) performing in-vitro validation of the cluster of candidate molecules comprising following steps: i. performing Thin layer chromatography (TLC) based assay to determine activity of ArgJ and to perform an activity-based screen; ii. performing ANS (hydrophobic dye) based dye-displacement assay to determine binding affinity of candidate molecules selected from the cluster; and iii.
  • TLC Thin layer chromatography
  • ANS hydrophobic dye
  • TSA Thermal Shift Assay
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • the computerized representation of ArgJ enzyme comprises an allosteric binding pocket wherein, the allosteric binding leads to inhibition of growth of Mycobacterium species.
  • a method for identifying a candidate molecule capable for binding to ArgJ enzyme of Mycobacterium species comprising: (a) obtaining a computerized representation of FDA (Food and Drug Administration) approved small molecule drug library comprising candidate molecules; (b) obtaining a computerized representation of a reference molecule, wherein the reference molecule is ANS (8-anilinonaphthalene sulfonate); (c) performing in-silico screening with a computerized representation of ArgJ enzyme involving multiple steps comprising: i. flexible docking and filtering the candidate molecules of library to obtain first phase candidate molecules; ii.
  • the five parameters are (1) Violation of Lipinski’s rule of five, (2) Binding free energies, (3) Ligand strain, (4) Change in Solvent accessible surface area (ASASA), and (5) Gap index, (d) performing in-vitro validation of the cluster of candidate molecules comprising following steps: i. performing Thin layer chromatography (TLC) based assay to determine activity of ArgJ and to perform an activity-based screen; ii. performing ANS (hydrophobic dye) based dye-displacement assay to determine binding affinity of candidate molecules selected from the cluster; and iii.
  • TLC Thin layer chromatography
  • ANS hydrophobic dye
  • Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
  • the computerized representation of ArgJ enzyme comprises an allosteric binding pocket wherein, the allosteric binding leads to inhibition of growth of Mycobacterium species.
  • the ArgJ enzyme has an amino acid sequence as set forth in SEQ ID NO: 2, encoded by a gene having a nucleic acid sequence as set forth in SEQ ID NO: 1.
  • compositions as described herein for use in treating infections caused by Mycobacterium species wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • Mycobacterium species is Mycobacterium tuberculosis.
  • compositions as described herein for use in treating infections caused by a pathogen wherein the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
  • the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
  • composition comprising: (a) pranlukast; and (b) rifampicin, for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid, for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • composition comprising: (a) sorafenib; and (b) rifampicin, for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid, for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • a method for treating a subject suffering from tuberculosis comprising administering to the subject therapeutically effective amount of the composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid.
  • a method for treating a subject suffering from tuberculosis comprising administering to the subject therapeutically effective amount of the composition comprising: (a) pranlukast; and (b) rifampicin.
  • a method for treating a subject suffering from tuberculosis comprising administering to the subject therapeutically effective amount of the composition comprising: (a) pranlukast; and (b) a standard of care anti-TB drug.
  • a method for treating a subject suffering from tuberculosis comprising administering to the subject therapeutically effective amount of the composition comprising: (a) sorafenib; and (b) rifampicin.
  • a method for treating a subject suffering from tuberculosis comprising administering to the subject therapeutically effective amount of the composition comprising: (a) sorafenib; and (b) a standard of care anti-TB drug.
  • a method for treating a subject suffering from tuberculosis comprising administering to the subject therapeutically effective amount of the composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid.
  • a method for treating a subject suffering from tuberculosis comprising administering to the subject therapeutically effective amount of the composition as described herein.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition as described herein; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition as described herein; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject, wherein the subject is suffering from a Mycobacterial infection caused by any one of the pathogens selected from a group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition comprising pranlukast, and rifampicin; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition comprising pranlukast, rifampicin, and isoniazid; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition comprising pranlukast, rifampicin, and at least one standard of care drug for treatment of tuberculosis; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition comprising pranlukast, and at least one standard of care drug for treatment of tuberculosis; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition comprising sorafenib, and rifampicin; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition comprising sorafenib, rifampicin, and isoniazid; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition comprising sorafenib, rifampicin, and at least one standard of care drug for treatment of tuberculosis; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • a method for treating a subject suffering from Mycobacterial infection comprising: (a) obtaining a composition comprising sorafenib, and at least one standard of care drug for treatment of tuberculosis; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
  • Arginine biosynthesis pathway is essential for the survival and pathogenesis of Mycobacterium tuberculosis ( Mtb ). Despite the acknowledged significance of arginine biosynthesis in Mtb, inhibitors to target this pathway remain to be discovered. The enzymes involved in this pathway are promising targets for anti-TB drug development (Gordhan, 2002; Mdluli & Spigelman, 2006). Moreover, inhibitors of this pathway may provide novel insights to the significance of arginine biosynthesis in Mtb associated stress responses and persistence.
  • MtArgJ Ornithine acetyltransferase
  • MtArgJ catalyzes the transfer of acetyl moiety from N-acetyl ornithine to glutamate, thereby producing ornithine and N-acetyl glutamate for next round of arginine biosynthesis (Xu et al, 2007) (Fig. la and Fig. lb).
  • MtArgJ an exciting target for drug development.
  • tuberculosis argj gene (SEQ ID No: 1) is encoded by the ORF Rvl653, and belongs to the N-terminal nucleophile fold family of enzymes (Xu et al, 2007).
  • the crystal structure of MtArgJ in native form and in complex with ornithine has been determined at 1.7 A... and 2.4 A... respectively (Sankaranarayanan et al, 2010).
  • the present disclosure discloses a previously unknown allosteric site on MtArgJ and report the discovery of a novel inhibitor that binds and impedes the catalytic efficiency of MtArgJ.
  • the selectivity and specificity of this inhibitor lies in its ability to allosterically modulate the substrate -binding interface.
  • the present disclosure concludes the potency of the compounds pranlukast and sorafenib as drug candidates against Mtb survival and pathogenesis.
  • the present disclosure demonstrates the extraordinarier potency of the inhibitors against arginine biosynthesis in Mtb thereby abating the pathogen survival, in both in vitro and in-vivo infection models.
  • this disclosure discloses a novel metabolic inhibitor of Mtb, and its potential for improved combinatorial therapy against tuberculosis.
  • the present disclosure is intended to cover the use of prnalukast alone or in any combination with known standard of care drugs for anti-TB. Specific examples have been shown in the present disclosure that includes pranlukast and its combination with rifampicin and isoniazid. It is contemplated that the following combinations obtained with pranlukast: pranlukast+ethambutol, pranlukast+isoniazid, pranlukast+drug selected from the group consisting of pyrazinamide, bedaquiline (TMC-207), PA-824, AZD5847, linezolid, moxifloxacin, rifapentine, and BTZ043 is covered by the present disclosure.
  • Example 1 any combination obtained using pranlukast for inhibiting growth of Mycobacterium species is intended to be covered by the present disclosure. Similarly, any combination obtained using sorafenib for inhibiting growth of Mycobacterium species is intended to be covered by the present disclosure.
  • MtArgJ structure (PDB Id: 3IT6) was probed for surface cavity predictions (by MetaPocket server and CastP analysis) and a well- defined pocket of area 2019.7A...2 and volume 3104.8A...3 located in-between the two active sites was discovered (Fig. 2a).
  • This pocket comprises of 48% hydrophobic amino-acid residues and 24% and 28% polar and charged residues respectively (Fig. 2a inset).
  • This large pocket comprised of four loops, four helices and one I 2 strand contributed by each protomer (AB) of A2B2 tetramer. However, two flanking loops positioned at the interface of the substrate binding pockets on either side were observed.
  • a fluorescent dye 8-anilinonaphthalene sulfonate (ANS) was used. It has specificity for the hydrophobic regions of a protein and shows a characteristic fluorescence at 470 nm upon binding. This property of ANS to probe the novel pocket on MtArgJ was utilized. As shown in Fig. 2b, ANS showed a concentration-dependent increase in binding to MtArgJ, manifested as a steady rise in fluorescence intensity. The kinetic analysis determined a K d of 937 I1 ⁇ 4M (ANS binding to MtArgJ) (Fig. 2c).
  • a novel screening strategy was designed to cull-off the non-specific compounds from the best hit molecules to augment in silico hit discovery against the novel drug target - ArgJ of M. tuberculosis.
  • the applied hierarchical strategy included embedding multiple scoring and filtering functions that analyzed the conformational space, non-covalent interactions energies and receptor-ligand interactions to identify ideal hits against MtArgJ.
  • Example 3
  • the PRK and SRB impart thermal stability to MtArgJ in a concentration dependent manner.
  • TSA Thermal Shift Assay
  • Binding pockets in Mycobacterium tuberculosis ornithine acetyltransferase (MtArgJ) were predicted using a web server MetaPocket 2.0 (http://prffiects.hiotec.tu- dresden.de/metapocket). Metapocket 2.0 uses a consensus method, eight different methods were deployed to predict the binding sites with high accuracy. Additionally, CASTp Webserver was employed to calculate the area and volume of different binding clefts. Pockets are concavities on a protein surface into which solvent (probe sphere 1.4 A) can gain access, i.e., these concavities have mouth openings connecting their interior with the outside bulk solution.
  • the binding site predicted was formed due to protein complexation at the interface of chains B and D essentially from the two monomers, which was distinct from the substrate binding pockets. This well-defined pocket was found to be a large cleft of area 2019.7A 2 and volume 3104.8A 3 .
  • the large interface pocket comprised of four loops flanking b4 a1, b8 b9, a5 b10, a9 b12, four helices - a5, a7, a8, a ⁇ q and one b15 strand contributed by each monomer. Also noticed, were that the loops flanking between b8 b9 and a5 b10 were positioned at the interface of the substrate binding pocket and the unfilled pocket.
  • the allosteric site was highly hydrophobic in nature and comprised of 48% hydrophobic amino acid residues and 24 and 28% polar and charged residues, respectively.
  • This large pocket comprised of four loops, four helices, and one beta strand contributed by each protomer (AB) of A2B2 tetramer.
  • the tetrazole ring of PRK interacts with Asp234 and Ser3 l0 respectively from chain B and chain D while chromene ring showed interactions with Ser3l0 of chain B and benzamide group nitrogen with Gln305 of chain B on the protein (Fig. 4a, b).
  • Sorafenib showed interactions via amino groups to Gln305 and Arg308 of Chain D and carbonyl group to Arg308 of Chain B (Fig. 4c, d).
  • PRK exhibited more number of interactions with the allosteric pocket than SRB; asserting higher affinity of PRK for MtArgJ.
  • the data shows that PRK and SRB both bind to the allosteric pocket on MtArgJ, however PRK binds with higher affinity than SRB (Fig. 3 c-j).
  • PRK showed an MIC90 of 15 I1 ⁇ 4g/ml and 25 I1 ⁇ 4g/ml for both Jal226l and Jal2287 strains, respectively (Fig. 5f and Fig. 5g). However, SRB demonstrated comparatively higher MIC (Fig. 5h and Fig. 5i) for both the strains. The results showed the promising effect of PRK on pathogenic Mtb including MDR strains.
  • the combination of SRB+RH shows increase in inhibiting Mtb as compared to the combination of RHE, although the effect of SRB+RH is lesser that PRK+RH but is still more than the effect of RHE.
  • the new combinations of RH+PRK and RH+SRB is significantly effective against Mtb survival than the pre-existing RHE combination and holds a potential for improved therapy regimen.
  • PRK significantly inhibits the survival of Mtb in macrophage infection model without affecting the host cell THP-l cells, a human monocytic cell line, stimulated for differentiation by PMA treatment were infected with pathogenic Mtb (H37Rv).
  • the Mtb infected THP-l cells were treated with varying inhibitor (PRK or SRB) concentrations or DMSO (control) at different time -points. Once treated, the cells were lysed at desired time points (0, 12, 24 and 48 hrs) and plated for colony formation assays.
  • the CFU colony forming unit
  • Examples 1 to 9 demonstrate the potency of pranlukast (PRK) and sorafenib (SRB) as a promising anti -tubercular molecule with no deleterious effect on the host cell survival. Also, the enhanced effect of PRK alone and in combination with the therapy drugs, on the macrophage internalized Mtb is interesting and beneficial from the host’ s perspective.
  • PRK pranlukast
  • SRB sorafenib
  • PRK is a known inhibitor of Cysteinyl Leukotriene Receptor- 1 (CysLTRl), on the mammalian cells and is used for treatment of Asthma. Macrophages also express CysLTRl in response to various inflammatory stimuli, including pathogenic bacterial colonization. Also, studies have shown that in macrophages and dendritic cells, PRK acts through a yet another mechanism, wherein it targets the leukotriene and prostaglandin (eicosanoids) biosynthesis, which are ligands for CysLTR’s (Theron et al, 2014). It does so by affecting 5 -Lipoxygenase (5-LO) signalling in the host.
  • 5-LO 5 -Lipoxygenase
  • Divangahi et al. have shown that M. tuberculosis infection activates the 5- lipoxygenase pathway, that facilitates the host cell necrosis thereby helping the pathogen dissemination. This also prevents the cross-antigen presentation by dendritic cells, thereby inhibiting the induction of T cell immunity (Divangahi et al, 2010).
  • the present disclosure depicts the dual role of PRK, it can be appreciated from Pig. 8a-8f that PRK apart from inhibiting function of ArgJ enzyme, inhibits the 5-LO signalling in the macrophages infected with Mtb, thereby reducing the pathogen survival in the host.
  • the present disclosure discloses inhibition of Mtb infection by dual targeting of arginine biosynthesis in the pathogen and 5-LO signalling in the host, mediated by PRK treatment.
  • 5 -lipoxygenase and the associated genes involved in eicosanoid biosynthesis were significantly down- regulated upon PRK treatment but not by Rif treatment.
  • the PRK treatment reduces the infection-associated apoptosis in the host. It has been reported that during the early phase of Mtb infection, macrophages undergo apoptosis as an innate defense mechanism, thereby increasing the levels of pro- apoptotic proteins like Caspase 3 and 8. Hence, the effect of PRK on infection- associated apoptosis in the host was explored. To examine this, caspase-3 dependent apoptosis in Mtb infected macrophages was monitored. As shown in Fig. 6 (h-j) treatment with increasing concentrations of PRK lead to reduced extracellular caspase-3 level in the Mtb infected human as well as murine macrophages (THP- 1 and Raw 264.7 respectively).
  • the PRK treatment reduces the Mtb burden and tubercular granulomas from the lungs of Mtb infected mice.
  • Chronic Mtb infection is characterized by the formation of lung associated granulomas, an organized aggregate of immune cells with infected macrophages at the core.
  • BALB/c mice were used and were infected with M. tuberculosis through aerosol (Fig. 7a).
  • PRK 40 mg/kg body weight
  • Rif (10 mg/kg body weight)
  • combination of PRK with Rif were treated with PRK (40 mg/kg body weight) or combination of PRK with Rif.
  • mice were sacrificed at three time points (0 day, 12 days and 24 days) and lung-associated granulomas were analyzed (Fig. 8 b, c, d and e).
  • Rif + PRK combination had practically no visible granulomas in the mice lungs, post 24 days of treatment (Fig. 7 d,e).
  • the mice lungs were homogenized and plated at different dilutions on 7H11 solid media supplemented with OADC and PANTA.
  • PRK showed most remarkable effect in combination with Rifampicin. Moreover, there was no splenic or hepatic cytotoxicity at the administered dosages, as shown in the detailed histopathological H staining images, analyzed by expert pathologists. The results demonstrate the potency of PRK in combating Mtb infection and its improved efficiency in combination with Rif, thereby proving its in-vivo efficacy on the pre-clinical model of tuberculosis.
  • the present example clearly depicts the possibility of using a combination of PRK with rifampicin for treatment of tuberculosis. Since, SRB also has a similar mechanism of action for inhibiting the activity of ArgJ as of PRK, thus it can be contemplated from the present Example that a combination of SRB and rifampicin can also be used to treat tuberculosis infection.
  • Example 12
  • ArgJ protein of Mtb similar to ArgJ protein of other pathogens
  • pranlukast and sorafenib binds to a novel allosteric binding pocket of ArgJ enzyme of Mtb. It can be contemplated that pranlukast and sorafenib can be used to bind to ArgJ protein of other pathogens having high similarity and identity to that of ArgJ of Mtb.
  • Table 1 depicts the comparative analysis of percentage sequence identity of ArgJ from M. tuberculosis ⁇ Mtb), M. avium (Mav), M. leprae (Mlp).
  • ArgJ enzyme of Mtb was also compared to that of other pathogens.
  • Table 2 depicts the percentage identity data for ArgJ of other significant pathogens with that of Mtb.
  • the present disclosure discloses the composition comprising pranlukast, and the composition comprising sorafenib in inhibiting the growth of Mycobacterium species.
  • the compositions have been obtained by using a novel method for screening various candidate molecules for binding to a novel allosteric binding pocket of ArgJ enzyme of Mtb. The binding inhibits the growth of Mtb, thus the present composition is highly useful in treating tuberculosis apart from other infections caused by Mycobacterium species.
  • ArgJ enzyme of Mtb lacks a homolog in humans thus any candidate molecules capable of binding to the enzyme can be used to prepare medicaments for treating infections.
  • Pranlukast and Sorafenib being FDA approved drugs display a potential for development of advanced combinational therapy against pathogenic tuberculosis including multi-drug resistant (MDR) Mtb.
  • MDR multi-drug resistant
  • the method as described herein can also be used to screen candidate molecules for binding to ArgJ enzyme of other pathogens selected from the group consisting of Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
  • the method as described in the present disclosure can also be used to screen candidate molecules to treat infections arising from Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.

Abstract

The present disclosure discloses a composition comprising pranlukast or sorafenib. The present disclosure further discloses use of pranlukast or sorafenib for treating Mycobacterium infections. The present disclosure also discloses a method to identify candidate molecules that are capable of binding to ornithine acetyltransferase (ArgJ) enzyme.

Description

ANTI-TUBERCULAR COMPOSITION, AND COMBINATORIAL IMPLEMENTATIONS THEREOF
FIELD OF INVENTION
[001] The present disclosure broadly relates to a composition for inhibiting growth of pathogens, and particularly relates to a composition comprising panlukast or sorafenib for inhibiting growth of Mycobacterium species.
BACKGROUND OF THE INVENTION
[002] Infections caused by microorganisms and challenges in treating such infections is a perennial challenge pan globe. Adding to the existing list of problems, is the challenge of antimicrobial resistance posed by the microbial community. The drugs previously known to treat the infections caused by certain pathogens have now become ineffective owing to the development of resistance to the drugs. Such problems have necessitated the need to explore new targets in pathogens. The revolution associated with genome sequencing technology has facilitated the opportunity to explore new avenues to address new challenges.
[003] The genome sequencing has led to deciphering of new genes and delineating genomic environment of the pathogens. The genomic data has made it feasible to identify new target sites in a challenging pathogen. The identification of such targets has facilitated drug discovery to a great extent which can provide solution to the challenge posed by new resistant and multidmg resistant strains of a pathogen. Consecutively, identification and validation of new targets in a pathogen is crucial for drug discovery projects. Similarly, screening of candidate molecules for binding and inhibiting of said targets is in itself a distinct challenge. Identification and use of a novel target in a pathogen is a crucial first step to addressing the present challenge. Extensive in-silico studies coupled with in-vitro and in-vivo validation to identify certain candidate molecules is an approach that is used to tackle the problem.
[004] Thus, constant pursuit to come up with a solution to address the problem of pathogens is topmost priority. One such disease that has involved time, money, and humongous efforts from scientific and medical community all over the world is tuberculosis. Tuberculosis (TB) is an airborne infectious disease caused by organisms of the Mycobacterium tuberculosis complex. Although primarily a pulmonary pathogen, M. tuberculosis can cause disease in almost any part of the body. The existing treatment regime to tackle tuberculosis is not adequate and novel therapeutic interventions are required to target Mycobacterium tuberculosis pathogenesis. Most of the targets identified in the pathogen have significant similarity to the human counterparts, thus adding to the woes. Therefore, there is a dearth of a novel target which lacks a human counterpart and also not many compositions are available which binds to such targets to intervene with the growth of M. tuberculosis.
[005] Novel compositions are required that act against several targets to provide a solution to tackle the present problem.
[006] US5439891A discloses a new pharmaceutical composition for the treatment of tuberculosis and leprosy, said composition comprising piperine in combination with known antituberculosis or antileprosy drugs or the mixtures thereof.
[007] W02014170820A3 discloses a pharmaceutical combination comprising therapeutically effective amounts of nano curcumin and isoniazide, for treating tuberculosis in a subject, is provided herein. Nano curcumin and isoniazide of the pharmaceutical combination of the present invention may be administered simultaneously, separately or sequentially or as a single fixed dose combination. Pharmaceutical compositions comprising nano curcumin and isoniazide are also described herein.
SUMMARY OF INVENTION
[008] In an aspect of the present disclosure, there is provided a composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species. The Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. [009] In an aspect of the present disclosure, there is provided a composition comprising: (a) pranlukast; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species. The Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0010] In an aspect of the present disclosure, there is provided a composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species. The Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0011] In an aspect of the present disclosure, there is provided a composition comprising: (a) sorafenib; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species. The Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0012] In an aspect of the present disclosure, there is provided a composition comprising pranlukast, for use in inhibiting growth of Mycobacterium species. The Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0013] In an aspect of the present disclosure, there is provided a composition comprising sorafenib, for use in inhibiting growth of Mycobacterium species. The Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0014] In an aspect of the present disclosure, there is provided a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen, said method comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ; and (b) predicting the binding of the candidate molecules to ArgJ to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen. The pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
[0015] In an aspect of the present disclosure, there is provided a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen, said method comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ ; (b) predicting the binding of the candidate molecules to identify selected candidate molecules; and (c) performing in-vitro validation of the selected candidate molecules, to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen. The pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
[0016] In an aspect of the present disclosure, there is provided a method for identifying a candidate molecule capable of binding to ArgJ enzyme of Mycobacterium species, said method comprising:
a) obtaining a computerized representation of FDA approved small molecule drug library comprising candidate molecules;
b) obtaining a computerized representation of a reference molecule, wherein the reference molecule is ANS (8-anilinonaphthalene sulfonate);
c) performing in-silico screening with a computerized representation of ArgJ enzyme involving multiple steps comprising: i. flexible docking and filtering the candidate molecules of library to obtain first phase candidate molecules;
ii. grid scoring of the first phase candidate molecules based on electrostatic energies within a range of 0.59 to -34.39 kcal/mol and van der Waals energies within a range of -5.43 to -109.25 kcal/mol, and selecting from the first phase candidate molecules based on comparison with the reference molecule to obtain second phase candidate molecules;
iii. amber based rescoring and selecting from the second phase candidate molecules based on comparison with the reference molecule to obtain third phase candidate molecules; and
iv. screening the third phase candidate molecules based on five parameters to obtain a cluster of candidate molecule, wherein the five parameters are (1) Violation of Lipinski’s rule of five (2) Binding free energies (3) Ligand strain (iv) Change in Solvent accessible surface area (ASASA) and (5) Gap index, and
(d) performing in-vitro validation of the cluster of candidate molecules comprising following steps:
i. performing Thin layer chromatography (TLC) based assay to determine activity of ArgJ and to perform an activity-based screen;
ii. performing ANS (hydrophobic dye) based dye-displacement assay to determine binding affinity of candidate molecules selected from the cluster; and
iii. performing Thermal Shift Assay (TSA) to determine apparent binding affinity of candidate molecules selected from the cluster, to identify the candidate molecule capable for binding to ArgJ enzyme of Mycobacterium species. The Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0017] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0018] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0019] Figure 1 illustrates arginine biosynthesis in Mycobacterium tuberculosis (a) Arginine biosynthesis pathway in M. tuberculosis in detail; (b) Reaction catalysed by MtA rg J (. Mycobacterium tuberculosis ArgJ), in accordance with an embodiment of the present disclosure.
[0020] Figure 2 illustrates characterization of allosteric site on M/ArgJ surface and its role in enzyme activity (a) schematic representation of M/ArgJ with the large pocket (allosteric site) represented in wheat color, with one active site on either side. Inset shows the composition of hydrophobic, polar and charged amino acid residues in the pocket (PDB iD 3IT6); (b) Fluorescence based assay; The graph shows increase in fluorescence upon ANS binding to M/ArgJ, in a concentration dependent manner (ANS = 10 mM - 10 mM); (c) Dose dependent net change in relative fluorescence of ANS upon MtA rgj binding plotted against ANS concentration for Kd determination; (d) Decrease in Mr ArgJ activity in the presence of ANS as determined by TLC based assay; (e) In silico screen for allosteric modulators: The hyperbolic graph (circular dendrogram) showing hierarchical clustering of selected compounds based on the five parameters represented as green (Violation of Lipinski’s rule of five), maroon (free binding energies), cyan (ligand strain energy), black (percentage change in total solvent accessible surface area) and purple (gap index) determining the surface complementarity in receptor ligand complexes in accordance with an embodiment of the present disclosure. [0021] Figure 3 illustrates in-vitro kinetics and binding studies of inhibitory compounds (PRK and SRB) M/ArgJ. Chemical structures of (a) Pranlukast (PRK) and (b) Sorafenib (SRB). Saturation curve fit using Michaelis-Menten plot for MtA rg J activity at two different inhibitor concentrations for (c) PRK and (d) SRB, quantified 26 by TLC based assay. Dixon plot analysis performed by plotting l/v against varying inhibitor concentration, at three different substrate concentrations for (e) PRK and (f) SRB. Fluorescence based dye (ANS) displacement assay to determine the binding of (g) PRK and (h) SRB into the hydrophobic pocket on M/ArgJ. Kd values for both were determined by plotting net change in relative fluorescence upon inhibitor binding as a function of inhibitor concentration. Thermal
Shift Assay (TSA) to determine apparent Kd for inhibitor binding to MtA rg J : with increasing concentration of (i) PRK and (j) SRB to examine the shift in melting temperature (Tm) of M/ArgJ. Apparent Kd values calculated by plotting net change in melting temperature upon inhibitor binding as a function of inhibitor concentration, in accordance with an embodiment of the present disclosure.
[0022] Figure 4 illustrates binding interactions of PRK and SRB with the allosteric pocket of M/ArgJ. (a) PRK (orange) interacting with the Asp234 and Gln305 of B chain, Ser3l0 of B and D chains in the allosteric pocket of the M/ArgJ (b) LIGPLOT for the interaction of PRK with the allosteric pocket of M/ArgJ (c) SRB (magenta) interacting with Gln305 of D chain and Arg308 of B and D chains in the allosteric pocket of the MtA rg J (d) LIGPLOT for the interaction of SRB with the allosteric pocket of MtA rg J , in accordance with an embodiment of the present disclosure.
[0023] Figure 5 illustrates effect of PRK and SRB on Mtb H37Rv survival (a) Representative image of Alamar Blue assay employed for MIC determination (b, c) Mtb H37Rv cells were treated with varying concentration of inhibitors and cell viability was determined using Alamar Blue assay. MIC90 was calculated by plotting cell viability (%) against increasing concentration of inhibitors PRK and SRB. (d, e) Hill’s plot analysis of decline in Alamar Blue fluorescence with increasing 51 concentration of PRK and SRB, for IC50 determination. Effect of PRK and SRB on the Multi drug resistant strains of Mtb. Mycobacterium tuberculosis MDR strains Jal 2261 and Jal 2287 were treated with varying concentration of inhibitors and cell viability was determined using Alamar blue assay. MIC was calculated by plotting cell viability (%) against increasing concentration of inhibitors: (f). Effect of PRK on Jal226l (g). Effect of PRK on Jal2287. (h) Effect of SRB on Jal 2261 (i) Effect of SRB on Jal2287 MDR strains (j, k) CFU analysis of Mtb treated with PRK/SRB alone or a cocktail of Rif, Inh and Emb abbreviated as RHE and compared with a new combination of RH+PRK and RH+SRB, at two concentrations of PRK and SRB (0.1 mM and 1 mM) respectively. The x-axis represents the time points post treatment with the inhibitors and all the experiments were performed in triplicates and confirmed with biological duplicates at least (****p< 0.0001, ***p< 0.001, **p< 0.01, *p< 0.1).
[0024] Figure 6 illustrates effect of PRK and SRB on the macrophage internalized Mtb. (a, b) THP1 (human monocytic cell-line) cells and Raw264.7 (mouse macrophage cell line) cells were infected with Mtb H37Rv followed by treatment with PRK (5 pM and 25 pM) and SRB (10 pM). CFU of internalized Mtb plotted at defined time points (c, d) CFU analysis of THP1 and Raw264.7 internalized Mtb upon treatment with a cocktail of Rif, Inh and Emb abbreviated as RHE and compared with a new combination of RH+PRK (0.5 pM) and RH+SRB (0.5 pM). (e, f) Flow cytometry analysis of macrophage internalized Mtb H37Rv-GFP at 4, 8, 16 and 32 hrs post treatment with PRK (5 pM). (g) Flow cytometry analysis of Mtb (H37Rv-GFP) infected macrophages stained with Propidium Iodide (PI) dye to determine macrophage cell death upon Mtb infection and PRK treatment (h, i) Effect of PRK treatment on infection induced apoptosis in macrophages by monitoring active Caspase 3 levels in supernatant media of Mtb infected THP1 and Raw264.7 macrophages in presence and absence of PRK (1 pM and 10 pM). (j) Relative decrease in chemiluminescence as a measure of extra-cellular Caspase 3 levels upon PRK treatment. All the experiments were performed in triplicates and confirmed with biological duplicates at least. (****p< 0.0001, ***76 p< 0.001, **p< 0.01, *p< 0.1), in accordance with an embodiment of the present disclosure. [0025] Figure 7 illustrates PRK treatment reduces the lung-associated granuloma from Mtb infected mice (a) Lung images of mice infected with chronic Mtb infection through aerosol and treated with PRK, intra-peritonealy, and/or Rif, orally, for 24 days; (b) Lungs of mice treated with PBS and (c) PRK, post 12 and 24 days, respectively. The white spot (cyst like) depicted with an arrow corresponds to tubercular granulomas; (d) Lungs of mice treated with Rif alone and (e) Rif + PRK post 12 and 24 days of infection; (f) CFU analysis of Mtb from lungs of infected mice treated with PBS or PRK at 0 day, 15 day and 24 days of treatment; (g) CFU analysis of Mtb from lungs of infected mice treated with Rif and Rif + PRK at 0 day, 15 day and 24 days of treatment. Appropriate negative and positive controls taken as PBS and Rif treated mice, respectively. (n=6, six mice per condition, for all the time points and dosages and **p< 0.01 is significant); (h) Histopathology based granuloma analysis (blind) of mice lung tissues was done and the number of granulomas per tissue section was plotted for PBS treated, PRK, Rif and Rif + PRK treated mice; (i) Histopathology based H&E staining of the lung tissue sections of mice treated with PBS vs PRK, Rif and Rif +PRK treatments (representative images, detailed images in Appendix fig. S10 & Sl l). (Abbreviations: L=Lymphocytes; FC = Foamy macrophages; PMNs = Polymorphonuclear cells) 97 (****p< 0.0001, ***p< 0.001, **p< 0.01, *p< 0.1), in accordance with an embodiment of the present disclosure.
[0026] Figure 8 illustrates the targeting of arginine biosynthesis by PRK in Mtb and
5 -lipoxygenase signalling in the Mtb infected host macrophages (a) Q-PCR analysis of the genes CysLTRl, 5-LO, FLAP, COX-2 and MCP1 in the Mtb infected vs uninfected macrophages (Raw 264.7); Q-PCR analysis of the genes (b) 5-LO; (c) COX-2; (d) FLAP; (e) CysLTRl and (f) MCP1 upon PRK treatment in the infected macrophages, DMSO and Rif treatment as controls, in accordance with an embodiment of the present disclosure.
SEQUENCES: [0027] SEQ ID NO: 1 depicts the nucleic acid sequence of the gene encoding ArgJ enzyme (ornithine acetyltransferase) of Mycobacterium tuberculosis.
[0028] GTGACCGACCTGGCCGGCACCACCCGGCTGCTGCGCGCTCAGGGC GTCACCGCCCCGGCCGGCTTTCGGGCCGCCGGCGTCGCCGCCGGGATCAA GGCCTCCGGTGCGCTGGATCTGGCGCTGGTGTTCAACGAGGGACCCGACT ACGCCGCCGCCGGGGTGTTCACCCGCAACCAGGTCAAGGCGGCGCCGGTG CTGTGGACCCAGCAAGTGCTGACCACCGGGCGGCTGCGCGCGGTGATCCT CAACTCCGGCGGCGCCAATGCCTGCACCGGGCCGGCCGGCTTCGCCGACA CCCACGCCACCGCGGAGGCGGTGGCCGCGGCGTTGTCGGACTGGGGAACC GAGACCGGGGCCATCGAGGTCGCCGTCTGCTCCACCGGGCTGATCGGCGA CCGGCTGCCGATGGACAAGCTGCTCGCCGGCGTCGCCCACGTGGTGCACG AGATGCATGGCGGGCTGGTCGGCGGCGATGAAGCCGCCCACGCCATCATG ACCACCGACAACGTGCCCAAACAGGTTGCGCTGCACCATCACGACAACTG GACGGTCGGCGGCATGGCCAAAGGCGCGGGCATGCTGGCGCCGTCGTTGG CCACCATGCTGTGCGTGCTCACCACCGACGCGGCCGCCGAGCCGGCCGCA CTCGAGCGGGCGCTGCGCCGCGCCGCCGCGGCCACGTTCGACCGGCTCGA CATCGACGGCAGCTGCTCCACCAACGACACCGTGCTGCTGCTGTCGTCCG GGGCCAGTGAAATCCCCCCTGCCCAGGCCGATCTCGACGAGGCCGTGCTA CGGGTCTGCGACGATTTGTGCGCCCAGCTGCAGGCCGACGCCGAAGGCGT CACCAAACGCGTCACCGTGACCGTGACCGGGGCCGCCACCGAAGACGACG CGCTGGTCGCCGCCCGCCAGATCGCCCGCGACAGCCTGGTCAAGACCGCG CTGTTCGGGTCCGACCCGAACTGGGGACGGGTGCTCGCCGCCGTCGGGAT GGCACCGATCACCCTCGACCCGGATCGAATCAGCGTGTCGTTCAACGGTG CCGCGGTGTGTGTGCACGGTGTCGGCGCTCCCGGTGCGCGCGAGGTGGAC CTGTCGGACGCGGACATCGATATCACCGTCGACCTCGGCGTCGGCGACGG GCAGGCGAGGATCCGAACCACTGATCTGTCGCATGCCTACGTCGAAGAGA ACTCGGCCTACAGCTCATGA.
[0029] SEQ ID NO: 2 depicts the amino acid sequence of ArgJ enzyme of M. tuberculosis. [0030] MTDLAGTTRLLRAQGVTAPAGFRAAGVAAGIKASGALDLALVFNEG PDYAAAGVFTRNQVKAAPVLWTQQVLTTGRLRAVILNSGGANACTGPAGFA DTHATAEAVAAALSDWGTETGAIEVAVCSTGLIGDRLPMDKLLAGVAHVVH EMHGGLV GGDEA AH AIMTTDN VPKQ V ALHHHDNWT V GGM AKG AGMLAPS LATMLCVLTTDAAAEPAALERALRRAAAATFDRLDIDGSCSTNDTVLLLSSG ASEIPPAQADLDEAVLRVCDDLCAQLQADAEGVTKRVTVTVTGAATEDDAL VAARQIARDSLVKTALFGSDPNWGRVLAAVGMAPITLDPDRISVSFNGAA VCVHGVGAPGAREVDLSDADIDITVDLGVGDGQARIRTTDLSHAYVEENSAY
ss
DETAILED DESCRIPTION OF THE INVENTION
[0031] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0032] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
[0033] The articles“a”,“an” and“the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
[0034] The terms“comprise” and“comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as“consists of only”. [0035] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as“comprises” and“comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.
[0036] The term “including” is used to mean “including but not limited to”.
“Including” and“including but not limited to” are used interchangeably.
[0037] Standard of care drugs for treatment of tuberculosis or standard of care anti- TB drugs refers to drugs which are recommended by World Health Organisation (WHO) for the treatment of tuberculosis. The list is constantly updated by the WHO; thus, it is contemplated that the term‘standard of care drugs’ is referred to cover all the drugs listed out timely by the WHO.
[0038] For the purposes of the present document, pranlukast as used in the compositions disclosed herein is intended to cover all the possible analogs of pranlukast. Similarly, sorafenib as used in the compositions disclosed herein is intended to cover all the possible analogs of sorafenib.
[0039] For the purposes of the present document, in-silico screening refers to screening for candidate molecules using computer or computer simulation. The expression is construed to cover all the simulation programmes known in the art to cover such screening using computer simulation.
[0040] For the purposes of the present document, in-vitro screening refers to screening for candidate molecules, wherein such screening takes place outside living biological organisms, i.e., in an environment that is distinct from their living environment. In-vitro screening can take place in a test-tube, petri-dish, cell lines, or microorganisms in a lab environment. It is intended to cover all the techniques known in the art to perform in-vitro studies.
[0041] For the purposes of the present document, in-vivo screening refers to screening for candidate molecules in which the effect is studied on whole living organisms, usually animals such as, mice, rat, rabbit, guinea pig, monkey and other lab animal models. [0042] For the purposes of the present document, candidate molecules are referred to any drugs, small molecules, analogs, novel structures that are used for testing their possible effects on controlling a diseased condition by performing specific biological function.
[0043] For the purposes of the present document,“therapeutically effective amount” refers to an amount of a respective composition which would be required to decrease the infection caused by the causative microorganism. The amount refers to any amount which is required to treat a subject suffering from infection caused by the causative microorganism. For the purposes of the present document,“modulates” refer to either an increase or decrease in activity of a concerned molecule in presence of any other molecule. The increase or decrease is with respect to the activity exhibited by the concerned molecule in the absence of any other molecule. The term “Mycobacterial infection” refers to any infection caused by a species belonging to the genus Mycobacterium. It is intended to cover an infection caused by one or more of species belonging to Mycobacterium. The term“subject” refers to any vertebrate, including human being.
[0044] The technical terms used in the present disclosure like, “Violation of Lipinski’s rule of five”,“Binding free energies”,“Ligand strain”,“Change in Solvent accessible surface area (ASASA)”, “Gap index”, “Thermal Shift Assay”, “ANS (hydrophobic dye) based dye-displacement assay”, “Thin Layer Chromatography (TLC)” are as per the well-defined terminologies in the art.
[0045] For the purposes of the present document, excipient include substances that serve as mechanisms to improve the delivery and the effectiveness of drugs. A diluent (also referred to as filler, dilutant, or thinner) is a diluting agent. An excipient is an inactive substance that serve as the vehicle or medium for a drug or other active substance. Excipients include colouring agents, humectants, preservatives, emollients, and combinations thereof.
[0046] Mycobacterium genus belongs to Phylum Actinobacteria, Order Actinomycetales, and Family Mycobacteriaceae. The present document depicts Mycobacterium species which is intended to cover all the species belonging to the genus Mycobacterium.
[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0048] Although there are several targets been identified in Mycobacterium, but there are no available targets that include the crucial arginine biosynthesis pathway of the pathogen. Also, there is a dearth of compositions which target novel enzyme pathway, particularly arginine biosynthesis pathway of a pathogen. The present disclosure reveals the use of ornithine acetyltransferase enzyme - ArgJ as a target for identifying candidate molecules for binding to ArgJ, wherein binding of the candidate molecules to said target leads to inhibition of growth of the respective pathogen harbouring the enzyme. Most importantly, said enzyme lacks a homolog in humans, said so the candidate molecule identified using ArgJ as a target will tend to have high selectivity to the ArgJ of the pathogen, thereby leading to minimum cross reactivity and minimum side effects. The pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
[0049] The present disclosure provides a composition to inhibit growth of Mycobacterium species by targeting ArgJ enzyme of the pathogen. The ArgJ enzyme is essential for survival, growth, and pathogenesis of M. tuberculosis. The present disclosure exploits the use of ArgJ enzyme in identifying a candidate drug capable of binding to a novel allosteric binding pocket of the enzyme, thereby inhibiting growth of the pathogen. Two such candidate drugs have been identified as part of the present disclosure namely, pranlukast and sorafenib. The composition comprising a combination of pranlukast or sorafenib with standard care of drugs for tuberculosis treatment has been shown to inhibit growth of M. tuberculosis in an enhanced manner as compared to the standard care of drugs used alone for tuberculosis treatment. Following combinations like: pranukast and rifampicin; pranlukast, rifampicin, and isoniazid; sorafenib and rifampicin; sorafenib, rifampicin, and isoniazid have been shown to inhibit growth of M. tuberculosis in an enhanced manner as compared to the combination of standard care drugs (rifampicin, isoniazid, and ethambutol).
[0050] The present disclosure also discloses a screening method for identifying candidate molecules for allosterically binding to a novel pocket in ArgJ enzyme. The screening method involves a combination of in-silico, in-vitro, and in-vivo methods to select the candidate molecules capable of binding to the ArgJ enzyme. The identified novel allosteric binding pocket can be used for identifying candidate molecules for binding to the ArgJ enzyme, thereby inhibiting growth of pathogen harbouring said enzyme.
[0051] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
[0052] In an embodiment of the present disclosure, there is provided a composition comprising: (a) pranlukast or sorafenib; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species.
[0053] In an embodiment of the present disclosure, there is provided a composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis. [0054] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid, said method comprising: (i) obtaining pranlukast; (ii) obtaining rifampicin; (iii) obtaining isoniazid; and (iv) contacting pranlukast, rifampicin, and isoniazid to obtain the composition.
[0055] In an embodiment of the present disclosure, there is provided a composition comprising: (a) pranlukast, and analogs thereof; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium,
Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0056] In an embodiment of the present disclosure, there is provided a composition comprising: (a) pranlukast; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0057] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) pranlukast; and (b) rifampicin, said method comprising: (i) obtaining pranlukast; (ii) obtaining rifampicin; and (iii) contacting pranlukast, and rifampicin to obtain the composition.
[0058] In an embodiment of the present disclosure, there is provided a composition comprising: (a) pranlukast, and analogs thereof; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0059] In an embodiment of the present disclosure, there is provided a composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0060] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid, said method comprising: (i) obtaining sorafenib; (ii) obtaining rifampicin; (iii) obtaining isoniazid; and (iv) contacting sorafenib, rifampicin, and isoniazid to obtain the composition.
[0061] In an embodiment of the present disclosure, there is provided a composition comprising: (a) sorafenib, and analogs thereof; (b) rifampicin; and (c) isoniazid, wherein the composition inhibits growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0062] In an embodiment of the present disclosure, there is provided a composition comprising: (a) sorafenib; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis. [0063] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) sorafenib; and (b) rifampicin, said method comprising: (i) obtaining sorafenib; (ii) obtaining rifampicin; and (iii) contacting sorafenib, and rifampicin, to obtain the composition.
[0064] In an embodiment of the present disclosure, there is provided a composition comprising: (a) sorafenib, and analogs thereof; and (b) rifampicin, wherein the composition inhibits growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0065] In an embodiment of the present disclosure, there is provided a composition comprising pranlukast, for use in inhibiting growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0066] In an embodiment of the present disclosure, there is provided a composition comprising sorafenib, for use in inhibiting growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0067] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition further comprises ethambutol.
[0068] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition further comprises a standard of care drug for treatment of tuberculosis. [0069] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition further comprises a drug selected from the group consisting of pyrazinamide, bedaquiline (TMC-207), PA-824, AZD5847, linezolid, moxifloxacin, rifapentine, and BTZ043.
[0070] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition further comprises: at least one excipient; at least one carrier; and at least one diluent.
[0071] In an embodiment of the present disclosure, there is provided a method for preparing the composition as described herein. The preparation method includes obtaining of relevant components and contacting the components to obtain respective composition.
[0072] In an embodiment of the present disclosure, there is provided a use of pranlukast for inhibiting growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0073] In an embodiment of the present disclosure, there is provided a use of pranlukast in combination with standard of care drugs for treatment of tuberculosis, for inhibiting growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0074] In an embodiment of the present disclosure, there is provided a use of sorafenib for inhibiting growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0075] In an embodiment of the present disclosure, there is provided a use of sorafenib in combination with standard of care drugs for treatment of tuberculosis, for inhibiting growth of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0076] In an embodiment of the present disclosure, there is provided a use of ArgJ
(ornithine acetyl transferase) enzyme of Mycobacterium species as a target for identifying candidate molecules for binding to ArgJ enzyme, wherein the binding modulates function of ArgJ enzyme thereby inhibiting growth of Mycobacterium species, and wherein Mycobacterium species is selected from group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In another embodiment of the present disclosure, Mycobacterium species is Mycobacterium tuberculosis.
[0077] In an embodiment of the present disclosure, there is provided a use of ArgJ (ornithine acetyl transferase) enzyme of a microorganism as a target for identifying candidate molecules for binding to ArgJ enzyme, wherein the binding modulates function of ArgJ enzyme thereby inhibiting growth of the microorganism, and wherein the microorganism is selected from group consisting of Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
[0078] In an embodiment of the present disclosure, there is provided a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen, said method comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ; and (b) predicting the binding of the candidate molecules to ArgJ to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen. In another embodiment of the present disclosure, the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
[0079] In an embodiment of the present disclosure, there is provided a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen, said method comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ ; (b) predicting the binding of the candidate molecules to identify selected candidate molecules; and (c) performing in-vitro validation of the selected candidate molecules, to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen, and wherein the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis. In another embodiment of the present disclosure, the pathogen is Mycobacterium species, wherein the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis. In yet another embodiment, the binding of the candidate molecule comprises allosteric binding to the ArgJ enzyme, wherein the allosteric binding inhibits growth of the pathogen.
[0080] In an embodiment of the present disclosure, there is provided a method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen, said method comprising: (a) performing in-silico screening of a library of candidate molecules for binding to ArgJ ; (b) predicting the binding of the candidate molecules to identify selected candidate molecules; (c) performing in-vitro validation of the selected candidate molecules, to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen; and (d) performing an in-vivo validation of the selected candidate molecules, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen, and wherein the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis. In another embodiment of the present disclosure, the pathogen is Mycobacterium species, wherein the Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis. In yet another embodiment, the binding of the candidate molecule comprises allosteric binding to the ArgJ enzyme, wherein the allosteric binding leads to inhibition of growth of Mycobacterium species.
[0081] In an embodiment of the present disclosure, there is provided a method for identifying a candidate molecule capable for binding to ArgJ enzyme of
Mycobacterium species, said method comprising: (a) obtaining a computerized representation of FDA (Food and Drug Administration) approved small molecule drug library comprising candidate molecules; (b) obtaining a computerized representation of a reference molecule, wherein the reference molecule is ANS (8-anilinonaphthalene sulfonate); (c) performing in-silico screening with a computerized representation of ArgJ enzyme involving multiple steps comprising: i. flexible docking and filtering the candidate molecules of library to obtain first phase candidate molecules; ii. grid scoring of the first phase candidate molecules based on electrostatic energies within a range of 0.59 to -34.39 kcal/mol and van der Waals energies within a range of -5.43 to -109.25 kcal/mol, and selecting from the first phase candidate molecules based on comparison with the reference molecule to obtain second phase candidate molecules; iii. amber based rescoring and selecting from the second phase candidate molecules based on comparison with the reference molecule to obtain third phase candidate molecules; and iv. screening the third phase candidate molecules based on five parameters to obtain a cluster of candidate molecule, wherein the five parameters are (1) Violation of Lipinski’s rule of five, (2) Binding free energies, (3) Ligand strain, (4) Change in Solvent accessible surface area (ASASA), and (5) Gap index, and (d) performing in-vitro validation of the cluster of candidate molecules comprising following steps: i. performing Thin layer chromatography (TLC) based assay to determine activity of ArgJ and to perform an activity-based screen; ii. performing ANS (hydrophobic dye) based dye-displacement assay to determine binding affinity of candidate molecules selected from the cluster; and iii. performing Thermal Shift Assay (TSA) to determine apparent binding affinity of candidate molecules selected from the cluster, to identify the candidate molecule capable for binding to ArgJ enzyme of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis. In an alternate embodiment, the computerized representation of ArgJ enzyme comprises an allosteric binding pocket wherein, the allosteric binding leads to inhibition of growth of Mycobacterium species.
[0082] In an embodiment of the present disclosure, there is provided a method for identifying a candidate molecule capable for binding to ArgJ enzyme of Mycobacterium species, said method comprising: (a) obtaining a computerized representation of FDA (Food and Drug Administration) approved small molecule drug library comprising candidate molecules; (b) obtaining a computerized representation of a reference molecule, wherein the reference molecule is ANS (8-anilinonaphthalene sulfonate); (c) performing in-silico screening with a computerized representation of ArgJ enzyme involving multiple steps comprising: i. flexible docking and filtering the candidate molecules of library to obtain first phase candidate molecules; ii. grid scoring of the first phase candidate molecules based on electrostatic energies within a range of 0.59 to -34.39 kcal/mol and van der Waals energies within a range of -5.43 to -109.25 kcal/mol, and selecting from the first phase candidate molecules based on comparison with the reference molecule to obtain second phase candidate molecules; iii. amber based rescoring and selecting from the second phase candidate molecules based on comparison with the reference molecule to obtain third phase candidate molecules; and iv. screening the third phase candidate molecules based on five parameters to obtain a cluster of candidate molecule, wherein the five parameters are (1) Violation of Lipinski’s rule of five, (2) Binding free energies, (3) Ligand strain, (4) Change in Solvent accessible surface area (ASASA), and (5) Gap index, (d) performing in-vitro validation of the cluster of candidate molecules comprising following steps: i. performing Thin layer chromatography (TLC) based assay to determine activity of ArgJ and to perform an activity-based screen; ii. performing ANS (hydrophobic dye) based dye-displacement assay to determine binding affinity of candidate molecules selected from the cluster; and iii. performing Thermal Shift Assay (TSA) to determine apparent binding affinity of candidate molecules selected from the cluster; and (e) performing in-vivo validation of the candidate molecules selected from the cluster, to identify the candidate molecule capable of binding to ArgJ enzyme of Mycobacterium species. In another embodiment of the present disclosure, Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In yet another embodiment, Mycobacterium species is Mycobacterium tuberculosis. In an alternate embodiment, the computerized representation of ArgJ enzyme comprises an allosteric binding pocket wherein, the allosteric binding leads to inhibition of growth of Mycobacterium species. [0083] In an embodiment of the present disclosure, there is provided a method as described herein, wherein the ArgJ enzyme has an amino acid sequence as set forth in SEQ ID NO: 2, encoded by a gene having a nucleic acid sequence as set forth in SEQ ID NO: 1.
[0084] In an embodiment of the present disclosure, there is provided a candidate molecule identified by any of the methods as described herein.
[0085] In an embodiment of the present disclosure, there is provided a candidate molecule identified by any of the methods as described herein, wherein the candidate molecule is pranlukast.
[0086] In an embodiment of the present disclosure, there is provided a candidate molecule identified by any of the methods as described herein, wherein the candidate molecule is sorafenib.
[0087] In an embodiment of the present disclosure, there is provided a composition as described herein for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. In another embodiment, Mycobacterium species is Mycobacterium tuberculosis.
[0088] In an embodiment of the present disclosure, there is provided a composition as described herein for use in treating infections caused by a pathogen, wherein the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
[0089] In an embodiment of the present disclosure, there is provided a composition comprising: (a) pranlukast; and (b) rifampicin, for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0090] In an embodiment of the present disclosure, there is provided a composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid, for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0091] In an embodiment of the present disclosure, there is provided a composition comprising: (a) sorafenib; and (b) rifampicin, for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0092] In an embodiment of the present disclosure, there is provided a composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid, for use in treating infections caused by Mycobacterium species, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
[0093] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from tuberculosis, said method comprising administering to the subject therapeutically effective amount of the composition comprising: (a) pranlukast; (b) rifampicin; and (c) isoniazid.
[0094] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from tuberculosis, said method comprising administering to the subject therapeutically effective amount of the composition comprising: (a) pranlukast; and (b) rifampicin.
[0095] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from tuberculosis, said method comprising administering to the subject therapeutically effective amount of the composition comprising: (a) pranlukast; and (b) a standard of care anti-TB drug.
[0096] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from tuberculosis, said method comprising administering to the subject therapeutically effective amount of the composition comprising: (a) sorafenib; and (b) rifampicin.
[0097] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from tuberculosis, said method comprising administering to the subject therapeutically effective amount of the composition comprising: (a) sorafenib; and (b) a standard of care anti-TB drug.
[0098] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from tuberculosis, said method comprising administering to the subject therapeutically effective amount of the composition comprising: (a) sorafenib; (b) rifampicin; and (c) isoniazid.
[0099] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from tuberculosis, said method comprising administering to the subject therapeutically effective amount of the composition as described herein.
[00100] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition as described herein; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00101] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition as described herein; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject, wherein the subject is suffering from a Mycobacterial infection caused by any one of the pathogens selected from a group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus. [00102] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition comprising pranlukast, and rifampicin; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00103] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition comprising pranlukast, rifampicin, and isoniazid; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00104] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition comprising pranlukast, rifampicin, and at least one standard of care drug for treatment of tuberculosis; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00105] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition comprising pranlukast, and at least one standard of care drug for treatment of tuberculosis; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00106] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition comprising sorafenib, and rifampicin; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00107] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition comprising sorafenib, rifampicin, and isoniazid; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00108] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition comprising sorafenib, rifampicin, and at least one standard of care drug for treatment of tuberculosis; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00109] In an embodiment of the present disclosure, there is provided a method for treating a subject suffering from Mycobacterial infection, said method comprising: (a) obtaining a composition comprising sorafenib, and at least one standard of care drug for treatment of tuberculosis; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
[00110] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES
[00111] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
[00112] The examples section is intended to give a detailed explanation of the procedures followed in the present disclosure. It sequentially discloses the importance of using the ornithine acetyltransferase (OAT) - ArgJ enzyme as a target followed by identification of a previously unknown, thus novel allosteric binding pocket of the enzyme. The allosteric pocket was further used to screen for candidate molecules from FDA approved drugs by following in-silico, in-vitro, and in-vivo screening methods. The compositions as disclosed in the present disclosure was shown to exhibit enhanced inhibitory effects on the growth of M. tuberculosis treatment.
[00113] Arginine biosynthesis pathway is essential for the survival and pathogenesis of Mycobacterium tuberculosis ( Mtb ). Despite the acknowledged significance of arginine biosynthesis in Mtb, inhibitors to target this pathway remain to be discovered. The enzymes involved in this pathway are promising targets for anti-TB drug development (Gordhan, 2002; Mdluli & Spigelman, 2006). Moreover, inhibitors of this pathway may provide novel insights to the significance of arginine biosynthesis in Mtb associated stress responses and persistence. One of the crucial enzyme from this pathway, Ornithine acetyltransferase (ArgJ) from Mtb has been implicated as essential gene for the survival and virulence of the pathogen (Sassetti et al, 2003; Sassetti & Rubin, 2003). MtArgJ catalyzes the transfer of acetyl moiety from N-acetyl ornithine to glutamate, thereby producing ornithine and N-acetyl glutamate for next round of arginine biosynthesis (Xu et al, 2007) (Fig. la and Fig. lb). Significantly, the absence of a homologous protein in human genome makes MtArgJ an exciting target for drug development. M. tuberculosis argj gene (SEQ ID No: 1) is encoded by the ORF Rvl653, and belongs to the N-terminal nucleophile fold family of enzymes (Xu et al, 2007). The crystal structure of MtArgJ in native form and in complex with ornithine has been determined at 1.7 A... and 2.4 A... respectively (Sankaranarayanan et al, 2010). The present disclosure discloses a previously unknown allosteric site on MtArgJ and report the discovery of a novel inhibitor that binds and impedes the catalytic efficiency of MtArgJ. The selectivity and specificity of this inhibitor lies in its ability to allosterically modulate the substrate -binding interface. Through a series of in silico, biochemical and biological approaches, the present disclosure concludes the potency of the compounds pranlukast and sorafenib as drug candidates against Mtb survival and pathogenesis. The present disclosure demonstrates the exquisite potency of the inhibitors against arginine biosynthesis in Mtb thereby abating the pathogen survival, in both in vitro and in-vivo infection models. Significant effect of the inhibitors independently in combination with the standard-of-care therapeutic regimen, attests to its promise for inclusion in our armamentarium against tuberculosis. Taken together, this disclosure discloses a novel metabolic inhibitor of Mtb, and its potential for improved combinatorial therapy against tuberculosis.
[00114] The present disclosure is intended to cover the use of prnalukast alone or in any combination with known standard of care drugs for anti-TB. Specific examples have been shown in the present disclosure that includes pranlukast and its combination with rifampicin and isoniazid. It is contemplated that the following combinations obtained with pranlukast: pranlukast+ethambutol, pranlukast+isoniazid, pranlukast+drug selected from the group consisting of pyrazinamide, bedaquiline (TMC-207), PA-824, AZD5847, linezolid, moxifloxacin, rifapentine, and BTZ043 is covered by the present disclosure. Also, any combination obtained using pranlukast for inhibiting growth of Mycobacterium species is intended to be covered by the present disclosure. Similarly, any combination obtained using sorafenib for inhibiting growth of Mycobacterium species is intended to be covered by the present disclosure. Example 1
Identification of a potential ligand binding pocket on MtArgJ surface
[00115] The quest to discover a small molecule inhibitor of MtArgJ started by investigating the protein surface. MtArgJ structure (PDB Id: 3IT6) was probed for surface cavity predictions (by MetaPocket server and CastP analysis) and a well- defined pocket of area 2019.7A...2 and volume 3104.8A...3 located in-between the two active sites was discovered (Fig. 2a). This pocket comprises of 48% hydrophobic amino-acid residues and 24% and 28% polar and charged residues respectively (Fig. 2a inset). This large pocket comprised of four loops, four helices and one I2 strand contributed by each protomer (AB) of A2B2 tetramer. However, two flanking loops positioned at the interface of the substrate binding pockets on either side were observed.
[00116] To experimentally validate the hydrophobicity of this pocket, a fluorescent dye, 8-anilinonaphthalene sulfonate (ANS) was used. It has specificity for the hydrophobic regions of a protein and shows a characteristic fluorescence at 470 nm upon binding. This property of ANS to probe the novel pocket on MtArgJ was utilized. As shown in Fig. 2b, ANS showed a concentration-dependent increase in binding to MtArgJ, manifested as a steady rise in fluorescence intensity. The kinetic analysis determined a Kd of 937 I¼M (ANS binding to MtArgJ) (Fig. 2c). Also, in blind docking experiment with whole molecule of MtArgJ, 2000 final conformations were generated, and it was observed that all the conformers of ANS were sitting and interacting exclusively with this novel pocket (data not shown). In concordance with MetaPocket analysis, these results further validate the presence of a major cavity on MtArgJ surface, which is considerably hydrophobic in nature. Characterization of this pocket was performed and as shown in Fig. 2d, addition of ANS lead to a decrease in the catalytic activity of MtArgJ. However, the effect was only marginal, which could be attributed to the relatively smaller size of ANS with respect to the binding pocket. Based on the initial results, it was contemplated that a suitable ligand bound to this pocket may cause inhibition of the enzymatic activity of the protein. Conventional substrate analogs (as inhibitors) may cross-react with other cellular proteins with similar ligands, giving rise to unwanted side effects. The initial results gave a lead to further probe this pocket for inhibitor development against MtArgJ.
Example 2 In-silico Screen of FDA -Approved drug library against MtArgJ
[00117] A high-throughput in-silico screen of a small molecule drug library to determine their binding to the major pocket on MtArgJ was performed. Importantly, chosen library was FDA approved therefore, was already cleared for cytotoxicity tests and poised for therapeutic repurposing. From the dataset of 1556 FDA-approved drugs, a total of 1417 compounds were prepared and sourced into the virtual screening pipeline along with the reference molecule ANS.
[00118] Initially flexible docking filtered 1340 compounds based on their internal degrees of freedom. Subsequently, grid scoring based on electrostatic energies (0.59 to -34.39 kcal/mol) and van der Waals energies (-5.43 to -109.25 kcal/mol) were applied. This bivariate histogram-based partitioning selected 743 compounds which showed better binding to MtArgJ than ANS.
[00119] In the second layer of screening, Amber based rescoring obtained 203 compounds that outperformed ANS. Next, screening based on five parameters, namely (i) Violation of Lipinski’s rule of five (Lipinski et al, 1997), (ii) Binding free energies, (iii) Ligand strain, (iv) Change in Solvent accessible surface area (ASASA), and (v) Gap index, segregated these 204 ligands (203+ANS) into various clusters (Fig. 2e). From these clusters, four major clusters were selected, A (0000), B (10000), C (01000) and D (11000), comprising of 43 molecules, with best possible set of scores for all five parameters mentioned above. Hydrogen bond interactions for these 43 receptor-ligand complexes were computed and tabulated for the assessment of their binding strength to MtArgJ. The results obtained from in-silico high-throughput screen paved the way for secondary validation screens.
[00120] A novel screening strategy was designed to cull-off the non-specific compounds from the best hit molecules to augment in silico hit discovery against the novel drug target - ArgJ of M. tuberculosis. The applied hierarchical strategy included embedding multiple scoring and filtering functions that analyzed the conformational space, non-covalent interactions energies and receptor-ligand interactions to identify ideal hits against MtArgJ. Example 3
In-vitro validation of in-silico predictions
To validate the virtual screening approach developed here, compounds from each sub- class were selected for experimental testing. Testing included performing an extensive enzyme kinetic study with selected compounds by evaluating their potential to inhibit MtArgJ activity. The activity-based screen led to the identification of two potent compounds, Pranlukast (PRK) and Sorafenib (SRB) (Fig. 3a, b). To determine the mechanism of inhibition, their effect on Km (Michaelis constant) and Vmax (maximum velocity) for the MtArgJ activity assay was assessed. The kinetic parameters were determined by varying substrate - nitroalkane oxidase (NAO) concentration at multiple inhibitor (PRK/SRB) concentration during enzyme catalysis. In the absence of inhibitor, the Vmax was calculated to be 22.4 I¼g/min. Increasing inhibitor (PRK/SRB) concentration lowered the Vmax of the reaction without affecting the apparent Km (Fig. 3 c, d). These results demonstrate a non-competitive mode of inhibition of MtArgJ, orchestrated by PRK and SRB. Next, Dixon plot analysis was performed for l/V versus inhibitor concentration (PRK/SRB) at three different substrate concentrations (O.lmM, 0.5mM and ImM NAO). The data revealed a Ki value of 139 I¼M for PRK mediated MtArgJ inhibition while that of SRB was calculated to be 244 I¼M (Fig. 3 e, f). These results establish a non-competitive mode of MtArgJ inhibition by PRK and SRB. Further, the data indicates PRK to be an efficient inhibitor of MtArgJ activity than SRB. The results are consistent with the rationale of probing the allosteric site for inhibition of MtArgJ. A negative validation of the in-silico screening strategy was performed by testing 10 compounds from the non-selected group (filtered out) and none of them could inhibit the MtArgJ activity in vitro. PRK and SRB bind to a novel allosteric pocket identified on the surface of MtArgJ
Example 4 Determination of the binding site of PRK and SRB on MtArg J
[00121] An ANS based fluorescence titration assay (Iyer et al, 2016) was designed. The MtArgJ, saturated with ANS gives characteristically high fluorescence intensity at 470nm, and any molecule that competes for ANS binding site should result in a dose dependent decrease in fluorescence. As shown in Fig. 3(g, h), addition of PRK/SRB lead to diminution in fluorescence intensity at 470 nm in a dose dependent manner. The data indicate binding of PRK and SRB to the allosteric pocket by competitive displacement of ANS from the MtArgJ complex. Both PRK and SRB were spectroscopically inert in this region. Further, net change in the relative fluorescence unit (RFU) was plotted as a function of ligand concentration to calculate binding constants. The dissociation constant (Ki) for PRK induced displacement of ANS from MtArgJ was calculated to be 115 I¼M, whereas that of SRB was 312 I¼M (Fig. 3g, h: inset). The Kd values thus obtained for both the compounds are about 10 times lesser than that for ANS, which establishes their significant binding ability for this region. These results demonstrate the suggestive affinity and specificity of PRK and SRB for the allosteric pocket disclosed here, at the surface of MtArgJ.
The PRK and SRB impart thermal stability to MtArgJ in a concentration dependent manner.
[00122] To further establish the binding affinity of PRK and SRB to MtArgJ, Thermal Shift Assay (TSA) was employed, it is a method orthologous to isothermal titration calorimetry (ITC) (Iyer et al, 2016) and is being productively used for drug discovery. MtArgJ with varying concentration of PRK/SRB was subjected to gradually increasing temperature, and the shift in melting temperature (Tm) was calculated. The extent of change in Tm is indicative of the ligand’s affinity for protein. As shown in Fig. 3 (i, j), thermal stability of MtArgJ demonstrate positive correlation with increasing concentration of both the inhibitors (PRK and SRB). However, the increase in Tm was relatively higher in case of PRK, consistent with its higher affinity for the protein. The apparent dissociation constant (Kd) for PRK and SRB, calculated by plotting net change in Tm versus inhibitor concentration, was 126 I¼M and 281 I¼M, respectively (Fig. 3i, j: inset). These results are consistent with PRK to have higher affinity for MtArgJ than SRB, thereby imparting enhanced thermal stability to the protein. Importantly, Kd values determined by TSA agreed with the enzyme kinetics and fluorescence spectroscopy data (Fig. 3 c-h). These results validated the significant affinity of both the inhibitors for MtArgJ. However, PRK induces a more positive shift in thermal stability as compared to SRB at same concentrations. Based on the data so far, it was evident that PRK is a better inhibitor of MtArgJ and has higher affinity for the protein, than SRB.
Residues involved in allosteric binding
[00123] Molecular Dynamic (MD) simulation results decipher a proposed mode of
PRK/SRB binding to the allosteric pocket on MtArgJ. Based on the promising results obtained through biochemical analysis, the molecular basis of PRK mediated inhibition of MtArgJ through computational approaches was sought for. Molecular Dynamic (MD) simulation was performed to examine the possible mode of PRK/SRB mediated allosteric inhibition of MtArgJ. Hydrogen bonds contribute to the directionality and stabilization of protein-ligand complexes. Hence, the occurrence of hydrogen bonds between substrate bound MtArgJ and PRK/SRB were examined. Binding pockets in Mycobacterium tuberculosis ornithine acetyltransferase (MtArgJ) were predicted using a web server MetaPocket 2.0 (http://prffiects.hiotec.tu- dresden.de/metapocket). Metapocket 2.0 uses a consensus method, eight different methods were deployed to predict the binding sites with high accuracy. Additionally, CASTp Webserver was employed to calculate the area and volume of different binding clefts. Pockets are concavities on a protein surface into which solvent (probe sphere 1.4 A) can gain access, i.e., these concavities have mouth openings connecting their interior with the outside bulk solution.
[00124] The binding site predicted was formed due to protein complexation at the interface of chains B and D essentially from the two monomers, which was distinct from the substrate binding pockets. This well-defined pocket was found to be a large cleft of area 2019.7A2 and volume 3104.8A3. The large interface pocket comprised of four loops flanking b4 a1, b8 b9, a5 b10, a9 b12, four helices - a5, a7, a8, aΐq and one b15 strand contributed by each monomer. Also noticed, were that the loops flanking between b8 b9 and a5 b10 were positioned at the interface of the substrate binding pocket and the unfilled pocket. The allosteric site was highly hydrophobic in nature and comprised of 48% hydrophobic amino acid residues and 24 and 28% polar and charged residues, respectively. This large pocket comprised of four loops, four helices, and one beta strand contributed by each protomer (AB) of A2B2 tetramer. While nestled in the allosteric pocket of MtArgJ, the tetrazole ring of PRK interacts with Asp234 and Ser3 l0 respectively from chain B and chain D while chromene ring showed interactions with Ser3l0 of chain B and benzamide group nitrogen with Gln305 of chain B on the protein (Fig. 4a, b). Sorafenib, on the other hand, showed interactions via amino groups to Gln305 and Arg308 of Chain D and carbonyl group to Arg308 of Chain B (Fig. 4c, d). PRK exhibited more number of interactions with the allosteric pocket than SRB; asserting higher affinity of PRK for MtArgJ. The data shows that PRK and SRB both bind to the allosteric pocket on MtArgJ, however PRK binds with higher affinity than SRB (Fig. 3 c-j).
Example 5
In-vitro determination of pranlukast and sorafenib induced inhibition of M. tuberculosis
[00125] So far, characterization of the affinity parameters and the possible mechanism involved in the PRK/SRB based inhibition of MtArgJ was depicted. Next, determination of the efficacy of these inhibitors was performed on pathogenic strain of Mtb, H37Rv. H37Rv cells were exposed to varying concentrations of PRK and SRB. The Microplate Alamar Blue Assay (MABA) was employed to determine the MIC90 (Minimum Inhibitory Concentration- 90% inhibition in cell survival) of the inhibitors. Alamar Blue (AB) is an oxidation/reduction indicator dye that has been widely used to measure the sensitivity of mycobacteria to anti-TB drugs (Franzblau, 2000). A color transition from non-fluorescent blue to fluorescent pink indicating reduction of AB dye occurs during mycobacterial growth. Inhibitor mediated depletion in growth interferes with AB reduction and subsequent color development. Administering pathogenic Mtb (H37Rv) with increasing concentration of PRK or SRB resulted in decreased fluorescence intensity (Fig. 5a). Rifampicin (Rif) was taken as a positive control. The minimum inhibitory concentration (MIC90) was calculated by plotting cell viability (%) against inhibitor concentration. Based on the MABA assay, the calculated MIC90 for PRK and SRB against Mtb H37Rv are 5I¼g/ml and lOI¼g/ml respectively (Fig. 5b, c). Next, the Hill’s plot analysis of fluorescence intensity versus inhibitor concentration revealed the IC50 of PRK and SRB mediated inhibition of Mtb survival to be 3.02 I¼g/ml and 6.7 I¼g/ml respectively (Fig. 5d, e). The results suggest the potential anti-tubercular properties of lead compounds. However, it also indicated the superiority of PRK over SRB in inhibiting the growth and survival of Mtb. The efficacy of PRK and SRB was also tested on the MDR strains isolated from tuberculosis patient: Jal226l and Jal2287. PRK showed an MIC90 of 15 I¼g/ml and 25 I¼g/ml for both Jal226l and Jal2287 strains, respectively (Fig. 5f and Fig. 5g). However, SRB demonstrated comparatively higher MIC (Fig. 5h and Fig. 5i) for both the strains. The results showed the promising effect of PRK on pathogenic Mtb including MDR strains. Example 6
Determination of inhibitory effect of combination comprising pranlukast or sorafenib along with a standard of care anti-TB drugs
[00126] After establishing the efficacy of PRK and SRB as potent inhibitor of mycobacterial growth, their efficacy in combination with the standard-of-care anti-TB drugs (Rifampicin (R), Isoniazid (H) and Ethambutol (E)) were tested. Mtb H37Rv cells were seeded in a 96 well plate for 48 hours at 37A°C and then treated with combination of inhibitors. The inhibitory properties of RHE combination was compared with novel combination of RH+PRK, wherein Ethambutol was replaced with PRK. This enabled to compare the efficacy of the inhibitor against a standard of care metabolic drug of Mtb, i.e., ethambutol. A combination of rifampicin (40 ng/ml), isoniazid (30 ng/ml) and ethambutol (1.5 I¼g/ml) was used as a reference control to compare the efficacy of novel combination of rifampicin and isoniazid with PRK/SRB. The results indicate a 10-fold decrease in CFU upon RH+PRK treatment at the end of 24 hrs (Fig. 5j). On the other hand, RH+SRB showed a decrease of 0.2 log unit CFU (Fig. 5k). These results demonstrate that PRK effectively inhibits Mtb survival and works very efficiently in combination with the standard-of-care anti-TB drugs (Rifampicin and Isoniazid). Also, the combination of SRB+RH shows increase in inhibiting Mtb as compared to the combination of RHE, although the effect of SRB+RH is lesser that PRK+RH but is still more than the effect of RHE. The new combinations of RH+PRK and RH+SRB is significantly effective against Mtb survival than the pre-existing RHE combination and holds a potential for improved therapy regimen. Example 7
Effect of pranlukast or sorafenib in reducing mycobacterial burden on infected macrophages
[00127] The efficacy of PRK/SRB on macrophage infection model of Mtb was tested. PRK significantly inhibits the survival of Mtb in macrophage infection model without affecting the host cell THP-l cells, a human monocytic cell line, stimulated for differentiation by PMA treatment were infected with pathogenic Mtb (H37Rv). The Mtb infected THP-l cells were treated with varying inhibitor (PRK or SRB) concentrations or DMSO (control) at different time -points. Once treated, the cells were lysed at desired time points (0, 12, 24 and 48 hrs) and plated for colony formation assays. The CFU (colony forming unit) is calculated and plotted against time at three different inhibitor concentrations. As shown in Fig. 6a, treatment of Mtb infected THP-l cells with PRK (5 I¼g/ml) lead to lOO-fold reduction in CFU within 48 hrs. Moreover, identical assay with mouse macrophages (Raw264.7) treated with PRK lead to about 200-fold decrease in CFU (Fig. 6b). In comparison, SRB was less potent and shows about 10 to 30-fold decrease in CFU for human and mouse macrophage cell lines respectively (Fig. 6a, b). These results demonstrate the superiority of PRK over SRB in reducing the mycobacterial burden from the infected macrophages. Referring to Examples 6 and 7, although, SRB is less potent but is still an effective alternate that can be used in combination with standard of care drugs for tuberculosis treatment.
Example 8
Determination of effect of combination comprising pranlukast or sorafenib along with a standard of care anti-TB drugs in reducing mycobacterial burden on infected macrophages
[00128] The Mtb infected macrophages were treated with either pre-existing RHE combination or novel R/H/PRK and R/H/SRB cocktails. Interestingly, treatment with RH+PRK combination exhibited almost 40 to 50-fold decrease in CFU from that of parent combination (RHE) in both human and murine macrophage cell lines (Fig. 6c, d). However, RH+SRB lead to only 0.2-0.3 log unit decrease in CFU signifying its reduced efficiency towards combination therapy (Fig. 6c, d). These results demonstrate the efficacy of PRK in reducing the Mtb burden from host macrophages and its enhanced efficiency in combination with standard-of-care drugs.
Example 9
Effect of PRK treatment on macrophage internalized Mtb
[00129] The active effect of PRK treatment on macrophage internalized Mtb and its possible side effects on the host cell survival was determined. Macrophages were infected with GFP-tagged Mtb H37Rv {Mtb- GFP henceforth) followed by treatment with PRK at varying time points. Cells were harvested and flow-sorted based on GFP expression followed by assessment of macrophage viability by PI (Propidium Iodide) staining. The PRK treatment leads to diminished GFP intensity with time, suggesting reduced Mtb burden in infected macrophages (Fig. 6e, f). However, host cell (macrophage) viability remains unaffected as determined by PI staining (Fig. 6g).
[00130] Thus, appreciating the results, Examples 1 to 9 demonstrate the potency of pranlukast (PRK) and sorafenib (SRB) as a promising anti -tubercular molecule with no deleterious effect on the host cell survival. Also, the enhanced effect of PRK alone and in combination with the therapy drugs, on the macrophage internalized Mtb is interesting and beneficial from the host’ s perspective.
Dual role of pranlukast (PRK)
[00131] PRK is a known inhibitor of Cysteinyl Leukotriene Receptor- 1 (CysLTRl), on the mammalian cells and is used for treatment of Asthma. Macrophages also express CysLTRl in response to various inflammatory stimuli, including pathogenic bacterial colonization. Also, studies have shown that in macrophages and dendritic cells, PRK acts through a yet another mechanism, wherein it targets the leukotriene and prostaglandin (eicosanoids) biosynthesis, which are ligands for CysLTR’s (Theron et al, 2014). It does so by affecting 5 -Lipoxygenase (5-LO) signalling in the host. Divangahi et al. have shown that M. tuberculosis infection activates the 5- lipoxygenase pathway, that facilitates the host cell necrosis thereby helping the pathogen dissemination. This also prevents the cross-antigen presentation by dendritic cells, thereby inhibiting the induction of T cell immunity (Divangahi et al, 2010). The present disclosure depicts the dual role of PRK, it can be appreciated from Pig. 8a-8f that PRK apart from inhibiting function of ArgJ enzyme, inhibits the 5-LO signalling in the macrophages infected with Mtb, thereby reducing the pathogen survival in the host. Thus, the present disclosure discloses inhibition of Mtb infection by dual targeting of arginine biosynthesis in the pathogen and 5-LO signalling in the host, mediated by PRK treatment. As observed (from Pig. 5a-5f), 5 -lipoxygenase and the associated genes involved in eicosanoid biosynthesis were significantly down- regulated upon PRK treatment but not by Rif treatment.
Example 10 Determination of effect of pranlukast on apoptosis
[00132] The PRK treatment reduces the infection-associated apoptosis in the host. It has been reported that during the early phase of Mtb infection, macrophages undergo apoptosis as an innate defense mechanism, thereby increasing the levels of pro- apoptotic proteins like Caspase 3 and 8. Hence, the effect of PRK on infection- associated apoptosis in the host was explored. To examine this, caspase-3 dependent apoptosis in Mtb infected macrophages was monitored. As shown in Fig. 6 (h-j) treatment with increasing concentrations of PRK lead to reduced extracellular caspase-3 level in the Mtb infected human as well as murine macrophages (THP- 1 and Raw 264.7 respectively). However, uninfected macrophages showed no increase in caspase 3 levels while the infected macrophages showed a gradual increase in caspase 3 with time (up to 48 hrs). The results demonstrate decrease in Mtb associated macrophage apoptosis upon PRK treatment in both human and mouse macrophage cell lines. The above results suggest that PRK not only targets the macrophages associated Mtb but also limits the infection induced host cell apoptosis.
Example 11
In-vivo studies to determine the effects of pranlukast
[00133] The PRK treatment reduces the Mtb burden and tubercular granulomas from the lungs of Mtb infected mice. Chronic Mtb infection is characterized by the formation of lung associated granulomas, an organized aggregate of immune cells with infected macrophages at the core. To investigate the effect of PRK on chronic Mtb exposure, BALB/c mice were used and were infected with M. tuberculosis through aerosol (Fig. 7a). One-month post successful establishment of infection, mice were treated with PRK (40 mg/kg body weight), Rif (10 mg/kg body weight) or combination of PRK with Rif. Next, these mice were sacrificed at three time points (0 day, 12 days and 24 days) and lung-associated granulomas were analyzed (Fig. 8 b, c, d and e). A marked reduction in the tubercular granulomas post 12 and 24 days of treatment with PRK in comparison to PBS (phosphate buffer saline) treated mice (Fig. 7 b,c) was observed. Notably, Rif + PRK combination had practically no visible granulomas in the mice lungs, post 24 days of treatment (Fig. 7 d,e). To determine the lung associated bacterial burden, the mice lungs were homogenized and plated at different dilutions on 7H11 solid media supplemented with OADC and PANTA. The plates were incubated for 21 days and colonies were analyzed for CFU count. Significant reduction in Mtb burden was observed, with a 0.5 log unit decrease in CFU, in PRK treated mice as compared to PBS control (Fig. 7 f). Notably, PRK in combination with standard-of-care anti-TB drug, Rif, showed improved results with maximum diminution of lung-associated Mtb burden and a decrease in CFU by 1 log unit as compared to treatment with Rif alone (Fig. 7 g). The number of granulomas per tissue section of the mice lungs were calculated by the H staining analysis of the lung slides (Fig. 7 h, i). There was a significant decrease in the tubercular granulomas in the PRK treated mice. However, PRK showed most remarkable effect in combination with Rifampicin. Moreover, there was no splenic or hepatic cytotoxicity at the administered dosages, as shown in the detailed histopathological H staining images, analyzed by expert pathologists. The results demonstrate the potency of PRK in combating Mtb infection and its improved efficiency in combination with Rif, thereby proving its in-vivo efficacy on the pre-clinical model of tuberculosis.
[00134] The present example clearly depicts the possibility of using a combination of PRK with rifampicin for treatment of tuberculosis. Since, SRB also has a similar mechanism of action for inhibiting the activity of ArgJ as of PRK, thus it can be contemplated from the present Example that a combination of SRB and rifampicin can also be used to treat tuberculosis infection. Example 12
ArgJ protein of Mtb similar to ArgJ protein of other pathogens
[00135] Since, pranlukast and sorafenib binds to a novel allosteric binding pocket of ArgJ enzyme of Mtb. It can be contemplated that pranlukast and sorafenib can be used to bind to ArgJ protein of other pathogens having high similarity and identity to that of ArgJ of Mtb. Table 1 depicts the comparative analysis of percentage sequence identity of ArgJ from M. tuberculosis {Mtb), M. avium (Mav), M. leprae (Mlp). It can be inferred from Table 1, that ArgJ of Mav and Mlp are more than 80% identical to that of Mtb hence it would be prudent to say that the compositions comprising either pranlukast or sorafenib can be used for treating infections caused by Mav and Mlp. Table: 1
Figure imgf000045_0001
[00136] Similarly, ArgJ enzyme of Mtb was also compared to that of other pathogens. Table 2 depicts the percentage identity data for ArgJ of other significant pathogens with that of Mtb.
Table 2:
M yco ba cteri y m_tu bercu !osis
Figure imgf000046_0001
Mycobacteriumjeprae 80,66
IVIycobacteriym_tubercylosis 100
Leptospira iiterrogans 41,32
Figure imgf000046_0002
Neisseria gonorrhoeae 52,61
Neisseria_meningtt¾dis 52.89
[00137] It can be inferred from Table 2, that as ArgJ of some of the significant pathogens depicted above have more than 40% identity to ArgJ, allosteric binding pocket of ArgJ can be diligently used to screen various candidate molecules for binding to respective ArgJ and inhibiting the growth of respective pathogens.
Advantages of the present disclosure:
[00138] The present disclosure discloses the composition comprising pranlukast, and the composition comprising sorafenib in inhibiting the growth of Mycobacterium species. The compositions have been obtained by using a novel method for screening various candidate molecules for binding to a novel allosteric binding pocket of ArgJ enzyme of Mtb. The binding inhibits the growth of Mtb, thus the present composition is highly useful in treating tuberculosis apart from other infections caused by Mycobacterium species. ArgJ enzyme of Mtb lacks a homolog in humans thus any candidate molecules capable of binding to the enzyme can be used to prepare medicaments for treating infections. Pranlukast and Sorafenib being FDA approved drugs display a potential for development of advanced combinational therapy against pathogenic tuberculosis including multi-drug resistant (MDR) Mtb. Also, the method as described herein can also be used to screen candidate molecules for binding to ArgJ enzyme of other pathogens selected from the group consisting of Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis. Thus, significance of the present disclosure lies in the ability of using ArgJ enzyme in screening candidate molecules. Therefore, the method as described in the present disclosure can also be used to screen candidate molecules to treat infections arising from Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
References:
1. Divangahi M, Desjardins D, Nunes-Alves C, Remold HG & Behar SM (2010) Eicosanoid pathways regulate adaptive immunity to Mycobacterium tuberculosis. Nature Immunology 11: 751-758.
2. Gordhan BG (2002) Construction and Phenotypic Characterization of an Auxotrophic Mutant of Mycobacterium tuberculosis Defective in L- Arginine Biosynthesis. Infection and Immunity 70: 3080-3084.
3. Iyer D, Vartak SV, Mishra A, Goldsmith G, Kumar S, Srivastava M, Hegde M, Gopalakrishnan V, Glenn M, Velusamy M, Choudhary B, Kalakonda N, Karki SS, Surolia A & Raghavan SC (2016) Identification of anovel BCL2- specific inhibitor that binds predominantly to the BH1 domain. The FEBS Journal 283:3408-3437
4. Lipinski CA, Lombardo F, Dominy BW & Feeney PJ (1997) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews 23: 3- 25.
5. Mdluli K & Spigelman M (2006) Novel targets for tuberculosis drug discovery. Current Opinion in Pharmacology 6: 459-467.
6. Sassetti CM & Rubin EJ (2003) Genetic requirements for mycobacterial survival during infection. Proceedings of the National Academy of Sciences 100: 12989-12994
7. Sassetti CM, Boyd DH & Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Molecular Microbiology 48: 77- 84.
8. Theron AJ, Steel HC, Tintinger GR, Gravett CM, Anderson R & Feldman C (2014) Cysteinyl leukotriene receptor- 1 antagonists as modulators of innate immune cell function. Journal of Immunology Research 2014: 60893-16. Xu Y, Labedan B & Glansdorff N (2007) Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms. Microbiology and Molecular Biology Reviews 71: 36-47.

Claims

I/We Claim:
1. A composition comprising:
a) pranlukast or sorafenib; and
b) rifampicin,
wherein the composition inhibits growth of Mycobacterium species.
2. The composition as claimed in claim 1, wherein the composition optionally comprises isoniazid.
3. A composition comprising pranlukast, for use in inhibiting growth of Mycobacterium species.
4. A composition comprising sorafenib, for use in inhibiting growth of Mycobacterium species.
5. The composition as claimed in any one of the claims 1-4, wherein the composition further comprises ethambutol.
6. The composition as claimed in claim 1, wherein the composition further comprises a standard of care drug for treatment of tuberculosis.
7. The composition as claimed in claim 1, wherein the composition further comprises a drug selected from the group consisting of pyrazinamide, bedaquiline (TMC-207), PA-824, AZD5847, linezolid, moxifloxacin, rifapentine, and BTZ043.
8. The composition as claimed in any one of the claims 1-4, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
9. Use of ArgJ (ornithine acetyl transferase) enzyme of a microorganism as a target for identifying candidate molecules for binding to ArgJ enzyme, wherein the binding modulates function of ArgJ enzyme thereby inhibiting growth of the microorganism, and wherein the microorganism is selected from group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
10. A method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen, said method comprising:
a) performing in-silico screening of a library of candidate molecules for binding to ArgJ ; and
b) predicting the binding of the candidate molecules to ArgJ to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen.
11. A method for identifying a candidate molecule for binding to ArgJ enzyme of a pathogen, said method comprising:
a) performing in-silico screening of a library of candidate molecules for binding to ArgJ ;
b) predicting the binding of the candidate molecules to identify selected candidate molecules; and
c) performing in-vitro validation of the selected candidate molecules, to identify the candidate molecule capable of binding to ArgJ enzyme of the pathogen, wherein the binding is capable of modulating function of ArgJ enzyme for inhibiting growth of the pathogen.
12. The method as claimed in claim 11, wherein the method further comprises performing an in-vivo validation of the selected candidate molecules.
13. The method as claimed in any of the claims 10 and 11, wherein the pathogen is selected from the group consisting of Mycobacterium species, Corynebacterium diphtheriae, Nocardia mikamii, Leptospira interrogans, Clostridium botulinum, Listeria monocytogenes, Burkholderia pseudomallei, Staphylococcus aureus, Bordetella pertussis, Neisseria gonorrhea, and Neisseria meningitidis.
14. A method for identifying a candidate molecule capable for binding to ArgJ enzyme of Mycobacterium species, said method comprising:
a) obtaining a computerized representation of FDA approved small molecule drug library comprising candidate molecules;
b) obtaining a computerized representation of a reference molecule, wherein the reference molecule is ANS (8-anilinonaphthalene sulfonate);
c) performing in-silico screening with a computerized representation of ArgJ enzyme involving multiple steps comprising:
i. flexible docking and filtering the candidate molecules of library to obtain first phase candidate molecules;
ii. grid scoring of the first phase candidate molecules based on electrostatic energies within a range of 0.59 to -34.39 kcal/mol and van der Waals energies within a range of -5.43 to -109.25 kcal/mol, and selecting from the first phase candidate molecules based on comparison with the reference molecule to obtain second phase candidate molecules;
iii. amber based rescoring and selecting from the second phase candidate molecules based on comparison with the reference molecule to obtain third phase candidate molecules; and iv. screening the third phase candidate molecules based on five parameters to obtain a cluster of candidate molecule, wherein the five parameters are (1) Violation of Lipinski’s rule of five (2) Binding free energies (3) Ligand strain (iv) Change in Solvent accessible surface area (ASASA) and (5) Gap index, and (d) performing in-vitro validation of the cluster of candidate molecules comprising following steps: i. performing Thin layer chromatography (TLC) based assay to
determine activity of ArgJ and to perform an activity-based screen; ii. performing ANS (hydrophobic dye) based dye-displacement assay to determine binding affinity of candidate molecules selected from the cluster; and
iii. performing Thermal Shift Assay (TSA) to determine apparent binding affinity of candidate molecules selected from the cluster, to identify the candidate molecule capable for binding to ArgJ enzyme of Mycobacterium species
15. The method as claimed in any one of the claims 13 and 14, wherein Mycobacterium species is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
16. The method as claimed in claim 14, wherein the computerized representation of ArgJ enzyme comprises an allosteric binding pocket.
17. The method as claimed in any one of the claims 10-11, and 14, wherein the binding of the candidate molecule comprises allosteric binding to the ArgJ enzyme.
18. The method as claimed in claim 14, wherein the ArgJ enzyme has an amino acid sequence as set forth in SEQ ID NO: 2, encoded by a gene having a nucleic acid sequence as set forth in SEQ ID NO: 1.
19. A candidate molecule as identified by a process as claimed in any one of the claims 10-11 and 14.
20. The candidate molecule as claimed in claim 19, wherein the candidate molecule is pranlukast.
21. The candidate molecule as claimed in claim 19, wherein the candidate molecule is sorafenib.
22. The method as claimed in claim 14, wherein the method further comprises in- vivo validation of the candidate molecule.
23. A method for treating a subject suffering from a Mycobacterial infection, said method comprising: (a) obtaining a composition as claimed in any one of the claims 1-8; and (b) administering a therapeutically effective amount of the composition to the subject, for treating the subject.
24. The method as claimed in claim 23, wherein the subject is suffering from a Mycobacterial infection caused by any one of the pathogens selected from a group consisting of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium marinum, and Mycobacterium abscessus.
25. A method for preparing a composition as claimed in claim 1 , said method comprising: (a) obtaining pranlukast or sorafenib; (b) obtaining rifampicin; and (c) contacting pranlukast or sorafafenib with rifampicin, to obtain the composition.
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Citations (3)

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WO2012143144A1 (en) * 2011-04-21 2012-10-26 Origenis Gmbh Pyrazolo [4, 3-d] pyrimidines useful as kinase inhibitors
US20140171445A1 (en) * 2011-08-04 2014-06-19 The Usa, As Represented By The Secretary, Department Of Health And Human Services Treatment and prevention of diseases mediated by microorganisms via drug-mediated manipulation of the eicosanoid balance
WO2015011163A1 (en) * 2013-07-26 2015-01-29 Sanofi Antitubercular composition comprising rifampicin, isoniazid, ethambutol and pyrazinamide and its process of preparation.

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012143144A1 (en) * 2011-04-21 2012-10-26 Origenis Gmbh Pyrazolo [4, 3-d] pyrimidines useful as kinase inhibitors
US20140171445A1 (en) * 2011-08-04 2014-06-19 The Usa, As Represented By The Secretary, Department Of Health And Human Services Treatment and prevention of diseases mediated by microorganisms via drug-mediated manipulation of the eicosanoid balance
WO2015011163A1 (en) * 2013-07-26 2015-01-29 Sanofi Antitubercular composition comprising rifampicin, isoniazid, ethambutol and pyrazinamide and its process of preparation.

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