DESC:FIELD OF THE INVENTION
[0001] The present disclosure pertains to therapeutic agents. In particular, the present disclosure pertains to method of identifying molecules capable of inhibiting activity of Mycobacterium tuberculosis topoisomerase I (MttopoI) and other related topoisomerase I. The present disclosure further relates to pharmaceutical compositions which can have a potential for treating mycobacterial infections in a subject.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Mycobacterium tuberculosis (Mtb) is the etiological agent of the dreaded disease, tuberculosis (TB) which claims 1.4 million lives annually (Eurosurveillance editorial, 2013) with 9 million new cases being added every year (Zumla et al., 2013). The long duration of treatment, that extends anywhere between 6 months to two years (Blumberg et al., 2003; Zumla et al., 2013), often leads to patient noncompliance to the treatment regimen. The problem is compounded by the emergence of multidrug and extremely drug resistant TB. A co-occurrence of AIDS and TB further worsens the situation. Considering the present scenario, the need of the hour is to come up with therapeutics that can target essential proteins of the dreaded pathogen that intelligently inhabits and survives in an otherwise hostile host environment (Behar et al., 2010; Flynn and Chan, 2003). Further, bacterial resistance to antibiotics has become an important public health problem, and hence there is a continuing need to develop newer and more potent antibiotics.
[0004] DNA topoisomerases constitute an essential class of enzymes which function to maintain topological homeostasis within the cell during a variety of DNA transaction processes such as replication, transcription and chromosome segregation (Champoux, 2001). On the basis of structure and mechanism, topoisomerases are broadly classified as type I and type II (Corbett and Berger, 2004; Forterre et al., 2007). Bacterial type I DNA topoisomerases belong to the type IA sub class based on their reaction characteristics. Several molecules which hamper the resealing of topoisomerase II mediated DNA breaks have been characterized and clinically validated as drugs (Collin et al., 2011; Pommier, 2013; Pommier et al., 2010). For example, DNA gyrase has been extensively exploited to develop anti-bacterial agents, further fluoroquinolones class of compounds stabilize protein-DNA cleavage complex to induce severe cytotoxicity (Maxwell, 1997). However, there is dearth of such inhibitors of bacterial topoisomerase I. The mechanism of topoisomerase I (Topo I) comprises DNA binding, DNA break, strand passage followed by resealing of cleaved DNA ends. Since Topo I is essential for Mtb growth, it offers an opportunity to be exploited as a drug target. Moreover, trapping Topo I-DNA complex intermediate (formed during the catalysis) would generate lesions in the bacterial genome leading to cytotoxicity. Thus, molecules that trap TopoI-DNA covalent complex are need of hour to combat tuberculosis (TB).
[0005] Accordingly, there exists a need in the art for small molecules, which selectively inhibit mycobacterial enzymes involved in maintenance of topological homeostasis. Further, there is a need in the art for method of identifying small molecules that can inhibit DNA relaxation activity of mycobacterial topoisomerase I. There is also a need for small molecules that inhibit Mtb cells with advantageous toxicological properties to combat mycobacterial infections.
[0006] The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION
[0007] It is an object of the present disclosure to provide inhibitors of Mycobacterium tuberculosis topoisomerase I (MttopoI).
[0008] It is a further object of the present disclosure to identify molecules that can inhibit DNA relaxation activity of topoisomerase I from Mycobacterium tuberculosis (Mtb) and other related bacteria.
[0009] It is another object of the present disclosure to provide a method for identifying specific small molecule inhibitors of MttopoI.
[0010] It is another object of the present disclosure to identify molecules that can target metal binding site of topoisomerase I enzyme to inhibit the enzyme activity.
[0011] It is another object of the present disclosure to identify small molecule inhibitors of MttopoI, wherein the inhibitors can be used as anti-tuberculosis agents.
[0012] It is another object of the present disclosure to identify small molecule inhibitors that can potentially be used in manufacture of pharmaceutical composition for use in the treatment of mycobacterial infections in a subject.
[0013] It is another object of the present disclosure to provide a method for inhibiting DNA relaxation activity of MttopoI and mycobacterial cell growth using small molecule inhibitors.

SUMMARY OF THE INVENTION
[0014] In an aspect, the present disclosure provides a method for identifying molecules capable of inhibiting Mycobacterium tuberculosis topoisomerase I (MttopoI), wherein the method can include the steps of: a) providing at least one homology model of MttopoI; b) in silico docking a plurality of test molecules into the homology model; and c) selecting test molecule(s) having a desired in silico LibDock docking score. The method can further include a step of testing the molecules that exhibit desired in silico docking score in vitro for assessing in vitro inhibition of MttopoI.
[0015] In an embodiment, the in silico docking of the test molecules into the homology model of MttopoI can be performed using automated docking algorithms, and the in silico LibDock docking score can preferably range from 46.4 to 126.3.
[0016] In another aspect, the present disclosure provides a method of inhibiting DNA relaxation activity of MttopoI by therapeutic agents such as imipramine, norclomipramine, amodiaquine, chloroquine, nortryptiline, berberine, moexipril and tianeptine.
[0017] In another aspect, the present disclosure provides a potential method for treating infection caused by Mtb, wherein the method can include administering to a subject in need of such treatment an antidepressant.
[0018] In an embodiment, the antidepressant agent that can be used to treat mycobacterial infection in a human can preferably be selected from the group consisting of imipramine, norclomipramine, nortryptiline, tianeptine, berberine and derivatives thereof.
[0019] In another aspect, the present disclosure provides a potential method for treating infection caused by Mtb, wherein the method can include administering to a subject in need of such treatment an antidepressant in combination with an existing anti-tuberculosis agent or a fluoroquinolone agent.
[0020] In an embodiment, mycobacterial infection in a human can be treated by administering an antidepressant, preferably imipramine or norclomipramine, in combination with either rifampicin or moxifloxacin.
[0021] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0023] FIG. 1 shows chemical structures of norclomipramine, imipramine, amodiaquine, chloroquine, nortryptyline, berberine, moexipril and tianeptine.
[0024] FIGs. 2A and 2B illustrate docking of norclomipramine (libdock score = 95.2) and imipramine (LibDock score = 103) on in silico model of MttopoI respectively, wherein docking is shown in yellow in the topoI homology model with DNA fragment (blue) and Mg2+ (green), in accordance with embodiments of the present disclosure.
[0025] FIGs. 2C and 2D illustrate interaction map of norclomipramine and imipramine respectively, which shows the region harbors the residues involved in Mg2+ coordination, in accordance with embodiments of the present disclosure.
[0026] FIG. 3 illustrates effect of the identified small molecules on DNA relaxation activity of MttopoI, in accordance with embodiments of the present disclosure.
[0027] FIGs. 4A-E illustrate effect of norclomipramine and imipramine on mycobacterial cell growth, in accordance with embodiments of the present disclosure.
[0028] FIGs. 4F-G are exemplary graphs illustrating cell killing potential of norclomipramine and imipramine, in accordance with embodiments of the present disclosure.
[0029] FIGs. 5A and 5B illustrate effect of imipramine and norclomipramine on DNA cleavage activity of MttopoI, in accordance with embodiments of the present disclosure.
[0030] FIGs. 6A and 6B are exemplary graphs illustrating effect of norclomipramine on growth of mycobacterial cells over expressing topoisomerase I, in accordance with embodiments of the present disclosure.
[0031] FIGs. 6C and 6D are exemplary graphs illustrating effect of imipramine on growth of mycobacterial cells over expressing topoisomerase I, in accordance with embodiments of the present disclosure.
[0032] FIG. 7A illustrates effect of imipramine on DNA relaxation activity of DxDxE mutants, in accordance with embodiments of the present disclosure.
[0033] FIG. 7B illustrates effect of norclomipramine on DNA relaxation activity of DxDxE mutants, in accordance with embodiments of the present disclosure.
[0034] FIGs. 8A and 8B illustrate effect of combining imipramine with rifampicin (8A) or moxifloxacin (8B) on the cell viability of Mtb, in accordance with embodiments of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0035] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0036] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.
[0037] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0038] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0039] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
[0040] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[0041] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0042] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0043] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0044] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0045] In an aspect, the present disclosure provides a method for identifying molecules capable of inhibiting MttopoI, wherein the method can include the steps of: a) providing at least one homology model of MttopoI b) in silico docking a plurality of test molecules into the homology model; and c) selecting test molecule(s) that exhibit a desired in silico LibDock docking score.
[0046] In an embodiment, the in silico docking of test molecules into a homology model of MttopoI can be performed using automated docking algorithms, and the in silico LibDock docking score can preferably range from 46.4 to 126.3.
[0047] The docking score resulting from the interaction of test molecules with the modeled MttopoI structure can be interpreted and used to identify possible inhibitors of topoisomerase I enzyme.
[0048] In an embodiment, docked test molecules that exhibit desired in silico docking score can be tested in vitro for assessing in vitro inhibition of MttopoI.
[0049] In an embodiment, possible molecules capable of targeting MttopoI can be identified by in silico docking of test molecules into the binding cavity of topoisomerase I enzyme model. In a more specific embodiment, test molecules can be screened in silico by docking the molecules near the metal binding site of the enzyme.
[0050] In an exemplary embodiment, in silico docking of imipramine or norclomipramine into homology model of MttopoI indicate that the test molecules can bind near the metal binding site of the enzyme.
[0051] The present disclosure further encompasses molecules identified using the present in silico docking method. Examples of possible inhibitors identified by the present in silico docking method are given in following Table 1 along with their inhibitory potential against MttopoI.

Table 1
Molecule Inhibition (µM)
Imipramine 0.1
Norclomipramine 0.1
Amodiaquine 10-25
Chloroquine 10
Moexipril 80 (partial)
Tianeptine 10 (partial)
Nortryptiline 10 (partial)
Berberine 10 (partial)

[0052] The identified molecules can inhibit DNA relaxation reactions catalysed by topoisomerase I from Mtb and from other related bacteria. Further, these molecules can perturb the cleavage-religation equilibrium of the relaxation reaction catalysed by topoisomerase I and can be active in a whole-cell assay against Mtb and Mycobacterium smegmatis (Msm). For example, imipramine and norclomipramine can arrest the topoisomerase reaction after first transesterification reaction leading to the accumulation of DNA breaks. The derivatives of these molecules with increased potency to cause double strand breaks can be synthesized to evaluate their effect on MttopoIand Mtb growth.
[0053] According to embodiments, the identified molecules can affect the growth of cells overexpressing the topoisomerase I enzyme. For example, norclomipramine and imipramine can inhibit the growth of Msm cells expressing normal levels of topoisomerase I at 30 µM and 125 µM respectively. However, cells over-expressing the enzyme can be more susceptible to the identified molecules, and the cell growth can be inhibited at lower concentrations of the molecules. Similarly Mtb cells expressing normal levels of topoisomerase I can exhibit minimum inhibitory concentration (MIC) values of 125 µM for norclomipramine and 250 µM for imipramine, while cells over-expressing the enzyme can exhibit MIC values of 60 µM for norclomipramine and 125 µM for imipramine. The decreased MIC of the molecules in topoisomerase I overexpressing cells can be due to stimulation of topoisomerase I mediated DNA cleavage in vivo leading to cell death, indicating that the enzyme is the cellular target for the molecules.
[0054] The present disclosure further encompasses the use of molecules identified by the present in silico docking methods for the preparation of potential medicaments for the treatment of infections caused by Mtb.
[0055] In another aspect, the present disclosure provides methods for screening possible therapeutic antidepressant, antimalarial and/or antihypertensive compounds that can help alleviate, treat and/or prevent mycobacterial infections, especially in humans, the method can include the steps of in silico docking a plurality of test molecules selected from antidepressant, antimalarial and/or antihypertensive compounds into the homology model of MttopoI, and selecting the molecules that exhibit desired in silico LibDock docking score which can preferably range from 46.4 to 126.3.
[0056] In an embodiment, the present disclosure provides a potential method of treating infection caused by Mtb, wherein the method can include administering to a subject in need of such treatment an antidepressant.
[0057] In an embodiment, the antidepressant agent that can be potentially used to treat mycobacterial infection in a human can preferably be selected from the group consisting of imipramine, norclomipramine, nortryptiline, tianeptine, berberine and derivatives thereof.
[0058] In another aspect, the present disclosure provides a potential method of treating infection caused by Mtb, wherein the method can include administering to a subject in need of such treatment an antidepressant in combination with an antituberculosis agent or a fluoroquinolone agent.
[0059] In an embodiment, mycobacterial infection in a human can be treated by administering an antidepressant, preferably imipramine or norclomipramine, in combination with either rifampicin or moxifloxacin. The combining of imipramine or norclomipramine with rifampicin or moxifloxacin can lower the MIC value due to enhancement of DNA breaks resulting from the combination of topoisomerase poisons targeting both enzymes.

EXAMPLES
[0060] The present disclosure is further explained in the form of following examples. However, it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
[0061] Homology modeling and in silico docking of molecules:
To build the homology model of MttopoI, three bacterial structures from Protein Data Bank (PDB) were used: 1ECL (closed state, no DNA or Mg2+ bound), 1MW8 (closed state with non-covalent DNA bound, no Mg2+ bound) and 1MW9 (closed state, no DNA or Mg2+ bound). A homology model of the MttopoI in a closed state, no DNA or Mg2+ bound (A2VM29 based on 1ECL/1MW9) was also available in ModBase. The bacterial topo II structure 2RGR and the topo III structure 1I7D were also available. A homology model for the EctopoI was first created with the site open and with Mg2+ bound. This was done by aligning topo I subdomains with topo III subdomains. The residue coordinates from topo III were used to generate the Mg2+ site. The MttopoI homology model with gate open and Mg2+ bound was created by using the same sequence alignment as used for 1ECL in the ModBase and the Ectopo I homology model as a scaffold. A homology model with Mg2+ and a covalently bound DNA fragment was made using this model where the position of the DNA is based on the DNA position in the EctopoII crystal structure 2RGR and was achieved using pyMOL.
[0062] The MttopoI homology model with Mg2+ and a DNA fragment bound in the open state was used for docking using LibDock (Discovery Studio, Biovia, San Diego CA). The proposed binding site was centered on Mg2+ with 8 Å diameter. 10 hotspots and docking tolerance (0.25) were included in the protocol. The FAST conformation method was also used along with steepest descent minimization with CHARMm. Further parameters followed the default settings. A set of FDA approved drugs was collected and exported from the Collaborative Drug Discovery Database (Burlingame, CA). This as well as other previously described sets of drugs approved by the FDA [SCUT database] were used for docking in the homology model. The molecules that scored well were visualized, and their two-dimensional (2D) interaction plots were generated and selected for follow-up. Examples of inhibitors identified by the present in silico docking method are given in above Table 1 along with their inhibitory potential against MttopoI, and their chemical structures are shown in FIG. 1. FIGs. 2A and 2B illustrate docking of norclomipramine (LibDock score of 95.2) and imipramine (LibDock score of 103) on in silico model of MttopoI respectively, wherein docking is shown in yellow in the topoI homology model with DNA fragment (blue) and Mg2+ (green). FIGs. 2C and 2D illustrate interaction map of norclomipramine and imipramine respectively, which shows the region harbors the residues involved in Mg2+ coordination.
[0063] Evaluation of inhibition of DNA relaxation activity of MttopoI by the identified molecules:
In order to evaluate the inhibitory potential of the small molecules, DNA relaxation inhibition assays were carried out. Enzyme inhibition assays were carried out with pre-incubation of the enzyme and increasing concentration of the compounds at 37oC for 15 min, followed by the addition of the substrate DNA. After incubating at 37oC for 30 min, the samples were electrophoresed in a 1.2% agarose gel for 12 hr at 2.5 V/cm, stained with ethidium bromide (0.5 ug/mL), and the DNA bands were visualized using a gel documentation system (Bio-Rad, Hercules, CA, USA).
[0064] In a particular experiment, 1 unit of MttopoI was incubated with various concentrations of the molecules (norclomipramine, imipramine, amodiaquine, chloroquine, tianeptine, moexipril, nortryptiline and berberine) at 37oC for 15 min following which, 500 ng of supercoiled pUC18 was added. The incubation was further continued at 37oC for 30 min and the reactions were terminated by addition of 0.6% SDS-agarose dye. The reaction products were resolved on a 1.2 % agarose gel followed by staining with ethidium bromide. The results are shown in FIG. 3. Lane 1, supercoiled pUC18 ; lane 2, relaxation reaction in absence of the compounds; lanes 3-5, various concentrations (50-300nM) of norclomipramine; lanes 6-8 , various concentrations (50-300nM) of imipramine.
[0065] Evaluation of growth inhibitory and cell killing potential of imipramine and norclomipramine:
The effect of imipramine and norclomipramine on the growth of mycobacterial cells was assayed by monitoring the growth profiles of the mycobacterial cells in presence of these molecules. The effect of exposing cells over expressing topoI to imipramine and norclomipramine was also assessed. Mycobacterial cells (expressing normal levels or over expressing wild type mycobacterial topoI) were grown to 0.6 O.D. in Middlebrook 7H9 broth supplemented with 0.2 % glycerol and 0.05 % Tween-80. The culture was diluted to a final O.D. of 0.05 with fresh media and aliquoted into a 100-well growth plate. Serial dilutions of the compounds were added to the culture. Untreated cultures were taken as a control. The growth was monitored at 595 nm with continuous shaking at 200 rpm. The readings were analyzed by GraphPad Prism software (version 5.0). To determine the MIC values of the compounds, resazurin reduction microplate assay (REMA) was carried out. The cultures were grown for 2 days (Msm) or 7 days for Mtb in presence of the compounds. Following this, resazurin dye was added to the cultures at a final concentration of 0.02 % and further incubated for 1 hr for Msm or for 14 hr for Mtb. To assess the cell lethality of imipramine and norclomipramine, Msm cells were grown to O.D.595nm =0.6–0.8 and treated with 1X, 2.5X and 5X MIC concentrations of the molecules for 12 hr. Following this treatment, serial dilutions of the cells were plated on Middlebrook 7H9 agar and CFU/ml was determined. Untreated culture was taken as control.
[0066] Inhibition of mycobacterial growth by norclomipramine and imipramine and determination of MIC value: Msm cells were grown in presence of various concentrations of norclomipramine (FIG. 4A) or imipramine (FIG. 4B). The growth was followed over a period of 48 hr with O.D. being measured every 2 hr. The growth curve was plotted. Sterile medium and untreated culture were used as control. Msm or (FIG. 4C) Mtb (FIG. 4D) H37 Ra cells were grown in presence of various concentrations of the compounds. Resazurin dye was added to a final concentration of 0.02% to each well. The plate was incubated at 37oC to determine the MIC values (FIG. 4E). Sterile medium and untreated culture were used as control. Msm cells in the log phase were exposed to 1X, 2.5X and 5X MIC concentrations of imipramine and norclomipramine and the results are shown in FIG 4F and 4G. Untreated cells were taken as control. Serial dilutions of the treated and untreated cells were plated on Middlebrook 7H9 agar plate and CFU/ml was determined. The values were plotted as log (CFU/ml) vs. concentration as shown in FIGs. 4F and 4G. Error bars indicate the standard deviation obtained in three independent experiments.
[0067] Oligonucleotide cleavage assay:
In order to determine if the molecules could act as topoisomerase poisons, cleavage assays were carried out with double stranded oligonucleotides harboring the Strong Topoisomerase Site (STS). The double stranded substrate was pre-incubated with MttopoI in a buffer containing 40 mM Tris-HCl (pH 8.0), 20 mM NaCl, 1 mM EDTA and 5 mM MgCl2 on ice for 15 min. Following this, various concentrations of imipramine or norclomipramine were added and the reactions were incubated at 37oC for 30 min. The reactions were stopped with 45% formamide and heating at 95oC for 2 min. The products were resolved in a 12% denaturing PAGE and analyzed by phosphorimager.
[0068] In a particular experiment, MttopoI was incubated with 5’end labelled specific 32-mer annealed to a complimentary sequence on ice for 15 min following which various concentrations of imipramine or norclomipramine were added and the reaction was allowed to proceed at 37oC for 30 min. The reactions were terminated by the addition of 45% formamide and heating at 950C for 2 min. The products were resolved on a 12% denaturing PAGE and analyzed by phosphorimager. FIG. 5A illustrates effect of imipramine or norclomipramine on DNA cleavage activity of MttopoI. Lane 2 (C) indicates cleavage reaction by the enzyme in absence of compound and lanes 3 and 7 indicate compound control (CC) for imipramine and norclomipramine respectively. FIG. 5B depicts quantification of cleavage products. Error bars represent the standard deviation obtained in three experiments. The results clearly indicate that imipramine and norclomipramine stimulate the DNA cleavage activity.
[0069] Determination of cytotoxicity: Msm cells over expressing (OE) MstopoI or normal level (N) of MstopoI were grown in presence of various concentrations (0 – 500 µM) of norclomipramine and imipramine separately. The growth was followed over a period of 40 hr with O.D. being measured every 2 hr. The growth curve was plotted using GraphPad Prism v5.0. Sterile medium and untreated culture were used as control. FIGs. 6A and 6B show the effect of norclomipramine on growth of mycobacterial cells over expressing topoisomerase I, and FIGs. 6C and 6D show effect of imipramine on growth of mycobacterial cells over expressing topoisomerase I. It was observed from the graphs that the molecules exhibited increased cytotoxicity upon topoI over expression in Msm.
[0070] Effect of imipramine or norclomipramine on metal binding mutants of MstopoI: 1 unit of D108A (lane 5) or E112A (lane 9) was incubated with various concentrations of imipramine (FIG. 7A) or norclomipramine (FIG. 7B) (lanes 6, 7 and 10, 11 respectively) at 37oC for 15 min. Following this, 500 ng of supercoiled pUC18 was added to each reaction tube and incubated for additional 30 min. The reactions were terminated by addition of 0.6% SDS-agarose dye. The reaction products were resolved on a 1.2 % agarose gel followed by staining with EtBr. mAb - 2F3G4, a monoclonal antibody that inhibits the DNA relaxation reaction of mycobacterial topoisomerase I was used as a positive control. FIG. 7A and 7B illustrate the effect of imipramine or norclomipramine on DNA relaxation activity of DxDxE mutants respectively.
[0071] Effect of combination of imipramine and rifampicin or moxifloxacin on the viability of Mycobacterium tuberculosis cells
In order to assess the effect of combining imipramine with known anti-TB drugs i.e. either rifampicin or moxifloxacin, the Mtb cells were grown in the presence of sub-MIC concentrations of rifampicin or moxifloxacin and various concentrations of imipramine. The cells were cultured at 37oC for 9 days following which resazurin dye was added to the cultures at a final concentration of 0.02% and further incubated for 14 hr and conversion of resazurin (blue) to rezorufin (pink) was monitored to score the viability of the cells.
[0072] In a particular experiment, sub MIC concentrations of rifampicin or moxifloxacin were combined with various concentrations of imipramine (0 - 500 µM). The M. tuberculosis cells were grown in the presence of these combinations at 37oC for 9 days after which resazurin dye was added to the cultures and further incubated at 37oC for 14 hrs. Cells grown in the presence of only imipramine or only rifampicin or only moxifloxacin were used as controls. The results are provided in FIGs. 8A and 8B, which clearly illustrate the effect of combining imipramine with rifampicin (8A) or moxifloxacin (8B) on the cell viability of Mtb.
,CLAIMS:1. A method for identifying inhibitors of Mycobacterium tuberculosis topoisomerase I (MttopoI), the method comprising the steps of:
a) providing at least one homology model of MttopoI;
b) in silico docking a plurality of test molecules into the homology model; and
c) selecting test molecule(s) having a desired in silico LibDock docking score.

2. The method according to claim 1, further comprising testing in vitro the selected test molecules(s) from step (c) for assessing in vitro inhibition of MttopoI I.

3. The method according to claim 1, wherein the in silico LibDock docking score ranging from 46.4 to 126.3.

4. The method according to claim 1, wherein the in silico docking in step (b) is performed using automated docking algorithms.

5. The method according to claim 1, wherein the plurality of test molecules are selected from the group consisting of antidepressant agents, antihypertensive agents and antimalarial agents.

6. Use of a therapeutic agent or a derivative thereof for the manufacture of a medicament for use in treating mycobacterial infection in a subject.

7. The use according to claim 6, wherein the therapeutic agent is selected from the group consisting of imipramine, norclomipramine, nortryptiline, tianeptine, berberine, amodiaquine, chloroquine, moexipril and derivatives thereof.

8. The use according to claim 6, wherein the mycobacterial infection is caused by Mycobacterium tuberculosis or Mycobacterium smegmatis.

9. The use according to claim 6, wherein the subject is an animal or a human.

10. The use according to claim 6, wherein the therapeutic agent is used in combination with at least one anti-tuberculosis agent.

11. The use according to claim 6, wherein the therapeutic agent is used in combination with rifampicin or a derivative thereof.

12. The use according to claim 6, wherein the therapeutic agent is used in combination with at least one fluoroquinolone antibacterial agent.

13. The use according to claim 6, wherein the therapeutic agent is used in combination with moxifloxacin or a derivative thereof.

14. A pharmaceutical composition comprising an antidepressant and a therapeutic agent selected from the group consisting of anti-tuberculosis agent and fluoroquinolone antibacterial agent, for the treatment of mycobacterial infection.

15. A method of treating mycobacterial infection, the method comprising administering to a subject in need of such treatment an antidepressant.

16. The method according to claim 15, wherein the antidepressant is a tricyclic antidepressant or a derivative thereof.

17. A method of treating mycobacterial infection, the method comprising administering to a subject in need of such treatment an antidepressant in combination with an anti-tuberculosis agent or a fluoroquinolone antibacterial agent.

18. The method according to claim 17, wherein the antidepressant is administered in combination with rifampicin or a derivative thereof.

19. The method according to claim 17, wherein the antidepressant is administered in combination with moxifloxacin or a derivative thereof.

20. The method according to any one of claims 15 to 19, wherein the mycobacterial infection is caused by Mycobacterium tuberculosis or Mycobacterium smegmatis.