Identification of Mycobacterium tuberculosis BioA inhibitors by using structure-based virtual screening.

BACKGROUND: 7,8-Diaminopelargonic acid synthase (BioA), an enzyme of biotin biosynthesis pathway, is a well-known promising target for anti-tubercular drug development. METHODS: In this study, structure-based virtual screening was employed against the active site of BioA to identify new chemical entities for BioA inhibition and top ranking compounds were evaluated for their ability to inhibit BioA enzymatic activity. RESULTS: Seven compounds inhibited BioA enzymatic activity by greater than 60% at 100 µg/mL with most potent compounds being A36, A35 and A65, displaying IC50 values of 10.48 µg/mL (28.94 µM), 33.36 µg/mL (88.16 µM) and 39.17 µg/mL (114.42 µM), respectively. Compounds A65 and A35 inhibited Mycobacterium tuberculosis (M. tuberculosis) growth with MIC90 of 20 µg/mL and 80 µg/mL, respectively, whereas compound A36 exhibited relatively weak inhibition of M. tuberculosis growth (83% inhibition at 200 µg/mL). Compound A65 emerged as the most potent compound identified in our study that inhibited BioA enzymatic activity and growth of the pathogen and possessed drug-like properties. CONCLUSION: Our study has identified a few hit molecules against M. tuberculosis BioA that can act as potential candidates for further development of potent anti-tubercular therapeutic agents.

Virtual Screening, pharmacophore development and structure based similarity search to identify inhibitors against IdeR, a transcription factor of Mycobacterium tuberculosis.

ideR, an essential gene of Mycobacterium tuberculosis, is an attractive drug target as its conditional knockout displayed attenuated growth phenotype in vitro and in vivo. To the best of our knowledge, no inhibitors of IdeR are identified. We carried out virtual screening of NCI database against the IdeR DNA binding domain followed by inhibition studies using EMSA. Nine compounds exhibited potent inhibition with NSC 281033 (I-20) and NSC 12453 (I-42) exhibiting IC50 values of 2 µg/ml and 1 µg/ml, respectively. We then attempted to optimize the leads firstly by structure based similarity search resulting in a class of inhibitors based on I-42 containing benzene sulfonic acid, 4-hydroxy-3-[(2-hydroxy-1-naphthalenyl) azo] scaffold with 4 molecules exhibiting IC50 ≤ 10 µg/ml. Secondly, optimization included development of energy based pharmacophore and screening of ZINC database followed by docking studies, yielding a molecule with IC50 of 60 µg/ml. More importantly, a five-point pharmacophore model provided insight into the features essential for IdeR inhibition. Five molecules with promising IC50 values also inhibited M. tuberculosis growth in broth culture with MIC90 ranging from 17.5 µg/ml to 100 µg/ml and negligible cytotoxicity in various cell lines. We believe our work opens up avenues for further optimization studies.

Disruption of mycobactin biosynthesis leads to attenuation of Mycobacterium tuberculosis for growth and virulence.

BACKGROUND: Low iron availability in the host upregulates the mbt gene cluster of Mycobacterium tuberculosis, which is responsible for mycobactin biosynthesis. However, the biological significance of mycobactins in the growth of this pathogen and in disease progression has not been elucidated. METHODS: We have disrupted the mbtE gene (Rv2380c) in the mbt cluster to evaluate the importance of mycobactin biosynthesis in the growth and virulence of M. tuberculosis. RESULTS: The mbtE mutant (MtbΔmbtE) was unable to synthesize mycobactins, displayed an altered colony morphology, and was attenuated for growth in broth culture and in macrophages. Transmission electron microscopy revealed that MtbΔmbtE displayed an altered cell wall permeability. The growth characteristics and colony morphology of MtbΔmbtE were similar to wild type when the medium was supplemented with mycobactins or when MtbΔmbtE was genetically complemented with the mbtE gene. Moreover, guinea pigs infected with MtbΔmbtE exhibited a significantly reduced bacillary load and histopathological damage in the organs, in comparison to M. tuberculosis-infected animals. CONCLUSIONS: This study highlights the importance of mycobactins in the growth and virulence of M. tuberculosis and establishes the enzymes of mycobactin biosynthesis as novel targets for the development of therapeutic interventions against tuberculosis.

A booster vaccine expressing a latency-associated antigen augments BCG induced immunity and confers enhanced protection against tuberculosis.

BACKGROUND: In spite of a consistent protection against tuberculosis (TB) in children, Mycobacterium bovis Bacille Calmette-Guerin (BCG) fails to provide adequate protection against the disease in adults as well as against reactivation of latent infections or exogenous reinfections. It has been speculated that failure to generate adequate memory T cell response, elicitation of inadequate immune response against latency-associated antigens and inability to impart long-term immunity against M. tuberculosis infections are some of the key factors responsible for the limited efficiency of BCG in controlling TB. METHODS/PRINCIPAL FINDINGS: In this study, we evaluated the ability of a DNA vaccine expressing α-crystallin--a key latency antigen of M. tuberculosis to boost the BCG induced immunity. 'BCG prime-DNA boost' regimen (B/D) confers robust protection in guinea pigs along with a reduced pathology in comparison to BCG vaccination (1.37 log(10) and 1.96 log(10) fewer bacilli in lungs and spleen, respectively; p<0.01). In addition, B/D regimen also confers enhanced protection in mice. Further, we show that B/D immunization in mice results in a heightened frequency of PPD and antigen specific multi-functional CD4 T cells (3(+)) simultaneously producing interferon (IFN)γ, tumor necrosis factor (TNF)α and interleukin (IL)2. CONCLUSIONS/SIGNIFICANCE: These results clearly indicate the superiority of α-crystallin based B/D regimen over BCG. Our study, also demonstrates that protection against TB is predictable by an increased frequency of 3(+) Th1 cells with superior effector functions. We anticipate that this study would significantly contribute towards the development of superior booster vaccines for BCG vaccinated individuals. In addition, this regimen can also be expected to reduce the risk of developing active TB due to reactivation of latent infection.

Crystal structure of Bfr A from Mycobacterium tuberculosis: incorporation of selenomethionine results in cleavage and demetallation of haem.

Emergence of tuberculosis as a global health threat has necessitated an urgent search for new antitubercular drugs entailing determination of 3-dimensional structures of a large number of mycobacterial proteins for structure-based drug design. The essential requirement of ferritins/bacterioferritins (proteins involved in iron storage and homeostasis) for the survival of several prokaryotic pathogens makes these proteins very attractive targets for structure determination and inhibitor design. Bacterioferritins (Bfrs) differ from ferritins in that they have additional noncovalently bound haem groups. The physiological role of haem in Bfrs is not very clear but studies indicate that the haem group is involved in mediating release of iron from Bfr by facilitating reduction of the iron core. To further enhance our understanding, we have determined the crystal structure of the selenomethionyl analog of bacterioferritin A (SeMet-BfrA) from Mycobacterium tuberculosis (Mtb). Unexpectedly, electron density observed in the crystals of SeMet-BfrA analogous to haem location in bacterioferritins, shows a demetallated and degraded product of haem. This unanticipated observation is a consequence of the altered spatial electronic environment around the axial ligands of haem (in lieu of Met52 modification to SeMet52). Furthermore, the structure of Mtb SeMet-BfrA displays a possible lost protein interaction with haem propionates due to formation of a salt bridge between Arg53-Glu57, which appears to be unique to Mtb BfrA, resulting in slight modulation of haem binding pocket in this organism. The crystal structure of Mtb SeMet-BfrA provides novel leads to physiological function of haem in Bfrs. If validated as a drug target, it may also serve as a scaffold for designing specific inhibitors. In addition, this study provides evidence against the general belief that a selenium derivative of a protein represents its true physiological native structure.

Oral silibinin inhibits lung tumor growth in athymic nude mice and forms a novel chemocombination with doxorubicin targeting nuclear factor kappaB-mediated inducible chemoresistance.

The acute and cumulative dose-related toxicity and drug resistance, mediated via nuclear factor kappaB (NFkappaB), of anthracycline anticancer drugs pose a major problem in cancer chemotherapy. Here, we report that oral silibinin (a flavanone) suppresses human non-small-cell lung carcinoma A549 xenograft growth (P = 0.003) and enhances the therapeutic response (P < 0.05) of doxorubicin in athymic BALB/c nu/nu mice together with a strong prevention of doxorubicin-caused adverse health effects. Immunohistochemical analyses of tumors showed that silibinin and doxorubicin decrease (P < 0.001) proliferation index and vasculature and increase (P < 0.001) apoptosis; these effects were further enhanced (P < 0.001) in combination treatment. Pharmacologic dose of silibinin (60 mumol/L) achieved in animal study was biologically effective (P < 0.01 to 0.001, growth inhibition and apoptosis) in vitro in A549 cell culture together with an increased efficacy (P < 0.05 to 0.001) in doxorubicin (25 nmol/L) combination. Furthermore, doxorubicin increased NFkappaB DNA binding activity as one of the possible mechanisms for chemoresistance in A549 cells, which was inhibited by silibinin in combination treatment. Consistent with this, silibinin inhibited doxorubicin-caused increased translocation of p65 and p50 from cytosol to nucleus. Silibinin also inhibited cyclooxygenase-2, an NFkappaB target, in doxorubicin combination. These findings suggest that silibinin inhibits in vivo lung tumor growth and reduces systemic toxicity of doxorubicin with an enhanced therapeutic efficacy most likely via an inhibition of doxorubicin-induced chemoresistance involving NFkappaB signaling.

Synergistic anti-cancer effects of grape seed extract and conventional cytotoxic agent doxorubicin against human breast carcinoma cells.

With an approach to enhance the efficacy of chemotherapy agents against breast cancer treatment, here, we investigated the anti-cancer effects of grape seed extract (GSE) and doxorubicin (Dox), either alone or in combination, in estrogen receptor-positive MCF-7 and receptor-negative MDA-MB468 human breast carcinoma cells. GSE (25-200 micro g/ml) treatment of cells resulted in 16-72% growth inhibition and 9-33% cell death, in a dose- and a time-dependent manner. In other studies, Dox (10-100 nM) treatment showed 23-96% growth inhibition and 10-55% cell death. Based on these results, several combinations of GSE (25-100 micro g/ml) with Dox (10-75 nM) were next assessed for their synergistic, additive and/or antagonistic efficacy towards cell growth inhibition and death. In both MCF-7 and MDA-MB468 cells, a combination of 100 micro g/ml GSE with 25-75 nM Dox treatment for 48 h showed a strong synergistic effect [combination index (CI) < 0.5] in cell growth inhibition, but mostly an additive effect (CI approximately 1) in cell death. In cell-cycle progression studies, GSE plus Dox combination resulted in a moderate increase in G1 arrest in MCF-7 cells compared to each agent alone. GSE plus Dox combination showed a very strong and significant G1 arrest in MDA-MB468 cells when compared with Dox alone, however, it was less than that observed with GSE alone. In quantitative apoptosis studies, GSE and Dox alone and in combination showed comparable apoptotic death of MCF-7 cells, however, a combination of the two was inhibitory to Dox induced apoptosis in MDA-MB468 cells. This was further confirmed in another estrogen receptor-negative MDA-MB231 cell line, in which GSE and Dox combination strongly inhibited cell growth but did not show any increase in apoptotic cell death caused by Dox. Together, these results suggest a strong possibility of synergistic efficacy of GSE and Dox combination for breast cancer treatment, independent of estrogen receptor status of the cancer cell.

Grape seed extract inhibits advanced human prostate tumor growth and angiogenesis and upregulates insulin-like growth factor binding protein-3.

Dietary intake of many fruits and vegetables has been shown to be associated with reduced risk of cancer. We investigated the in vivo efficacy of grape seed extract (GSE, patented as Traconol) against prostate cancer (PCA) and associated molecular events. Athymic nude mice were implanted with hormone-refractory human prostate carcinoma DU145 cells and fed with 100 and 200 mg/kg/day (5 days/week) doses of GSE for 7 weeks. At the end of experiment, tumors were immunohistochemically analyzed for cell proliferation, apoptosis and angiogenesis. Our data show that GSE feeding strongly inhibited tumor growth that accounted for 59-73% (p < 0.001) inhibition in tumor volume and 37-47% (p < 0.05) decrease in tumor weight at the end of the experiment. It did not show any significant change in body weight gain profile and diet consumption. Immunohistochemical analysis of tumors showed that GSE decreases proliferation index by 51-66% (p < 0.001) and increases apoptotic index by 3-4-fold (p < 0.001). CD31 staining for endothelial cells, showed decrease in intratumoral microvasculature in GSE-fed group of mice. Control tumors showed 64.0 +/- 1.6 CD31 positive cells/400x field compared to 23.2 +/- 0.9 and 15.7 +/- 0.08 (p < 0.001) CD31 positive cells in 100 and 200 mg/kg doses of GSE-treated tumors, respectively. GSE strongly inhibited (47-70%, p < 0.05) vascular endothelial growth factor (VEGF) secretion in conditioned medium by DU145 cells. Recently, the circulating level of insulin-like growth factor binding protein (IGFBP)-3 is shown to inversely related with PCA risk, growth and prognosis. Consistent with this, we observed 6-7-fold (p < 0.001) increase in tumor-secreted levels of IGFBP-3 after GSE feeding. In other immunohistochemical studies, compared to controls, tumor xenografts from GSE-fed groups of mice showed a moderate decrease in VEGF but an increase in IGFBP-3 levels. These findings suggest that GSE possesses in vivo anticancer efficacy against hormone-refractory human PCA, which is associated with its antiproliferative, proapoptotic and antiangiogenic activities together with upregulation of IGFBP-3.

Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels.

We have reported recently the anticancer effect of flavonoid antioxidant silymarin, the major part of milk thistle extract, against advanced human prostate carcinoma DU145 cells (X. Zi et al., Cancer Res., 58: 1920-1929, 1998) and later identified that silibinin is the main active component in silymarin responsible for its effect in cell culture studies. On the basis of these observations, here we assessed in vivo growth inhibitory potential of silibinin against advanced human prostate cancer (PCA). Dietary feeding of silibinin at 0.05 and 0.1% doses (w/w) for 60 days, 24 h after s.c. DU145 tumor xenograft implantation in athymic male nude mice, significantly inhibited tumor volume by 35 and 58% (P < 0.05), and wet weight of tumor by 29 and 40% (P < 0.05), respectively. In a second experiment where mice were fed with these test diets for 3 weeks before tumor xenograft implantation and continued on these diets for a total of 63 days, tumor volume and wet weight of tumor were reduced by 53-64% (P < 0.001-0.05) and 31-52% (P < 0.05), respectively. In both studies, animals did not show weight loss or reduced food consumption. These in vivo anticancer effects of silibinin were associated with an increased accumulation (up to 5.8 fold; P < 0.05) of human insulin-like growth factor-binding protein-3 in mouse plasma. In additional studies assessing biological availability of silibinin in nude mice and its antiproliferative activity at such doses in DU145 cells in culture, silibinin levels in plasma and prostate were found to be in the range of 7-13 microg/ml and 3.7-4.6 microg/g, respectively. At these biologically achievable silibinin concentrations, increased IGFBP-3 level in DU145 cell culture medium and a strong DU145 cell growth inhibition were observed that were irreversible in the absence of silibinin in culture medium. These findings extend and translate our observations on in vitro anticancer effect of silibinin/silymarin to an in vivo preclinical PCA model, which may form the basis for a Phase I clinical trial in PCA patients.

Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogen-activated protein kinases and induction of apoptosis.

This study reports in vivo therapeutic efficacy of silymarin against skin tumors with mechanistic rationale. 7,12-Dimethylbenz[a]anthracene-12-O-tetradecanoyl-phorbol-13-acetate (DMBA-TPA)-induced established skin papilloma (tumor)-bearing SENCAR mice were fed with 0.5% silymarin in AIN-93M-purified diet (w/w), and both tumor growth and regression were monitored during 5 weeks of feeding regimen. Silymarin feeding significantly inhibited (74%, P < 0.01) tumor growth and also caused regression (43%, P < 0.01) of established tumors. Proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling immunohistochemical staining of tumors showed that silymarin decreases proliferation index by 48% (P < 0.001) and increases apoptotic index by 2.5-fold (P < 0.001), respectively. Skin tumor growth inhibition and regression by silymarin were also accompanied by a strong decrease (P < 0.001) in phospho-ERK1/2 levels in tumors from silymarin-fed mice compared with controls. In the studies evaluating bioavailability and physiologically achievable level of silymarin (as silibinin) in plasma, skin tumor, skin, liver, lung, mammary gland and spleen, we found 10, 6.5, 3.1, 13.7, 7.7, 5.9 and 4.4 microg silibinin/ml plasma or per gram tissue, respectively. In an attempt to translate these findings to human skin cancer and to establish biological significance of physiologically achievable level, effect of plasma concentration of silibinin was next examined in human epidermoid carcinoma A431 cells. Silibinin treatment of cells in culture at 12.5, 25 (plasma level) and 50 microM doses resulted in 30-74% (P < 0.01-0.001) growth inhibition and 7-42% death of A431 cells in a dose- and time-dependent manner; apoptosis was identified as a cell death response by silibinin. Similar silibinin treatments also resulted in a significant decrease in phospho-mitogen-activated protein kinase/extracellular signal-regulated protein kinase 1/2 (MAPK/ERK1/2) levels, but an up-regulation of stress-activated protein kinase/jun NH(2)-terminal kinase (SAPK/JNK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) activation in A431 cells. The use of MEK1 inhibitor, PD98059, showed that inhibition of ERK1/2 signaling, in part, contributes to silibinin-caused cell growth inhibition. Together, the data suggest that an inhibition of ERK1/2 activation and an increased activation of JNK1/2 and p38 by silibinin could be possible underlying molecular events involved in inhibition of proliferation and induction of apoptosis in A431 cells. These data suggest that silymarin and/or its major active constituent silibinin could be an effective agent for both prevention and intervention of human skin cancer.