In silico discovery of potential drug molecules to improve the treatment of isoniazid-resistant Mycobacterium tuberculosis

The emergence of multidrug-resistant Mycobacterium tuberculosis (M.tb) has become one of the major hurdles in the treatment of tuberculosis (TB). Drug-resistant M.tb has evolved with various strategies to avoid killing by the anti-tubercular drugs. Thus, there is a rising need to develop effective anti-TB drugs to improve the treatment of these strains. Traditional drug design approach has earned little success due to time and the cost involved in the process of development of anti-infective drugs. Numerous reports have demonstrated that several mutations in the drug target sites cause emergence of drug-resistant M.tb strains. In this study, we performed computational mutational analysis of M.tb inhA, fabD, and ahpC genes, which are the primary targets for first-line isoniazid (INH) drug. In silico virtual drug screening was performed to identify the potent drugs from a ChEMBL compound library to improve the treatment of INH-resistant M.tb. Further, these compounds were analyzed for their binding efficiency against active drug binding cavity of M.tb wild-type and mutant InhA, FabD and AhpC proteins. The drug efficacy of predicted lead compounds was verified by molecular docking using M.tb wild-type and mutant InhA, FabD and AhpC protein template models. Different in silico and pharmacophore analysis predicted three potent lead compounds with better drug-like properties against both M.tb wild-type and mutant InhA, FabD, and AhpC proteins as compared to INH drug, and thus may be considered as effective drugs for the treatment of INH-resistant M.tb strains. We hypothesize that this work may accelerate drug discovery process for the treatment of drug-resistant TB.

Communicated by Ramaswamy H. Sarma     

Exploratory algorithm to devise multi-epitope subunit vaccine by investigating Leishmania donovani membrane proteins

Visceral leishmaniasis (VL) is a deadly parasitic infection which affects poorest to poor population living in the endemic countries. Increasing resistant to existing drugs, disease burden and a significant number of deaths, necessitates the need for an effective vaccine to prevent the VL infection. This study employed a combinatorial approach to develop a multi-epitope subunit vaccine by exploiting Leishmania donovani membrane proteins. Cytotoxic T- and helper T-lymphocyte binding epitopes along with suitable adjuvant and linkers were joined together in a sequential manner to design the subunit vaccine. The occurrence of B-cell and IFN-γ inducing epitopes approves the ability of subunit vaccine to develop humoral and cell-mediated immune response. Physiochemical parameters of vaccine protein were also assessed followed by homology modeling, model refinement and validation. Moreover, disulfide engineering was performed for the increasing stability of the designed vaccine and molecular dynamics simulation was performed for the comparative stability purposes and to conform the geometric conformations. Further, molecular docking and molecular dynamics simulation study of a mutated and non-mutated subunit vaccine against TLR-4 immune receptor were performed and respective complex stability was determined. In silico cloning ensures the expression of designed vaccine in pET28a(+) expression vector. This study offers a cost-effective and time-saving way to design a novel immunogenic vaccine that could be used to prevent VL infection.

Communicated by Ramaswamy H. Sarma     

Phosphorylation of profilin regulates its interaction with actin and poly (L-proline)

Activation of bovine platelets with thrombin and phorbol 12,13-dibutyrate (PDBu) resulted in phosphorylation of profilin on serine. The phosphorylation was inhibited when platelets were pretreated with the PI 3-kinase inhibitor, LY294002, indicating that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation. Phosphorylation of profilin resulted in significant decrease in actin polymerization (16.5%), indicating an increased affinity of phosphoprofilin towards actin. The critical actin monomer concentration (Cc) increased to 260 nM in the presence of phosphoprofilin in comparison with 200 nM in the presence of profilin. The interaction of phosphoprofilin with phosphatidylinositol 4,5-bisphosphate [PI (4,5)-P2] and poly (L-proline) (PLP) was examined by monitoring the quenching of tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no difference in PI (4,5)-P2 binding between profilin and phosphoprofilin (Kd=20.4 microM), while poly (L-proline)-binding studies indicated a sixfold decrease (27.34 microM for profilin and 4.73 microM for phosphoprofilin) in Kd with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85alpha, the regulatory subunit of PI 3-kinase with phosphoprofilin over profilin. Overall, the data presented conclude that profilin phosphorylated under in vivo conditions and phosphorylation depends upon activation of PI 3-kinase. Phosphoprofilin exhibited increased affinity to poly (L-proline) sequences both in vitro and in vivo.     

In vitro Activation of Protein Kinase C by β-N-oxalyl-l-α, β-diaminopropionic acid,the Lathyrus sativus Neurotoxin

-N-oxalyl-l-,-diaminopropionic acid (l-ODAP) toxicity has been associated with lathyrism; a spastic paraparesis caused by excessive dietary intake of the pulse Lathyrus sativus. We investigated the effect of Lathyrus neurotoxin l-ODAP on protein kinase C (PKC) activity under in vitro conditions. l-ODAP activated phosphorylation activity of purified chick brain PKC. Both lysine-rich (histone III-S) and arginine-rich (protamine sulfate) substrate phosphorylation was enhanced in the presence of l-ODAP. The activation is concentration dependent, and maximal activation is observed at 100 M concentration. Protamine sulfate phosphorylation was enhanced by 47%, whereas histone III-S phosphorylation was enhanced by 50% over PS/PDBu/Ca²⁺ dependent activity. The nontoxic d-isomer (d-ODAP) did not affect both histone III-S and protamine sulfate phosphorylation activity. These results indicate that l-ODAP taken up by neuronal cells could also contribute to PKC activation and so be associated with toxicity.     

Mycobacterium leprae binds to a major human peripheral nerve glycoprotein myelin P zero (P0)

We have previously shown that a major phosphorylated 25-kDa glycoprotein of the human peripheral nerve binds to Mycobacterium leprae. In the present study, we confirm that the 25-kDa glycoprotein of the human peripheral nerve is myelin P zero (P₀) by immunoprecipitation and Western blot experiments using monoclonal antibodies to myelin P₀. Immunohistochemical studies on human nerve using these antibodies to myelin P₀ exhibited a strong immunoreactivity to the myelin and Schwann cells. Myelin P₀ is a peripheral nerve specific protein; therefore it could likely be one of the key target molecules for M. leprae binding/internalization or even contact-dependent demyelination. This finding of M. leprae binding to myelin P₀ adds to the present understanding on neural predilection of M. leprae.     

Cisplatin-induced genotoxic effects and endogenous glutathione levels in mice bearing ascites Dalton's lymphoma

cis-Diaminedichloroplatinum(II), commonly known as cisplatin, treatment of mice for 24-96, 30 h and 10 days caused the development of chromosomal aberrations in bone marrow cells as well as in Dalton's lymphoma (DL) cells, micronuclei (MN) in bone marrow cells and abnormalities in sperm heads, and it indicates the genotoxic potential of cisplatin in the host. Cisplatin exerts differential effects on the chromosomes of the bone marrow and tumor cells. Combination treatment of cisplatin with L-buthionine(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, enhanced these cisplatin-induced genotoxic effects, but supplementing glutathione level with cysteine, its precursor, reduced the cisplatin-induced genotoxicity. The reduction in cellular glutathione level may facilitate increased intracellular accumulation and binding of drug to DNA to enhance the frequency of genotoxicity parameters. These findings support the possible involvement of glutathione as an important intracellular protective agent and suggest that differences in its levels may be one of the factors in the varying sensitivity of cells to cisplatin-induced genotoxic effects in the mice bearing ascites Dalton's lymphoma.     

Mutagenic effects of carbosulfan,a carbamate pesticide

The genotoxic effects of carbosulfan were evaluated using chromosome aberration (CA), bone marrow micronucleus (MN) and sperm abnormality assays in mice. All the three acute doses (1.25, 2.5 and 5mg/kg) of carbosulfan induced significant dose-dependent increase in the frequency of CA (P<0.02), micronucleated polychromatic erythrocytes (PCEs) (P<0.05) and sperm head abnormalities (P<0.05) but did not affect the total sperm count. The highest acute dose of carbosulfan induced >7-fold increase in the frequency of CA, >3.5-fold increase in the frequency of micronucleated PCEs and >4.6-fold increase in the frequency of sperms with abnormal head morphology following intraperitoneal exposure as compared to the untreated controls. The present findings suggest that carbosulfan is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.     

Synthesis,Characterization, and Biological Activities of Pendant Arm‐Pyridyltetrazole Copper(II) Complexes: DNA Binding/Cleavage Activity and Cytotoxic Studies

2‐(1H‐Tetrazol‐5‐yl)pyridine ( L ) has been reacted separately with Me₂NCH₂CH₂Cl?HCl and ClCH₂CH₂OH to yield two regioisomers in each case, N,N‐dimethyl‐2‐[5‐(pyridin‐2‐yl)‐1H‐tetrazol‐1‐yl]ethanamine ( L1 )/N,N‐dimethyl‐2‐[5‐(pyridin‐2‐yl)‐2H‐tetrazol‐2‐yl]ethanamine ( L2 ) and 2‐[5‐(pyridin‐2‐yl)‐1H‐tetrazol‐1‐yl]ethanol ( L3 )/2‐[5‐(pyridin‐2‐yl)‐2H‐tetrazol‐2‐yl]ethanol ( L4 ), respectively. These ligands, L1 – L4 , have been coordinated with CuCl₂?H₂O in 1 : 1 composition to furnish the corresponding complexes 1 – 4 . EPR Spectra of Cu complexes 1 and 3 were characteristic of square planar geometry, with nuclear hyperfine spin 3/2. Single X‐ray crystallographic studies of 3 revealed that the Cu center has a square planar structure. DNA binding studies were carried out by UV/VIS absorption; viscosity and thermal denaturation studies revealed that each of these complexes are avid binders of calf thymus DNA. Investigation of nucleolytic cleavage activities of the complexes was carried out on double‐stranded pBR322 circular plasmid DNA by using a gel electrophoresis experiment under various conditions, where cleavage of DNA takes place by oxidative free‐radical mechanism (OH ^? ). In vitro anticancer activities of the complexes against MCF‐7 (human breast adenocarcinoma) cells revealed that the complexes inhibit the growth of cancer cells. The IC₅₀ values of the complexes showed that Cu complexes exhibit comparable cytotoxic activities compared to the standard drug cisplatin.