Chandan Kishor

Design and synthesis of pyrazole-oxindole conjugates targeting tubulin polymerization as new anticancer agents.

A series of twenty one compounds with pyrazole and oxindole conjugates were synthesized by Knoevenagel condensation and investigated for their antiproliferative activity on different human cancer cell lines. The conjugates are comprised of a four ring scaffold; the structural isomers 12b and 12c possess chloro-substitution in the D ring. Among the congeners 12b, 12c, and 12d manifested significant cytotoxicity and inhibited tubulin assembly. Treatments with 12b, 12c and 12d resulted in accumulation of cells in G2/M phase, disruption of microtubule network, and increase in cyclin B1 protein. Zebrafish screening revealed that 12b, and 12d caused developmental defects. Docking analysis demonstrated that the congeners occupy the colchicine binding pocket of tubulin.

Synthesis of chalcone-amidobenzothiazole conjugates as antimitotic and apoptotic inducing agents

A series of chalcone-amidobenzothiazole conjugates (9a-k and 10a,b) have been synthesized and evaluated for their anticancer activity. All these compounds exhibited potent activity and the IC(50) of two potential compounds (9a and 9f) against different cancer cell lines are in the range of 0.85-3.3 μM. Flow cytometric analysis revealed that these compounds induced cell cycle arrest at G2/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay (IC(50) of 9a is 3.5 μM and 9f is 5.2 μM) and immuofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Annexin staining also suggested that these compounds induced cell death by apoptosis. Moreover, docking experiments have shown that they interact and bind efficiently with tubulin protein. Overall, the current study demonstrates that the synthesis of chalcone-amidobenzothiazole conjugates as promising anticancer agents with potent G2/M arrest and apoptotic-inducing activities via targeting tubulin.

3‐Substituted 2‐Phenylimidazo[2,1‐b]benzothiazoles: Synthesis, Anticancer Activity, and Inhibition of Tubulin Polymerization

A new series of 3‐substituted 2‐phenylimidazo[2,1‐b]benzothiazoles (3 a–h) were synthesized by C‐arylation of 2‐arylimidazo[2,1‐b]benzothiazoles using palladium acetate as catalyst, and the resulting compounds were evaluated for their anticancer activity. Compounds 3 a, 3 e, and 3 h exhibited good antiproliferative activity, with GI50 values in the range of 0.19–83.1 μM. Compound 3 h showed potent anticancer efficacy against 60 human cancer cell lines, with a mean GI50 value of 0.88 μM. This compound also induced cell‐cycle arrest in the G2/M phase and inhibited tubulin polymerization followed by activation of caspase‐3 and apoptosis. A high‐throughput tubulin polymerization assay showed that the level of inhibition for compound 3 h is similar to that of combretastatin A‐4. Molecular modeling studies provided a molecular basis for the favorable binding of compounds 3 a, 3 e, and 3 h to the colchicine binding pocket of tubulin.

Synthesis and biological evaluation of combretastatin-amidobenzothiazole conjugates as potential anticancer agents

A series of combretastatin-amidobenzothiazole conjugates have been synthesized and evaluated for their anticancer activity. All these compounds exhibited significant anticancer activity and the most potent compound (11a) showed GI(50) values ranging 0.019-11 μM. Biological studies such as cell cycle distribution, effect on tubulin polymerization and effect on ERK signalling pathway have been examined in MCF-7 cell line. FACS analysis revealed that these compounds induced cell cycle arrest at G2/M phase. Compound 11a showed significant effect on tubulin polymerization and affected the ERK signalling pathway that result in the decreased levels of ERK1/2, p-ERK and c-Jun proteins. Docking experiments have shown that the active molecules interact and bind well in the ATP binding pocket of ERK protein.

Synthesis and structure–activity relationships of pyridinyl-1H-1,2,3-triazolyldihydroisoxazoles as potent inhibitors of tubulin polymerization

Three series of compounds; pyridinyl-1H-1,2,3-triazoles, pyridinyl-1H-1,2,3-triazolylisoxazoles and pyridinyl-1H-1,2,3-triazolyldihydroisoxazoles with TMP moiety were designed, synthesized and screened for their anti-cancer and anti-tubulin properties. By sequentially designing three series of compounds comprising of dihydroisoxazole in the linker, a small substituent like chlorine on one side (R(1)) and aromatic group (R) on the pyridine ring, we have optimized the anti-cancer as well as anti-tubulin activity. Pyridinyl-1H-1,2,3-triazolyldihydroisoxazoles 28b and 28c were found to be potent anti-cancer agents against all the cell lines tested with a concomitant accumulation of cells in the G2/M phase of the cell cycle. Molecular modeling suggests that the trimethoxyphenyl ring in 28b and 28c occupies the cholchicine binding domain of β-tubulin, whereas, the dihydroisoxazole extends towards the interface of α,β-tubulin.

Identification, Biochemical and Structural Evaluation of Species-Specific Inhibitors against Type I Methionine Aminopeptidases

Methionine aminopeptidases (MetAPs) are essential enzymes that make them good drug targets in cancer and microbial infections. MetAPs remove the initiator methionine from newly synthesized peptides in every living cell. MetAPs are broadly divided into type I and type II classes. Both prokaryotes and eukaryotes contain type I MetAPs, while eukaryotes have additional type II MetAP enzyme. Although several inhibitors have been reported against type I enzymes, subclass specificity is scarce. Here, using the fine differences in the entrance of the active sites of MetAPs from Mycobacterium tuberculosis , Enterococcus faecalis , and human, three hotspots have been identified and pyridinylpyrimidine-based molecules were selected from a commercial source to target these hotspots. In the biochemical evaluation, many of the 38 compounds displayed differential behavior against these three enzymes. Crystal structures of four selected inhibitors in complex with human MetAP1b and molecular modeling studies provided the basis for the binding specificity.

Glu121-Lys319 salt bridge between catalytic and N-terminal domains is pivotal for the activity and stability of Escherichia coli aminopeptidase N.

Escherichia coli aminopeptidase N (ePepN) belongs to the gluzincin family of M1 class metalloproteases that share a common primary structure with consensus zinc binding motif (HEXXH‐(X18)‐E) and an exopeptidase motif (GXMEN) in the active site. There is one amino acid, E121 in Domain I that blocks the extended active site grove of the thermolysin like catalytic domain (Domain II) limiting the substrate to S1 pocket. E121 forms a part of the S1 pocket, while making critical contact with the amino‐terminus of the substrate. In addition, the carboxylate of E121 forms a salt bridge with K319 in Domain II. Both these residues are absolutely conserved in ePepN homologs. Analogous Glu‐Asn pair in tricon interacting factor F3 (F3) and Gln‐Asn pair in human leukotriene A4 hydrolase (LTA4H) are also conserved in respective homologs. Mutation of either of these residues individually or together substantially reduced or entirely eliminated enzymatic activity. In addition, thermal denaturation studies suggest that the mutation at K319 destabilizes the protein as much as by 3.7°C, while E121 mutants were insensitive. Crystal structure of E121Q mutant reveals that the enzyme is inactive due to the reduced S1 subsite volume. Together, data presented here suggests that ePepN, F3, and LTA4H homologs adopted a divergent evolution that includes E121‐K319 or its analogous pairs, and these cannot be interchanged.

Synthesis of tetrazole–isoxazoline hybrids as a new class of tubulin polymerization inhibitors

A new series of tetrazole based isoxazolines (4a–l) was synthesized and evaluated for their anticancer potential against two cancer cell lines. All these compounds exhibited profound cytotoxicity with IC50 values ranging from 1.22 to 3.62 μM and compounds 4h, 4i showed prominent anticancer efficacy with IC50 values of 1.51, 1.49 μM in A549 and 2.83, 2.40 μM in MDA-MB-231 cell lines. Further, these compounds (4h, 4i) induced apoptotic cell death by inhibition of tubulin polymerization leading to cell cycle arrest at G2/M phase of the cell cycle followed by caspase-3 activity. Moreover, the level of tubulin inhibition by these compounds was examined by in vitro HTS tubulin polymerization assay. Docking of compound 4h and 4i to the active site of tubulin revealed that the trimethoxy ring of the compounds occupies the colchicine binding site of tubulin, whereas the isoxazoline moiety moves towards the interface of α–β tubulin and involves a series of hydrogen bonds with αTyr224 and αSer178.

Synthesis, cytotoxicity and hDHFR inhibition studies of 2H-pyrido[1,2-a]pyrimidin-2-ones

Synthesis of the titled scaffolds was achieved by the condensation of Baylis–Hillman acetates with 2-aminopyridines under solvent-free conditions. Resulting compounds were evaluated for anticancer activity against five different cancer cell lines. Compounds 3c–g displayed low-micromolar inhibition with IC50 values ranging from 0.86 to 0.94 μM, and 3b, 3h, 3i and 3j between 8.6 and 9.8 μM against a neuroblastoma cell line (SK-n-SH). 3b, 3i and 3j inhibited the proliferation of breast cancer cells (MCF-7) at 10 μM. hDHFR inhibitory studies produced IC50 values of 2.7 and 3.1 μM for 3i and 3j, and 8.7 μM for 3o. Molecular docking studies established the mode of binding of these compounds into the methotrexate binding pocket of hDHFR. Structure–activity relationship studies indicate a clear preference for some substitutions over others.

Discovery of a New Genetic Variant of Methionine Aminopeptidase from Streptococci with Possible Post-Translational Modifications: Biochemical and Structural Characterization

Protein N-terminal methionine excision is an essential co-translational process that occurs in the cytoplasm of all organisms. About 60-70% of the newly synthesized proteins undergo this modification. Enzyme responsible for the removal of initiator methionine is methionine aminopeptidase (MetAP), which is a dinuclear metalloprotease. This protein is conserved through all forms of life from bacteria to human except viruses. MetAP is classified into two isoforms, Type I and II. Removal of the map gene or chemical inhibition is lethal to bacteria and to human cell lines, suggesting that MetAP could be a good drug target. In the present study we describe the discovery of a new genetic variant of the Type I MetAP that is present predominantly in the streptococci bacteria. There are two inserts (insert one: 27 amino acids and insert two: four residues) within the catalytic domain. Possible glycosylation and phosphorylation posttranslational modification sites are identified in the ‘insert one’. Biochemical characterization suggests that this enzyme behaves similar to other MetAPs in terms of substrate specificity. Crystal structure Type Ia MetAP from Streptococcus pneumoniae (SpMetAP1a) revealed that it contains two molecules in the asymmetric unit and well ordered inserts with structural features that corroborate the possible posttranslational modification. Both the new inserts found in the SpMetAP1a structurally align with the P-X-X-P motif found in the M. tuberculosis and human Type I MetAPs as well as the 60 amino acid insert in the human Type II enzyme suggesting possible common function. In addition, one of the β-hairpins within in the catalytic domain undergoes a flip placing a residue which is essential for enzyme activity away from the active site and the β-hairpin loop of this secondary structure in the active site obstructing substrate binding. This is the first example of a MetAP crystallizing in the inactive form.