Benzimidazole derivatives as tubulin polymerization inhibitors: Design, synthesis and in vitro cytotoxicity studies.

A new class of benzimidazole derivatives as tubulin polymerization inhibitors has been designed and synthesized in this study. The in vitro anticancer profile of the developed molecules was reconnoitred on selected human cancer cells. The highest cytotoxicity was illustrated by compounds 7n and 7u with IC50 values ranging from 2.55 to 17.89 µM with specificity toward SK-Mel-28 cells. They displayed 5-fold less cytotoxicity towards normal rat kidney epithelial NRK52E cells, which implies that they are not harmful to normal, healthy cells. The cellular staining procedures like AO/EB, DCFDA, and DAPI were applied to comprehend the inherent mechanism of apoptosis which displayed nuclear and morphological alterations. The Annexin V binding and JC-1 studies were executed to evaluate the extent of apoptosis and the decline in mitochondrial transmembrane potential in SK-Mel-28 cell lines. Compound 7n dose-dependently arrested the G2/M phase of the cell cycle and the target-based outcomes proposed tubulin polymerization inhibition by 7n (IC50 of 5.05±0.13 µM). Computational studies were also conducted on the tubulin protein (PDB ID: 3E22) to investigate the stabilized binding interactions of compounds 7n and 7u with tubulin, respectively.

Synthesis and biological evaluation of 1-phenyl-4,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(1H)-one/thiones as anticancer agents.

Cancer is associated with uncontrolled cell proliferation invading adjoining tissues and organs. Despite the availability of several chemotherapeutic agents, the constant search for newer approaches and drugs is necessitated owing to the ever-growing challenge of resistance. Over the years, DNA has emerged as an important druggable therapeutic drug due to its role in critical cellular processes such as cell division and maintenance. Further, evading apoptosis stands out as a hallmark of cancer. Hence, designing new compounds that would target DNA and induce apoptosis plays an important role in cancer therapy. In the current work, we carried out the synthesis and anticancer evaluation of 1-aryl-4,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(1H)-ones/thiones (26 compounds) against selected human cancer cell lines. Among these, compounds 8ae, 8ad, 8cf, 10ad and Kenpaullone have shown good inhibitory properties against HeLa cells (IC50 < 2 µM) with good selectivity over the non-cancerous human embryonic kidney (Hek293T) cells. In cell cycle analysis, the compounds 8ad and 8cf have exhibited G2/M cell cycle arrest in HeLa cells. In addition, the compounds 8ad and 8cf induced apoptosis in a dose-dependent manner in the Annexin-V FITC staining assay. The DAPI staining clearly demonstrated the condensed and fragmented nuclei in 8ad, 8cf, 8ae and Kenpaullone-treated HeLa cells. In addition, these compounds strongly suppressed the healing after 48 h in in vitro cell migration assay. The DNA binding experiments indicated that compounds 8ae, 8cf, and 8ad as well as Kenpaullone interact with double-stranded DNA by binding in grooves which may interrupt the DNA replication and kill fast-growing cells. Molecular docking studies revealed the binding pose of 8ad and Kenpaullone at HT1 binding pocket of double-stranded DNA. Compounds 8ad and 8cf demonstrated moderate topo II inhibition which could be a possible reason for their anticancer properties. Compounds 8ad and 8cf may cause the topo II and DNA covalent complex, which leads to the inhibition of DNA replication and transcription. This eventually increases the DNA damage in cells and promotes cell apoptosis. With the above interesting biological profile, the new 1-aryl-2,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(4H)-one/thione derivatives have emerged as promising leads for the discovery of new anticancer agents.

Triazolo-linked benzimidazoles as tubulin polymerization inhibitors and DNA intercalators: Design, synthesis, cytotoxicity, and docking studies.

A simple "click" protocol was employed in the quest of synthesizing 1,2,3-triazole-linked benzimidazoles as promising anticancer agents on various human cancer cell lines such as A549, HCT116, SK-Mel-28, HT-29, and MCF-7. Compound 12j demonstrated significant cytotoxic potential towards SK-Mel-28 cancer cells (IC50 : 4.17 ± 0.09 µM) and displayed no cytotoxicity (IC50 : > 100 µM) against normal human BEAS-2B cells inferring its safety towards normal healthy cells. Further to comprehend the underlying apoptosis mechanisms, AO/EB, dichlorodihydrofluorescein diacetate (DCFDA), and 4',6-diamidino-2-phenylindole (DAPI) staining were performed, which revealed the nuclear and morphological alterations. Compound 12j displayed impairment in cellular migration and inhibited colony formation. The annexin V binding assay and JC-1 were implemented to evaluate the scope of apoptosis and the loss of the mitochondrial transmembrane potential in SK-Mel-28 cells. Cell-cycle analysis revealed that compound 12j arrested the cells at the G2/M phase in a dose-dependent manner. Target-based assays established the inhibition of tubulin polymerization by 12j at an IC50 value of 5.65 ± 0.05 µM and its effective binding with circulating tumor DNA as a DNA intercalator. The detailed binding interactions of 12j with tubulin and DNA were examined by docking studies on PDB ID: 3E22 and DNA hexamer (PDB ID: 1NAB), respectively.

Synthesis and cytotoxicity evaluation of DNA-interactive ß-carboline indolyl-3-glyoxamide derivatives: Topo-II inhibition and in silico modelling studies.

In a quest for effective cancer targeted drug therapy, a series of new ß-carboline tethered indole-3-glyoxylamide derivatives, conjoining salient pharmacophoric properties with prominent cytotoxicity, were synthesized. The in vitro cytotoxic ability of the compounds was established, and many of the compounds exhibited remarkable cytotoxicity (IC50 < 10 µM) on human cancer cell lines like HCT116, A549, SK-MEL-28, and MCF7. Precisely, compound 12x expressed the best cytotoxic potential against melanoma cancer cell line (SK-MEL-28) with an IC50 value of 4.37 µM. In addition, cytotoxicity evaluation against normal kidney cell line (NRK52E) entrenched the cytospecificity and selectivity index of 12x. The traditional apoptosis assays advised morphological and nuclear alterations such as apoptotic body formation, condensed/horseshoe-shaped/fragmented nuclei, and generation of ROS. The flow cytometric analysis revealed significant early and slight late-stage induction of apoptosis. The target-based physiochemical assays indicated the ability of compound 12x to bind with DNA and inhibition of Topoisomerase II. Moreover, molecular modeling studies affirm the excellent DNA intercalation potential and stabilized interactions of 12x with DNA base pairs. In silico prediction of physicochemical parameters revealed the promising drug-like properties of the synthesized derivatives.

Exome sequencing and functional analyses revealed CETN1 variants leads to impaired cell division and male fertility.

Human spermatogenesis requires an orchestrated expression of numerous genes in various germ cell subtypes. Therefore, the genetic landscape of male infertility is highly complex. Known genetic factors alone account for at least 15% of male infertility. However, ~40% of infertile men remain undiagnosed and are classified as idiopathic infertile men. We performed exome sequencing in 47 idiopathic infertile men (discovery cohort), followed by replication study (40 variants in 33 genes) in 844 infertile men and 709 controls using Sequenom MassARRAY® based genotyping. We report 17 variants in twelve genes that comprise both previously reported (DNAH8, DNAH17, FISP2 and SPEF2) and novel candidate genes (BRDT, CETN1, CATSPERD, GMCL1, SPATA6, TSSK4, TSKS and ZNF318) for male infertility. The latter have a strong biological nexus to human spermatogenesis and their respective mouse knockouts are concordant with human phenotypes. One candidate gene CETN1, identified in this study, was sequenced in another independent cohort of 840 infertile and 689 fertile men. Further, CETN1 variants were functionally characterized using biophysical and cell biology approaches. We demonstrate that CETN1 variant- p.Met72Thr leads to multipolar cells, fragmented nuclei during mitosis leading to cell death and show significantly perturbed ciliary disassembly dynamics. Whereas CETN1-5' UTR variant; rs367716858 leads to loss of a methylation site and increased reporter gene expression in vitro. We report a total of eight novel candidate genes identified by exome sequencing, which may have diagnostic relevance and can contribute to improved diagnostic workup and clinical management of male infertility.

Design, synthesis of DNA-interactive 4-thiazolidinone-based indolo-/pyrroloazepinone conjugates as potential cytotoxic and topoisomerase I inhibitors.

With the rising cancer incidence and mortality globally, there is a prerequisite for effective design strategies towards the discovery of newer small molecular entities in chemotherapy. Hence, a series of new thiazolidinone-based indolo-/pyrroloazepinone conjugates was designed, synthesized via molecular hybridization, and evaluated for their in vitro cytotoxicity potential and DNA topoisomerase I and II inhibition. Among this series, conjugate 11g emerged as the most active compound with an IC50 value of 1.24 µM against A549 and 3.02-10.91 µM in the other tested cancer cell lines. Gratifyingly, 11g displayed 43-fold higher selectivity towards A549 cancer cells as compared to the non-cancer cells. Subsequently, conjugate 12g also demonstrated significant cytotoxicity against SK-MEL-28 cells. Basing the in vitro cytotoxicity results, SAR was established. Later, the conjugates 11g and 12g were further evaluated for their apoptosis-inducing ability, which was quantified by flow cytometric analysis, DNA-binding, Topo I inhibitory activity and IC50 value calculation. Molecular modeling studies provided profound insights about the binding nature of these compounds with DNA-Topo I complex. In silico ADME/T and prediction studies corroborated the drug-likeness of the two investigated compounds. TOPKAT toxicity profiling studies demonstrated the compounds' safety in many animal models with a minimal toxicological profile. Encouraging results obtained from in vitro and in silico studies could put this series of conjugates at the forefront of cancer drug discovery.

Synthesis of indolo/pyrroloazepinone-oxindoles as potential cytotoxic, DNA-intercalating and Topo I inhibitors.

A series of 17 indolo/pyrroloazepinone-oxindole conjugates was synthesized and evaluated for their antiproliferative activity against a panel of selected human cancer cell lines including A549 (lung cancer), HCT116 (colon cancer), MCF7 (breast cancer), and SK-MEL-28 (melanoma). Among the synthesized molecules (14a-m and 15a-d), compound 14d displayed remarkable activity against A549, HCT116 and SK-MEL-28 cells with IC50 values < 4 µM with the best cytotoxicity and a 13-fold selectivity towards lung cancer cells (IC50 value of 2.33 µM) over the normal rat kidney cells (NRK). Further, 14d-mediated apoptosis affected the cellular and nuclear morphology of the cancer cells in a dose-dependent manner. Wound healing and clonogenic assays inferred the inhibition of cell growth and migration. Target-based studies of compound 14d corroborated its DNA-intercalative capability and Topo I inhibitory activity which have been fortified by molecular modeling studies. Finally, the drug-likeness of the potent compound was determined by performing in silico ADME/T prediction studies.

Conformational scanning of individual EF-hand motifs of calcium sensor protein centrin-1.

Centrin-1, a Ca2+ sensor protein of the centrin family is a crucial player for cell division in eukaryotes and plays a key role in the microtubule organising centre. Despite being regarded as a calcium sensor with a matched structure to calmodulin/troponin C, the protein undergoes mild changes in conformation and binds Ca2+ with moderate affinity. We present an in-depth analysis of the Ca2+ sensing by individual EF-hand motifs of centrin-1 and address unsolved questions of the rationales for moderate affinity and conformational transitions of the protein. Employing the more sensitive approach of Trp scanning of individual EF-hand motif, we have undertaken an exhaustive investigation of Ca2+ binding to individual EF-hand motifs, named EF1 to EF4. All four EF-hand motifs of centrin-1 are structural as all of them bind both Ca2+ and Mg2+. EF1 and EF4 are the most flexible sites as they undergo drastic conformational changes following Ca2+ binding, whereas EF3 responds to Ca2+ minimally. On the other hand, EF2 moves towards the protein surface upon binding Ca2+. The independent filling mode of Ca2+ to EF-hand motifs and lack of intermotif communication explain the lack of cooperativity of binding, thus constraining centrin-1 to a moderate affinity binding protein. Thus, centrin-1 is distinct from other calcium sensors such as calmodulin.

Optimization of purification method and characterization of recombinant human Centrin-1.

Centrins are acidic proteins, present in all eukaryotes to perform imperative roles in centrosome positioning and segregation. Existing methods for the purification of centrins for biophysical studies involves either multiple steps or yields protein with an affinity tag, which pins additional tag-cleavage step. Therefore, we have made an attempt to develop a simple and single step method for protein purification. We have performed categorical evaluation of existing methods, and describe a one-step procedure based on cleavable Intein-tag, which can be utilized for routine preparation of any isoform of centrins. Since human Centrin-1 and Centrin-2 are devoid of Trp, we exploit this feature to assess the purity of the protein using Tyr fluorescence; an essential point ignored generally. In addition, we report important spectral and hydrodynamic characteristics of human Centrin-1, accounting that HsCentrin-1 has moderate affinity for Ca(2+). Centrin-1 does not gain structure as seen by far- and near-UV circular dichroism, rather there is a loss of ellipticity, though inconsiderable upon binding Ca(2+).

Development of new genomic microsatellite markers from robusta coffee (Coffea canephora Pierre ex A. Froehner) showing broad cross-species transferability and utility in genetic studies.

BACKGROUND: Species-specific microsatellite markers are desirable for genetic studies and to harness the potential of MAS-based breeding for genetic improvement. Limited availability of such markers for coffee, one of the most important beverage tree crops, warrants newer efforts to develop additional microsatellite markers that can be effectively deployed in genetic analysis and coffee improvement programs. The present study aimed to develop new coffee-specific SSR markers and validate their utility in analysis of genetic diversity, individualization, linkage mapping, and transferability for use in other related taxa. RESULTS: A small-insert partial genomic library of Coffea canephora, was probed for various SSR motifs following conventional approach of Southern hybridisation. Characterization of repeat positive clones revealed a very high abundance of DNRs (1/15 Kb) over TNRs (1/406 kb). The relative frequencies of different DNRs were found as AT >> AG > AC, whereas among TNRs, AGC was the most abundant repeat. The SSR positive sequences were used to design 58 primer pairs of which 44 pairs could be validated as single locus markers using a panel of arabica and robusta genotypes. The analysis revealed an average of 3.3 and 3.78 alleles and 0.49 and 0.62 PIC per marker for the tested arabicas and robustas, respectively. It also revealed a high cumulative PI over all the markers using both sib-based (10-6 and 10-12 for arabicas and robustas respectively) and unbiased corrected estimates (10-20 and 10-43 for arabicas and robustas respectively). The markers were tested for Hardy-Weinberg equilibrium, linkage dis-equilibrium, and were successfully used to ascertain generic diversity/affinities in the tested germplasm (cultivated as well as species). Nine markers could be mapped on robusta linkage map. Importantly, the markers showed ~92% transferability across related species/genera of coffee. CONCLUSION: The conventional approach of genomic library was successfully employed although with low efficiency to develop a set of 44 new genomic microsatellite markers of coffee. The characterization/validation of new markers demonstrated them to be highly informative, and useful for genetic studies namely, genetic diversity in coffee germplasm, individualization/bar-coding for germplasm protection, linkage mapping, taxonomic studies, and use as conserved orthologous sets across secondary genepool of coffee. Further, the relative frequency and distribution of different SSR motifs in coffee genome indicated coffee genome to be relatively poor in microsatellites compared to other plant species.