Mukesh Chourasia

Synthesis, DNA-binding ability and anticancer activity of benzothiazole/benzoxazole-pyrrolo[2,1-c][1,4]benzodiazepine conjugates

A series of benzothiazole and benzoxazole linked pyrrolobenzodiazepine conjugates attached through different alkane or alkylamide spacers was prepared. Their anticancer activity, DNA thermal denaturation studies, restriction endonuclease digestion assay and flow cytometric analysis in human melanoma cell line (A375) were investigated. One of the compounds of the series 17d showed significant anticancer activity with promising DNA-binding ability and apoptosis caused G0/G1 phase arrest at sub-micromolar concentrations. To ascertain the binding mode and understand the structural requirement of DNA binding interaction, molecular docking studies using GOLD program and more rigorous 2 ns molecular dynamic simulations using Molecular Mechanics-Poisson-Boltzman Surface Area (MM-PBSA) approach including the explicit solvent were carried out. Further, the compound 17d was evaluated for in vivo efficacy studies in human colon cancer HT29 xenograft mice.

Remarkable enhancement in the DNA-binding ability of C2-fluoro substituted pyrrolo(2,1-c)(1,4)benzodiazepines and their anticancer potential

C2-Fluoro substituted DC-81, and its dimers that comprise of two C2-fluoro substituted DC-81 subunits tethered to their C8-position through simple alkane spacers as well as piperazine moiety side-armed with symmetrical alkyloxy spacers have been designed and synthesized. These fluoro substituted pyrrolo[2,1-c][1,4]benzodiazepines have shown remarkable DNA-binding ability and most of them possess promising anticancer activity, having GI(50) values in micromolar to nanomolar concentration range. DNA thermal denaturation studies show that some of these compounds (14a-c and 15) increase the DeltaT(m) values in the range of 28.9-38 degrees C, and this is further confirmed by the restriction endonuclease studies. This study illustrates the importance of introducing fluoro substitution at the C2-position apart from the incorporation of a piperazine ring in between the alkyloxy linker for enhancement of the DNA-binding ability in comparison to DSB-120 and SJG-136 (DeltaT(m)=10.2 and 25.7 degrees C). Moreover, the variations in the DNA-binding ability with respect to fluoro substitution in this class of dimers has been investigated by molecular modeling studies. Some representative C2-fluoro substituted dimers (8a and 14a) have also exhibited significant anticancer activity in the 60 cancer cell line assay of the National Cancer Institute (NCI).

Comparison of computational methods to model DNA minor groove binders.

There has been a profound interest in designing small molecules that interact in sequence-selective fashion with DNA minor grooves. However, most in silico approaches have not been parametrized for DNA ligand interaction. In this regard, a systematic computational analysis of 57 available PDB structures of noncovalent DNA minor groove binders has been undertaken. The study starts with a rigorous benchmarking of GOLD, GLIDE, CDOCKER, and AUTODOCK docking protocols followed by developing QSSR models and finally molecular dynamics simulations. In GOLD and GLIDE, the orientation of the best score pose is closer to the lowest rmsd pose, and the deviation in the conformation of various poses is also smaller compared to other docking protocols. Efficient QSSR models were developed with constitutional, topological, and quantum chemical descriptors on the basis of B3LYP/6-31G* optimized geometries, and with this ΔT(m) values of 46 ligands were predicted. Molecular dynamics simulations of the 14 DNA-ligand complexes with Amber 8.0 show that the complexes are stable in aqueous conditions and do not undergo noticeable fluctuations during the 5 ns production run, with respect to their initial placement in the minor groove region.

A single-site mutation (F429H) converts the enzyme CYP 2B4 into a heme oxygenase: a QM/MM study.

The intriguing deactivation of the cytochrome P450 (CYP) 2B4 enzyme induced by mutation of a single residue, Phe429 to His, is explored by quantum mechanical/molecular mechanical calculations of the O-OH bond activation of the (Fe(3+)OOH)(-) intermediate. It is found that the F429H mutant of CYP 2B4 undergoes homolytic instead of heterolytic O-OH bond cleavage. Thus, the mutant acquires the following characteristics of a heme oxygenase enzyme: (a) donation by His429 of an additional NH---S H-bond to the cysteine ligand combined with the presence of the substrate retards the heterolytic cleavage and gives rise to homolytic O-OH cleavage, and (b) the Thr302/water cluster orients nascent OH(•) and ensures efficient meso hydroxylation.

Carbazole–pyrrolo[2,1-c][1,4]benzodiazepine conjugates: design, synthesis, and biological evaluation

A series of carbazole–pyrrolobenzodiazepine conjugates (4a–g and 5a–f) have been designed, and synthesized as anticancer agents. These compounds are prepared by linking the C8-position of DC-81 with a carbazole moiety through simple alkane spacers as well as piperazine side-armed alkane spacers in good yields. The DNA binding ability of these conjugates has been determined by thermal denaturation studies and also supported by molecular docking studies. These conjugates showed potent anticancer activity with GI50 ranging from 5.27–0.01 μM. The FACS analysis and BrdU assay of selected conjugates (4c, 4f, 5a and 5f) on MCF-7 cell lines disclosed the increased G1 cell cycle arrest and one of the conjugates 5f has exhibited significant anticancer activity. The analysis of the intrinsic factors involved in causing the G1 arrest in MCF-7 cell lines by 5f conjugate has been demonstrated on the proteins which play a vital role in G1 arrest followed by apoptosis (Cyclin D1, CDK4, c-Jun, JunB, CREB, p53, JNK1/2, procaspase-7, cleaved PARP, pRb, and BAX). Thus, these PBD conjugates (in particular 5f) have promising potency for combating human carcinoma.

CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency

Significance The significance of the results reported is in two areas. (i) Because the cannabinoid receptor type 2 (CB2) agonists seem to be general protective agents, HU-433, a new specific CB2 agonist, may be of major therapeutic importance. (ii) Enantiomers usually have different activity profiles. We report now that HU-433 and its enantiomer HU-308 are both specific CB2 agonists, but whereas HU-433 is much more potent than HU-308 in the rescue of ovariectomy-induced bone loss and ear inflammation, its binding to the CB2 receptor (through which the activity of both enantiomers takes place) is substantially lower compared with HU-308. This situation questions the usefulness of universal radioligands for comparative binding studies. Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ9-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3–4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [3H]CP55,940 displacement and its effect on [35S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [35S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes.

Molecular insights into the differences in anti-inflammatory activities of green tea catechins on IL-1β signaling in rheumatoid arthritis synovial fibroblasts

&NA; In this study, we found that catechins found in green tea (EGCG, EGC, and EC) differentially interfere with the IL‐1&bgr; signaling pathway which regulates the expression of pro‐inflammatory mediators (IL‐6 and IL‐8) and Cox‐2 in primary human rheumatoid arthritis synovial fibroblasts (RASFs). EGCG and EGC inhibited IL‐6, IL‐8, and MMP‐2 production and selectively inhibited Cox‐2 expression. EC did not exhibit any inhibitory effects. When we looked at the expression of key signaling proteins in the IL‐1&bgr; signaling pathway, we found all the tested catechins could inhibit TAK‐1 activity. Therefore, the consumption of green tea offers an overall anti‐inflammatory effect. Molecular docking analysis confirms that EGCG, EGC, and EC all occupy the active site of the TAK1 kinase domain. However, EGCG occupies the majority of the TAK1 active site. In addition to TAK1 inhibition, EGCG can also inhibit P38 and nuclear NF‐&kgr;B expression whereas EC and EGC were not effective inhibitors. Our findings suggest one of the main health benefits associated with the consumption of green tea are due to the activity of EGCG and EGC which are both present at higher amounts. Although EGCG is the most effective catechin at inhibiting downstream inflammatory signaling, its effectiveness could be hindered by the presence of EC. Therefore, varying EC content in green tea may reduce the anti‐inflammatory effects of other potential catechins in green tea. Graphical abstract Figure. No caption available. HighlightsCatechins EC and EGC have anti‐inflammatory properties in synovial fibroblasts.EGCG is more effective at inhibiting downstream IL‐1&bgr; signaling than EGC and EC.EC content may modify green teas anti‐inflammatory effect by competing with EGCG.

HU-446 and HU-465, Derivatives of the Non-psychoactive Cannabinoid Cannabidiol, Decrease the Activation of Encephalitogenic T Cells.

Cannabidiol (CBD), the non‐psychoactive cannabinoid, has been previously shown by us to decrease peripheral inflammation and neuroinflammation in mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Here we have studied the anti‐inflammatory effects of newly synthesized derivatives of natural (−)‐CBD ((−)‐8,9‐dihydro‐7‐hydroxy‐CBD; HU‐446) and of synthetic (+)‐CBD ((+)‐8,9‐dihydro‐7‐hydroxy‐CBD; HU‐465) on activated myelin oligodendrocyte glycoprotein (MOG)35‐55‐specific mouse encephalitogenic T cells (TMOG) driving EAE/MS‐like pathologies. Binding assays followed by molecular modeling revealed that HU‐446 has negligible affinity toward the cannabinoid CB1 and CB2 receptors while HU‐465 binds to both CB1 and CB2 receptors at the high nanomolar concentrations (Ki = 76.7 ± 5.8 nm and 12.1 ± 2.3 nm, respectively). Both, HU‐446 and HU‐465, at 5 and 10 μm (but not at 0.1 and 1 μm), inhibited the MOG35‐55‐induced proliferation of autoreactive TMOG cells via CB1/CB2 receptor independent mechanisms. Moreover, both HU‐446 and HU‐465, at 5 and 10 μm, inhibited the release of IL‐17, a key autoimmune cytokine, from MOG35‐55‐stimulated TMOG cells. These results suggest that HU‐446 and HU‐465 have anti‐inflammatory potential in inflammatory and autoimmune diseases.

The Nucleotide, Inhibitor, and Cation Binding Sites of P-type II ATPases

P‐type ATPases constitute a ubiquitous superfamily of cation transport enzymes, responsible for carrying out actions of paramount importance in biology such as ion transport and expulsion of toxic ions from cells. The harmonized toggling of gates in the extra‐ and intracellular domains explain the phenomenon of specific cation binding in selective physiological states. A quantitative understanding of the fundamental aspects of ion transport mechanism and regulation of P‐type ATPases requires detailed knowledge of thermodynamical, structural, and functional properties. Computational studies have made significant contributions to our understanding of biological ion pumps. Various 3D structures of Ca2+‐ATPase between E1 and E2 transition states have given a impetus to the theorists to work on the Na+K+‐ and H+K+‐ATPase to address important questions about their function. The current review delineates the importance of cation, nucleotide, and inhibitor binding domains, with a focus on the therapeutic potential and biological relevance of the three P‐type II ATPases. This will give an insight into the ion selectivity and their conduction across the transmembrane helices of P‐type II ATPases, which may pave the way to a range of fundamental questions about the mechanism and aid in the efforts of structure‐ and analog‐based drug design.

Regulation of Transforming Growth Factor β-Activated Kinase Activation by Epigallocatechin-3-Gallate in Rheumatoid Arthritis Synovial Fibroblasts: Suppression of K63-Linked Autoubiquitination of Tumor Necrosis Factor Receptor-Associated Factor 6

Transforming growth factor β–activated kinase 1 (TAK1) is a key MAPKKK family protein in interleukin‐1β (IL‐1β), tumor necrosis factor (TNF), and Toll‐like receptor signaling. This study was undertaken to examine the posttranslational modification of TAK1 and its therapeutic regulation in rheumatoid arthritis (RA).