Prashant Joshi

Total synthesis and anti-cholinesterase activity of marine-derived bis-indole alkaloid fascaplysin

A short and efficient two-step total synthesis of marine-derived bis-indole alkaloid fascaplysin starting from commercially available tryptamine in 68% overall yield is reported. A key step involved in the present strategy is tandem dehydrative condensation between ortho-halo substituted glyoxal with tryptamine followed by dehydrogenation. Fascaplysin inhibited acetylcholinesterase (AChE) in non-competitive manner with IC50 and ki values of 1.49 and 2.28 μM respectively and with 60-fold selectivity for AChE versus butyrylcholinesterase. Molecular docking studies revealed that fascaplysin accommodates within a peripheral anionic site and inner linings of the AChE active site gorge.

Discovery of a marine-derived bis-indole alkaloid fascaplysin, as a new class of potent P-glycoprotein inducer and establishment of its structure-activity relationship.

The screening of IIIM natural products repository for P-gp modulatory activity in P-gp over-expressing human adenocarcinoma LS-180 cells led to the identification of 7 natural products viz. withaferin, podophyllotoxin, 3-demethylcolchicine, agnuside, reserpine, seseberecine and fascaplysin as P-gp inducers. Fascaplysin (6a), a marine-derived bis-indole alkaloid, was the most potent among all of them, showing induction of P-gp with EC50 value of 25 nM. P-gp induction is one of the recently targeted strategy to increase amyloid-β clearance from Alzheimer brains. Thus, we pursued a medicinal chemistry of fascaplysin to establish its structure-activity relationship for P-gp induction activity. Four series of analogs viz. substituted quaternary fascaplysin analogs, D-ring opened quaternary analogs, D-ring opened non-quaternary analogs, and β-carbolinium analogs were synthesized and screened for P-gp induction activity. Among the total of 48 analogs screened, only quaternary nitrogen containing analogs 6a-g and 10a, 10h-l displayed promising P-gp induction activity; whereas non-planar non-quaternary analogs 9a-m, 13a-n, 15a-h were devoid of this activity. The P-gp induction activity of best compounds was then confirmed by western-blot analysis, which indicated that fascaplysin (6a) along with 4,5-difluoro analog of fascaplysin 6f and D-ring opened analog 10j displayed 4-8 fold increase in P-gp expression in LS-180 cells at 1 μM. Additionally, compounds 6a and 6f also showed inhibition of acetylcholinestease (AChE), an enzyme responsible for neuronal loss in Alzheimers disease. Thus, fascaplysin and its analogs showing promising P-gp induction along with AChE inhibition at 1 μM, with good safety window (LS-180: IC50 > 10 μM, hGF: 4 μM), clearly indicates their promise for development as an anti-Alzheimer agent.

Natural alkaloids as P-gp inhibitors for multidrug resistance reversal in cancer

The biggest challenge associated with cancer chemotherapy is the development of cross multi-drug resistance to almost all anti-cancer agents upon chronic treatment. The major contributing factor for this resistance is efflux of the drugs by the p-glycoprotein pump. Over the years, inhibitors of this pump have been discovered to administer them in combination with chemotherapeutic agents. The clinical failure of first and second generation P-gp inhibitors (such as verapamil and cyclosporine analogs) has led to the discovery of third generation potent P-gp inhibitors (tariquidar, zosuquidar, laniquidar). Most of these inhibitors are nitrogenous compounds and recently a natural alkaloid CBT-01® (tetrandrine) has advanced to the clinical phase. CBT-01 demonstrated positive results in Phase-I study in combination with paclitaxel, which warranted conducting its Phase II/III trial. Apart from this, there exist a large number of natural alkaloids possessing potent inhibition of P-gp efflux pump and other related pumps responsible for the development of resistance. Despite the extensive contribution of alkaloids in this area, has never been reviewed. The present review provides a comprehensive account on natural alkaloids possessing P-gp inhibition activity and their potential for multidrug resistance reversal in cancer.

6-Aryl substituted 4-(4-cyanomethyl) phenylamino quinazolines as a new class of isoform-selective PI3K-alpha inhibitors

Isoform-selective inhibition of PI3K-α has been identified as one of the important strategy to discover effective and safer anticancer agents. Herein, we report discovery of quinazoline as a new chemotype for isoform-selective PI3K-α inhibitors. The indolyl substituted quinazoline 9u displayed selective inhibition of PI3K-α with IC50 value of 0.201xa0μM with >49.7 over PI3K-β, and δ-isoforms. Quinazoline 9u also inhibited PI3K-γ with IC50 value of 0.750 μM (3.7 fold selective for α-versus γ-isoform). The isoform-selective inhibition was also demonstrated at protein-expression level by western-blot analysis in MCF-7 and PC-3xa0cells. The isoform-selective inhibitor 9u also showed inhibition of phospho-Akt levels in these cells. Quinazoline 9u showed in-vitro cytotoxicity in MCF-7xa0cells with GI50 of 7xa0μM, which was highly selective for cancer cells, as it was non-toxic to normal cells fR2, HEK293 and hGF (GI50xa0>xa050xa0μM). Compound 9u at 25xa0mg/kg dose showed 62 and 37% TGI in Ehrlich Ascites Carcinoma and Ehrlich Solid Tumor mice models. In nutshell, our efforts to identify potent and efficacious PI3K inhibitors resulted in the discovery of a new class of isoform-selective PI3K-α inhibitors possessing promising in-vivo anticancer activity.

Meridianin derivatives as potent Dyrk1A inhibitors and neuroprotective agents.

Meridianins are a group of marine-derived indole alkaloids which are reported to possess kinase inhibitory activities. In the present Letter, we report synthesis of N1-substituted and C-ring modified meridianin derivatives and their evaluation as Dyrk1A inhibitors and neuroprotective agents. Among the library of 52 compounds screened, morpholinoyl linked derivative 26b and 2-nitro-4-trifluoromethyl phenyl sulfonyl derivative 29v displayed potent inhibition of Dyrk1A with IC50 values of 0.5 and 0.53 μM, respectively. The derivative 26b also inhibited Dyrk2 and Dyrk3 with IC50 values of 1.4 and 2.2 μM, respectively showing 2.2 and 4.4 fold selectivity for Dyrk1A with respect to Dyrk2 and Dyrk3. The compound 26b was not cytotoxic to human neuroblastoma SH-SY5Y cells (IC50>100 μM) and it displayed significant neuroprotection against glutamate-induced neurotoxicity in these cells at 10 μM. Molecular modelling studies of compound 26b led to identification of key interactions in the binding site of Dyrk1A and the possible reasons for observed Dyrk1A selectivity over Dyrk2.

Osthol and curcumin as inhibitors of human Pgp and multidrug efflux pumps of Staphylococcus aureus: reversing the resistance against frontline antibacterial drugs

The in-house IIIM natural product repository of 302 small molecules was screened for their ability to inhibit P-glycoprotein (Pgp) in Pgp-overexpressing human adenocarcinoma LS-180 cells. The screening has identified 13 natural products displaying significant Pgp-inhibition activity, which include praeruptorin B, curcumin, imperatorin, osthol, 5,7-diacetoxy-8-(3-methyl-2-butenyl)-coumarin, 5,7-dihydroxy-8-(3-methyl-2-butenyl) coumarin, pongamol, phellopterin, tangeretin, 3-(2-methyl but-3-en-2-yl) xanthyletin, 7-demethyl osthol, allorottlerin and tetrahydroangeolide. These natural products were then screened for their effects on bacterial efflux pump inhibition activity against NorA (Staphylococcus aureus), MdeA (S. aureus Mupr-1), TetK (S. aureus SA-K2192), and MsrA (S. aureus SA-K2191) efflux pumps. Curcumin and osthol showed significant inhibition of the S. aureus NorA efflux pump with 8- and 4-fold reductions in the MIC of ciprofloxacin at 25 μM. The molecular docking studies of curcumin and osthol with the human Pgp and S. aureus NorA efflux pump identified plausible binding modes and binding sites for these natural products.

Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization

The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G0/G1 phase of the cell cycle, the block occurs primarily at the G2/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC50 <1u2009μM. The pharmacokinetic study in BALB/c mice indicated good plasma exposure after intravenous administration. It was found to be efficacious at 1/10th the maximum-tolerated dose (1000u2009mg/kg) against human tumours derived from HCT-116 (colon) and NCI-H460 (lung) cells in SCID (severe-combined immunodeficient) mice models. BPT is a relatively better anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G0/G1. Its ability to effectively halt tumour growth in human tumour-bearing mice would suggest that BPT has the potential to be a candidate for further clinical development.

Identification of ZINC02765569: a potent inhibitor of PTP1B by vHTS

The present study describes identification of a novel lead molecule ZINC02765569 for inhibition of protein tyrosine phosphatase 1B (PTP1B) enzyme by a high-throughput virtual screening of Zinc database against catalytic domain of PTP1B employing docking algorithm Glide. The identified hit molecule ZINC02765569 was synthesized and evaluated for in vitro PTP1B enzyme inhibition, in vitro cellular glucose uptake assay, and animal models of hyperglycemia. ZINC02765569 shows promising inhibition of PTP1B enzyme at 10xa0μm assay, positively up-regulate the cellular glucose uptake in skeletal cell muscle myotubes and SLM/STZ hyperglycemic animal experiments. The novel hit reported here should provide a platform for the further development of its analogs as potential PTP1B enzyme inhibitors.

Biphenyl urea derivatives as selective CYP1B1 inhibitors

Highly selective CYP1B1 inhibitors have potential in the treatment of hormone-induced breast and prostate cancers. Mimicry of potent and selective CYP1B1 inhibitors, α-naphthoflavone and stilbenes, revealed that two sets of hydrophobic clusters suitably linked via a polar linker could be implanted into a new scaffold biphenyl ureas to create potentially a new class of CYP1B1 inhibitors. A series of sixteen biphenyl ureas were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes™, yeast-derived recombinant microsomal enzymes. The most active human CYP1B1 inhibitors were further studied for their selectivity against human CYP1A1, CYP1A2, CYP3A4 and CYP2D6 enzymes. The meta-chloro-substituted biphenyl urea 5h was the most potent inhibitor of CYP1B1 with IC50 value of 5 nM. It displayed excellent selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 (IC50 >10 μM in the four CYP assays, indicating >2000-fold selectivity). Similarly, two methoxy-substituted biphenyl ureas 5d and 5e also displayed potent and selective inhibition of CYP1B1 with IC50 values of 69 and 58 nM, respectively, showing >62 and >98-fold selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 enzymes. In order to probe if the relatively insoluble biphenyl ureas were cell permeable and if they could at all be used for future cellular studies, their CYP1B1 inhibition was investigated in live recombinant human and yeast cells. Compound 5d displayed the most potent inhibition with IC50s of 20 nM and 235 nM, respectively, in the two cell-based assays. The most potent and selective CYP1B1 inhibitor (compound 5h) from Sacchrosomes, also displayed potent inhibition in live cell assays. Molecular modeling was performed to understand the trends in potency and selectivity observed in the panel of five CYP isoenzymes used for the in vitro studies.

Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic chalcones: Overcoming cisplatin resistance in CYP1B1-overexpressing lines

The structure of alpha-napthoflavone (ANF), a potent inhibitor of CYP1A1 and CYP1B1, mimics the structure of chalcones. Two potent CYP1B1 inhibitors 7k (DMU2105) and 6j (DMU2139) have been identified from two series of synthetic pyridylchalcones. They inhibit human CYP1B1 enzyme bound to yeast-derived microsomes (Sacchrosomes™) with IC50 values of 10 and 9xa0nM, respectively, and show a very high level of selectivity towards CYP1B1 with respect to the IC50 values obtained with CYP1A1, CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19 Sacchrosomes™. Both compounds also potently inhibit CYP1B1 expressed within live recombinant yeast and human HEK293 kidney cells with IC50 values of 63, 65, and 4, 4xa0nM, respectively. Furthermore, the synthesized pyridylchalcones possess better solubility and lipophilicity values than ANF. Both compounds overcome cisplatin-resistance in HEK293 and A2780xa0cells which results from CYP1B1 overexpression. These potent cell-permeable and water-soluble CYP1B1 inhibitors are likely to have useful roles in the treatment of cancer, glaucoma, ischemia and obesity.