Sudhakar Manda

Synthesis, antileishmanial and antitrypanosomal activities of N-substituted tetrahydro-β-carbolines.

A series of N-substituted tetrahydro-β-carbolines were synthesized and screened for antileishmanial activity through an in vitro assay that involves promastigotes and axenic amastigotes of Leishmania donovani, the causative agent for visceral leishmaniasis. The thiophen-2-yl analogs 9b and 11f and naphthyl analog 11h were found to show significant activity against promastigotes with IC50 values of 12.7, 9.1 and 22.1 μM, respectively. Analogs 9b and 11h were also effective against axenic amastigotes with IC50 values of 62.8 and 87.6 μM, respectively. The antileishmanial activity of analogs was then tested in human macrophage cell line infected with L. donovani amastigotes and 2-naphthyl linked analog 11h was found to be effective with IC50 value of 28.3 μM. Several analogs also displayed antitrypanosomal activity against Trypanosoma brucei, the causative agent for human African trypanosomiasis. Compounds 11e, 11f and 11h were more effective than others with IC50 values of 1.0, 8.9 and 10.2 μM, respectively. All synthesized analogs were not cytotoxic towards mammalian cell lines including Vero (monkey kidney fibroblasts), HEPG2 (human hepatoma cells), LLC-PK1 (pig kidney epithelial cells) and THP-1 (human macrophages).

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.

Fascaplysin Induces Caspase Mediated Crosstalk Between Apoptosis and Autophagy Through the Inhibition of PI3K/AKT/mTOR Signaling Cascade in Human Leukemia HL-60 Cells

In this study, we for the first time explored the cellular and molecular mechanism of anticancer properties of fascaplysin, a marine sponge‐derived alkaloid. Our study demonstrated that fascaplysin induced a cooperative interaction between apoptotic and autophagic pathways to induce cytotoxicity in HL‐60 cells. Fascaplysin treatment not only activated pro‐apoptotic events like PARP‐1 cleavage and caspase activation but also triggered autophagy signaling as shown by the increased expression of LC3‐II, ATG7and beclin. Interestingly, it was found that use of pan‐caspase inhibitor completely reversed the fascaplysin mediated cell death as analyzed by MTT and cell cycle assays. It was observed that cell death as well as the expression of pro‐death proteins was partially reversed, when key autophagy mediators ATG7 was silenced by siRNA in fascaplysin treated cells. Cooperative involvement of autophagy and apoptotic signaling in cytotoxicity was confirmed when combined silencing of pro‐apototic (PARP‐1) and autophagic (ATG‐7) signaling by respective siRNAs lead to substantial rescue of cell death induced by fascaplysin. Although, apoptosis and autophagy are two independent cell death pathways, our findings provide detailed insight by which both the pathways acted cooperatively to elicit fascaplysin induced cell death in HL‐60 cells. Our findings provide molecular insight into the anti‐cancer potential of fascaplysin by showing that both autophagic and apoptotic signaling can work together in the induction of cell death. J. Cell. Biochem. 116: 985–997, 2015.

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 <1 μM. 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 (1000 mg/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.

Biphenyl-4-carboxylic Acid [2-(1H-Indol-3-yl)-ethyl]-methylamide (CA224), a Nonplanar Analogue of Fascaplysin, Inhibits Cdk4 and Tubulin Polymerization: Evaluation of in Vitro and in Vivo Anticancer Activity

Biphenyl-4-carboxylic acid-[2-(1H-indol-3-yl)-ethyl]-methylamide 1 (CA224) is a nonplanar analogue of fascaplysin (2) that specifically inhibits Cdk4-cyclin D1 in vitro. Compound 1 blocks the growth of cancer cells at G0/G1 phase of the cell cycle. It also blocks the cell cycle at G2/M phase, which is explained by the fact that it inhibits tubulin polymerization. Additionally, it acts as an enhancer of depolymerization for taxol-stabilized tubulin. Western blot analyses of p53-positive cancer cells treated with compound 1 indicated upregulation of p53, p21, and p27 proteins together with downregulation of cyclin B1 and Cdk1. Compound 1 selectively induces apoptosis of SV40 large T-antigen transformed cells and significantly reduces colony formation efficiency, in a dose-dependent manner, of lung cancer cells. It is efficacious at 1/10th of the MTD against human tumors derived from HCT-116 and NCI-H460 cells in SCID mouse models. The promising efficacy of compound 1 in human xenograft models as well as its excellent therapeutic window indicates its potential for clinical development.

A marine sponge alkaloid derivative 4-chloro fascaplysin inhibits tumor growth and VEGF mediated angiogenesis by disrupting PI3K/Akt/mTOR signaling cascade

Tumor angiogenesis and PI3K/Akt/mTOR pathway are two major molecular objectives for the treatment and management of breast cancer. Here we first time report the molecular mechanism of a marine sponge alkaloid derivative 4-chloro fascapysin (4-CF) for its anticancer and antiangiogenesis potential. It simultaneously targets multiple cancer and angiogenesis dynamics, such as proliferation, chemotaxis cell migration, and invasion, growth factors signaling cascade, autophagy and apoptosis in HUVEC and MDAMB-231 breast cancer cells. It inhibited the VEGF mediated microvessel sprouting and blood vessel formation in the matrigel plug of C57/BL6J mice. It inhibits the tumor growth in ET (solid) mouse tumor model. It significantly inhibited cell survival through PI3K/Akt/mTOR pathway, with attendant effects on key pro-angiogenesis factors like HIF-1α, eNOS and MMP-2/9. The cytotoxicity of 4-CF was reversed by co-treatment with the VEGF and Akt inhibitors sunitinib and perifosine, respectively or by the addition of neutralizing VEGF antibodies. The apoptotic potential of 4-CF was through mitochondrial dependent as illustrated through loss of mitochondrial membrane potential. The safety profile of 4-CF was acceptable as it exhibits five times high cytotoxic IC50 value in normal cells as well as no apparent toxicities in experimental tumor mice at therapeutic doses.

Design, synthesis and P-gp induction activity of aryl phosphonate esters: identification of tetraethyl-2-phenylethene-1,1-diyldiphosphonate as an orally bioavailable P-gp inducer

The clearance of amyloid-beta is mediated by the P-glycoprotein (P-gp) transporter pump located at the blood brain barrier. Therefore, the induction of P-gp has been considered as a potential therapeutic strategy for the treatment of Alzheimers disease. The expression of P-gp is regulated through a nuclear receptor, pregnane X receptor (PXR). Thus, herein we investigated the potential of a known PXR activator, diphosphonate ester SR12813 (6a), for P-gp induction activity and further studied its structure–activity relationship. The diphosphonate ester SR12813 along with three series of analogs, viz. aryl alkylidene, aryl alkynyl, and aryl α-amino phosphonate esters, were synthesized and screened for P-gp induction activity in P-gp overexpressing adenocarcinoma LS180 cells using rhodamine 123 efflux assay. The parent compound SR12813 along with several new analogs displayed P-gp induction activity at 5 μM. Western blot analysis indicated that tetraethyl-2-phenylethene-1,1-diyldiphosphonate (6c) and tetraethyl-2-(anthracene-10-yl)ethene-1,1-diyldiphosphonate (6s) showed 7–8-fold increase in P-gp expression in LS180 cells. The diphosphonate ester 6c displayed excellent aqueous solubility, no cytochrome P450 inhibition liability and no efflux pump substrate liability. Furthermore, it exhibits an excellent oral pharmacokinetic profile in BALB/c mice with AUC0–∞ of 2067 ng h mL−1 and 37.6% oral bioavailability. The results presented here clearly indicate the potential of this scaffold to increase the clearance of brain Aβ across the BBB and thus its promise for development as a potential anti-Alzheimer agent.