Abstract
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.
Highlights
As Cdk[4] inhibitors target a pathway that links pRb, p16INK4A, cyclin D1 and Cdk[4], it makes inhibition of Cdk4-cyclin
BPT was tested against a panel of 58 representative kinases including Cdk5-p35, Cdk6-cyclin D1, Cdk7-cyclin H, EGFR, GSK3β, MAPK1, MEK1, PDGFR, Plk[3], PKA, PKCα, IGF-1R, and so on, wherein it does not inhibit any kinase to any appreciable degree at 10 μM (Table 1)
To understand the observed selectivity towards Cdk4-cyclin D1 versus Cdk2-cyclin A, molecular modelling studies were performed.[33]. These two Cdks share 45% sequence homology; they differ by three peptidic sequences including 94–97 (Glu-His-Val-Asp)Cdk4/81–84 (Glu-Phe-LeuHis)Cdk[2, 101–102] (Arg-Thr)Cdk4/88–89 (Lys-Lys)Cdk[2] and Glu144Cdk4/Gln131Cdk[2]
Summary
As Cdk[4] inhibitors target a pathway that links pRb, p16INK4A, cyclin D1 and Cdk[4], it makes inhibition of Cdk4-cyclin. It has been suggested that fascaplysin’s planar structure is the possible explanation for its ability to intercalate d-s DNA and its unusual toxicity at the cellular level To overcome this unusual toxicity, recently we reported CA224 (2), a non-planar analogue of fascaplysin exhibiting selective Cdk[4] inhibition with no DNA-intercalating property.[29] In continuation to these efforts, we report identification of tryptoline-based compounds CA198 (3), CA199 (4), CA211 (5) and N-(biphenyl-2-yl)-tryptoline (BPT, 6) as selective Cdk[4] inhibitors with no DNA-intercalating property. Based on the molecular modelling design, a number of non-planar analogues of fascaplysin were synthesized They show specificity towards Cdk4-cyclin D1 enzyme activity and blocks the growth of cancer cells at the G0/G1 phase.
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