Structural optimization on a virtual screening hit of smoothened receptor.

The Hedgehog (Hh) pathway plays a critical role during embryonic development by controlling cell patterning, growth and migration. In adults, the function of Hh pathway is curtailed to tissue repair and maintenance. Aberrant reactivation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. The Smoothened (Smo) receptor, a key component of the Hh pathway which is central to the signaling transduction, has emerged as an attractive therapeutic target for the treatment of human cancers. Taking advantage of the availability of several crystal structures of Smo in complex with different antagonists, we have previously conducted a molecular docking-based virtual screening to identify several compounds which exhibited significant inhibitory activity against the Hh pathway activation (IC50 < 10 µM) in a Gli-responsive element (GRE) reporter gene assay. The most potent compound (ChemDiv ID C794-1677: 47 nM) showed comparable Hh signaling inhibition to the marketed drug vismodegib (46 nM). Herein, we report our structural optimization based on the virtual screening hit C794-1677. Our efforts are aimed to improve potency, decrease cLogP, and remove potentially metabolic labile/toxic pyrrole and aniline functionalities presented in C794-1677. The optimization led to the identification of numerous potent compounds exemplified by 25 (7.1 nM), which was 7 folds more potent compared with vismodegib. In addition, 25 was much less lipophilic compared with C794-1677 and devoid of the potentially metabolic labile/toxic pyrrole and aniline functional groups. Furthermore, 25 exhibited promising efficacy in inhibiting Gli1 mRNA expression in NIH3T3 cells with either wildtype Smo or D473H Smo mutant. These results represented significant improvement over the virtual screening hit C794-1677 and suggested that compound 25 can be used as a good starting point to support lead optimization.

Discovery of potent and novel smoothened antagonists via structure-based virtual screening and biological assays.

The Hedgehog (Hh) signaling pathway plays a critical role in controlling patterning, growth and cell migration during embryonic development. Aberrant activation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. As a key member of the Hh pathway, the Smoothened (Smo) receptor, a member of the G protein-coupled receptor (GPCR) family, has emerged as an attractive therapeutic target for the treatment and prevention of human cancers. The recent determination of several crystal structures of Smo in complex with different antagonists offers the possibility to perform structure-based virtual screening for discovering potent Smo antagonists with distinct chemical scaffolds. In this study, based on the two Smo crystal complexes with the best capacity to distinguish the known Smo antagonists from decoys, the molecular docking-based virtual screening was conducted to identify promising Smo antagonists from ChemDiv library. A total of 21 structurally novel and diverse compounds were selected for experimental testing, and six of them exhibited significant inhibitory activity against the Hh pathway activation (IC50 < 10 µM) in a GRE (Gli-responsive element) reporter gene assay. Specifically, the most potent compound (compound 20: 47 nM) showed comparable Hh signaling inhibition to vismodegib (46 nM). Compound 20 was further confirmed to be a potent Smo antagonist in a fluorescence based competitive binding assay. Optimization using substructure searching method led to the discovery of 12 analogues of compound 20 with decent Hh pathway inhibition activity, including four compounds with IC50 lower than 1 µM. The important residues uncovered by binding free energy calculation (MM/GBSA) and binding free energy decomposition were highlighted and discussed. These findings suggest that the novel scaffold afforded by compound 20 can be used as a good starting point for further modification/optimization and the clarified interaction patterns may also guide us to find more potent Smo antagonists.

Discovery of novel dihydrobenzofuran cyclopropane carboxylic acid based calcium sensing receptor antagonists for the treatment of osteoporosis.

In a search for novel small molecule calcium-sensing receptor (CaSR) antagonists as oral bone anabolic agents, we discovered dihydrobenzofuran cyclopropane carboxylic acid derivatives, such as 12f (IC50=27.6nM), are highly potent calcium-sensing receptor antagonists. Studies in rats established that compound 12f stimulates parathyroid hormone (PTH) release in a fast-acting, pulsatile manner.

Epigallocatechin gallate (EGCG), a major component of green tea, is a dual phosphoinositide-3-kinase/mTOR inhibitor.

The PI3K signaling pathway is activated in a broad spectrum of human cancers, either directly by genetic mutation or indirectly via activation of receptor tyrosine kinases or inactivation of the PTEN tumor suppressor. The key nodes of this pathway have emerged as important therapeutic targets for the treatment of cancer. In this study, we show that (-)-epigallocatechin-3-gallate (EGCG), a major component of green tea, is an ATP-competitive inhibitor of both phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) with K(i) values of 380 and 320nM respectively. The potency of EGCG against PI3K and mTOR is within physiologically relevant concentrations. In addition, EGCG inhibits cell proliferation and AKT phosphorylation at Ser473 in MDA-MB-231 and A549 cells. Molecular docking studies show that EGCG binds well to the PI3K kinase domain active site, agreeing with the finding that EGCG competes for ATP binding. Our results suggest another important molecular mechanism for the anticancer activities of EGCG.

Characterization of PI3K class IA isoforms with regulatory subunit p55alpha using a scintillation proximity assay.

Differential activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway has been linked to cancer. Activation occurs through gene amplification and activating mutations. High-frequency mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been observed in a variety of tumors including colon, brain, breast, ovarian, and gastric. Inhibition of PI3K kinase activity may provide a specific way to treat multiple types of human cancer. A scintillation proximity assay (SPA) was developed to detect phosphatidylinositol 3-kinase catalytic activity. Using this assay format, steady-state kinetic parameters were compared for the PI3K class IA enzymes p110alpha, p110beta, and p110delta, each coexpressed with the regulatory subunit p85alpha or splice variant p55alpha. Inhibition by the natural product wortmannin and LY294002 was detected with potencies consistent with alternate assay formats. Other biochemical assay formats have been described for phosphoinositide 3-kinases but each has its unique limitations. The simple, inexpensive, sensitive high-throughput nature of the SPA format has advanced our knowledge of isoform-specific enzymology and will facilitate the discovery of novel PI3K inhibitors.