Hsp90 as a promising therapeutic target for the treatment of cancer has received great attention. Many Hsp90 inhibitors such as BIIB021 and CUDC-305 have been in clinical. In this paper shape-based similarity screening through ROCS overlays on the basis of CUDC-305, BIIB021, PU-H71 and PU-3 were performed to discover HSP90 inhibitors. A set of 19 novel pyrazolopyrimidine analogues was identified and evaluated on enzyme level and cell-based level as Hsp90 inhibitors. The compound HDI4-04 with IC50 0.35 mu M in the Hsp90 ATP hydrolysis assay exhibited potent cytotoxicity against five human cancer cell lines. Western blot analysis and Hsp70 luciferase reporter assay further confirmed that HDI4-04 targeted the Hsp90 protein folding machinery. And according to the biological assay, the SAR was discussed and summarized, which will guide us for further optimization of these compounds.

Rapid Overlay of Chemical Structures (ROCS), which can rapidly identify potentially active compounds by shape comparison, is recognized as a powerful virtual screening tool. By ROCS, a class of novel Hsp90 inhibitors was identified. The calculated binding mode of the most potent hit 36 guided us to design and synthesize a series of analogs (57a-57h). Over 100-fold improvement was achieved in the target-based assay. The most potent compound 57h inhibited Hsp90 with IC50 0.10 +/- 0.01 mu M. It also showed much improved cell potency and ligand efficiency. Our study showed that ROCS is efficient in the identification of novel cores of Hsp90 inhibitors. 57h can be ideal leads for further optimization. (C) 2014 Published by Elsevier Masson SAS.

Herein we first reported hierarchical structure-based virtual screening utilizing the receptor–ligand binding model of Nrf2–Keap1. The most promising compound, 15, which is one of the most potent direct PPI inhibitors of Nrf2–Keap1 reported so far, can effectively disrupt the Nrf2–Keap1 interaction with the in vitro EC50 of 9.80 μM in the fluorescence polarization (FP) assay. 15 can also activate the Nrf2 transcription activity in the cell-based ARE–luciferase reporter assays in a dose-dependent manner. The compound can serve as a promising starting point for the discovery of potent inhibitors of Nrf2–Keap1 interaction.

E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein–protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin–RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design.

Several chemical fragments have been confirmed as highly efficient cores for the design of Hsp90 inhibitors. Molecular hybridization of potent fragments has been widely used as a rational drug discovery strategy. In this study, a novel class of hybrids of benzofuran, a privileged core from natural products, and 2,4-dihydroxy-5-isopropyl phenyl, an efficient fragment in Hsp90 inhibitors, were de-signed and synthesized. Subsequent evaluation confirmed they inhibited cell proliferation and regulated the level of client proteins through Hsp90 inhibition. Some of the hybrids can serve as leads to obtain novel chemotypes of Hsp90 inhibitors. The methods reported here may expand the range of known structural types accommodated by the ATP binding site of Hsp90.