Pancreatic cancer is an aggressive disease with multiple biochemical and genetic alterations. Thus, a single agent to hit one molecular target may not be sufficient to treat this disease. The purpose of this study is to identify a novel Hsp90 inhibitor to disrupt protein-protein interactions of Hsp90 and its cochaperones for down-regulating many oncogenes simultaneously against pancreatic cancer cells. Here, we reported that celastrol disrupted Hsp90-Cdc37 interaction in the superchaperone complex to exhibit antitumor activity in vitro and in vivo. Molecular docking and molecular dynamic simulations showed that celastrol blocked the critical interaction of Glu 33 (Hsp90) and Arg 167 (Cdc37). Immunoprecipitation confirmed that celastrol (10 Mmol/L) disrupted the Hsp90-Cdc37 interaction in the pancreatic cancer cell line Panc-1. In contrast to classic Hsp90 inhibitor (geldanamycin), celastrol (0.1-100 Mmol/L) did not interfere with ATP binding to Hsp90. However, celastrol (1-5 Mmol/L) induced Hsp90 client protein degradation (Cdk4 and Akt) by 70% to 80% and increased Hsp70 expression by 12-fold. Celastrol induced apoptosis in vitro and significantly inhibited tumor growth in Panc-1 xenografts. Moreover, celastrol (3 mg/kg) effectively suppressed tumor metastasis by more than 80% in RIP1-Tag2 transgenic mouse model with pancreatic islet cell carcinogenesis. The data suggest that celastrol is a novel Hsp90 inhibitor to disrupt Hsp90-Cdc37 interaction against pancreatic cancer cells.

Abstract:Cocaine is recognized as the most reinforcing of all drugs of abuse. There is no anticocaine medication available. The disastrous medical and social consequences of cocaine addiction have made the development of an anticocaine medication a high priority. It has been recognized that an ideal anticocaine medication is one that accelerates cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., cocaine hydrolysis catalyzed by plasma enzyme butyrylcholinesterase (BChE). However, wild-type BChE has a low catalytic efficiency against the abused cocaine. Design of a high-activity enzyme mutant is extremely challenging, particularly when the chemical reaction process is rate-determining for the enzymatic reaction. Here we report the design and discovery of a high-activity mutant of human BChE by using a novel, systematic computational design approach based on transition-state simulations and activation energy calculations. The novel computational design approach has led to discovery of the most efficient cocaine hydrolase, i.e., a human BChE mutant with an ∼2000-fold improved catalytic efficiency, promising for therapeutic treatment of cocaine overdose and addiction as an exogenous enzyme in human. The encouraging discovery resulted from the computational design not only provides a promising anticocaine medication but also demonstrates that the novel, generally applicable computational design approach is promising for rational enzyme redesign and drug discovery.

The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC50s of 1.24, 2.93 and 2.78 μM) in pancreatic cancer cell lines Panc-1, MiaPaca2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-Biotin pull-down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90, which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 μM) disrupted Hsp90-Cdc37 complexes from 1–24 hour post treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6 mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global crisis. There is no therapeutic treatment specific for COVID-19. It is highly desirable to identify potential antiviral agents against SARS-CoV-2 from existing drugs available for other diseases and thus repurpose them for treatment of COVID-19. In general, a drug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. Here we report a virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions and its use in identifying drugs targeting SARS-CoV-2 main protease (Mpro). The accurate FEP-ABFE predictions were based on the use of a restraint energy distribution (RED) function, making the practical FEP-ABFE−based virtual screening of the existing drug library possible. As a result, out of 25 drugs predicted, 15 were confirmed as potent inhibitors of SARS-CoV-2 Mpro. The most potent one is dipyridamole (inhibitory constant Ki = 0.04 µM) which has shown promising therapeutic effects in subsequently conducted clinical studies for treatment of patients with COVID-19. Additionally, hydroxychloroquine (Ki = 0.36 µM) and chloroquine (Ki = 0.56 µM) were also found to potently inhibit SARS-CoV-2 Mpro. We anticipate that the FEP-ABFE prediction-based virtual screening approach will be useful in many other drug repurposing or discovery efforts.