In Silico Screening of Inhibitors of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.

The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.B79) is essential for viral replication. High throughput in silico/in vitro screening using a focused set of known cysteine protease inhibitors identified two epoxysuccinyl prodrugs, E64d and CA074 methyl ester (CA074me) and a reversible oxindole inhibitor. Here, we determined the X-ray crystal structure of the CA074-inhibited nsP2 protease and compared it with our E64d-inhibited structure. We found that the two inhibitors occupy different locations in the protease. We designed hybrid inhibitors with improved potency. Virus yield reduction assays confirmed that the viral titer was reduced by >5 logs with CA074me. Cell-based assays showed reductions in viral replication for CHIKV, VEEV, and WEEV, and weaker inhibition of EEEV by the hybrid inhibitors. The most potent was NCGC00488909-01 which had an EC50 of 1.76 µM in VEEV-Trd-infected cells; the second most potent was NCGC00484087 with an EC50 = 7.90 µM. Other compounds from the NCATS libraries such as the H1 antihistamine oxatomide (>5-log reduction), emetine, amsacrine an intercalator (NCGC0015113), MLS003116111-01, NCGC00247785-13, and MLS00699295-01 were found to effectively reduce VEEV viral replication in plaque assays. Kinetic methods demonstrated time-dependent inhibition by the hybrid inhibitors of the protease with NCGC00488909-01 (Ki = 3 µM) and NCGC00484087 (Ki = 5 µM). Rates of inactivation by CA074 in the presence of 6 mM CaCl2, MnCl2, or MgCl2 were measured with varying concentrations of inhibitor, Mg2+ and Mn2+ slightly enhanced inhibitor binding (3 to 6-fold). CA074 inhibited not only the VEEV nsP2 protease but also that of CHIKV and WEEV.

Vinyl Sulfone-Based Inhibitors of Nonstructural Protein 2 Block the Replication of Venezuelan Equine Encephalitis Virus.

Emerging infectious diseases like those caused by arboviruses such as Venezuelan equine encephalitis virus (VEEV) pose a serious threat to public health systems. Development of medical countermeasures against emerging infectious diseases are of utmost importance. In this work, an acrylate and vinyl sulfone-based chemical series was investigated as promising starting scaffolds against VEEV and as inhibitors of the cysteine protease domain of VEEV's nonstructural protein 2 (nsP2). Primary screen and dose response studies were performed to evaluate the potency and cytotoxicity of the compounds. The results provide structural insights into a new class of potent nonpeptidic covalent inhibitors of nsP2 cysteine protease represented by compound 11 (VEEV TrD, EC50 = 2.4 µM (HeLa), 1.6 µM (Vero E6)). These results may facilitate the evolution of the compounds into selective and broad-spectrum anti-alphaviral drug leads.