8T8N

Venezuelan Equine Encephalitis Virus (VEEV) Nonstructural Protein 2 (nsP2) Cysteine Protease Inhibited with CA074

Summary for 8T8N

• Entry DOI: 10.2210/pdb8t8n/pdb
• Related: 5EZS
• Related PRD ID: PRD_001032
• Descriptor: Protease nsP2, [PROPYLAMINO-3-HYDROXY-BUTAN-1,4-DIONYL]-ISOLEUCYL-PROLINE (3 entities in total)
• Functional Keywords: cysteine protease, ca074, alphavirus, covalent, inhibitor, viral protein
• Biological source: Venezuelan equine encephalitis virus
• Total number of polymer chains: 1
• Total formula weight: 38727.25

Authors

Compton, J.R.,Legler, P.M. (deposition date: 2023-06-22, release date: 2023-08-09, Last modification date: 2024-10-23)

Primary citation

Hu, X.,Morazzani, E.,Compton, J.R.,Harmon, M.,Soloveva, V.,Glass, P.J.,Garcia, A.D.,Marugan, J.J.,Legler, P.M.
In Silico Screening of Inhibitors of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.
Viruses, 15:-, 2023

PubMed Abstract: 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 EC of 1.76 µM in VEEV-Trd-infected cells; the second most potent was NCGC00484087 with an EC = 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 (K = 3 µM) and NCGC00484087 (K = 5 µM). Rates of inactivation by CA074 in the presence of 6 mM CaCl, MnCl, or MgCl were measured with varying concentrations of inhibitor, Mg and Mn slightly enhanced inhibitor binding (3 to 6-fold). CA074 inhibited not only the VEEV nsP2 protease but also that of CHIKV and WEEV.

PubMed: 37515189
DOI: 10.3390/v15071503

Experimental method

X-RAY DIFFRACTION (2.32 Å)