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Venezuelan Equine Encephalitis Virus (VEEV) Nonstructural Protein 2 (nsP2) Cysteine Protease Inhibited with CA074
Structural highlights
FunctionPOLN_EEVVT P123 and P123' are short-lived polyproteins, accumulating during early stage of infection. P123 is directly translated from the genome, whereas P123' is a product of the cleavage of P1234. They localize the viral replication complex to the cytoplasmic surface of modified endosomes and lysosomes. By interacting with nsP4, they start viral genome replication into antigenome. After these early events, P123 and P123' are cleaved sequentially into nsP1, nsP2 and nsP3/nsP3'. This sequence of delayed processing would allow correct assembly and membrane association of the RNA polymerase complex (By similarity). nsP1 is a cytoplasmic capping enzyme. This function is necessary since all viral RNAs are synthesized in the cytoplasm, and host capping enzymes are restricted to the nucleus. The enzymatic reaction involves a covalent link between 7-methyl-GMP and nsP1, whereas eukaryotic capping enzymes form a covalent complex only with GMP. nsP1 capping would consist in the following reactions: GTP is first methylated and then forms the m7GMp-nsP1 complex, from which 7-methyl-GMP complex is transferred to the mRNA to create the cap structure. Palmitoylated nsP1 is remodeling host cell cytoskeleton, and induces filopodium-like structure formation at the surface of the host cell (By similarity). nsP2 has two separate domain with different biological activities. The N-terminal section is part of the RNA polymerase complex and has RNA trisphosphatase and RNA helicase activity. The C-terminal section harbors a protease that specifically cleaves and releases the four mature proteins (By similarity). nsP3 and nsP3' are essential for minus strand and subgenomic 26S mRNA synthesis (By similarity). nsP4 is a RNA dependent RNA polymerase. It replicates genomic and antigenomic RNA by recognizing replications specific signals. Transcribes also a 26S subgenomic mRNA by initiating RNA synthesis internally on antigenomic RNA. This 26S mRNA encodes for structural proteins. nsP4 is a short-lived protein regulated by several ways: the opal codon readthrough and degradation by ubiquitin pathway (By similarity). Publication Abstract from PubMedThe 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(50) of 1.76 microM in VEEV-Trd-infected cells; the second most potent was NCGC00484087 with an EC(50) = 7.90 microM. 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(i) = 3 microM) and NCGC00484087 (K(i) = 5 microM). Rates of inactivation by CA074 in the presence of 6 mM CaCl(2), MnCl(2), or MgCl(2) were measured with varying concentrations of inhibitor, Mg(2+) and Mn(2+) slightly enhanced inhibitor binding (3 to 6-fold). CA074 inhibited not only the VEEV nsP2 protease but also that of CHIKV and WEEV. In Silico Screening of Inhibitors of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.,Hu X, Morazzani E, Compton JR, Harmon M, Soloveva V, Glass PJ, Garcia AD, Marugan JJ, Legler PM Viruses. 2023 Jul 4;15(7):1503. doi: 10.3390/v15071503. PMID:37515189[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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