Skipping Purification to Find Chikungunya Antivirals Faster

Review written by: Megan Mauriello

Chikungunya virus (CHIKV) is a +ssRNA alphavirus in the Togaviridae which is primarily transmitted by Aedes aegypti and Aedes albopictus mosquitoes. Chikungunya fever can have acute effects lasting months to years, and can rarely cause neurological complications, severe febrile illness, and death. It is one of the most widely distributed arboviruses globally, owing largely to the expansion of A. aegypti and A. albopictus geographic ranges and adaptation to urban settings. The CHIKV genome encodes a nonstructural protein 2 (nsP2) with an N-terminal RNA-specific helicase domain, a papain-like cysteine protease domain, and a C-terminal methyltransferase domain. nsP2 processes the viral nonstructural polyprotein to produce nsP1-4 and suppresses host antiviral activity. The nsP2 protease domain and the polyprotein cleavage sites are highly conserved. Previous studies have identified RA-0002034 and Cmp10 as potent nsP2 inhibitors, each with an internal beta-amidomethyl vinyl sulfone reactive warhead.

A direct-to-biology (D2B) platform of in silico covalent docking, rapid synthesis, crude-mixture enzymatic screening, and structure-guided optimization was used to create a series of candidate nsP2 inhibitors with diverse chemical scaffolds, potent enzymatic inhibition (as determined by a FRET assay), and demonstrated antiviral activity via the cytopathic effect assay.

Figure. Virtual screening plus direct to biology synthesis yields a structurally diverse set of nsP2 inhibitors. (A) The pipeline, from virtual library to covalent docking to synthesis to testing against CHIKV nsP2. (B) Predicted binding poses overlaid on RA-0002034, with reaction conversion, enzyme inhibition by both purified and crude material, antiviral activity against CHIKV 181/25, and cytotoxicity. (C) Potency from crude mixtures tracks potency from purified compounds.

The two strategies for structure-based design of inhibitors were: 1) extending the substituents on the phenyl ring, and 2) inserting a flexible benzyl ether linker in between the 5-membered heterocycle and the distal aromatic substituent. For strategy 1, substitution with 3,5-dimethyl, 3-cyano, and 3-methyl groups best improved inhibitor activity. For strategy 2, bicyclic aromatic groups and 2F or 3F substitution best improved activity and reduced cytotoxicity. Validation experiments found a strong positive correlation (R²=0.71) between enzymatic inhibition results from crude and purified samples, justifying the skip of the purification steps for faster turnaround.

nsP2 kinetics were calculated using the Michaelis-Menten equation for a catalytic efficiency (k_cat/K_m) of 16,194 M^-1s^-1. RA-0002034 and Cmp10 were used as positive controls for inhibition, and in the tested conditions their half-maximal inhibitory concentrations (IC₅₀) were 0.41 and 0.35 μM, respectively. RA-0002034 had a half maximal effective concentration (EC₅₀) for antiviral potency of 5.6 μM. Cmp10 had a covalent inhibition efficiency (k_intact/K_i) of 2,215 M^-1s^-1. Overall, the most promising candidate was compound 53, which had an IC₅₀ value of 0.27 μM, a EC₅₀ of 2.2 μM, and a k_intact/K_i of 2,514 M^-1s^-1. 53 also demonstrated greater plaque formation inhibition than RA-0002034 and Cmp10 for CHIKV. 53 was also effective at inhibiting o’nyong−nyong virus (ONNV), Mayaro virus (MAYV), and Ross River virus (RRV), although not Sindbis virus (SINV). The nsP2 protease domains of the 5 viruses showed that SINV had a seven-amino acid insertion in the flexible loop at the drug-binding site of SINV, which is a likely explanation for the lack of SINV inhibition.

This study developed a streamlined approach to covalent inhibitor development which proved to function well for nsP2 inhibitors and will likely be useful for development of other inhibitors. In conventional approaches, compound synthesis and purification is a major time sink, but the approach presented here avoids these steps without much effect on downstream analysis.

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READ MORE: Cai Z, Li K, Sridhar S, Tan H, Demssie H, Fan G, Zhang W, Herrera BB, Wang J. Development of Covalent Inhibitors of Chikungunya Virus nsP2 Cysteine Protease Enabled by Direct-to-Biology Synthesis and Screening. J Med Chem. 2026 Sep 10;69(17):20138-20159. doi: 10.1021/acs.jmedchem.5c03672. PMID: 42720489; PMCID: PMC13576448.