Development of (<i>E</i>)‑2-((1,4-Dimethylpiperazin-2-ylidene)amino)-5-nitro‑<i>N</i>‑phenylbenzamide, ML336: Novel 2‑Amidinophenylbenzamides as Potent Inhibitors of Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit <b>1</b> with promising cellular antiviral activity (EC₅₀ = 0.8 μM), limited cytotoxic liability (CC₅₀ > 50 μM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 μM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound <b>45</b>, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC₅₀ = 0.02–0.04 μM, CC₅₀ > 50 μM) while limiting in vitro viral replication (EC₉₀ = 0.17 μM). Brain exposure was observed in mice with <b>45</b>. Significant protection was observed in VEEV-infected mice at 5 mg kg^–1 day^–1 and viral replication appeared to be inhibited through interference of viral nonstructural proteins

Spectrum of antiviral activity of CID15997213.

<p>IC₅₀ measured in a cell-based CPE assay (µM) with triplicate data points for VEEV 3526, TrD, CHIKV and RSV. IC₅₀ v*alue presented here for VEEV TC-83 is the mean from 17 independent experiments.</p>†<p>Log difference in progeny virus titers between in the absence/presence of the compound at 5 µM was >6. 0.05 MOI of VEEV TC-83 was used for infection.</p>‡<p>IC₅₀ measured in Neuro 2A cell line.</p

HTS of 348K compounds and identification of the hit compound.

<p>A flow diagram of various assays used in the screen. The number of hits remaining after each run is indicated in bold.</p

CID15997213 targets viral nsP2.

<p>(<b>A</b>) Time of addition study. Test compound, CID15997213, was added to the designated wells by replenishing the culture media with fresh culture media containing 5 µM of the compound at the time points denoted on the x axis. The graph denotes the virus titers at 16 hours post-infection from various time of addition points. Each data point is the mean from two independent replicates with duplication in titration. (<b>B</b>) Location of the mutations in the CID15997213 resistant viruses. The mutations mapped within the N-terminus of nsP2 protein (pink). There were no missense mutations in either nsP1, nsP3 or nsP4 genes. * Methyl-transferase like domain. (<b>C</b>) Sequence alignment of nsP2 alphaviruses. Amino acid sequences of nsP2 of following alphaviruses were aligned with Clustal W (<a href="http://www.clustal.org" target="_blank">www.clustal.org</a>): VEEV (L01442.2, GenBank Access No. hereafter), EEEV (NC_003899), WEEV (NC_003908), Fort Morgan virus (FMV, NC_013528), Ross River virus (RRV, NC_001544), Semliki Forest virus (SFV, NC_003215), O'nyong-nyong virus (ONYV, NC_001512.1), CHIKV (L37661.3), Sindbis virus (SINV, NP_740671.1). Y102 position is highlighted in red.</p

Antiviral activity of CID15997213 with VEEV and VEEV mutants.

<p>TC-83 Y102C and D116N were isolated by passaging of VEEV TC-83 in the presence of CID15997213. V3526 Y102C and V3526 D116N were generated by site-directed mutagenesis of pV3526 and virus was generated from synthetic RNA. The amino acid positions in the table refer to the position of the amino acid in the nsP2 protein.</p