Novel isomannide-based peptide mimetics containing a tartaric acid backbone as serine protease inhibitors

Abstract

Hepatitis C viral infection is a cause of chronic liver disease, and current therapies are only effective in 50 % of patients. Serine proteases, which are present in both hepatitis C virus (HCV) and the dengue virus, are the most studied class of proteolytic enzymes and are the primary targets for drug development in this field. In this paper, we describe the synthesis of a novel class of isomannide-based peptide mimetic compounds based on a tartaric acid backbone. Our data showed that substitutions at position 168 (D168A) and 170 (V170A) conferred lowlevel resistance against compound 5a3, whereas substitutions at position 155 (R155K) and 156 (A156V) conferred no resistance. These data suggest that even though compound 5a3 is a noncompetitive inhibitor; it is able to interact with important residues located near the catalytic site. In addition, this novel compound class exhibits potent antiviral activity against variants carrying resistance mutations to boceprevir and telaprevir. Our docking studies showed important interactions, including hydrogen bonds and a p-p interaction, between compound 5a3 and residues of the allosteric site of NS3/4A. Biological and theoretical results indicate that 5a3 is a promising lead compound for the development of new drugs targeting HCV infection.

Development of a rapid phenotypic test for HCV protease inhibitors with potential use in clinical decisions

“…In order to produce a recombinant HCV NS3-4A similar to its biological equivalent, an expression plasmid was constructed encoding the NS4A central peptide GSGVVIVGRILLS (NS4A aa 21-32) covalently joined to the NS3 protease domain (aa 1-182) via an amino acid linker GSGS ( Abrahim-Vieira et al , 2014 ). The fragment encoding the single-chain protease derived from the pFKI389 Luc-ubi-neo/NS3-3'ET replicon was inserted into a pET21d expression vector between the Bam HI and Hind III sites.…”

“…This fragment was cloned into a pET21dhis-TEV bacterial expression vector (a pET21d plasmid, which was modified to codify a 6x histidine tag followed by a cleavage site recognized by the Tobacco Etch virus nTev protease at the protein N-terminus). The single chain NS3-4A protease contains the protease domain (182 amino acids) and a 20-aa portion of the cofactor NS4A ( Abrahim-Vieira et al , 2014 ). Multidrug resistant variants (T54A, V36M, V170I, T54S + V170I, V158I) were generated by Phusion Site-Directed Mutagenesis Kit (Thermo Scientific) and all the constructs were sequenced to confirm the presence of the desired mutations.…”

“…Protein expressions were carried out as described previously with some modifications to simplify techniques and to perform the protocol in a smaller inoculum volume ( Abrahim-Vieira et al , 2014 ). Transformed BL21 (λDE3) cells were grown at 37°C in 500 mL of LB medium containing 100 mg/mL ampicilin until an OD 600 of 0.6-0.8 was reached.…”

Statine-based peptidomimetic compounds as inhibitors for SARS-CoV-2 main protease (SARS-CoV‑2 Mpro)

“…HCl) 43 , by employing the classical N-(3-dimethylaminopropyl) N'-ethylcarbodiimide/1-hydroxy-benzotriazole/ N-methylmorpholine (EDC/HOBt/NMM) protocol (Fig. 2) 18,44 .…”