Investigating the Origin of the Slow-Binding Inhibition of HCV NS3 Serine Protease by a Novel Substrate Based Inhibitor

Abstract

Indandiones were identified as a novel class of small molecule inhibitors of hepatitis C virus NS3 serine protease from high throughput screening. We further studied the structure activity relationships and the mechanisms of inhibition for this class of compounds. Our studies revealed two similar, yet different, mechanisms accounting for the apparent indandione inhibition of HCV NS3 protease. In one case, the apparent inhibition results from the chemical breakdown of the parent compound and the subsequent redox chemistry of the compound. Oxidation of the cysteine containing substrate A to a disulfide-linked dimer converts this substrate to a potent, slow-binding inhibitor with a K(i) value of 170 nM. The second class of indandiones appears to react directly with the substrate to form an S-phenyl disulfide adduct with the P1 cysteine. This modification converts the substrate to a slow-binding inhibitor with a K(i) value of 110 nM, a k(on) = 2370 M(-1) s(-1), and k(off) = 2.5 x 10(-4) s(-1). A stable analogue of this latter compound was synthesized that contained a CH(2)-S linkage instead of the S-S linkage. The CH(2)-S compound showed no inhibition at concentrations as high as 40 microM, which suggests an important role for the S-S linkage in the inhibitory mechanism. Cysteine 159, which lies near the active site of the HCV protease, was mutated to serine. The C159S mutant displayed wild-type catalytic activity and susceptibility to inhibition by the S-S linked inhibitor. This result argues against a mechanism involving disulfide exchange between the inhibitor and the sulfhydryl group of C159. The mechanism of inhibition for this S-S linked substrate based inhibitor is likely due to oxidation of cysteines involved in chelation of the structural zinc atom.

Scintillation Proximity Assays in High-Throughput Screening

“…We also like to add a short preincubation step of compound with enzyme or receptor prior to the initiation of the reaction to allow for slow-binding inhibitors, which require a conformational change, in order to form a tight complex with the target. [114][115][116][117] Longer incubation steps can also add significant amounts of plate processing time in HTS automation because incubation time often represents the rate-limiting step in the HTS process. Reaction rates can be increased by increasing the enzyme concentration or temperature, and, in this fashion, incubation times can be reduced.…”

Discovery of novel West Nile Virus protease inhibitor based on isobenzonafuranone and triazolic derivatives of eugenol and indan-1,3-dione scaffolds

“…Indan-1,3-diones derivatives have been specifically associated with antiviral activity. Studies have indicated efficient action of indan-1,3-diones against the enzyme integrase of the HIV-1 virus [36]; against the structure of human papillomavirus (HPV) [37–39]; and also against the Hepatitis C virus (HCV) protease [40], and recently for WNV protease [41]. Eugenol has been tested against the Herpes virus (HSV), HSV-1 (five viral isolates) and HSV-2 (five viral isolates), providing complete protection against one isolate of each type, HSV-1 and 2, and protection between 16.5% and 87.7% for the remaining isolates [21–23].…”

Synthesis, Antimicrobial, and Anticoagulant Activities of 2-(Arylsulfonyl)indane-1,3-diones

“…(b) The molecule offers wide scope for the study of physiochemical properties such as 1,3-indandione tautomerism, dual reactivity, electrochemical redox properties, unique property of polycrystalline films, and quantum mechanical calculations, and so forth [3,4]. (c) A wide range of biological properties covering antimicrobial, antitumor, anti-inflammatory, antiviral, antihepatitis, anticoagulant, rodenticidal, herbicidal, and insecticidal properties were associated with indane-1,3-dione derivatives [5][6][7][8][9][10].…”