High-Throughput Screening (HTS) and Hit Validation to Identify Small Molecule Inhibitors with Activity against NS3/4A proteases from Multiple Hepatitis C Virus Genotypes
Figure 2
Primary HTS results and SPR validation binding assay results.
(A) Replicate plot of the screening of 40,967 compounds from structurally diverse in-house, Prestwick FDA-approved drug, Maybridge, and Life Chemicals libraries. The blue box indicates hit compounds with over 50% inhibition at 50 µM compound concentration. Initial percent inhibitions of the two confirmed hits are shown in pink (Compound 12) and cyan (Compound 13). (B) Bar graphs of IC50 values and the dissociation equilibrium constants (KD) of 15 initial hit compounds. KD SR is single referenced KD values with a blank channel immobilized with a small molecule ethanolamine, whereas KD DR represents double referenced with ethanolamine and an unrelated reference protein (B. anthracis PyrC, ∼95 kDa as a dimer) immobilized in two different channels. All data were normalized for immobilization levels of target and reference proteins. Bars that reached the top of the graph represent KD values of over 200 µM (no binding). (C) The fitting curve of compound 6 with a single rectangular hyperbolic equation (See methods). The determined KD of compound 6 was 8.3±1.9 µM, similar to its IC50 value (7.6±0.6 µM). (D) Response units of compound 4 (mitoxantrone from the Prestwick) at a series of increasing concentrations (0–200 µM), showing a non-specific binding pattern. (E) Response units of compound 15 at various concentrations (0–200 µM), showing lack of binding to NS3/4A.