Figure 1.
Background information and sequence alignment.
(A) Schematic of the HCV polyprotein with cleavage sites of the two proteases, NS2 and NS3. (B) Structures of two FDA-approved NS3/4A inhibitors. (C) Structure of the NS3/4A serine protease, with the NS3 protease domain colored in cyan, and the co-factor NS4A (beta strand) shown in red. The active site residues, S139, H57 and D81, sit on the protein-protein interaction surface and are shown as stick figures in green. The amino acids prone to mutation in the binding site enabling drug resistance against both Telaprevir and Boceprevir are shown as stick figures in magenta (V36, F43, T54, R155 and A156). Images were prepared using Chimera v1.6.1, UCSF, 2012 [37]. (D) Sequence alignment of NS3 proteases from four HCV 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.
Table 1.
Statistical parameters of all screened compounds from four libraries.
Figure 3.
Mechanism of enzyme inhibition of 12 and 13.
Dixon plot for competitive inhibition of Compound 12 (A) and Compound 13 (B) with respect to the substrate Ac-DE-Dap(QXL520)-EE-Abu-ψ-[COO]AS-C(5-FAMsp)-NH2. Determined Ki values of Compound 12 and 13 were 3.5 µM and 19.1 µM, respectively. A known competitive inhibitor BILN-2061 was used as a control. Four equations (See Experimental section) in SigmaPlot Enzyme Kinetics Module 1.3 were used to fit the experimental data. The competitive inhibition model was the best fit for both compounds and for the control.
Table 2.
Comparison of the inhibitory activities of 12 with a control.
Figure 4.
IC50 value comparisons of two confirmed hit compounds.
Bar graphs are shown with IC50 values of two identified compounds against NS3/4As from four HCV genotypes, genotypes 1a (GT1a), 1b (GT1b), 2a (GT2a), and 4d (GT4d), and five mutants from genotype 1b. IC50 determination was done in triplicate with a total of 32 positive and 32 negative controls in a plate. IC50 values were calculated by fitting the data to the three parameter Hill equation with OriginPro 8.5 (See Methods). Bars that reached the top represent IC50 values of over 100 µM (no inhibitory effect).
Figure 5.
Comparison of the docking poses with NS3 proteases from sub-genotype 1a and 1b.
(A) Docking poses of 12 colored in pink with the sub-genotype 1b NS3 protease (colored in tan). Compound 12 is colored in cyan when it was docked with the sub-genotype 1a NS3 protease (colored in cyan). (B) Docking poses of compound 13 colored in cyan with sub-genotype 1a (also colored in cyan). The histidine 57 residue is shown in green, and a hydrogen bond with 13 is shown as a black dotted line. Compound 13 is colored in pink when it was docked with sub-genotype 1b (colored in tan) NS3 proteases. Figure prepared with Chimera v1.6.1, UCSF, 2012 [37].
Figure 6.
Substrate binding sites and docking studies.
(A) The surface of the NS3 protease substrate binding sub-sites. The S4-S2’ binding pockets are labeled in red. (B) Active-site view of docked 12 (colored in green) and 13 (colored in magenta) overlaid with a known macrocyclic inhibitor ITMN191 (colored in tan) from crystal structure (PDB code: 4A92). Figure prepared with Chimera v1.6.1, UCSF, 2012 [37].
Table 3.
Selectivity and microsomal stability of selected compounds.
Figure 7.
Preliminary Structure and Activity Relationship (SAR) of Compound 12.
Out of 25 structurally similar Life Chemicals compounds and 28 additional compounds from a scaffold search based on 12, % inhibition and IC50 values of 13 compounds are shown for comparison. IC50 values were determined from two to four independent assays. The percent inhibitions of each compound are shown in parenthesis in cases of IC50 value above 100 µM at 50 µM compound concentration.
Figure 8.
Preliminary Structure and Activity Relationship (SAR) map of compound 12.
A total of 53 similarly structured compounds were analyzed to generate this map (See Figure 7 for structures and IC50 values for 13 compounds).
Table 4.
Inhibitory activity of 12 and a control with HCV subgenomic replicon cell lysates.