Discovery of an allosteric mechanism for the regulation of HCV NS3 protein function

Here we report the discovery of a highly conserved novel binding site located at the interface between the protease and helicase domains of the Hepatitis C Virus (HCV) NS3 protein. Using a chemical lead, identified by fragment screening and structure-guided design, we demonstrate that this site has a regulatory function on the protease activity via an allosteric mechanism. We propose that compounds binding at this allosteric site inhibit the function of the NS3 protein by stabilising an inactive conformation and thus represent a new class of direct acting antiviral agents.


Protein stability and susceptibility
The stability of the HCV NS3/4a protein was tested by incubating samples of the protein at a concentration of 16.5uM at RT in the presence and absence of 12 fold molar excess of compound 5 for up to 17h 30min. Identical NS3/4a samples were incubated over the same time course with sequencing grade trypsin and chymotrypsin at 1:60 protease to NS3/4a ratios using buffer B 50mM Hepes pH 7.6, 150mM NaCl, 10% glycerol and D 50mM Hepes pH 7.6, 30mM NaCl, 10% glycerol. Controls to rule out inhibition of the proteases by compound 5 were run alongside, using lyzozyme as the substrate. Samples were taken at t=0, 10, 20, 45, 90, 180 and after overnight incubation t=17 hours 30min and run on SDS 4-20% gradient polyacrylamide gels (Novex).
Size Exclusion Chromatography Analytical size exclusion runs were performed on a Superdex 200 analytical column (GE Healthcare) at 4 o C under varying buffer conditions. The NS3/4a protein at a concentration of 6mg/ml was ultracentrifuged prior to injection on to the column equilibrated in one of the following buffers: (A) 50mM HEPES, pH7.6, 1M NaCl, 10% glycerol, 1mM TCEP; (B) 50mM HEPES, pH7.6, 0.15M NaCl, 10% glycerol, 1mM TCEP (C) 50mM HEPES, pH7.6, 0.15M NaCl, 1mM TCEP or (E) 50mM HEPES, pH7.6, 0.15M NaCl, 1mM TCEP, 1uM Compound 3. The column calibrated using the 1.35-670kD molecular weight standards from BioRad. Flow rate of the column was adjusted for the presence of glycerol in the buffers. Representative chromatograms of the elution profiles are shown in supplementary figure 4. Sedimentation Velocity Analysis Sedimentation Velocity Experiments were done at CAMIS, New Hampshire, USA (C. May, T. Laue). NS3/4a protein was ultracentrifuged prior to buffer exchange into the appropriate buffer using Microspin columns (GE Healthcare). The protein was diluted using the same buffer to achieve a final concentration of 0.5mg/ml. Choice of buffer conditions was aimed to mimic crystallisation, physiological and bioassay conditions, respectively: (A) 50mM HEPES, pH7.6, 1M NaCl, 10% glycerol; (B) 50mM HEPES, pH7.6, 0.1M NaCl, 10% glycerol or (D) 50mM HEPES, pH7.6, 0.03M NaCl. Experiments were performed at 25 °C, in a Beckman Optima XL-I analytical ultracentrifuge. Spin Analytical charcoal epon 12 mm double-sector centerpieces were used in an eight-hole AN 50 Ti rotor at 42,000 rpm. Radial absorption scans were measured at λ = 280 nm. Sedfit 12.0 was used for data analysis, with a 68% confidence limit. Sedimentation coefficients were corrected to standard s 20,w conditions using the experimental buffer density and buffer viscosity as calculated with SEDNTERP. Results of this analysis are shown on supplementary figure 5.
Dynamic Light Scattering Fixed angle light scattering experiments were performed on an Avid Nano W130i instrument (Avid Nano, High Wycombe, UK). The NS3/4a protein was ultracentrifuged prior to buffer exchange into the same buffers as specified for sedimentation velocity. The final protein concentration was 5mg/ml. The experiments were performed at room temperature using the disposable BladeCells from AvidNano. The data was analysed using system software i-Size version 1.3.0.3 which allowed for correction to solvent viscosity from glycerol or ionic strength. A negligible degree of error will have been introduced by not correcting for both simultaneously. Repeating the measurement but analysing the data using the alternative solvent correction showed not effect to the margin of error.
Replicon Assay Direct determination of HCV replicon RNA levels in HCV replicon bearing huh-7 cells was done by seeding 100,000 cells/well in a 6 well tissue culture plates and allowing them to attach overnight before compound addition at a final DMSO concentration of 0.1%. At 72 hours post compound addition RNA was extracted using a Qiagen RNeasy kit (Qiagen) according to manufacturer's instructions. All samples were normalized for total RNA concentration. Quantitative RT-PCR was carried out using the following HCV NS5B gene specific primers: HCV5BF: CTCCATGGCCTTAGCGCATTT and HCV5BR: AAAAAACAGGATGGCCTATTGG in a one-step reaction using the Quantitect SYBR Green RT-PCR kit (Qiagen) following manufacturer's instructions. Briefly 2 ng of sample RNA was combined with the NS5B primers listed above at a final concentration of 1µM and an equal volume of 2x Quantitect SYBR Green RT-PCR Master Mix. Reactions were transferred to a thin walled 96 well plate and the RT reaction was carried out using the MX3005p (Stratagene) instrument at 50 o C for 30 minutes, followed by a denaturation step at 94°C for 15 min. The PCR amplification was conducted in 45 cycles, each of which was 94°C for 15 s followed by 59°C for 30 seconds, then 72 o C for 2 minutes. Amplification of HPRT RNA, as an internal RNA normalization control, for each sample was determined in separate reactions. The amount of input RNA from the untreated control sample was varied in order to generate a standard curve by which the relative levels of replicon RNA from each treated sample could be expressed as fold changes relative to the untreated control sample.
Western blot analysis of polyprotein processing Cell lysates from replicon bearing Huh-7 cells were treated for 24 hours with the stated compounds at 10 x EC 50 concentrations. Samples were resolved on 4-12% NuPage gels (Life Technologies) and immunoblotted with antibodies specific for (A) HCV NS5b (ab65410, Abcam) or (B) GAPDH (Chemicon Intl.). Untreated cell lysates were used as controls for cell lysates that were treated for different time intervals with compound 6, telaprevir or GS-7977 respectively. Molecular weight markers ran alongside (Rainbow markers, GE Healthcare) HCV NS5b and GAPDH were followed by infrared dye labelled anti-rabbit or anti-mouse antibodies (Licor Bioscience, Lincoln, NE, USA). Blots were scanned to detect infrared fluorescence on the Odyssey Infrared Imaging System (Licor Bioscience).

Cytotoxicity Assay
In this assay 4 x 10 -3 huh-7 cells persistently infected with the HCV subgenomic replicon construct pFKI3889luc-ubi-neo/NS3-3'/ET were plated /well in a 96 well tissue culture plate and allowed to attach overnight in DMEM medium supplemented with 10% FBS 1% NEAA, and 250 µg/ml gentamicin. The medium was replaced with fresh medium as described above lacking gentamicin. Semilog dilutions of the compound in medium were added to triplicate wells to give a 0.1% DMSO final concentration. Plates were incubated at 37 o C in 5% CO 2 and air for 72 h. Following the 72 h incubation, the test compound's CC 50 value was determined by adding Alamar Blue™ (Biosource International, Camarillo, CA, USA) and incubating for 6 h. The plate was read at 535 nm (excitation) and 590 nm (emission) on a SpectraMax Gemini reader (Molecular Devices) to determine the number of viable cells by measuring the conversion of rezasurin (Alamar blue) to resorufin in response to mitochondrial activity.

Antiviral Resistance Selection
Compound 5 resistant mutations in the subgenomic replicon (pFKI3889luc-ubi-neo/NS3-3'/ET) were isolated by culturing Huh-7 cells persistently infected with this subgenomic replicon construct in the presence of medium containing 500 g/L G418 and 3 M compound 5 for 11 days, followed by 10 M of compound 5 to for a further 28 days. Individual G418 resistant colonies were isolated and resistance to compound 5 was confirmed by determining their EC 50 by Luciferase activity. RNA was isolated from each clonal population of cells using a Qiagen RNeasy kit (Qiagen) according to manufacturer's instructions. The RNA for each sample was reverse transcribed by priming 1 g of the isolated colony RNA with the NS4AR primer (listed below) in a reaction using GoScript reverse transcriptase reagents (Promega) as described in manufacturer's instructions. The resulting first strand cDNA was denatured at 95 o C for 15 min, followed by PCR amplification of the corresponding NS3 protein using the following primers: NS3F: ATGGCGCCTATTACGGCCTAC NS4AR: CTCCGCTTGCTTGGTGGCTG The PCR amplification was conducted in 30 cycles (94°C for 15 s followed by 55°C for 30 s, then 72 o C for 1 min). Amplified PCR products were cloned into the pCR4-TOPO vector using manufacturer's instructions for the TopoTA Cloning Kit for Sequencing (Invitrogen). Sequencing of the cloned PCR products was performed using vector specific primers.
Validation of cellular target: Compound 5 dose-dependently inhibited HCV NS3 target function at 0.4µM in a genotype 1b HCV replicon bearing Huh-7 cell line as determined by luminesce activity from the replicon encoded luciferase gene (Suppl Fig. 6). Replication of the replicon construct and therefore luciferase expression levels are known to be dependent on HCV NS3 protease activity, amongst other viral protein activities. Compound 5 was also demonstrated to bind full length HCV NS3/4a by ITC (Table 1) and direct structural determination ( Fig. 3c-e). Furthermore compound 5 was demonstrated to inhibit the protease activity of full length NS3/4a in a FRET-based assay (Table 1). Notably selection pressure placed upon cells persistently infected with the GT1b replicon by compound 5, resulted in the of development resistance mutations that mapped to regions of HCV NS3/4a shown by x-ray structural analyses to affect the compound 5 binding site (Fig 3f). At concentrations of compound 5 that rapidly lead to a greater than 3 log decline in replicon RNA levels the health of the cell measured as measured by an Alamar blue assay was unaffected.

A B
Supplementary figure 1 Polyprotein processing and western blot analysis Cell lysates from replicon bearing Huh-7 cells were treated for 24 hours with the stated compounds at 10 x EC 50 concentrations. Samples were immunoblotted with antibodies specific for (A) HCV NS5b or (B) GAPDH. Untreated cell lysates were used as a control and loaded in the first lane, followed by cell lysates treated with compound 6, telaprevir or GS-7977 respectively. Molecular weight markers are shown as labelled. Panel A shows the amount of processed viral protein present in the cell culture in the presence of our allosteric inhibitor, the active site inhibitor telaprevir or the HCV polymerase inhibitor GS7977. The strongest band on the blot, running below the 76kD molecular weight marker is consistent with the NS5b protein, with a molecular weight of 66kD. The bands at around150kD are consistent with the molecular weight expected for unprocessed NS4a-NS5b protein (~153kD) and NS4b-NS5b (~149kD) can be gleaned in the control sample and increases in intensity in the samples treated with compound 6 and telaprevir, yet is absent in the nucleoside analogue GS7977 samples. The band running above 102kD may be an artefact. The nature of this experiment does not allow us to unambiguously separate the effect of the compounds on the inhibition of viral replication from that of polyprotein processing, despite the absence of the bands in the GS7977 sample. Panel B is a guide for the normalisation of sample loading.

A B
Supplementary Figure 2 Titration Curves of the wildtype (A) and V630L mutant (B) replicons.
Clonal populations of Huh-7 cells bearing replicons with the V630L mutation were selected and identified following selective pressure from compound 5. EC 50 values were determined in cells carrying WT vs the V630L mutant replicon using the active site protease inhibitor telaprevir as a control. Triplicate wells were analysed using a 10 point dilution series to generate the curves shown. Three independent experiments were run with representative graphs for one such experiment shown here with error bars representing the standard deviation from the means for each point. The graphs show no change within experimental error in the EC 50 for telaprevir against the WT and V630L mutant replicon. The EC 50 values for compound 5 show a 30 fold drop in the V630L mutant compared to WT. This result confirms that the cell based antiviral activity of compound 5 is mediated by the compound binding at the newly discovered allosteric site on the HCV NS3/4a protein.
conditions, suggesting that the extended conformation is required for function 31, 12 . Analysis of protein samples by SDS page incubated under the same conditions for extended periods of time gave no evidence of proteolytic degradation, thus confirming that the signals observed where due to different conformations of the HCV NS3/4a protein.

A B Supplementary Figure 4 Size Exclusion Chromatography
A. Superposition of the chromatograms of the WT protein using an analytical superdex 200 column (GE Healthcare) in 50mM HEPES, pH7.6, 1M NaCl, 10% glycerol, 1mM TCEP blue curve, 50mM HEPES, pH7.6, 0.15M NaCl, 10% glycerol, 1mM TCEP, green curve and 50mM HEPES, pH7.6, 0.15M NaCl, 1mM TCEP red curve. At 1M NaCl the protein appears as a homogeneous sharp peak of molecular weight consistent with a monomeric species. At 0.15M NaCl and 10% glycerol, the peak begins to broaden suggesting more conformational heterogeneity. Removal of the glycerol (red curve) shows the emergence of a shoulder moving ahead of the main peak, suggesting a different conformation of the protein. Based on the calibration of the column the faster moving species is not likely to be a dimer, but rather an extended monomer. No dimers are observed under high ionic strength conditions (dimers were seen to be predominant by sedimentation velocity) which suggest that the interactions are weak enough to be disrupted by the frictional flow present in the column. B. The chromatograms of the WT protein in 0.15M NaCl and in the absence of glycerol reproducibly showed the presence of a faster moving shoulder ahead of the main peak as shown by the blue curve. The red curve shows the effect of the presence of 1uM of compound 5 (10-fold excess over IC 50 ) to the elution of the WT protein under otherwise identical conditions. The shoulder is less obvious consistent with a more homogeneous closed conformation of the protein.
Supplementary Figure 5 Graph of observed thermodynamic trends for 230 compounds binding to HCV NS3/4a Enthalpic ( H) and entropic (-T S) values are plotted against the free energy of binding ( G). The graph clearly shows that enthalpy is the major driving force for improving compound potency and that improving potency correlates well with a more favourable enthalpy and a less favourable entropy. Similar thermodynamic trends have been observed for a large and varied number of in-house targets, indicating that such trends are typical of Astex drug design and not necessarily target related. WT and mutant proteins were purified as describe in the materials and methods. Lanes 1 and 6 show Molecular weight markers (Rainbow markers, GE Healthcare); lane 2 is WT, lanes 3 and 4 are two different site directed mutants (T631A and T631C) of the full length NS3/4a not discussed in the text. Lane 5 is empty. The gel was stained with Coomassie blue and is representative of the quality of protein used for crystallisation and characterisation. Mass Spectrometry analysis of protein resulted in a mass of 70663D (expected 70794D) and indicated the loss of the N-terminal methionine, which was confirmed by peptide sequencing. Aggregation was observed upon incubation of the protein at concentrations exceeding 25uM at room temperature for periods of times exceeding 2 or more hours.

B.
WT protein at 16.5uM concentration was incubated at room temperature in the presence and absence 12 molar excess of compound 5 for up to 17.5 hours. No evidence of proteolytic degradation was observed in either sample during the duration of the experiments. Identical samples were subjected to proteolysis using sequencing grade trypsin and chymotrypsin at a ratio of 1:60 in both cases. Inhibition of either protease by compound 5 was ruled out by using lysozyme as the substrate in a parallel experiment. The gel shows the samples from the trypsin susceptibility experiment taken at t=0min (lanes 2, 3,4), 10min (lanes 5,6), 20min (lanes 7,8), 45min (lanes 9,10), 90min (lanes 11,12), 180 min (lanes 13,14) and after overnight incubation t=17.5 hours (lanes 15, -17) and run on SDS 4-20% gradient polyacrylamide gels (Novex). Lane 1 are the molecular weight markers, lanes 2 and 17 show the t=0 and t=17.5h samples of the HCV NS3/4a protein alone respectively. No evidence of auto-degradation was observed over the entire sampling period in the NS3/4a samples regardless of whether compound 5 was present or not (lanes 2 and 17). The presence of the allosteric compound 5 in the NS3/4a protein samples delays but does not prevent the fragmentation of the HCV NS3/4a protein by either trypsin or chymotrypsin. This result is consistent with the model in which compound 5 binds at the interface between the protease and helicase domains and stabilises the closed, more compact conformation of the protein, thus protecting it from proteolytic degradation.
Compound 5 was prepared in 100% DMSO, with a final experimental DMSO concentration between 1 and 3%. The figure shows a typical curve in which compound 5 was titrated into the sample cell containing the WT protein. The compound bound to the NS3/4a protein in 1:1 stoichiometry (N=0.9) with an affinity of 60nM and confirms that the protein in solution can adopt the closed conformation observed in the crystal structure.