binding of a noncovalent inhibitor exploiting the s′ region stabilizes the hepatitis c virus ns3...

doi:10.1016/j.jmb.2008.11.017 J. Mol. Biol. (2009) 385, 1142–1155

Available online at www.sciencedirect.com

Binding of a Noncovalent Inhibitor Exploiting the S′region Stabilizes the Hepatitis C virus NS3 ProteaseConformation in the Absence of Cofactor

Mariana Gallo1†, Matteo Pennestri1†, Matthew James Bottomley2,Gaetano Barbato2, Tommaso Eliseo1, Maurizio Paci1, Frank Narjes2,Raffaele De Francesco2, Vincenzo Summa2, Uwe Koch2,Renzo Bazzo1,2 and Daniel O. Cicero1⁎

1Department of ChemicalScience and Technology,Università di Roma “TorVergata” Via della RicercaScientifica 1, 00133 Rome, Italy2Istituto di Ricerche di BiologiaMolecolare P. Angeletti,Pomezia, Rome, Italy

Received 3 July 2008;received in revised form17 October 2008;accepted 12 November 2008Available online24 November 2008

*Corresponding author. E-mail [email protected].† M. Gallo and M. Pennestri contr

work.Abbreviations used: NS3p, NS3 pr

C virus; ORF, open reading frame; 32D, two-dimensional; NOE, nuclearenhancement; HSQC, heteronuclearcoherence; NOESY, NOE spectrosco

0022-2836/$ - see front matter © 2008 E

We present the first structure of a noncovalent inhibitor bound to theprotease domain of hepatitis C virus NS3 protein (NS3p), solved by NMR.The inhibitor exploits interactions with the S′ region of NS3p to form a long-lived complex, although the absence of negative charges strongly reducesthe association rate. The inhibitor stabilizes the N-terminal domain of NS3pand the substrate-binding site, and correctly aligns catalytic His-Aspresidues. These actions were previously attributed exclusively to thecofactor NS4A, which interacts with the N-terminal domain of the NS3pand functions as an activator in vivo. The structure of the inhibitor/NS3pcomplex is very similar to that of the NS3p–NS4A complex, showing thatbinding of the NS4A cofactor is not the only event leading to a stable active-site conformation.

© 2008 Elsevier Ltd. All rights reserved.

Edited by A. G. Palmer III
Keywords: Hepatitis C virus; NS3 protease; inhibitors; cofactor; NMR
Introduction

Hepatitis C virus (HCV), a small enveloped RNAvirus belonging to the Hepacivirus genus of theFlaviviridae family, has been recognized as a majorcause of chronic liver disease and affects approxi-mately 200 million people worldwide.1 Chronichepatitis, hepatic steatosis, cirrhosis, and hepatocel-lular carcinoma are the main diseases associated withpersistent infection with HCV.2 The HCV genomeconsists of a single-stranded positive-sense RNA ofapproximately 9.6 kb, which contains an open read-ing frame (ORF) encoding a polyprotein precursor of

ess:

ibuted equally to this

otease; HCV, hepatitisD, three-dimensional;Overhausersingle quantumpy.

lsevier Ltd. All rights reserve

approximately 3000 residues flanked by untranslatedregions at both ends.3 Cleavage of the precursoryields at least 10 different proteins: the structuralproteins Core, E1, E2, and p7, and the nonstructuralproteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B.The HCV NS3 protein is a 69-kDa hydrophobic

bifunctional protein with protease and helicaseactivities. The two enzymatic activities are situatedin separated domains of the protein: the helicase islocated in the C-terminal two-thirds region of theprotein, while the serine protease (NS3p) is encodedby the N-terminal one-third region. NS3p is onecomponent of a heterodimeric serine protease thatrequires the noncovalently associated viral proteincofactor NS4A for optimal catalytic activity.4 Theactivity of the NS3–NS4A complex is responsible forthe proteolytic cleavage of the viral polyprotein.5 Inaddition to its essential role in the generation of theviral RNA replication machinery, it has beenrecently shown that NS3 is a major factor bywhich the virus antagonizes the host cell innateimmune response. The NS3–NS4A complex isresponsible for the cleavage of CARDIF and TRIF,two critical components by which cells sense

d.

1143A Noncovalent Inhibitor/HCV NS3 Protease Complex Structure

invasion by viral pathogens, thus triggering induc-tion of the antiviral state.6,7

The general topology of the protease domain ofHCV NS3p consists of two β-barrels, with thecatalytic triad composed ofH57, D81, and S139 sittingona crevice between the twoβ-barrels. In the complexNS3p–NS4A, the cofactor inserts itself into the proteinstructure and completes a large β-sheet, makingsignificant contacts with several NS3p regions.8

Structural studies on NS3p have shown that thesubstrate recognition site lacks several surface loopsthat typically contribute to the formation of thewall ofthe substrate-binding pocket in other members of thechymotrypsin protease family. As a consequence, thesubstrate-binding site of the NS3–NS4A serine pro-tease appears to be relatively flat, featureless, andsolvent-exposed. In the full-length protein, the heli-case domain slightly protects the P-side region fromexposition to the solvent and interacts with someresidues of the substrate recognition site.9 The

Fig. 1. Inhibitors of NS3–NS4A serine protease and titratio(compound 1) and hexapeptide DEDifEChaC-OH (compoundof NS3p showing the well-resolved R123 NH signal after threspectively.

unusual characteristics of the NS3p substrate-bindingsite account for the enzyme's requirement of 10-amino-acid-long peptide substrates, spanning fromP6andP4′,10 and constitute one of themajor problemsin the development of small-molecule inhibitors of theenzyme.An additional particular feature of the HCV NS3p

with respect to other serine proteases is that N-terminal cleavage products are inhibitors of theenzyme itself.11 This has been exploited for thecreation of a variety of potent peptide-basedinhibitors. Three compounds, which have beendesigned with such lead compounds as startingpoints, have shown proof-of-concept in clinicaltrials: telaprevir (VX-950; Vertex), boceprevir (SCH-503034; Schering-Plough), and ciluprevir (BILN-2061; Boehringer-Ingelheim).12 It is interesting tomention that while VX-950 and SCH-503034 arecovalent reversible inhibitors, BILN-2061 is a non-covalent product-based inhibitor. An optimized

n experiments. (a) Chemical structures of phenethylamide2) inhibitors. (b and c) Selected region of the HSQC spectrae addition of different amounts of compounds 1 and 2,

1144 A Noncovalent Inhibitor/HCV NS3 Protease Complex Structure

hexapeptide product inhibitor that serves as thebasis of several inhibitors is depicted in Fig. 1a(compound 2), which was shown to have a Ki of40 nM.13 This compound uses a dual anchor—an“acid anchor” at the N-terminus and a “P1 anchor”at the C-terminal part of the molecule—for optimalbinding.14 However, a major limitation of peptidesas drug candidates, especially those containingmultiple negatively charged carboxylate groups, isthat they are unlikely to have the desired pharma-ceutical properties, particularly with respect tobioavailability.15 Therefore, significant effort bymedicinal chemists was aimed at decreasing thesize and charge of these initial compounds. Aninteresting approach in this sense led to thediscovery of phenethylamide inhibitors (such ascompound 1, Fig. 1a; Ki of 0.6 μM).16–18 In this typeof inhibitors, phenethylamide moiety was designedto extend the interaction to the cavities present in theS′ region of the protein. This extra interactionallowed us to reduce the length of the inhibitorfrom a hexapeptide (residues P1–P6) to a tetrapep-tide (P1′–P3) (Fig. 1a), with a 250-Da consequentreduction in molecular mass.In the present work, we have studied the interac-

tion of the phenethylamide inhibitor compound 1and compared its kinetics of interaction with that ofthe hexapeptide inhibitor compound 2, which onlyoccupies the S region. The high-resolution structureof the complex between NS3p belonging to thegenotype 1b and compound 1 revealed particularfeatures that explain the very stable complexformed. Moreover, formation of the complex isaccompanied by a large conformational stabilizationof the N-terminal domain and the active site thatstrongly resembles the effect of the cofactor binding.From these results, we demonstrate that inhibitorsoccupying, at least partially, the S′ region of theprotein may induce a final structure of the complexthat is, in many respects, similar to that of the activeNS3–NS4A complex.

Results

Interaction of phenethylamide (compound 1)with NS3p shows a slow koff

Figure 1b and c show the change in the R123backbone amide NH chemical shift resonance ofNS3p when titrated with compounds 1 and 2,respectively. As shown in this figure, the exchangebetween free and bound protein states occurs withdifferent timescales for the two compounds. At0.5 Eq of compound 2, we observed a peak with anaverage position between that of the free proteinand that of the bound protein (Fig. 1c). In general,the effect of the exchange between the free form andthe bound form of the proteins on the spectrumdepends on an exchange constant k:

k = koff 1 + xEL=xEð Þ ð1Þ

where koff is the dissociation rate constant, and xELand xE are the molar fractions of the complex and thefree protein, respectively. The lack of line broad-ening due to exchange indicates that k is muchhigher (at least 50 times) than the difference inchemical shift between the resonance of the freespecies and the resonance of the bound species. Byusing Eq. (1), a lower limit for koff on the order of3700 s−1 can be estimated, considering the largestchemical shift difference between the two signals(150 Hz in the 15N dimension). On the other hand,the exchange observed in the case of compound 1 issignificantly slower because, with 0.3 Eq of theinhibitor, the two forms of the protein are detectedseparately. As in the first case, no line broadeningdue to exchange was observed, indicating that k ismuch lower than the difference in chemical shift.Analysis of this result with Eq. (1) and consideringthe smallest chemical shift difference (25 Hz in the1H dimension) lead to an upper limit for koff on theorder of 1 s−1. Estimation using other well-resolvedpeaks showed similar values for the two titrationexperiments (data not shown).The calculated koff values imply very different

lifetimes for the two complexes (less than 1 ms forNS3p/compound 2 and longer than 1 s for NS3p/compound 1). Unexpectedly, the longer lifetime isobserved for the complex formed by the compoundpresenting the lower inhibitory potency (compound1 shows a 15-fold larger Ki). The ionic strength usedin the NMR experiments (100 mM NaCl) is knownto destabilize the interaction of compound 2 withNS3p: the IC50 measured at 150 mMNaCl is 40 timeshigher than that measured in the absence of salt,13

explaining the short lifetime observed for the NS3p/compound 2 complex. On the other hand, experi-ments performed in the presence of the NS4Acofactor show that compound 1 quantitativelydisplaced an active-site fluorescent probe withkoff=3.2 s−1,16 indicating a lifetime for the NS3p/compound 1 complex very similar to that estimatedunder our conditions. Both the NMR experimentsand the activity assay measurements show thatcompound 1 forms a very stable complex withNS3p, in spite of its moderate inhibitory potency.

The phenethyl ring is highly complementary tothe S1′ pocket

The high-resolution structure of the complexbetween NS3p and compound 1 was determinedby using standard NMR procedures. A good finalconvergence was obtained (see Fig. 2a and Table 1).This structure represents the first available structureof a complex of the NS3 protease with a noncovalentinhibitor solved by NMR (Protein Data Bank 2K1Q).Actually, a crystallographic structure with a non-covalent macrocyclic inhibitor has indeed beenpresented.19 However, atomic coordinates are notavailable at the present time.The solution structure was determined for resi-

dues 22–186 of NS3p, since residues 1–21 were notdetected in any of the three-dimensional (3D)

Fig. 2. High-resolution structure of the NS3p/compound 1 complex derived by NMR. (a) Stereoview of thesuperposition of the 20 lowest-energy structures and (b) a ribbon model of the structure. Compound 1 backbone is shownin green. Only residues 22–186 are included, since residues 1–21 are not assigned. (c) The intermolecular interface is color-coded to indicate electrostatic potential (negative charge: red; positive charge: blue). (d) Schematic representations of theN-terminal domain in the NS3–NS4A (left) and the NS3/compound 1 (right) complexes are shown to illustrate the changein topology caused by the insertion of the cofactor NS4A.


experiments. As already reported for free NS3p20

and the α-ketoacid complex,21 the N-terminalresidues only show up in the two-dimensional(2D) 1H–13C experiments, but their HN nucleicould not be observed in any of the 2D and 3Dexperiments. The two β-barrels defining the N-terminal (residues 32–86) and the C-terminal (resi-

dues 103–181) domains are equally well defined inthe structure superposition. This observation shouldbe contrasted with a previous result relative to thesolution structure of NS3p, for which a remarkablybetter resolution was obtained for the C-domainwhen compared to the N-domain.20 In the NS3p/compound 1 complex, the secondary elements and

Table 1. Experimental restraints and structural statisticsfor the 20 lowest-energy structures of the NS3p/compound 1 complex

Number of experimental restraints 2978Distance restraints from NOEs 2622Intraresidue 1519Interresidue 1013Intermolecular 90

Hydrogen-bond distancerestraintsa

118

Dihedral angle restraints 238Phi constraints 119Psi constraints 119

Average number of restraintsper residue (32–181)

18.6

Xplor energies (kcal/mol)Etotal 1317.4±11.7Ebond 21.0±0.7Eangle 201.9±2.9Eimproper 28.9±1.0EvdW 66.2±2.9Ecdih 1.6±0.5Erama 982.7±9.0ENOE 15.1±2.9rmsd from experimental restraintsAverage distance restraintsviolation (Å)

0.017±0.002

Average dihedral angle restraintsviolation (°)

0.33±0.05

rmsd from idealized covalentgeometry

Bond (Å) 0.00286±0.00004Angle (°) 0.535±0.004Improper (°) 0.383±0.007Ramachandran analysisResidues in favored regions (%) 91.5±1.3Residues in additionally allowedregions (%)

6.9±1.3

Residues in generously allowedregions (%)

1.6±0.0

Residues in disallowedregions (%)

0.0±0.0

Coordinates precision(residues 32–181)

Backbone (Å) All heavyatoms (Å)

0.33±0.05 0.90±0.05a HN–O and N–O distances were constrained to 2.1±0.5 and

3.0±0.5 Å, respectively.


overall topology of the C-terminal domain (Fig. 2band c) are in complete agreement with all the NS3pstructures reported to date. On the other hand, thetopology of the N-terminal domain is different fromthat observed in the presence of the cofactor (Fig.2d), although the seven β-strands are constituted bythe same residues. In fact, the D1-E1a β-sheet isshifted towards the A1-B1 β-sheet, allowing thehydrophobic surface of A1 to be shielded from thesolvent. In the NS3p–NS4A complex, the cofactorcompletes a larger β-sheet making contacts bothwith A1 from one side and with A0 and D1 from theother side (Fig. 2d).Ninety intermolecular nuclear Overhauser enhan-

cements (NOEs) were detected through isotope-filtered and isotope-edited experiments, and used todefine the relative position and conformation ofcompound 1 in the active site. In particular, a largenumber ofNOEswere observedbetween the aromatic

protons of the phenethylamide ring and the side chainof K136. Extensive stacking between the long sidechain of K136 and the phenyl ring of the phenethy-lamide explains the modest twofold decrease inpotency for the K136M mutant.16 The para-positionof the carboxyl group also plays a role in thisinteraction. In the structure, it is close to both K136and R109, suggesting an electrostatic interaction thatfavors binding (Fig. 2c). This interaction explains anumber of observations: (i) the ortho-CO2H com-pound showed a reduction in inhibitory potency ofmore than 3 orders ofmagnitude compared to the paraisomer;16 (ii) mutation of K136 into arginine does notchange the inhibitor potency of compound 1; and (iii)the corresponding methyl ester is inactive.18 K136 isalso important for the interaction with P3 side chain,as it is placed equidistantly from the phenethyl andthe P3 carboxylate groups. Other hydrophobic inter-actions with the phenethyl ring involve the side chainof Q41, which, together with K136, forms a shallowcavity that is used to bind the aromatic ring tightly,probably contributing to the very long lifetime of thecomplex, as indicated by exchange data (Fig. 1b). Theortho-chlorine atom of the phenyl ring projects out ofthe active-site cavity to avoid steric clashes with F43.Only P1 and the phenyl ring of the phenethylamidemoiety are bound insidedeep cavities,whereas P2, P3,and theN-terminal capping group lie on a flat surface.The aromatic ring clearly contributes to bindingenergy by exploiting the S1′ pocket.

Binding of compound 1 provokes an active-sitestructural rearrangement similar to that causedby NS4A

A comparison with the crystal structures of NS3p,obtained in the absence22 or in the presence of theNS4A cofactor8,23 (presented in Fig. 3a), showspairwise Cα–Cα distances measured after the super-position of the NS3p/compound 1 and free NS3p(red) or the NS3p–NS4A complex (blue). Therelative positions of the three members of thecatalytic triad are very similar in the three structures.With respect to NS3p, the NS3p/compound 1complex shows large differences mainly in threeregions: in the α1-D1 loop, in the E1b strand, and inthe E1b–F1 loop and the α3 helix, yielding an overallRMSD of 2.7 Å for Cα atoms (residues 32–181). Onthe other hand, the NS3p/compound 1 complex ismore similar to NS3p–NS4A. The overall RMSD forCα atoms is 1.9 Å, which drops to 0.9 Å if residues63–72, belonging to the D1-E1a β-sheet, areexcluded.Superposition of the NS3p/compound 1 complex

onto a number of covalent-inhibitor/NS3p–NS4Aternary complexes (Fig. 3b) shows a high degree ofsimilarity, with an average RMSD of 0.9 Å forbackbone atoms, excluding the D1-E1a β-sheet.After shifting with respect to the other complexstructures, the D1 strand occupies a position veryclose to the cofactor. Figure 3c shows the relativeposition of the inhibitors and the His-Asp dyadobtained after superimposing only the protein

Fig. 3. Comparison of the NMR solution structure of the NS3p/compound 1 complex with existing NS3p crystalstructures. (a) Comparison with crystal structures of NS3p and NS3p–NS4A. After superposition of structures, distancesbetween corresponding α carbon atom nuclei in the two structures are plotted as a function of the residue number (NS3p/compound 1 versus NS3p, in red; NS3p/compound 1 versus NS3p–NS4A, in blue). At the top the secondary structure,elements of the NS3p/compound 1 complex are reported. (b) Superposition of the NS3p/compound 1 NMR structure(green) onto a series of covalent inhibitor/NS3p–NS4A ternary crystal structures (red). The following structures wereused for comparison: 1RGQ, 2A4Q, 2A4R, 2F9V, 2FM2, 2GVF, 2F9U, 1RTL, 1DY8, 1DY9, 1N1L, and 1W3C. (c) Relativeposition of the different inhibitors, including compound 1. The relative positions of the His-Asp dyad in the differentstructures are also included: NS3p/compound 1 (green) and inhibitor/NS3p–NS4A (red).


backbone. The absence of the cofactor in the NS3p/compound 1 complex does not have any significantinfluence on the relative orientation of the inhibitoror on the position of the catalytic residue side chains.An analysis of induced changes in chemical shift

upon compound 1 binding further supports theobservation that the structure of the catalytic site isvery close to that observed for the cofactor complex.Using chemical shift perturbation, it was concludedthat interaction of NS3p with the cofactor results inlarge stabilization of the catalytic triad.24,25 Largechanges in the chemical shift of the H57 amidegroup are detected, and the observed 1H chemicalshift (around 11 ppm) probably reflects a hydrogenbond to the D81 Oδ1 side-chain atom.8,23,26 Theinteraction of NS3p with compound 1 also causes asignificant change in the 1H–15N heteronuclearsingle quantum coherence (HSQC) spectrum, as

observed in Fig. 4a. Most notably, the observedchemical shifts for the HN groups of residues 56–59are very similar to those reported for the two NS4Acomplexes (Fig. 4a, red and green). The chemicalshift differences between free NS3p and the NS3p/compound 1 complex are illustrated in Fig. 4b,where a negative index corresponds to residues forwhich the HN group resonance assignment isavailable for the complex with compound 1, butnot for the free protein. The highest difference inchemical shift belongs to H57, and the large down-field shift observed for the proton, as described, is astrong indication of the formation of a hydrogenbond with the close D81 carboxylate (see Fig. 3c). Inaddition to the expected chemical shift differencesfor the S-binding region of the C-terminal domain,there are several regions of the N-domain that areperturbed by the binding of compound 1 and

Fig. 4. The effect of the formation of the complex on the resonance frequency of several diagnostic peaks in the 1H–15NHSQC spectrum. (a) The free NS3p protein and the corresponding NS3p/compound 1 complex are shown in blue andblack, respectively. The complexes scNS3p–NS4A and NS3p(2a)–NS4A are shown in red and green, respectively. Thecomparison between blue and black peaks shows chemical shift perturbation upon compound 1 binding. (b) Chemicalshift differences measured in the 1H–15N spectrum between free NS3p and the NS3p/compound 1 complex using theparameter Δδ=sqrt(ΔδH

2 +ΔδN2 /25). A negative index is used for residues not assigned in the free NS3p spectrum and

observed and assigned in the NS3p/compound 1 complex, due to the general structure stabilization induced by theformation of the complex.


involve strand E1b, strand F1, and the α1-D1 loop.The position of the first two strands defines theorientation of the catalytic aspartate, which islocated at the beginning of the F1 strand. Moreover,most of the new assignments in the NS3p/compound 1 complex correspond to the N-terminaldomain and are concentrated in the two regionsaround the catalytic His and Asp residues. Theappearance of these new peaks is another line ofevidence for a more structured situation around thecatalytic site and in the N-terminal domain ingeneral, induced by inhibitor binding.

The catalytic histidine is hydrogen-bonded toD81 in the NS3p/compound 1 complex

A sharp signal for a proton at 14.9 ppm is readilyobservable for the NS3p/compound 1 complex,which could be assigned to H57 Hδ1, based on the15N chemical shift (Fig. 5a and b) and the NOEsobtained in a 2D NOE spectroscopy (NOESY)spectrum (Fig. 5c). In the 1H–15N long-rangeHSQC spectrum, the catalytic histidine showstypical signals for a δ1 protonated tautomer (Fig.5b); at the pH used in this study (pH 6.8), it is fully in

Fig. 5. Histidine H–N resonancecharacterization. (a and b) 1H–15Ncorrelation experiments for theNS3p/compound 1 complex show-ing characteristic signals for thethree histidines. In particular, H57shows a pattern typical of a δ1protonated tautomer in the long-range 1H–15N HSQC spectrum (b).The δ1 nitrogen shows correlationwith a proton at 14.9 ppm (d) in the1H–15N HSQC spectrum (a). Two-dimensional NOESY experiment (c)showing short interproton dis-tances that allow unambiguousassignment of the resonance at14.9 ppm. (e) The relative positionsof D81, H57, and leucine P2 sidechains, as derived from NOErestraints (dotted lines).


the neutral state, indicative of a low pKa (b6). Thechemical shifts for both the Nδ1 nucleus (178.6 ppm)and the Hδ1 nucleus (14.9 ppm) are indicative of astrong hydrogen bond, whereas that of the non-protonated Nɛ2 (253.2 ppm) indicates that it is not anacceptor of hydrogen bonding.27

The His Hδ1 signal remains sharp in a very broadtemperature range (from 5 to 35 °C.) Its chemical shiftis very similar to that already observed for an α-ketoacid complex,21 but the fact that the peak is easilyobservable at 35 °C indicates a reduced exchangewiththe solvent with respect to the α-ketoacid situation(for the latter, the corresponding peak disappeared attemperatures above 20 °C). This difference isobserved even if the P2 side chain (a leucine) looksat the same position in both complexes, indicatingthat further stabilization of the active site is achievedin the NS3p/comp1 complex, probably due to thebinding of the aromatic ring in the S′ region.An empirical correlation between the low-field

proton chemical shift and the N–O distance can beused to gain insights into the strength of thehydrogen bond between H57 and D81.28 In thisway, a distance of 2.67 Å can be calculated from theobserved catalytic histidine Hδ1 chemical shift(14.9 ppm), which is somewhat longer than expectedfor a strong and short hydrogen bond (2.45–2.65 Å).29 Strong and short hydrogen bonds arenormally formed when the catalytic histidine isprotonated,28 which is not the case for the NS3p/compound 1 complex. However, the estimateddistance is shorter than that of a normal weak

hydrogen bond (2.75–3.00 Å)29 and that of the vander Waals contact distance between nitrogen andoxygen (2.9 Å), indicating the significant strength ofthe His-Asp interaction and the stabilization of theactive-site dyad by inhibitor binding.

Backbone dynamics and water exchangeprofiles indicate stabilization of the N-domainupon binding of compound 1

Backbone dynamics (via the study of 15N relaxa-tion) and amide proton–water exchange kineticswere measured to obtain further evidences ofdifferences in the overall compactness of thestructure induced by compound 1 binding. Regard-ing the 15N relaxation measurements, 15N T2,

15N T1,and 1H–15NNOEwere measured for both NS3p andthe NS3p/compound 1 complex, with 4% glycerol atpH 6.8 and at a temperature of 25 °C (Fig. S1 inSupplemental Data). These conditions, dictated bystability considerations of free NS3p,20 significantlyinfluence the overall motion of the molecule due tothe high viscosity of the solution caused by thepresence of glycerol.Interpretation of these relaxation data using the

model-free approach30 is complicated by the factthat we cannot safely assume an isotropic motion forNS3p or for the NS3p/inhibitor complex, mainlybecause the first 22 residues are not observed andprobably disordered, leading to a large deviationfrom isotropic tumbling.31 For this reason, thespectral density mapping (J-mapping) approach is


to be preferred, particularly in its reduced version.32

From the 15N relaxation data measured for the twoproteins, the corresponding spectral density func-

Fig. 6. Comparison of the backbone dynamics of the NS3p/exposure factors. (a) Spectral density correlation J(0.87ωH)/J(0) gNS3p and (b) the NS3p/compound 1 complex. Error bars araccording to their mobility (yellow, orange: residues with picosmotions; magenta, blue: residues with microsecond-to-millisecoRelative solvent exposure factors for the NS3p/compound 1 copositive values indicate a lower solvent exchange rate (i.e., redcompared to the complex. The reverse applies to negative valuesthe N-terminal domain upon compound 1 binding.

tions at the three appropriated frequencies werecalculated at a 1H frequency of 700 MHz (Fig. S2 inSupplemental Data).

compound 1 complex and free NS3p, and relative solventraphs derived from 15N relaxationmeasurements for the freee included in both dimensions. Residues are color-codedecond motions; black: residues with no significant internalndsmotion), and their location on the structure is shown. (c)mplex and the free NS3p (ΔζNOE=ζNOE

NS3p−ζNOENS3p/compound):

uced solvent exposure) of NH protons for the free protein.Data showageneralizeddecrease in the solvent exposure of


One way to detect the presence of motions ondifferent timescales is to analyze correlation graphsbetween J(0.87ωH) and J(0), as depicted in Fig. 6aand b. In these figures, residues characterized bymotions in the picosecond timescale appear on theupper-left corner of the plot, as a consequence ofhigh values for J(0.87ωH) and low values for J(0). Onthe other hand, residues with motions in themicrosecond-to-millisecond timescale appear onthe lower-right corner of the plot due to high valuesfor J(0). Residues in structured regions of the proteinand lacking internal motions are clustered at thebottom center of the plot.Analysis of the plot obtained for free NS3p (Fig.

6a) shows a larger scatter, as compared to the oneobtained for the NS3p/compound 1 complex (Fig.6b). This is the consequence of a series of internalmotions present in the free protein and quenchedupon formation of the complex. As expected, thelysine tail (residues 183–186) added to the protein toincrease its solubility is the most disordered regionin both cases. Another region showing fast motionsis that of residues 23–30, but it is clearly moredisordered in the complex. NS3p presents severalresidues with motions in the microsecond-to-milli-second timescale that show lower-than-averagevalues for J(0). These residues are mainly locatedin the N-terminal domain and comprise E30, T38,N49, W53, K68, T76, N77, G84, I153, and T178.Figure 6b shows that, upon binding of compound 1,these slow motions disappear, and a certain degreeof flexibility is only conserved for residues 72 and 73,which are located in the E1a-E1b loop. In addition,comparison of Fig. 6a and b shows that the averageJ(0) value for residues not showing internal mobility(in black) moves from 8.9 to 8.2 ns/rad when goingfrom the apo form to the complex form of NS3p.Given that J(0)=2τc/5 (where τc is the globalcorrelation time) in the absence of internal flexibility,this shift indicates a reduction of about 1.8 ns in theτc values of the complex. Our interpretation of theseresults is that the N-domain is significantly morecompact in the complex, based on the distribution ofthe two spectral density functions plotted in Fig. 6aand b.These conclusions were further confirmed by an

analysis of the water exchange rates of amideprotons. These kinetic parameters were measuredusing the water–NOE experiment33 (see MaterialsandMethods). For short mixing times, the NOEwithbound water or with protons that exchange withwater is significantly less important than thechemical exchange (ζNOE values larger than 0.1 aredue almost exclusively to chemical exchange).Figure 6c shows the difference between the mea-sured ζNOE obtained for the NS3p/compound 1complex and the measured ζNOE obtained for thefree NS3p. Residues showing a positive ΔζNOE aremore protected with respect to solvent exchange inthe free protein, whereas the reverse is true for thoseshowing a negative ΔζNOE. For the C-domain, thedifference in water exchange, and hence in solventexposure, is not significant, whereas the N-domain

shows an overall decrease in solvent exposure dueto the formation of the complex. It is particularlyrelevant to the α1-E1a loop, but also to other strandsand loops close to the catalytic triad. This result isfurther evidence for the formation of a morecompact structure in the complex. The only excep-tion is represented by the 23–30 loop, which showshigher protection in the free NS3p. The reducedmobility (found in the analysis of 15N relaxationdata) and solvent exposure of the 23–30 tailobserved for the free protein are the consequenceof hydrophobic interactions in a more flexible N-terminal domain. When this domain becomes morecompact, these residues acquire higher flexibilityand, consequently, higher solvent exposure.

Discussion

Compound 1 exploits the binding of a phenyl ringin the S′-binding region, which increases signifi-cantly the conformational stability of the protein inthe complex. In addition, the para-carboxylate groupof the ring is favorably positioned to interact withR109 and K136, further enhancing the affinity of thecompound. Exchange kinetics indicates that thecomplex has a long half-life, on the order of secondsor longer. Remarkably, a more potent inhibitorbased only on the P-side of the substrate (compound2), under the same experimental conditions, showsthe formation of a considerably less long-livedcomplex. Both in the presence and in the absenceof the cofactor, compound 1, together with NS3p,forms a long-lived complex (with a lifetime ofaround 1 s) despite its modest inhibitory potency.16

This scenario strongly suggests that the moderatemicromolar affinity of compound 1 for NS3p is theresult of an inefficient association. In support of thisview, an association rate constant that is 3 orders ofmagnitude lower than that expected for a diffusion-controlled mechanism was observed in an activityassay (kon=3.5×10

6 M−1 s−1).16 This feature can beattributed to the absence of the negative charges thatthe substrate and the hexapeptide inhibitor com-pound 2 exhibit in P5 and P6 positions, ready tointeract with residues of the protein around R161.14

This “acidic anchor” is present in the hexapeptideinhibitor compound 2 and explains a faster associa-tion kinetics leading to a more potent inhibitor,although it forms a short-lived complex.The mechanism by which the NS4A cofactor

activates the proteolytic activity of NS3p is still amatter for debate. Although it enhances the activityof NS3p, the first cleavage of the enzyme isperformed without the cofactor. Hence, there is aresidual activity even in its absence—a behavior thatis not common to other NS3 proteases of viruses ofthe same family.34 Based on crystallographic andbiochemical evidence, the generally acceptedmechanism of activation by NS4A includes: (i)stabilization of the N-terminal domain; (ii) optimi-zation of the orientation of residues forming thecatalytic triad; and (iii) contribution to the formation


of the substrate recognition site.35 Remarkably, wefound all these effects, which had been previouslythought to be exclusively induced by the cofactor, tobe exerted by a noncovalent inhibitor in the absenceof NS4A.

Binding of compound 1 stabilizes the N-terminalconformation

It has been shown that binding of inhibitors basedexclusively on the S′ region of the protein producesgreat protection against proteolysis of the N-term-inal region of the protein.36 OurNMRmeasurementspresented here show that the N-terminal domain isflexible in the absence of the cofactor, substrate, orinhibitor (Fig. 6a). Both dynamical behavior andsolvent exposure of the N-domain show a significantdecrease upon binding of compound 1. The resultingstructure differs in one single region with respect tothat of the NS3p–NS4A complex, circumscribed tothe D1-E1a β-sheet (Fig. 3a, blue line). In this shiftedposition, there is an interaction between strands D1and A1, mimicking the presence of the cofactor (seeFig. 2d). This new topology in the absence of thecofactor forms a “closed” and stable conformationthat could be a less favorable starting point forcofactor binding than the “open” and flexibleconformation of the free NS3p.

Binding of compound 1 optimizes theorientation of active-site residues

Two of the catalytic triad residues belong to the N-domain (H57 and D81), and their correct orientationwas initially assumed to be correlated to NS4Abinding.22 However, the complex of NS3p with anα-ketoacid inhibitor showed the presence of adownfield peak at around 15.0 ppm, indicatingthat a strong hydrogen bond was formed betweenH57 and D81 in the absence of the cofactor.21 Weobserved a similar signal for the NS3p/compound 1complex, and the stability of the resulting H57-D81hydrogen bond was enhanced. The presence of thissignal, which was unambiguously assigned to theH57 Nδ1 proton, reveals that the H57-D81 iscorrectly aligned, even without NS4A. This conclu-sion agrees with the fact that the pH dependence ofthe NS3 hydrolysis reaction is not affected by thepresence of NS4A, indicating that the pKa of thecatalytic histidine, and hence its structural environ-ment, is not changed by the cofactor.37

Binding of compound 1 fully stabilizes thesubstrate-binding site

The substrate-binding site is constituted by an Sregion (located in the C-terminal β-barrel) and an S′region (located in the N-terminal β-barrel). The Sregion has a very defined structure, and substrate orinhibitor binding does not change it significantly(Fig. 3a). The S′ region is located in the N-domaincomprising residues 37–41,38 a region partly occu-pied by the phenyl ring of compound 1. Figure 3a

shows that the NS3p/compound 1 complex and theNS3–NS4A complex do not differ in the conforma-tion of this region. The S′ region of free NS3p, on theother hand, shows dynamical motions as suggestedby our relaxation data. This conformational fluctua-tion is partially maintained upon binding ofinhibitors based exclusively on the P side of thesubstrate, such as compound 2; in the absence of thecofactor, it shows more than one binding mode.39

The behavior of compound 1 in stabilizing the N-terminal domain of NS3p contrasts with the effect ofa boronic acid inhibitor.25 It was observed thatbinding of the boronic acid inhibitor in the absenceof the cofactor produced no changes in the chemicalshift of several residues located in the S-binding site,whereas in the presence of the cofactor, it produceda larger chemical shift perturbation, suggesting theformation of a more stable complex. The differencewith compound 1, which shows both the formationof a long-lived complex without the cofactor and thestabilization of the N-domain, may reside in the factthat the phenyl ring of compound 1 occupies the S1′binding pocket located in the N-terminal domain. Inthis respect, the compound 1 inhibitor better mimicsthe substrate, which binds also in the S′ bindingregion, and opens the question as to whether thesubstrate itself can have a role in the stabilization ofthe N-terminal domain. However, it is clear that, inthe absence of the cofactor, the substrate will find aflexible N-domain, and the transition towards amore structured N-domain represents a thermody-namic and kinetic barrier along the productivereaction pathway. This penalty explains the 80-folddecrease in affinity for NS3 protease in the absenceof NS4A shown by inhibitors carrying primeresidues, whereas the affinity of inhibitors withonly P residues is NS4A-independent.37

Conclusions

The development of small molecules that caninhibit the enzymatic activity of HCV NS3 proteaseis still a major challenge. The major problems relatedto the discovery of inhibitory compounds of lowmolecular mass reside particularly in the relativelyfeatureless nature of the P region of the substrate-binding site of the enzyme. One way to circumventthis problem is to exploit the characteristics of the S′site, as in the case of the noncovalent inhibitorcompound 1. The structure of the NS3p/compound1 complex shows that the para-substituted phenylring is both hydrophobically and electrostaticallycomplementary to the S1′ pocket of NS3p, leading toa very stable complex. The absence of the “acidicanchor,” however, reduces the association rate,diminishing the overall value of the associationconstant.The binding of an inhibitor exploiting the S′ region

induces structural changes in NS3p that are gen-erally attributed only to the NS4A cofactor binding,such as the stabilization the N-terminal domain ofNS3p and the substrate-binding site, and the correct


alignment of the catalytic His-Asp residues. In fact,the structure of the inhibitor/NS3p complex is verysimilar to that of the NS3p–NS4A complex. How-ever, the N-domain of the protein shows a significantshift of the D1-E1a β-sheet, which positions the D1strand in a region very similar to the one occupied bythe cofactor. This fact, together with a less flexiblestructure of the N-domain upon inhibitor binding,suggests that interaction with the cofactor to formthe ternary complex can become energetically lessfavorable. This opens the possibility for inhibitors,which are capable of exploiting the S′ region andstabilizing the N-domain of NS3p, to finally preventthe formation of the functional replication complexby inhibiting also the interaction with NS4A. Thisnovel mechanism of actionwas recently investigatedwith ACH-806 (GS-9132), an acylthiourea com-pound that binds the NS4A cofactor.40 This inhibitorshowed reversible nephrotoxicity that precludedfurther clinical development. However, the observedantiviral activity from Phase I studies validatedNS4A interaction as a therapeutic target.

Materials and Methods

NMR sample preparation

Uniformly 15N- or 15N/13C-labeled samples of the NS3pprotein (residues 1–180) with a solubilizing hexapeptidetail (ASKKKK) were expressed and purified as previouslydescribed.21 The chemical synthesis of compound 1 (thephenethylamide inhibitor) has also been describedpreviously.16 Final samples for NMR experiments con-tained 0.6–0.8 mM NS3p or NS3p/compound 1 complex,100 mM NaCl, 20 mM sodium phosphate (pH 6.8), 1 mMdithiothreitol, 0.3% deuterated n-octyl β-D-glucopyrano-side, and 0.01% sodium azide in a solution of 90% H2O/10% D2O or 100% D2O.

NMR spectroscopy

The NMR experiments were performed at 298 and 308 Kon Bruker Avance700, Avance600, Avance500, andAvance400 spectrometers equipped with triple-resonanceprobes incorporating shielded z-axis gradient coils. NMRdatawere processed on SiliconGraphicsworkstations usingNMRPipe41 and analyzed using NMRView software.42

Chemical shift assignment

The following standard set of triple-resonance spectrawas acquired in H2O: HNCO, HNCA, HN(CO)CA,CBCANH, and CBCA(CO)NH. For the sample dissolvedin D2O, the following experiments were performed: (H)CCH-COSY, (H)CCH total correlated spectroscopy, H(C)CH-COSY, and H(C)CH total correlated spectroscopy.Side-chain resonance assignment was achieved for almostall nuclei.

NOESY experiments

15N- and 13C-edited 3D NOESY experiments with amixing time of 150 ms were recorded in H2O. Intermole-

cular NOEs between the labeled protein and the unlabeledligand were detected using two F1-edited, F3-filtered 3DHMQC-NOESY spectra43 recorded in D2O with a mixingtime of 80 ms. A double-filtered [F1-C/N,F2-C/N]NOESYexperiment44 with a mixing time of 80 ms was performedto filter out NOEs originating from the 15N/13C-labeledprotein.

Exchange measurements

Exchange measurements were performed at 298 K forthe free NS3p and for the complex with compound 1 on aBruker Avance700 spectrometer using water–NOE pulsesequence. The experiment was performed in an inter-leaved manner to obtain two spectra, with watermagnetization oriented along the +z-axis and the −z-axis, respectively.33 The mixing time was 60 ms. In theexperiment with water magnetization oriented parallelwith the z-axis (parallel with amide proton magnetiza-tion), the ζ NOE effect (ζNOE) of water is added to theintrinsic intensity of the cross-peaks. In contrast, in theexperiment with water oriented anti-parallel with the z-axis, the ζNOE is subtracted from the intrinsic intensity ofthe cross-peak. Therefore, ζNOE can be calculated bytaking the difference between the cross-peak intensitiescorresponding to both experiments normalized by thesum of the intensities. For short mixing times, ζNOE is duealmost exclusively to chemical exchange.

15N relaxation measurements and backbonedynamics

Relaxation experiments were carried out at 298 K in abuffer solution containing 4% deuterated glycerol for thecomplex and the free-NS3p protein. Measurements of 15NT1, T2, and

1H–15N NOEwere performed in an interleavedmanner at a 15N frequency of 70.94 MHz using standardpulse schemes.45,46 For NS3p in complex with compound1, relaxation delays of 8, 25, 33, 42, 50, and 59 ms wereemployed for T2, and delays of 14, 561, 1122, 1682, 2243,and 2804 ms were employed for T1. For free NS3p,relaxation delays of 8, 25, 42, and 59mswere employed forT2, and delays of 16, 563, 1124, 2245, and 2806 ms wereemployed for T1. Data were fitted using the Rate Analysisroutine of NMRView.42 Heteronuclear NOE values weredetermined by the ratio of peak volumes of spectrarecorded with and without 1H saturation, employing a netrelaxation delay of 4 s for each scan in both experiments.Typically, errors were about 5% for T1, 6% for T2, and 5%for 1H–15N NOE measurements. Analysis of the 15Nrelaxation data for free NS3p and the NS3p/compound 1complex was performed using spectral density mappinganalysis.32

Structure calculation

Structure calculation of HCV NS3p 1b strain BKcomplexed to compound 1 was performed using Xplor-NIH.47 Topology, force fieldparameters, and coordinates forcompound 1 were created in-house. A set of 2442intraprotein and 90 protein/compound 1 distance restraintswas used, subdividing them into three groups: strong (1.8–3.3 Å), medium (1.8–4.5 Å), and weak (1.8–6.0 Å) NOEs.Backbone ϕ and ψ dihedral angles were constrained tovalues predicted by TALOS.48 Backbone hydrogen bondswere recognized by evaluating the spatial relationship ofamide protons with potential acceptors in the initial


structures produced without the use of hydrogen-bondconstraints. Two distance restraints were defined for eachhydrogen bond: 1.8–2.3 Å for the HN–O distance and 2.3–3.3 Å for the N–O distance. A statistic-derived database ofRamachandran plot dihedral angles was used to improvestructural convergence. The high-temperature annealingphase used 3000 steps of molecular dynamics at 2000 K.Then temperature was lowered to 100 K in 127 cycles ofcooling phase with 0.002-ps steps. Finally, 4000 steps ofPowell energy minimization were executed.The statistics of structural geometry and experimental

constraints employed in calculations are summarized inTable 1. The programs AQUA and PROCHECK49 wereused to analyze the structures.

Accession numbers

Coordinates and structure factors have been depositedin the Protein Data Bank with accession number 2K1Q.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.11.017

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binding of a noncovalent inhibitor exploiting the s′ region stabilizes the hepatitis c virus ns3...

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