Ligand Bioactive Conformation Plays a Critical Role in the Design of

Article pubs.acs.org/jmc

Ligand Bioactive Conformation Plays a Critical Role in the Design of Drugs That Target the Hepatitis C Virus NS3 Protease Steven R. LaPlante,*,†,§ Herbert Nar,‡ Christopher T. Lemke,† Araz Jakalian,† Norman Aubry,† and Stephen H. Kawai*,†,⊥ †

Department of Chemistry, Boehringer-Ingelheim (Canada) Ltd., Research and Development, Laval, Québec H7S 2G5, Canada Department of Medicinal Chemistry, Boehringer Ingelheim Pharma KG, 88397 Biberach an der Riss, Germany

‡

S Supporting Information *

ABSTRACT: A ligand-focused strategy employed NMR, Xray, modeling, and medicinal chemistry to expose the critical role that bioactive conformation played in the design of a variety of drugs that target the HCV protease. The bioactive conformation (bound states) were determined for key inhibitors identified along our drug discovery pathway from the hit to clinical compounds. All adopt similar bioactive conformations for the common core derived from the hit peptide DDIVPC. A carefully designed SAR analysis, based on the advanced inhibitor 1 in which the P1 to P3 side chains and the N-terminal Boc were sequentially truncated, revealed a correlation between affinity and the relative predominance of the bioactive conformation in the free state. Interestingly, synergistic conformation effects on potency were also noted. Comparisons with clinical and recently marketed drugs from the pharmaceutical industry showed that all have the same core and similar bioactive conformations. This suggested that the variety of appendages discovered for these compounds also properly satisfy the bioactive conformation requirements and allowed for a large variety of HCV protease drug candidates to be designed.

■

INTRODUCTION The binding of a ligand to a macromolecule may be viewed as involving the collision of two flexible objects whose shapes must be of sufficiently complementary to allow for the formation of an encounter complex. Subsequent mutual adaptations may then occur which stabilize the final ligand− protein complex.1 The shape and conformational properties of the ligand, in both the free and bound states, must be considered for a clear understanding of such biomolecular recognition processes.2 This said, deciphering the details of such factors governing binding events are often viewed as impractical in a drug discovery setting. Nonetheless, we believe that a lucid comprehension of the conformational and dynamic aspects of binding events, on the small molecule side, can be exploited to advance drug design and optimization efforts. The binding of peptidic species to proteins such as protease enzymes represents an especially problematic case. A protease generally binds its substrates or other peptide ligands through a number of important points of contact throughout a large active site. The peptides themselves exhibit rather complex conformational behavior that is highly dependent on the nature of the side chains. Such targets are well-known to be very frugal with respect to offering interesting non-peptidic compounds, even after the screening of very large compound collections. Moreover, even modest modifications to the peptide backbone are generally not well-tolerated. The NS3 protease of the hepatitis C virus (HCV), an enzyme which has been a target of © XXXX American Chemical Society

great interest to the pharmaceutical industry, clearly illustrates this. Inspection of the scientific and patent literature for inhibitors of this enzyme reveals only peptidic species. Furthermore, they are nearly all derived from the N-terminal product sequence DDIVPC. One can view DDIVPC as incorporating a flexible ‘scaffold’ consisting of a polypeptide backbone in which a particular residue is cyclized (i.e., the P2 proline). Whether carried out consciously or not, optimization in the varied directions reported to date has involved concurrent improvement of the individual subcontacts and maintenance of suitable conformational properties. In this work, the conformation and dynamic properties of the key peptidyl inhibitor 1 of HCV protease were systematically monitored by a number of NMR methods and through the use of a thorough truncation SAR (molecular dissection). The goal of this exercise was to acquire a clear understanding of the roles of various pharmacophores in maintaining the bioactive conformation. This represents an essential step to the design or further optimization of such therapeutic agents as it can greatly aid in separating conformational factors and subcontact aspects of the global binding event. Special Issue: HCV Therapies Received: August 30, 2013

A

dx.doi.org/10.1021/jm401338c | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Shown are the bound structures of DDIVPC (X-ray structure with inhibitor side chains labeled in yellow), BILN 127 (NMR and X-ray based model), 1 (X-ray structure), Faldaprevir (X-ray structure), and BILN 2061 (X-ray based model), all superimposed on the enzyme derived from the X-ray structure of the Faldaprevir complex. The ligand−enzyme complexes are displayed with atoms colored by atom -type except for the common scaffold, which is colored red. The complexes were derived by X-ray crystallography for DDIVPC (PDB code 4JMY) and 1 (where the urea N-terminus of 1′, PDB code 4K8B, was changed to a Boc), and the complex of BILN 127 was docked and consistent with NMR transferred NOESY data and X-ray structures of related compounds. The complex involving Faldaprevir was derived by X-ray crystallography (PDB code 3P8O). The complex involving BILN 2061 was based on the modification of an X-ray structure3f,6 of a related macrocyclic compound and energy minimization. See also Materials and Methods and Supporting Information. B



■

Article

(Faldaprevir) (“linear series”)8 and BILN 2061 (Ciluprevir) (“macrocycle series”).7 Both molecules successfully demonstrated a reduction in viral load in humans.9 As shown in Figure 1, these two molecules also maintain the same conformation of the peptide skeleton when protease-bound. The conformational features of the linear series is discussed in detail below, whereas the conformational optimization and design of the macrocycle series has been addressed elsewhere.2c,6,7a,b,9a Bound Conformation: Structure of the Inhibitor 1′: HCV Protease Complex. The conformation of inhibitor 1′ bound to the active site of the protease was unequivocally established by the X-ray structure of the complex (Figure 3). In this structure, π-stacking between the P2 quinolines of two complexed inhibitor molecules due to the face-to-face packing of the two protomers in the asymmetric unit was observed (see Supporting Information). It was therefore important to verify whether the P2 groups, when the complex is in solution, have the same conformation as in the crystal structure. NMR transferred nuclear Overhauser effect (transferred NOESY) experiments were employed to evaluate the protease-bound conformation of compound 1 in solution. The key interhydrogen distances from P2-Hγ to Q-H3 and from Q-H6 to the Boc were measured to be 1.8 and 2.9 Å, respectively. (The system used to designate atoms and amino acid positions10 in compounds 1 and 1′ are shown in Figure 2). In the crystal

RESULTS AND DISCUSSION Summary of SAR. Our drug discovery efforts with regard to HCV protease drew on our history of studying peptidyl inhibitors of another viral enzyme. We noted during our human cytomegalovirus (HCMV) campaign that, although very poor ligands, N-terminal cleavage products were complexed in a welldefined manner by this serine protease.4a This prompted us to investigate the N-terminal cleavage products of HCV protease. Using NMR,3a we discovered that the hexapeptide Asp-Asp-IleVal-Pro-Cys (DDIVPC; Figure 1) also binds its target in a welldefined way and were eventually found by us and others to be surprisingly potent inhibitors of the enzyme.3a,b,4b,5,7b Enzymatic assays subsequently found that the compound inhibited HCV protease with an IC50 of 71 μM (Ki = 19 μM).3a,b A recent publication speculates as to the nature of this exceptional product inhibition of a serine protease.3g As a lead molecule, DDIVPC was particularly attractive in view of the C-terminal carboxylate that imparts good solubility, as well as the dramatically improved specificity (notably with respect to elastase) since an activated ‘warhead’, commonly required for serine protease inhibition, is not present.2c The Xray structure of this key hexapeptide, complexed to HCV protease, is reported here (Figure 1; top right). The binding of the ligand occurs through the four C-terminal residues (IVPC) with the N-terminal aspartates solvent exposed, corroborating earlier NMR binding studies.3a,d The critical P1−P4 core binds in an extended conformation, a general feature of complexes of serine proteases and their peptidyl ligands. A summary of our extensive optimization effort is shown in Figure 1. A number of modifications,3b,c notably addition of the aryloxy group to the P2 residue, provided BILN 127 and a 10fold improvement in binding.3a NMR and modeling suggested that the introduced aryl moiety lies over part of the catalytic triad of the enzyme (vide infra).3a It was also apparent that the bound conformation is similar to that of DDIVPC (both displayed in red). A battery of NMR methods, including 13C T1 and transferred 13C T1 experiments,2c,d,3a found that important differences existed between the free and bound states of the this ligand (also see Supporting Information). These investigations formed the basis of a number of rational, structure-based optimization strategies including macrocyclization.6,7b N-Truncation coupled with further side chain and aryloxyoptimizations3c,d afforded the Boc-tripeptide 1 (BILN 1508) with a Ki of 0.020 μM.3 Shortly after, the X-ray structure of 1′, a very close urea analogue of carbamate 1 in complex with the protease, was solved. This complex is described in detail below and shows that the peptide scaffold is also bound in a very similar fashion as seen for DDIVPC and BILN 127. The large tricyclic P2-substituent indeed lies over part of the catalytic triad of the protease. Compound 1 became a molecule of central importance to our understanding of the SAR as crucial structural information concerning its binding to the target could be gleaned from the crystallographic structure of the protease complex with inhibitor 1′. Furthermore, the NMR spectra (1H and 13C) were very well-suited for a wide range of studies, notably ROESY and 13C NT1 analyses. It must be clarified that SAR had revealed that the carbamate-capped (Boc) tripeptides have better cell-based activity than their urea counterparts, which accounts for why the present studies (as well as optimization efforts) involved the former series. Subsequent efforts to improve potency and biopharmaceutical properties led to two series exemplified by BI 201335

Figure 2. Atom numbering used in this study. Inhibitor 1, X = O; inhibitor 1′, X = NH.

structure and after hydrogen atoms were added, the corresponding key distances were similar and were determined to be 2.0 and 3.2 Å. It was later found that similar distances were observed for compound 1′ using transferred NOESY experiments. Taken together, the NMR studies confirm that the bound crystal and solution structures correlate very well and that inhibitors 1 and 1′ are complexed, not surprisingly, in identical manners. As shown in Figure 3, compound 1′ is bound to the active site of HCV protease through an anti-parallel β-sheet between the inhibitor backbone (scaffold) and the E2-strand of the protease (Figure 3C and E). There are apparent hydrogen bonds between the P1-NH and the carbonyl of Arg155, as well as a pair of H-bonds between the P3 residue (carbonyl and NH) and Ala157 of the enzyme. The terminal urea-NH is also within H-bonding distance of the latter. These interactions are normally observed for peptide ligands complexed to serine C



Article

Figure 3. (A) Full view of the X-ray structure of HCV protease (green, with the catalytic triad colored magenta), the NS4A peptide cofactor (orange), and 1′ colored yellow and as atom-type (PDB code 4K8B). (B) Same as panel A but for apo HCV protease (PDB code 1BT7). (C) View showing the Cγ-exo conformation of the P2 proline ring and the orientation of the quinoline substituent, which lies over the protease catalytic acid side chains of Arg155 and Asp181. (D) View showing the shallow pocket of HCV protease with bound 1′ (green). (E) View of inhibitor 1′ (green) bound to the active site of HCV protease (white carbon atoms) showing canonical hydrogen bonds between the inhibitor backbone and the E2 strand (P3 to Ala157 and P1(NH) to Arg155). The side chain of Phe154 lies at the bottom of the S1 pocket. (F) View showing the interactions between the terminal carboxylate and the oxyanion hole, the latter composed of the backbone amides of residues 137−139. The hydrogen-bonding network involving the protease catalytic triad His57, Asp81, and Ser139 is also shown.

involved in binding of the inhibitor, from which it is oriented away. The P1 vinylcyclopropyl group protrudes into the shallow S1 pocket. The N-terminal portion of the peptide is conformationally extended, and with little S3 pocket to speak of, the P3 tert-butyl side chain lies on the surface of the protease and is solvent-exposed. The P2-quinoline6 moiety is oriented perpendicular to the gross plane of the proline ring. The latter is puckered such that the substituted γ-carbon is below the plane of the other pyrrolidine atoms (referred to as a Cγ-exo

protease active sites and are globally referred to as the ‘canonical’ binding mode. The terminal carboxylate forms a network of hydrogen bonds to the active site (Figure 3F). One of the oxygen atoms is within H-bonding distance of the backbone NH groups of Gly137, Ser138, and Ser139, which constitutes the oxyanion hole. The second oxygen is within H-bonding distance of the εproton of the catalytic His57. This residue is presumed to exist in the protonated state and is within H-bonding distance to Asp81.3f The catalytic Ser side chain does not appear to be D



Article

of the molecules studied herein have two well-defined sets of NMR resonances, arising from very slowly (on the NMR time scale) interconverting peptide conformers of the tertiary amide linkage between the P2 and P3 residues. However, the cisconformer exists as a minor population compared to that of the relevant trans-conformer which is present in 87%. Close analysis of the free-state data in Table 1 reveals that the predominant conformation of 1 greatly resembles that of the bound state. There are short P1-NH/P2-αH distances that are indicative of an extended, trans-peptide orientation for the P1P2 residues. The longer distance between the P3-NH and P3αH is consistent with the J-coupling data, indicating an extended orientation. For the Ψ-torsion angle, a key correlation was that observed between the P3-tert-butyl group (P3-Hγ) and the P1-HβC (3.3 Å), which also defines a primarily extended conformation. A number of crosspeaks between the Q-H3 and Q-H5 hydrogens and those of the P2 proline ring indicate that the predominant disposition of the quinoline group is such that the B-ring is oriented away from the peptide backbone. Key correlations also include an intense crosspeak between Q-H3 and P2Hγ as well as that between Q-H5 and P2Hα. The predominance of a Cγ-exo proline pucker (as observed in the bound state) is indicated by the strong P2HβA/P2HδA crosspeak (and corroborated by the lack of a peak between the corresponding B-ring hydrogens). Therefore, the ROESY data for inhibitor 1 points to the f ree molecule existing predominantly in a conformation closely resembling that when bound to the protease as indicated by the X-ray crystallographic structure of HCV protease−inhibitor 1′ complex described herein and the accompanying NMR work (Table 1). Systematic Truncation of Inhibitor 1. Knowing that the predominant free-state conformation of inhibitor 1 is very similar to that when bound by the enzyme, we focused on establishing which structural features of the ligand contribute to stabilizing this bioactive conformation. A systematic ‘molecular dissection’ approach was undertaken in which sequential removal of portions of molecule 1 was performed and the resulting effects on binding affinity monitored. While a particular group in the parent structure can obviously contribute to the total binding energy through either the stabilization of the bioactive conformation or the formation of favorable contacts with the active site, or both, we focused on the former, monitoring a number of parameters reflecting the conformational/dynamic behavior throughout the series of compounds. Sequential truncations targeted the P3 side chain and N-terminus (Table 2), the P2 aryloxy group (Table 3), and the P1 side chain (Table 4). The cropping steps were planned out such that no charged or highly polar groups would appear in the new compounds so that we could assume that all truncated molecules would bind in the same fashion as parent inhibitor 1. Nonetheless, a selection of truncated ligands were docked into the enzyme (see Supporting Information, Appendix 13). Free Conformations of P3 and N-Terminal Truncated Inhibitors. The free-state conformational properties of molecules 1−3 were first investigated by an array of NMR methods. All of the present NMR studies were carried out under identical conditions in DMSO-d6, a solvent in which most compounds are soluble and for which conformational data compare well with those obtained in water, allowing for a convenient comparison of solution data. Perhaps the most expeditious measure12 of the flexibility of the Boc-P3 moiety, specifically the rotational freedom about the Φ angle, are the

conformation; see Figure 3C). Also shown in this view, the large P2 residue lies over the Asp-His catalytic pair,3 as well as the side chain of Arg155 of the E2 strand involved in canonical binding. These interactions appear largely hydrophobic, although some electrostatic interaction between the Arg155 guanidinium and the aryl group methoxy substituent likely contributes to binding. Free versus Bound Conformations of 1/1′. Having determined the bound conformation of inhibitor 1′ by X-ray crystallography (and establishing that it is also that of compound 1), comparisons could then be made with the predominant free-state conformation of molecule 1 using NMR rotating-frame nuclear Overhauser effect (ROESY) experiments11 (Table 1). It must be emphasized that although Table 1. Apparent Interproton Distances (Å) Derived the from ROESY of Free Inhibitor 1 and HCV Protease-Bound Inhibitor 1′ from X-ray Crystallographic Dataa crosspeak

free compound 1

bound compound 1′

P1-NH−P1-HβC P1-NH−P2-HβA P1-NH−P2-Hα P1-Hγ−P1-HβB P1-Hγ−P1-HβC P1-HδA−P1-HβC P1-HδA−P1-HδB P1-HδA−P1-HβB P2-Hα−P2-HβB P2-Hγ−P2-HβB P2-Hγ−P2-HβA P2-Hγ−P2-HδB P2-HδA−P2-HβA P3-NH−P3-Hα P3-NH−P2-HδB P3-Hγ−P1-HβC Q-H6−Q-OMe Q-H8−Q-OMe Q-H3−P2-HβB Q-H3−P2-HδB Q-H3−P2-HδA Q-H3−P2-Hγ Q-H5−P2-Hα Q-H5−P2-Hβb Q-H3−Boc P3-NH−Boc P2-HδA−Boc Q-H5−Boc

2.8 4.6 2.3 2.6 2.8 2.6 2.0 3.5 2.3 3.4 2.5 2.6 3.2 3.5 4.9 3.3 3.4 2.1 2.7 3.8 2.8 2.0 3.8 > 5.5 4.1 4.5 3.6

3.0 4.4 2.2 4.0 2.9 1.9 1.9 4.6 2.4 2.7 2.4 2.7 2.5 3.0 4.5 3.9 3.9 2.2 3.1 4.5 4.3 2.0 4.0 4.9 7.0 4.5 6.5 2.4

a Distances for free compound 1 were derived from 200 ms ROESY data (in DMSO-d6 solvent). Crosspeak volumes were scaled to the QH5−Q-H6 correlation assigned a distance of 2.4 Å. Scaling also included multiplying the volumes of crosspeaks that involved methyl and tert-butyl groups by factors of 0.66 and 0.5, respectively. “>” indicates no crosspeak observed. Distances for bound 1′ were derived from the X-ray crystal structure. Hydrogens were added to the crystal structure.

ROESY crosspeak volumes were converted to distances between hydrogen atoms, these values should not be taken as literal values (interatomic distances), but rather as a convenient means of identifying predominant solution conformations. Given a sufficient number of key ROESY crosspeaks, the predominant conformation was identified since crosspeak areas reflect interproton distances.11 It should also be noted that all E



Article

Table 2. Binding Parameters and Conformational Indices of HCV Protease Inhibitors 1−5a

a

The conserved bonds/atoms of the scaffold derived from the lead peptide DDIVPC are colored red, and all other bonds/atoms are colored black. Note that the inherent solubility imparted by the carboxylates made testing for activity feasible at high concentrations. Ki and ΔGbinding values were calculated from IC50 data as described in Materials and Methods. 1

hydrogens remained quite constant within the series. Differences, however, were observed for the Boc-P3 region of the molecules. Nearly all of the Boc-tert-butyl crosspeaks observed for inhibitor 1 were not observed in the case of the P3-Gly analogue 3. An indication of the greater mobility of the Boc-P3 portion of inhibitors 2 and 3 are the changes in P3-NH and P3αH ROESY distances, being shorter than those measured for 1. This is due, in part, to freer Φ-bond rotation (corroborated by the J-coupling data) that allows the protons to come into proximity, whereas they were held apart in the predominantly extended conformation assumed by compound 1. ROESY analysis was also carried out for the P3 tertbutylacetyl inhibitor 4. While the derived distances reflect generally similar conformational properties for the P1 and P2 residues (data not shown), two key points of comparison are notable with respect to the parent compound 1. The crosspeak corresponding to the P3-tert-butyl (Hγ’s) and the P1-HβC was not observed. Furthermore, a correlation (4.3 Å) between the tert-butyl and the quinoline-H6 is present. The equivalent ROESY peak is absent in the spectrum of 1. Thus, the terminal acyl group in 4 moves freely in contrast to 1, underscoring the role of the Boc-amino moiety in maintaining the latter in the bioactive extended conformation. This is clearly corroborated by NMR 13C spin-lattice relaxation times (13C T1) (vide infra and Tables S15−S17 in the Supporting Information). The above analysis exemplifies how ROESY data may be used not only to gain information concerning the preferred conformation of a molecule in solution but also to shed light on its dynamic properties relative to similar analogues. Another

H NMR JNH‑αH coupling constants, which are provided in Table 2. The value of 8.3 Hz observed for compound 1 indicates a predominantly extended conformation in solution, consistent with the X-ray derived bound conformation. A clear trend in the J values was observed, the coupling constant decreasing as bulk was removed from the side chain, indicating that other conformations are present in the P3-Ala analogue 2. The value of 5.6 Hz for molecule 3 is consistent with a freely rotating (on the NMR time scale) αC-NH bond. ROESY NMR data were also acquired for comparison of the free-state properties of compounds 1−3 and are provided in Table S14 in the Supporting Information. As mentioned earlier, the interproton distances must not be taken as literal values. Changes in the intensities of ROESY crosspeaks for equivalent groups of related molecules may reflect changes in the predominant conformation but can also arise from differences in the dynamic ensemble average about a predominant conformation. It must therefore be kept in mind that signals arising from conformers with a relatively low population may be emphasized in cases where two hydrogens are in very close proximity, owing to the exponential relationship between crosspeak intensity and interproton distance.11 Globally, the distances measured for inhibitors 1−3 were similar between protons within the P1 and P2 residues (Table S14 in the Supporting Information). However, there appear to be differences in the predominant orientation of the P1 unit with respect to the P2 residue (P1-NH/P2-HβA). The distances between the quinoline protons and the P2 proline ring F



Article

Table 3. Binding Parameters and Conformational Indices of HCV Protease Inhibitors 1 and 6−10a

The conserved bonds/atoms of the scaffold derived from DDIVPC are colored red, and all other bonds/atoms are colored black. Ki and ΔGbinding values were calculated from IC50 data as described in Materials and Methods. a

method of obtaining insight into the flexibilities or mobilities of molecules is through 13C T1 times by NMR.2a,c,3a,7b The chemical shifts of the trans-conformers of inhibitors 1−5 and relevant NT1 values are provided in Table S16 (Supporting Information). Care was taken to only use T1 values in cases where the trans- and cis-signals were adequately resolved. Resonance overlap was especially problematic in the case of molecule 2. Comparison of the data obtained for inhibitors 1 and 3 showed a slight increase in the NT1 values, indicative of a slightly greater degree of mobility for the P2 proline ring

carbons in the latter. A clear indication of increased mobility in the Boc-P3 portion with reduction of the size of the side chain was provided by the Boc-tert-butyl T1 values, which sequentially increase from 0.51 to 0.55 to 0.58 s in going from 1 to 3. The higher mobility of the tert-butylacetyl group in 4 as compared to 1 was clearly evidenced by comparison of the NT1 values for both the P3-Cα and P3-Cγ(Me) signals, which corroborated the ROESY data. Free Conformations of P2 and P1 Truncated Inhibitors. The conformational properties of free inhibitors G



Article

Table 4. Binding Parameters and Conformational Indices of HCV Protease Inhibitors 1, 11 and 12.a

The conserved bonds/atoms of the scaffold derived from DDIVPC are colored red, and all other bonds/atoms are colored black. Ki and ΔGbinding values were calculated from IC50 data as described in the Materials and Methods. a

NT1 data (Table S17, Supporting Information) also revealed that, even for the P1-glycyl inhibitor 12, the mobility of the rest of the molecule is quite similar to that of the parent compound 1. While analysis of the present series does not allow for any conclusions as to changes in the mobility of the P1-residue itself, the great decreases in binding are no doubt due to both the losses of S1−P1 contacts as well as increases in the mobility of the terminal carboxylate group. Relationship between Inhibitor Conformation and Binding. Taken together, the J-coupling, ROESY, and 13C NT1 data for the free inhibitors provides a mutually reinforcing picture of the conformational and dynamic consequences of removing steric bulk from the P3 side chains in the series of inhibitors 1−3. Sequential reduction of the size of the side chain from a tert-butyl to methyl to hydrogen results in stepwise losses of 1.6 and 1.3 kcal/mol. The large substituent in compound 1 constrains the Boc-P3 moiety to an extended fashion such that the NH and CO groups are correctly oriented to form the key hydrogen bonds to Ala157 of HCV protease, accounting for the reduced potencies of 2 and 3. This effect, however, is contingent on the presence of the bulky Bocgroup as evidenced by the greater degree of mobility of inhibitor 4, whose binding energy is 2.5 kcal/mol less than that of parent compound 1. We believe that these conformational factors can be invoked to explain, in large part, the observed changes in binding reflected in the Ki and ΔGbinding values for the present inhibitors. We have described a formal method for presenting SAR aimed at assessing the contributions of combinations of groups to biomolecular recognition events14 and have applied it to the P3 and N-terminal truncations described above. This “chemical double-mutant” analysis clearly showed that the binding contributions of the P3-tert-butyl and N-Boc moieties, when added to the ‘core’ consisting of inhibitor 5, are synergistic and believe that much of this synergy stems, ultimately, from conformational factors. Both the tert-butyl and Boc-amino groups in compound 1 play roles in holding the P3 H-bonding elements in the correct orientation with respect to each other,

6, 8, and 10, for which structural elements of the P2-aryloxy group have been sequentially removed, were also studied. A striking difference between this series and that described above is the invariance of the P3 JNH‑αH values (Table 3). In all cases, they remain within a range consistent with an extended Φangle. In addition, the trans-conformers of the P2-P3 peptide bond predominated in all cases, which greatly facilitated the analysis and allowed for far more complete 13C T1 data sets to be obtained (see Table S17, Supporting Information). Truncation of the P2-aryloxy group clearly resulted in an increase in the mobility of all regions of the molecules. For the series of compounds 1, 6, 8, and 10, the T1 values arose fairly evenly from 0.51 to 0.62 s for the Boc methyl carbons. The effect of the P2-aryl substituent on the motion of the proline ring is evident. While modest increases in NT1 values accompanied truncation to the pyridine 8, indicating an increase in the flexibility of the pyrrolidine, the unsubstituted prolyl inhibitor 10 exhibited much greater mobility, notably at the γ-position. In terms of ROESY analysis, only key crosspeaks were analyzed in a semiquantitative manner. For molecules 6 and 8, the key correlations defining the Cγ-exo proline ring pucker (P2-HβA−P2-HδA) and the proximity of the P3 and P1 side chains (P3-Hγ−P1-HβC) are present, being slightly less intense compared to the equivalent signals for compound 1. Analysis of the prolyl peptide 10 could not, however, be carried out due to extensive signal overlap, notably for the P1 and P2 residues. Examination of the P2Hα signal (1D spectrum) revealed a very different coupling pattern compared to all of the other molecules, indicating that the lack of a γ-substituent results in a much different conformational behavior. The influence of the P1 residue on the global conformational properties of the inhibitors was also studied (Table 4). As was observed for the truncation of the P2-aryl substituent, sequential deletion of elements of the P1 vinylcyclopropyl side chain13 had very little influence on either the cis/trans conformational equilibrium of the P2-P3 amide or the P3 Φangle as evidenced by the large coupling constants. The 13C H



Article

Figure 4. HCV NS3 protease inhibitors that are recently approved drugs for treating HCV infection, and other inhibitors currently in phase II and III clinical trials. The conserved bonds/atoms derived from DDIVPC or 1 are colored red, whereas new bonds/atoms are colored blue. The bottom view is a superposition of the complexes involving the above compounds that were either derived from X-ray crystallography or from the docking exercise described in Materials and Methods. All complexes are superimposed onto the X-ray structure of Faldaprevir (see Materials and Methods) where the Faldaprevir atoms are all colored blue.

number of groups who have sought to mimic the substructure with rigid scaffolds.15 The impact of γ-substitution of the P2-prolyl residue on the conformational behavior of the present molecules is clearly evidenced by the NMR data. The effect of rigidifying the pyrrolidine ring to a Cγ-exo pucker was consistent with studies of simple proline derivatives, which pointed to stereoelectronic factors being important in electronegative substituents conferring this preferred ring conformation. Expanding the aromatic system (B-ring) restricts O-aryl bond rotation to conformations similar to that observed in the X-ray structure and further rigidifies the molecule as a whole. Understanding

as well as the rest of the molecule. Global stabilization of the active site by the P3 to S3 backbone contacts likely strengthen other key remote interactions and vice versa. While favorable interaction between the P3-tert-butyl and the enzyme must contribute to binding, the X-ray structure shows that the hydrophobic contact area is small and that much of the group is exposed to solvent. NMR studies also argue against the interaction being very strong.2a,d,3a The nonlinear SAR described above serves to underline the shortcomings of assessing binding contributions of particular groups outside the context of the ligand as a whole. The importance of the extended P3 conformation described above has been noted by a I



■

the conformational effects of γ-proline substitution also clarifies the role of the P3 tert-butyl group of inhibitor 1. The invariance of the P3 JNH‑αH values points to the bulky side chain being the predominant factor in restricting the P3 Φ-angle to an extended conformation, as well as rendering the trans-P2-P3 rotamer much more stable than its cis-counterpart. The P2-aryloxy substituent clearly stabilizes the conformation of the Boc-P3 moiety as evidenced by the incremental increases in 13C NT1 values through the series 1, 6, 8, and 10. This was likely due, for the most part, to the restriction of the Ψ-angle. The similar changes in the mobility of both the Boc and P3 tert-butyl carbons are consistent with this view. Although seemingly remote from each other, the P2-aryloxy and Boc groups (provided a tert-butyl was present at P3) are in proximity to each other and may restrict each other’s motion. Unlike the role of the P2-aryl substituent, the vinylcyclopropyl moiety of the P1 residue does not appear to have a great influence on the dynamic behavior of the remainder of the molecule. The fact that complete removal of the side chain results in only small increases in mobility (NT1 data) is consistent with the notion that a bulky P3 side chain and large proline substituent are the major determinants in global rigidification of the inhibitors. Thus, the losses in potency observed within the sequence 1 to 11 to 12 are the result of local increases in flexibility at P1 and the loss of interactions within the S1 pocket of the active site.2c,7b The alteration of the bond angles about the P1 α-carbon upon introduction of the cyclopropyl ring has been discussed elsewhere.3g Bioactive Conformations of Clinical Drugs. A patent and literature search of HCV protease inhibitors/drugs showed that most, if not all, of the advanced compounds targeting this enzyme incorporate the identical peptide core structure derived from DDIVPC (see Appendix 12 of the Supporting Information). Recently approved drugs and other inhibitors currently in phase II and III clinical trials were docked into the enzyme active site and are superimposed at the bottom of Figure 4. Despite the fact that these compounds vary substantially with regard to their C-termini and side chain appendages (blue-colored atoms at the top in Figure 4), they all have the identical peptide core structure derived from DDIVPC (red-colored atoms in Figure 4). The remainder of the peptidic structures all consist of a modified P2 proline and a P3 residue with either a bulky tert-butyl side chain or one that is tethered to P1. The design and benefits of macrocyclization from P1 to P3 have been addressed elsewhere2c,6,7a,b,9a and resulted in the breakthrough compound BILN 2061 (Figure 1), the first proofof-concept for a new class of HCV antiviral. This inspired others to employ macrocyclization as a strategy for conformational restriction (see Figure 4 and Appendix 12 of the Supporting Information).16 Figure 4 shows how the P1 to P3 and the P2 to P4 macrocycles can bind to the HCV protease active site. It is also noteworthy that many compounds with proline modifications consist of the attachment of a γ-aryloxy or similar structure that would be expected to lock the pyrrolidine in the appropriate pucker (see Figure 4 and Appendix 12 of the Supporting Information). The fusion of a second ring, as in the cases of Boceprevir and Telaprevir, is another rigidification strategy that accomplishes the same conformational locking of the five-membered ring.

Article

CONCLUSIONS The present work underlines how optimization of the peptidic N-terminal product sequence DDIVPC has led to a number of marketed and advanced drug candidates targeting HCV protease, including Faldaprevir and BILN 2061 (by way of inhibitor 1, BILN 1508). Along the path to the latter, improvements to binding involved both stabilization of the bioactive conformation, as established by the current results, as well as the formation of better interactions between ligand and target, at times simultaneously. Although one cannot separate these two global factors in an unambiguous manner, we have demonstrated through a carefully designed truncation SAR (exemplified by the dissection of compound 1) coupled to a series of systematic NMR studies that rigidification of the peptide scaffold to the bioactive conformation is of central importance. Examination of an array of advanced molecules targeting HCV protease and which appear to all (present molecules included) bind in a single, canonical mode when one examines the conformation of the peptide core through molecular modeling indicates that stabilization of the bound conformation is globally relevant. The multidisciplinary approach described herein, which combines medicinal chemistry, NMR spectroscopy, molecular modeling, and X-ray crystallography, clearly demonstrates that systematic conformational analysis and investigation of dynamic behavior of a carefully chosen series of molecules can certainly provide important insights into the nature of small molecule− protein interactions that can be exploited for drug discovery purposes.

■

MATERIALS AND METHODS

Inhibitors. The purity of all inhibitors was determined to be >95% by HPLC and high-field NMR. Synthetic procedures and additional physical data are provided either in the Supporting Information or have been described elsewhere.3b,c,8 Enzyme Assay. The radiometric enzymatic assay13 was performed in 50 mM Tris-HCl, pH 8.0, 0.25 M sodium citrate, 0.01% n-dodecylβ-D-maltoside, 1 mM TCEP at 23 °C. Twenty-five micromolar concentration of the substrate DDIVPCSMSYTW, ∼1 nM biotinDDIVPCSMSY[125I]TW, and various concentrations of inhibitors (DMSO stock solutions) were incubated with 6 nM HCV NS3-NS4A (genotype 1b). Under these assay conditions, a KM of 9.1 μM was found for the substrate DDIVPCSMSYTW. At least two IC 50 determinations were carried out for each of two weightings for all of the inhibitors. Note that the inherent solubility imparted by the carboxylates for this series of compounds made testing for activity feasible at high concentrations. Ki values were calculated using eq 1:

IC50 = 0.5[Etotal ] + K i(1 + [S]/KM)

(1)

The free energies of binding were calculated using eq 2: ΔG binding = − RT ln(1/K i)

(2)

X-ray Crystallography. The X-ray structures of compounds DDIVPC (PDB code 4JMY) and 1′ (PDB code 4K8B) were determined as described in the Supporting Information. NMR Studies. NMR experiments were performed on a Bruker Avance spectrometer (400 or 600 MHz 1H frequencies) at 27 °C. Samples were prepared by dissolving inhibitors 1−12 in DMSO-d6 (13−16 mM) followed by deoxygenation of the solutions through several rounds of exposure to vacuum and purges with nitrogen gas. The glass NMR tubes were subsequently sealed using a flame. Assignment of the 1H resonances were secured by careful analysis of standard homonuclear COSY and ROESY11 experiments. Homonuclear ROESY data were acquired with a continuous spin-lock during J



Article

mixing (300 ms), a 2.5 kHz spin-lock field, the carrier set at 4.7 ppm, 256−400 t1 data points from the addition of 32−64 transients, a 5200 Hz sweep width, and a relaxation delay of 1.2 s. Similar ROESY data sets were also collected for comparison purposes where the offset was shifted to 0 or 10 ppm, the spin-lock time varied, and where the spinlock was applied by other methods. The existence of cis- and transrotamers was clearly identified through variable temperature studies that showed the coalescence of resonances at higher temperatures, as well as the observation of chemical exchange crosspeaks in the ROESY spectra. The resonances of the trans-rotamer were easily distinguished from those of the cis-rotamer based on comparison of distinctive ROESY crosspeak intensities (e.g., P2-Hδ/P3-Hα and P2-Hα/P3Hα). The 1H chemical shift and J-coupling values were easily extracted from high-resolution, one-dimensional spectra. In some instances, decoupling strategies (selective irradiation) were used to derive coupling constants. 13C resonance assignments were carried out using 1 H/13C correlation HMBC and HMQC experiments. HMBC spectra, providing long-range 1H/13C correlation data, were acquired with no decoupling during acquisition, 256−512 t1 data points from the addition of 64 transients, a relaxation delay of 1.5 s, and delays for evolution of long-range couplings corresponding to 5 or 8 Hz. HMQC spectra provided single-bond 1H/13C correlation data and were acquired with decoupling (GARP) during acquisition, 512 t1 points from the addition of 16−32 transients, a relaxation delay 1.5 s, and a delay for evolution of one-bond coupling corresponding to 140 Hz. Samples and experimental data acquisition for transferred NOESY experiments are described in ref 3a. Samples for the 13C T1 relaxation measurements were prepared by dissolving inhibitors 1−12 in DMSO-d6 (41−77 mM), adding EDTAd16 (3 mM) for the purpose of chelating any traces of contaminating paramagnetic metals, and degassing the solution by vacuum followed by purges with argon gas. The glass NMR tubes were immediately sealed using a flame. Relaxation data were acquired at 150 MHz (with cryoprobe) and 27 °C using the inversion recovery method with power-gated proton decoupling (WALTZ16) during acquisition. Twelve spectra were acquired corresponding to the τ delays (0.01, 0.1, 0.2, 0.37, 0.6, 0.65, 1.0, 1.5, 2.2, 3.0, 5.0, and 7.5 s). Each spectrum was typically acquired by adding 3000 transients and using a relaxation delay of 8.0 s. T1 relaxation curves were calculated based on resonance intensities using the WinNMR software (Bruker Canada, Milton, Ontario). Relaxation times reported here are derived from resonances that were clearly resolved (i.e., the cis- and trans-resonances were distinct) and for which the calculated T1 curves described the experimental data with relatively little deviation. Some samples were subjected to repeated data acquisition and calculations, and less than 5−10% variation was generally observed, as a result of good S/N obtained with a cryoprobe, and the fact that natural abundance 13C nuclei relax predominantly via the covalently attached hydrogen(s). All two-dimensional data sets were processed using XWinNMR (Bruker Canada, Milton, Ontario) and WinNMR software. Data sets were typically zero-filled to yield 2048 (f2) × 1024 (f1) points after Fourier transformation using a shifted sine-bell window function. 1H and 13C spectra were chemical shift calibrated relative to the standard values attributed to the DMSO peaks (2.5 and 39.51 ppm, respectively). ROESY crosspeak volumes were scaled and converted to apparent interproton distances. The derived distances were normalized to the Q-H5 to Q-H6 crosspeak, which corresponds to a fixed distance of 2.4 Å. Only well-resolved ROESY crosspeaks corresponding to the trans-conformers and which did not exhibit any visual artifacts stemming from COSY or HOHAHA contributions were used in the analysis. Data similar to that reported below were collected using other offsets and spin-lock sequences. Molecular Modeling and Docking. Protein−ligand complexes were derived in the following manner. If multiple chains were present in the deposited X-ray coordinates then Chain A was used by default. Crystallographic water molecules were kept. Hydrogen atoms were added using MOE’s Protonate 3D module. The Amber99 force field was applied with AM1-BCC charges for the ligands. In order to relieve any potential major strains between the protein and ligand, energy minimization was performed to an RMS gradient of 0.1 kcal/mol/

Awith MOE’s LigX module with receptor, ligand, and solvent atoms restrained. Energy cutoff range was set between 10 and 12 Å and the Born solvation model was used for implicit solvation. The binding models for 1, 3, 5, 10, and 12 were constructed based on the in-house X-ray structure of Faldaprevir (ref 3f, PDB 3P8O) in complex with the protease. A complex involving compound 1 was also derived from an X-ray structure of the highly related analogue 1′ (PDB code 4K8B) such that the urea was replaced by a carbamate (N atom to an O) then energy minimized. The X-ray structure of DDIVPC (PDB code 4JMY) was acquired as described in the Materials and Methods section. The complex involving BILN 2061 was based on the modification of an X-ray structure3f,6 of a related macrocyclic compound and energy minimization. The binding model for Boceprevir was created from PDB code 3LOX where the cyclohexyl was transformed to the tert-butyl. An inhouse X-ray structure was used for Faldaprevir (PDB 3P8O). PDB code 3KEE was used for Simeprevir, while PDB code 3M5L was used for Danoprevir. Vaniprevir was modeled starting from the X-ray structure of Danoprevir while the binding mode model of Asunaprevir was based on PDB code 3OYP. Telaprevir was modeled based on PDB code 3LOX with initial cyclopropyl orientation based on PDB code 3M5L. The superposition of the protein−ligand complexes was performed with MOE’s Superpose module. For Figure 4, the RMSD = 0.62 Å for all complexes. The surface was created with the coordinates of Faldeprevir (PDB code 3P8O) [LigX: unselect: Waters farther than 4.5 from ligand; select: Solvent restraint; fix atoms farther than 10 Å; refine to 0.1 kcal/mol/Å].

■

ASSOCIATED CONTENT

S Supporting Information *

Characterization information for novel compounds reported here, the structures of patented compounds that also have the DDIVPC critical scaffold, NMR resonance assignment tables, free vs bound differences, resistance suppression, free-state ROESY data and more. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Addresses §

NMX Solutions, 500 boulevard Cartier Ouest, Suite 6000, Laval, QC, Canada H7 V 5B7. ⊥ Department of Chemistry and Biochemistry, 7141 Sherbrooke Street West, SP 201.00, Montreal, QC, Canada H4B 1R6. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful for the valuable contributions and support provided by G. Kukolj, J. Gillard, P. Anderson, M. Bailey, M. Cordingley, R. Bethell, P. Edwards, D. Lamarre, M.-A. Poupart, P. White, M. Llinas-Brunet, J. Rancourt, A.-M. Faucher, and D. Thibeault. We also acknowledge the valuable teamwork and contributions of our colleagues at Boehringer Ingelheim.

■

ABBREVIATIONS USED Boc, tert-butoxycarbonyl; COSY, correlated spectroscopy; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; HCMV, human cytomegalovirus; HCV, hepatitis C virus; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid; HMBC, heteronuclear multiplebond correlation; HMQC, heteronuclear multiple quantum K



Article

LaPlante, S. R.; Maurice, R.; Poirier, M.; Poupart, M.-A.; Thibeault, D.; Wernic, D.; Lamarre, D. Peptide-based inhibitors of the hepatitis C virus serine protease. Bioorg. Med. Chem. Lett. 1998, 8, 1713−1718. (c) Llinàs-Brunet, M.; Bailey, M.; Fazal, G.; Ghiro, E.; Gorys, V.; Goulet, S.; Halmos, T.; Maurice, R.; Poirier, M.; Poupart, M.-A.; Rancourt, J.; Thibeault, D.; Wernic, D.; Lamarre, D. Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease: towards smaller inhibitors. Bioorg. Med. Chem. Lett. 2000, 10, 2267−2270. (d) LaPlante, S. R.; Aubry, N.; Bonneau, P.; Kukolj, G.; Lamarre, D.; Lefebvre, S.; Li, H.; Llinàs-Brunet, M.; Plouffe, C.; Cameron, D. R. NMR line-broadening and transferred NOESY as a medicinal chemistry tool for studying inhibitors of the hepatitis C virus NS3 protease domain. Bioorg. Med. Chem. Lett. 2000, 10, 2271−2274. (e) Goudreau, N.; Cameron, D. R.; Bonneau, P.; Gorys, V.; Plouffe, C.; Poirier, M.; Lamarre, D.; Llinàs-Brunet, M. NMR structural characterization of peptide inhibitors bound to the hepatitis C virus NS3 protease: Design of a new P2 substituent. J. Med. Chem. 2004, 47, 123−132. (f) Lemke, C. T.; Goudreau, N.; Zhao, S.; Hucke, O.; Thibeault, D.; Llinàs-Brunet, M.; White, P. W. Combined X-ray, NMR, and kinetic analyses reveal uncommon binding characteristics of the hepatitis C virus NS3-NS4A protease inhibitor BI 201335. J. Biol. Chem. 2011, 286, 11434−11443. (g) Kawai, S. H.; LaPlante, S. R.; Llinàs-Brunet, M.; Hucke, O. The effect of the P1 side-chain on the binding of optimized carboxylate and activated carbonyl inhibitors of the hepatitis C virus NS3 protease. Fut. Med. Chem. 2010, 2, 1073− 1081 and references therein. (4) (a) LaPlante, S. R.; Aubry, N.; Bonneau, P. R.; Cameron, D. R.; Lagacé, L.; Massariol, M.-J.; Montpetit, H.; Plouffe, C.; Kawai, S. H.; Fulton, B. D.; Chen, Z.; Ni, F. Biochemistry 1998, 37, 9793−9801. (b) Edwards, P. J.; LaPlante, S. R. Peptides as Leads for Drug Discovery, in Peptide Drug Discovery and Development; Castanho, M., Santos, N. C., Eds.; Wiley: New York, 2011; pp 3−55. (5) Nar, H.; Bauer, M.; Schmid, A.; Stassen, J.-M.; Wienen, W.; Priepke, H. W. M.; Kauffmann, I. K.; Ries, U. J.; Hauel, N. H. Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors. Structure 2001, 9, 29−37. (6) Tsantrizos, Y. S.; Bolger, G.; Bonneau, P.; Cameron, D.; Goudreau, N.; Kukolj, G.; LaPlante, S. R.; Llinas-Brunet, M.; Nar, H.; Lamarre, D. Macrocyclic inhibitors of the NS3 protease as potential therapeutic agents of hepatitis C virus infection. Angew. Chem., Int. Ed. 2003, 42, 1356−1360. (7) (a) Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger, G.; Bonneau, P.; Bös, M.; Cameron, D. R.; Cartier, M.; Cordingley, M. G.; Faucher, A.-M.; Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagace, L.; LaPlante, S. R.; Narjes, H.; Poupart, M.-A.; Rancourt, J.; Sentjens, R. E.; St George, R.; Simoneau, B.; Steinmann, G.; Thibeault, D.; Tsantrizos, Y. S.; Weldon, S. M.; Yong, C. L.; Llinas-Brunet, M. An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus. Nature 2003, 426, 186−189. (b) LaPlante, S. R.; Llinàs-Brunet, M. Dynamics and structure-based design of drugs targeting the critical serine protease of the hepatitis C virus - from a peptidic substrate to BILN 2061. Curr. Med. Chem.: Anti-Infect. Agents 2005, 4, 111−132. (8) Llinàs-Brunet, M.; Bailey, M. D.; Goudreau, N.; Bhardwaj, P. K.; Bordeleau, J.; Bös, M.; Bousquet, Y.; Cordingley, M. G.; Duan, J.; Forgione, P.; Garneau, M.; Ghiro, E.; Gorys, V.; Goulet, S.; Halmos, T.; Kawai, S. H.; Naud, J.; Poupart, M. A.; White, P. W. Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335). J. Med. Chem. 2010, 53, 6466−6476. (9) (a) Hinrichsen, H.; Benhamou, Y.; Wedemeyer, H.; Reiser, M.; Sentjens, R. E.; Calleja, J. L.; Forns, X.; Erhardt, A.; Cronlein, J.; Chaves, R. L.; Yong, C. L.; Nehmiz, G.; Steinmann, G. G. Short-term antiviral efficacy of BILN 2061, a hepatitis C virus serine protease inhibitor, in hepatitis C genotype 1 patients. Gastroenterology 2004, 127, 1347−1355. (b) Zeuzem, S.; Asselah, S.; Angus, P.; Zarski, J.-P.; Larrey, D.; Mullhaupt, H.; Gane, E.; Schuchmann, M.; Lohse, A.; Pol, S.; Bronowicki, J.-P; Roberts, S.; Arasteth, A.; Zoulim, A.; Heim, M.; Stern, J. O.; Kukolj, G.; Nehmiz, H.; Haefner, C.; Boecher, W. O. Efficacy of the protease inhibitor BI 201335, polymerase inhibitor BI

coherence; MES, 4-morpholineethanesulfonic acid; NMR, nuclear magnetic resonance; NS, nonstructural; ROESY, rotating-frame nuclear Overhauser effect; SAR, structure− activity relationship; TCEP, tris(2-carboxyethyl)phosphine; Tris, tris(hydroxymethyl)aminomethane; transferred NOESY, transferred nuclear Overhauser effect; T1, longitudinal relaxation time

■

REFERENCES

(1) (a) Teague, S. Implications of protein flexibility for drug discovery. Nat. Rev. Drug Discovery 2003, 2, 527−541. (b) Weikl, T. R.; von Deuster, C. Selected-fit versus induced-fit protein binding: Kinetic differences and mutational analysis. Proteins: Struct., Funct., Bioinf. 2009, 75, 104−110. (c) Hammes, G. G.; Chang, Y. C.; Oas, T. G. Conformational selection or induced fit: A flux description of reaction mechanism. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 13737− 13741. (d) Bakan, A.; Bahar, I. The intrinsic dynamics of enzymes plays a dominant role in determining the structural changes induced upon inhibitor binding. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 14349− 14354. (e) Lange, O. F.; Lakomek, N. A.; Fares, C.; Schroeder, G. F.; Gunnar, G.; Walter, K. F. A.; Becker, S.; Meiler, J.; Grubmueller, H.; Griesinger, C.; de Groot, B. L. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 2008, 320, 1471−1475. (f) Kern, D.; Zuiderweg, E. R. The role of dynamics in allosteric regulation. Curr. Opin. Struct. Biol. 2003, 13, 748−757. (g) Mittermaier, A.; Kay, L. E. New tools provide new insights in NMR studies of protein dynamics. Science 2006, 312, 224−228. (h) Koshland, D. The active site and enzyme action. Adv. Enzymol. Relat. Subj. Biochem. 1960, 22, 45−97. (2) (a) LaPlante, S. R.; Bonneau, P. R.; Aubry, N.; Cameron, D. R.; Déziel, R.; Grand-Maître, C.; Plouffe, C.; Tong, L.; Kawai, S. H. Characterization of the human cytomegalovirus protease as an induced-fit serine protease and the implications to the design of mechanism-based inhibitors. J. Am. Chem. Soc. 1999, 121, 2974−2986. (b) LaPlante, S. R.; Gillard, J. R.; Jakalian, A.; Aubry, N.; Coulombe, R.; Brochu, C.; Tsantrizos, Y. S.; Poirier, M.; Kukolj, G.; Beaulieu, P. L. Importance of ligand bioactive conformation in the discovery of potent indole-diamide inhibitors of the hepatitis C virus NS5B. J. Am. Chem. Soc. 2010, 132, 15204−15212. (c) LaPlante, S. R. Exploiting Ligand and Receptor Adaptability in Rational Drug Design Using Dynamics and Structure-based Strategies. Top. Curr. Chem. 2007, 272, 259−296. (d) LaPlante, S. R.; Aubry, N.; Deziel, R.; Ni, F.; Xu, P. Transferred 13C T1 relaxation at natural isotopic abundance:A practical method for determining site-specific changes in ligand flexibility upon binding to a macromolecule. J. Am. Chem. Soc. 2000, 122, 12530−12535. (e) Peng, J. W. New probes of ligand flexibility in drug design: transferred (13)C CSA-dipolar cross-correlated relaxation at natural abundance. J. Am. Chem. Soc. 2003, 125, 11116−11130. (f) Peng, W.; Wilson, B. D.; Namanja, A. T. Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance. J. Biomol. NMR 2009, 45 (1−2), 171−183. (g) Peng, J. W. Communication breakdown: Protein dynamics and drug design. Structure 2009, 17, 319−320. (h) Mauldin, R. V.; Carroll, M. J.; Lee, A. L. Dynamic dysfunction in dihydrofolate reductase results from antifolate drug binding: Modulation of dynamics within a structural state. Structure 2009, 17, 386−394. (i) Taylor, R. E.; Chen, Y.; Beatty, A.; Myles, D. C.; Zhou, Y. Conformation−activity relationships in polyketide natural products: A new perspective on the rational design of Epothilone analogues. J. Am. Chem. Soc. 2003, 125, 26−27. (j) Thoma, G.; Magnani, J. L.; Patton, J. T.; Ernst, B.; Jahnke, W. Preorganization of the bioactive conformation of sialyl LewisX analogues correlates with their affinity to E-selectin. Angew. Chem., Int. Ed. 2001, 125, 1941−1945. (3) (a) LaPlante, S. R.; Cameron, D. R.; Aubry, N.; Lefebvre, S.; Kukolj, G.; Maurice, R.; Thibeault, D.; Lamarre, D.; Llinàs-Brunet, M. Solution structure of substrate-based ligands when bound to hepatitis C virus NS3 protease domain. J. Biol. Chem. 1999, 274, 18618−18624. (b) Llinàs-Brunet, M.; Bailey, M.; Fazal, G.; Goulet, S.; Halmos, T.; L



Article

207127, and ribavirin in patients with chronic HCV infection. Gastroenterology 2011, 141, 2047−2055. (c) Zeuzem, S.; Soriano, V.; Asselah, T.; Bronowicki, J.-P.; Lohse, A.; Müllhaupt, B.; Schuchmann, M.; Bourliere, M.; Buti, M.; Roberts, S.; Gane, E.; Stern, J. O.; Kukolj, G.; Dai, L.; Böcher, W. O.; Mensa, F. J. SVR4 and SVR12 with an interferon-free regimen of BI201335 AND BI207127, ± ribavirin, in treatment-naive patients with chronic genotype-1 HCV infection: Interim results of SOUND-C2. J. Hepatol. 2012, 56 (Suppl. 2), S45. (d) Zeuzem, S.; Soriano, V.; Asselah, T.; Bronowicki, J.-P.; Lohse, A. W.; Müllhaupt, B.; Schuchmann, M.; Bourlière, M.; Buti, M.; Roberts, S. K.; Gane, E. J.; Stern, J. O.; Vinisko, R.; Kukolj, G.; Gallivan, J.-P.; Böcher, W.-O.; Mensa, F. J. Faldaprevir and Deleobuvir for HCV Genotype 1 Infection. N. Engl. J. Med. 2013, 369, 630−639. (10) The peptide/protease subsite nomenclature used is that of Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun. 1967, 27, 157−162 where the amino acid residues are denoted ...P4-P3-P2-P1P1′-P2′..., the scissile bond being situated between P1 and P1′. The notation ...S4-S3-S2... etc. is used to indicate the corresponding binding subsites on the enzyme.. (11) (a) Bax, A.; Davis, D. G. Practical aspects of two-dimensional transverse NOE spectroscopy. J. Magn. Reson. 1985, 63, 207−213. (b) Bax, A.; Grzesiek, S. In Encyclopedia of Nuclear Magnetic Resonance; Grant, D. M., Harris, R. K., Eds; Wiley: New York, 1996; pp 4157− 4167. (c) Hwang, T.-L.; Shaka, A. J. Cross relaxation without TOCSY: transverse rotating-frame Overhauser effect spectroscopy. J. Am. Chem. Soc. 1992, 114, 3157−3159. (d) Hwang, T.-L.; Shaka, A. J. Reliable two-dimensional rotating-frame cross-relaxation measurements in coupled spin systems. J. Magn. Reson., Ser. B 1993, 102, 155−165. (e) Baleja, J. D.; Moult, J.; Sykes, B. D. J. Magn. Reson. 1990, 87, 375− 384. (f) Neuhaus, D.; Williamson, M. P. The Nuclear Overhauser Effect in Structural and Conformational Analysis; VCH: New York, NY, 1989. (12) Wüthrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons: New York, NY, 1986. (13) Rancourt, J.; Cameron, D. R.; Gorys, V.; Lamarre, D.; Poirier, M.; Thibeault, D.; Llinàs-Brunet, M. Peptide-based inhibitors of the hepatitis C virus NS3 protease: Structure−activity relationship at the C-terminal position. J. Med. Chem. 2004, 47, 2511−2522. (14) Kawai, S. H.; Bailey, M. D.; Halmos, T.; Forgione, P.; LaPlante, S. R.; Llinas-Brunet, M.; Naud, J.; Goudreau, N. The use of chemical double-mutant cycles in biomolecular recognition studies: Application to HCV NS3 protease inhibitors. ChemMedChem 2008, 3, 1654−1657. (15) Kawai, S. H.; Aubry, N.; Duceppe, J.-S.; Llinàs-Brunet, M.; LaPlante, S. R. Dimethylthiazolidine carboxylic acid as a rigid P3 unit in inhibitors of serine proteases: Application to two targets. Chem. Biol. Drug Des. 2009, 74, 517−522. (16) Marsault, E.; Peterson, M. L. Macrocycles are great cycles: Applications, opportunities, and challenges of synthetic macrocycles in drug discovery. J. Med. Chem. 2011, 54, 1961−2004.

M


Ligand Bioactive Conformation Plays a Critical Role in the Design of

Recommend Documents