Article pubs.acs.org/jmc
Discovery of Novel P1 Groups for Coagulation Factor VIIa Inhibition Using Fragment-Based Screening Daniel L. Cheney,*,† Jeffrey M. Bozarth,† William J. Metzler,‡ Paul E. Morin,‡ Luciano Mueller,‡ John A. Newitt,‡ Alexandra H. Nirschl,† Alan R. Rendina,†,§ James K. Tamura,‡ Anzhi Wei,† Xiao Wen,† Nicholas R. Wurtz,† Dietmar A. Seiffert,†,⊥ Ruth R. Wexler,† and E. Scott Priestley† †
Bristol-Myers Squibb Co., Research and Development, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08543, United States ‡ Bristol-Myers Squibb, Research & Development, P.O. Box 4000, Princeton, New Jersey 08543-4000, United States S Supporting Information *
ABSTRACT: A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.
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INTRODUCTION
The S2 and S4 subsites are adjacent and largely hydrophobic, being defined by the side chain of Thr99, the surfaces of the His57 and Trp215 aryl rings, and the backbone atoms of Thr98−Thr99 (see Supporting Information). Site S1′, located on the substrate C-terminal side of the catalytic triad, is defined principally by the Cys42−Cys58 disulfide bridge. Site S1, the subsite of principal interest in this report, consists of a deep, narrow cavity, the sides of which are largely hydrophobic and defined by the side chain of Val213, and the backbone atoms (that is, the Cα’s and amide planes) of Trp215−Gly216 and Ser190−Lys192. By contrast, the bottom of the S1 pocket is highly polar, consisting of Asp189 and Ser190 side chains and a water molecule typically positioned above the Tyr228 aryl ring in the presence of basic P1 groups such as benzamidine and protonated 1-amino-isoquinolines.7 As a result, the factor VIIa S1 pocket, like that of factor Xa and thrombin, preferentially binds P1 groups consisting of both hydrophobic and cationic components, most notably aryl amidines and related organic bases, which unfortunately are associated with poor membrane permeability and oral absorption.8 To address the problem of poor pharmacokinetic (PK) properties associated with factor VIIa inhibitors containing cationic P1 groups, a fragment-based screening effort was undertaken to identify viable neutral P1 groups. In this report, we describe our strategy, challenges that were encountered and overcome, and the resulting discovery of several novel neutral P1 groups, one of which was demonstrated to be viable by incorporation into a phenylglycine chemotype (1).9
Arterial thrombosis remains a leading cause of morbidity and mortality worldwide.1 The existing standards of care, P2Y12 antagonists and aspirin, are effective in treating thromboembolic disorders; however, significant residual cardiovascular risk and bleeding side effects highlight the need for agents with an improved balance of efficacy, safety, and convenience.2 Results from recent clinical trials using novel antithrombotic agents continue to show limited incremental efficacy and significant bleeding liabilities.3 In recent years, a major pharmaceutical industry focus has been to identify a safe and efficacious oral anticoagulant with a broader therapeutic ratio than the current standard of care. Further investigation into the coagulation cascade has identified the tissue factor−factor VIIa proteolytic complex (TF−factor VIIa) as a potential biological target. In preclinical models, inhibition of this complex has demonstrated strong efficacy as well as low bleeding risk.4 This serine protease initiates the coagulation cascade by activating factors IX and X to IXa and Xa, respectively, leading to prothrombin activation. In turn, thrombin cleaves fibrinogen to fibrin and activates platelets, thus triggering a thrombotic event. Like the coagulation enzymes thrombin and factor Xa, the catalytic domain of factor VIIa is a serine protease containing a trypsin-like fold consisting of two antiparallel β-barrel structures. Optimal catalytic activity requires complexation with tissue factor, which is thought to induce subtle conformational changes of the substrate binding site.5 Several subsites in factor VIIa are implicated in binding to small molecule inhibitors and are designated using the Schechter and Berger nomenclature.6 © 2015 American Chemical Society
Received: December 22, 2014 Published: March 12, 2015 2799
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heavy atoms, and flexibility (≤3 rotatable bonds), yielding ∼18000 compounds.16 Subsequent selection of compounds for screening was performed either visually or with virtual screening, as described below. Following clustering using a proprietary atom pair-based similarity program,17 200 compounds were visually selected based on our understanding of the binding requirements of the factor VIIa S1 pocket, which typically prefers unsaturated mono- and bicyclic ring systems with polar substituents. An additional 250 compounds were selected on the basis of a second approach consisting of a virtual screening strategy using the molecular docking program Glide.18 Initial docking trials using Glide on a small test set of fragments revealed a strong dependency of score and binding mode on the particular (rigid) crystallographic structure of factor VIIa used. For example, docking the 1,6-diamino-isoquinoline and 3-amino-benzamide P1 substructures of inhibitors back into their cognate factor VIIa crystal structures reproduces the correct binding mode, whereas cross docking (docking each P1 into the other crystallographic structure of factor VIIa) gives mixed results, presumably due to induced fit effects (see Supporting Information). Furthermore, we observed this dependence of docking results on receptor conformation to increase with the size and complexity of the fragment (see Supporting Information). This problem, originating from protein flexibility, was addressed using protein ensemble docking, a methodology we had developed and reported earlier.19 In this methodology, a single ligand is docked not against one but against a representative set of conformations culled from available high quality crystallographic structures of a given protein target. The top-scoring docked pose is then used for the purpose of ranking. Thus, from among the 55 crystallographic structures of factor VIIa available to us at the time of this work (public and proprietary), an ensemble20 was created to qualitatively represent the range of observed structural variations of the S1 pocket, with emphasis on key P1 binding elements such as Asp189 and Ser190 side chains, the Cys191−Cys220 disulfide bridge, and Gly218 carbonyl, as well as the hydration state, that is, the number and positions of waters (Figure 2a; an RMSD matrix of the S1 pockets of the factor VIIa ensemble can be found in the Supporting Information). Various hydration states of the S1 pocket were represented: no waters; the presence of one water (W1) located above Tyr228 side chain; and the
Trypsin-like proteases may be differentiated on the basis of the S1 specificity arising from the amino acid at position 190. Ala190 serine proteases, such as thrombin and factor Xa, favor arginine at P1, while their Ser190 counterparts, such as factor VIIa, tryptase, urokinase, and trypsin, favor lysine.10,11 Examination of crystallographic structures of Ala190 serine proteases suggests that the bottom of the S1 pocket may be relatively more hydrophobic and open. Indeed, a number of factor Xa and thrombin inhibitors have been reported that incorporate substituted aryl P1 groups, wherein a small substituent such as a halogen atom, methyl, or methoxy group extends deep into the pocket displacing the aforementioned water molecule above the aryl ring of Tyr228.12 In the case of Ser190 serine proteases, the position of the oxygen atom of the Ser190 side chain is, in most crystal structures, oriented toward the S1 interior, constricting the pocket and making it more polar, leading us to speculate that deep binding of neutral groups such as aryl halides might be impeded.13 At the time of this work, only one example of a neutral P1 bound to factor VIIa, benzamide 2 (PDB ID 4JZF), had been reported.7a However, shortly after the completion of this work, Blaney and colleagues reported the discovery of an aryl halide factor VIIa P1 fragment.14
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RESULTS AND DISCUSSION The overall process of fragment screening and our results are outlined in Figure 1. The Available Chemicals Directory (ACD)15 was filtered on the basis of drug-likeness, number of
Figure 2. (a) The ensemble of 15 structures and hydration states used for the virtual screen of the S1 pocket. Benzamidine is shown as a reference P1. (b) Crystallographic structure of 2 bound in the factor VIIa S1 (PDB ID = 4JZF) depicting hydrogen bonding (yellow) and polar interactions (magenta) in the S1 pocket. Graphics were generated using the program PyMol.23
Figure 1. Overview of the protocol for the selection of 450 compounds for follow-up with assays and crystallography. 2800
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Journal of Medicinal Chemistry presence of a second water (W2) that is uniquely observed in the crystal structure consisting of benzamide 2 bound to factor VIIa. The latter hydration state was defined only for the structure bound to 2 (PDB ID 4JZF).21 In this structure (Figure 2b), the P1 amide is engaged in a hydrogen bond network with the side chains of Asp189 and Tyr228 and W2 water.7a The factor VIIa crystallographic structures chosen for this ensemble were tissue factor naive, that is, they were determined in the absence of tissue factor. Visual examination of factor VIIa structures from the PDB revealed no structural features in the vicinity of S1 that could be correlated with the presence (22 structures) or absence (19 structures) of tissue factor (see Supporting Information). While expanding the ensemble to include more instances of factor VIIa complexed with tissue factor is reasonable, it is not obvious that doing so would have altered the results in any systematic way.22 The set of approximately 18000 compounds described above (Figure 1) were docked into the S1 pockets of the factor VIIa ensemble, with each fragment ranked according to its best overall score. To bias our selection to include neutral and also smaller fragments, we scored neutral and cationic fragments separately and normalized scores by dividing by the solvent accessible volume or surface area (See Materials and Methods).16 From among the top scoring fragments, 250 were selected, and together with the 200 fragments already chosen from clustering, these were progressed into biochemical and NMR binding assays (Figure 1). The NMR fragment screen focused primarily on those compounds for which compound solubility and photometric interference with the biochemical assay readout led to lower confidence in the results. Fragment hits of interest in either the biochemical or NMR binding assays were submitted to X-ray crystallography for structure determination. Ki’s were determined for a number of fragments in the biochemical assay in the presence of tissue factor. Several of these fragment hits did not contain a strongly basic amine or heterocycle (Figure 3 and Supporting Information section). Of the 41 crystal structures attempted, 27 crystallographic structures were successfully determined with a mean resolution of 2.3 Å; 12 of these involved complexes with neutral P1 fragments. With respect to the structure determination of factor VIIa/ fragment complexes, the crystallographic experiments involved soaking the fragment of interest into a cocrystal of factor VIIa catalytic domain in which benzamidine was bound in the S1 pocket. It is possible, that this may have introduced a bias in terms of protein structure or hydration state, as to whether and in what manner fragments may have bound. Because alternative strategies for obtaining crystallographic structures were not investigated, this remains an open question. Given our initial concern that Ser190 might hinder access of extended P1 groups toward Tyr228 by constricting the S1 pocket, we were pleased to have discovered several active aryl halide P1 fragments such as 3 and 6. Substructure searches based on 6 yielded a number of additional P1 groups, some with functional handles for derivatization such as 15 (Ki = 3.5 mM) (Figure 4 and the Supporting Information). The structures for factor VIIa complexes with 6 and 15 were both determined at 2.1 Å resolution and warrant some discussion.24 Both aryl halides bind the S1 pocket in a manner similar to what has been observed in Ala190 serine proteases, such as factor Xa (see Supporting Information). In the crystal structure of 6 bound to the factor VIIa S1 pocket, two alternate
Figure 3. Representative structures and factor VIIa inhibitory potencies from fragment screening, including aryl halides, lactams, and amides.
side chain rotamers of Ser190 are observed in 1:1 proportion, placing the side chain oxygen atom either toward the S1 interior (X1 = −60°), which is more commonly observed, or away (X1 = +60°) (Figure 5).13 Furthermore, the P1 bromine atom has a partial occupancy of ∼50%, while the remaining atoms in P1 have full occupancy (see Supporting Information). Impurity of the material would seem an unlikely explanation for this phenomenon in that it would require both fragments to bind in an identical manner and in similar proportions. On the other hand, removal of bromine has been reported as a result of radiation damage to crystals due to its large absorptive cross-section.26 Radiationmediated cleavage of the carbon−bromine bond in some of the crystal sample may account for the observed partial occupancy. It is an open question whether in the presence of bromine the Ser190 side chain rotates away to avoid steric clash, rendering it “alanine”-like, similar to factor Xa or thrombin, which readily bind aryl halides, since the correlation of these two phenomena cannot be assessed based on our data. However, the crystal structure of 15 bound to factor VIIa indicates the presence of the same two side chain rotamers of Ser190 observed for 6 but with full occupancy of the fragment chlorine atom, suggesting that the orientation of the Ser190 side chain toward the S1 interior does not significantly hinder deep binding of aryl chlorides (Figure 5b). The utility of protein ensemble docking is evident in Figures 6 and 7. From Figure 6, the sensitivity of docking 6 to the crystal conformation and hydration state of factor VIIa S1 is reflected in the wide variation of binding poses and scores. Without the ensemble docking approach, it is less than certain whether this fragment would have been chosen for biochemical testing. 2801
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Figure 4. Substructure searches based on brom-phenol 6 yielded several active and novel P1 fragments.
Figure 5. Crystal structures of 6 (a) and 15 (b) bound in the factor VIIa S1 pocket, both determined at 2.1 Å. The initial Fo − Fc electron density map around each fragment is depicted at 3 RMSD (magenta). Alternative side chain forms for Ser190 are shown. Graphics were generated with Maestro v9.7.25
Figure 7. Several structural features in factor VIIa/6 binding were predicted by protein ensemble docking. The predicted binding pose is in magenta, the crystallographic structure that was subsequently solved is in green. Graphics were generated using the program PyMol.23
calculations suggest that a 40° twist incurs about 2.0 kcal of strain, and analysis of high resolution small molecule crystal structures in the Cambridge Structural Database27 reinforces this prediction with only 1.3% of anisoles having torsions of greater than or equal to 20° (see Supporting Information).27−29 Among the other P1 fragments that we discovered was lactam 7 (Figure 3). This fragment was intriguing in that it represented a cyclized version of the benzamide P1 of 2. The crystallographic structure of 7 bound to factor VIIa was determined at 2.15 Å and suggests that it binds in a similar orientation, forming a hydrogen bond with Gly218 oxygen (Figure 8a). An interesting feature shared by the binding modes of both benzamide of 2 and lactam 7 is the close contact formed by the amide carbonyl and the carbonyl carbon of Gly218. This polar carbonyl−-carbonyl interaction mimics the Dunitz angle of nucleophilic attack30 and has been described by Friesner,31 Diederich,32 and others.33 Furthermore, this interaction is not uncommon in protein−ligand complexes, being seen, for example, in several other drug complexes with serine proteases of the coagulation cascade involving the Gly216 carbonyl.34 That the lactam fragment 7 would be a viable P1 group was not anticipated, since placing the hydrophobic ethylene bridge in contact with the polar Asp189 carboxylate displaces both waters (W1 and W2; Figure 2b) observed in the crystal structure of 2, thereby disrupting the hydrogen bond network involving Asp189 and Tyr228.
Figure 6. Protein ensemble docking yields binding poses of fragment 6 in the factor VIIa S1 that vary widely in terms of orientation and score. Larger negative numbers represent more favorable scores.
Ranking the poses of 6, however, by score yields the factor VIIa S1/6 binding mode that captures several details of the crystal structure that was subsequently determined (Figure 7). These features include the hydrogen bond of 6 to Gly218 oxygen, expulsion of the water above the aryl ring of Tyr228, and the Ser190 rotamer. The one feature that significantly deviates from the crystallographic structure is the phenyl methoxy torsion, which is predicted to be about 40° but is approximately 0° in the crystal structure. Quantum chemical 2802
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CONCLUSIONS
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MATERIALS AND METHODS
In summary, we have implemented a fragment screening protocol, based on protein ensemble docking and utilizing biochemical and NMR assays, which has led to the discovery of several neutral factor VIIa P1 fragments, among them aryl halides and lactams. Crystallographic analysis showed that aryl halides bind factor VIIa in a manner similar to alanine 190 serine proteases such as factor Xa and thrombin. The lactam fragment 7 bound in a manner similar to the benzamide P1 of 2. Incorporation of 7 into a phenylglycine chemotype led to potent inhibitors with improved permeability. This demonstrates that neutral factor VIIa P1 groups are viable and that cationic P1 groups are not absolutely required for effective binding to factor VIIa.
Figure 8. (a) Overlay of crystallographic structures of 1 and 7 bound in factor VIIa. The crystal structure of 7 was determined at 2.2 Å resolution. The crystal structure of 1 was previously reported.9 (b) Crystallographic structure of 18 bound in factor VIIa (2.5 Å resolution). The initial Fo − Fc electron density map is depicted within 1 Å of ligand at a contour at 3.0 RMSD. Graphics were generated with Maestro v9.7.25
Enzymatic Assays. Assays were conducted using substrate and inhibitor levels that were in excess of enzyme (10 nM factor VIIa/ soluble tissue factor) as described in detail by Wong et al.4a with the following modifications. Soluble tissue factor (sTF), comprising residues 1 to 206 of full-length mature human tissue factor, was expressed in Origami B(DE3) cells based on methods previously described.37 Compound fragments were serially diluted in DMSO from a 500 mM DMSO stock solution to give final assay concentrations of 50, 20, 10, and 5 mM and 10% DMSO. Due to the high inhibitor concentrations being investigated, it was important to establish a chemical properties scoring system to identify compounds that exhibited either color or solubility related concerns (A. High, minimal color or solubility concerns; B. Medium, observed color or solubility concerns; C. Low, poorly soluble or highly colored). This ranking allowed us to quickly triage compounds for additional characterization studies. All assays were conducted at room temperature in 96-well microtiter plates using a spectrophotometer.38 Upon initiation of the assay, simultaneous measurements of enzyme activity in control (10% DMSO) and inhibitor-containing solutions were collected for a period of 30 min, and the velocities (rate of absorbance or change versus time) were measured. To capture color or solubility related liabilities within the assay, all plates were read at 405 and 595 nm prior to initiation. All assays were initiated by adding enzyme to a buffered solution containing substrate (D-Ile-Pro-Arg-pNA) in the presence or absence of inhibitor. Hydrolysis of the substrate resulted in the release of pNA (para-nitroaniline), which was monitored spectrophotometrically by measuring the increase in absorbance at 405 nm. because the rate of absorbance is proportional to enzyme activity, a decrease in the rate of absorbance in the presence of inhibitor is indicative of enzyme inhibition. The Michaelis constant, Km, for
Overlay of the crystallographic structures of the lactam 7 and one of our program chemotypes 1 (Figure 8a) suggested that this fragment could replace the basic P1 moiety with little or no perturbation of other key interactions.9 As a result, a number of analogues incorporating lactams were synthesized, many of which were demonstrated to have good inhibitory potency and improved permeability. Compound 18 serves as a representative example with the lactam moiety bound in the same manner as fragment 7 (Figure 8b; for the synthesis of 18, see the Supporting Information).24 While potency was acceptable, permeability as measured by PAMPA was markedly improved (Figure 9).4a,35 We were subsequently able to improve potency through further exploration of lactam analogues; details will be published in a subsequent report. Efforts to similarly incorporate the bromo methoxy phenol 6, or analogues thereof, into the scaffold of 1 were unsuccessful owing, we reasoned, to a geometric incompatibility between fragment and scaffold. It should be noted that while protein ensemble docking was of demonstrated utility in this work, its application has limitations. A large number of crystal structures are not available for most targets, particularly at early stages when a fragment screen might be most impactful. Second, it is unclear, if not doubtful, that any number of crystal structures would capture the full range of protein dynamics at room temperature in solution. Approaches are being explored by others by which to generate useful protein ensembles from MD trajectories.36
Figure 9. Biological and permeability data for compounds 17 and 18. 2803
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Journal of Medicinal Chemistry substrate hydrolysis for factor VIIa was determined in separate experiments by fitting the data at multiple substrate concentrations to the Michaelis−Menten equation, eq 1. Values of Ki were determined by allowing the protease to react with the substrate in the presence of the inhibitor, and eqs 2 and 3 were used to calculate Ki values assuming competitive inhibition. For many of the fragments, negligible inhibition was observed at the highest inhibitor concentration tested (50 mM). In these cases, the value assigned was the percent inhibition observed at the highest concentration evaluated. Fragments that demonstrated potency and possessed a chemical properties score of “A” or were confirmed by NMR were evaluated in a second tier study. For these studies, four concentrations of inhibitor, bracketing the Ki, were evaluated in the presence of four concentrations of substrate, bracketing its Km (0.25, 1, 2, and 4 mM). These experiments established the fragment kinetic mechanism of binding by following the same protocol as described above; however, the data was analyzed using global fits to equations for competitive, eq 4, or mixed noncompetitive inhibition, eq 5.
v=
Vmax[S] K m + [S]
(1)
vs B−A =A+ IC vo 1 + I50
n
( )
Ki =
v=
v=
of on-resonance and off-resonance spectra in order to minimize spectral artifacts. On- and off-resonance spectra were stored and processed separately. The final STD NMR spectrum was obtained by subtracting the individual on- and off-resonance spectra. To be classified as a hit, the observed STD signal intensity was required to be at least 1% of the signal intensity for the corresponding resonance in the unsaturated spectrum. Control STD NMR experiments were performed using an identical experimental setup but in the absence of the protein. Total acquisition time per compound was ∼22 min. We note the NMR screen did not utilize tissue factor, which is known to be required for activity; this exclusion may have biased or impacted the detection of hits. However, because there is no crystallographic evidence of structural changes in the S1 pocket induced by tissue factor (see Supporting Information), we believed it would be appropriate to perform the NMR fragment screening in the absence of TF, because all of the interactions that we would be monitoring are located in this pocket. Crystallization, Data Collection, and Structure Refinement. Recombinant, lyophilized factor VIIa protein (Novoseven, Novo Nordisk, Inc.) was purchased and reconstituted according to the manufacturer’s recommendations. This protein was processed to remove the Gla-domain based upon a method described previously.40 Briefly, recombinant factor VIIa was dissolved to 1 mg/mL in cold 50 mM Tris-HCl (pH 9.0 at 4 °C), 20 mM EDTA buffer. Cathepsin G (Human Neutrophil; Calbiochem) was added at 4 mU (∼2 μg) per milligram of factor VIIa, and the sample was incubated at 37 °C for 45 min. The reaction was stopped by the addition of 0.5 M CaCl2 to a final concentration of 20 mM and placing on ice. The sample was diluted 15-fold with 20 mM Tris-HCl, 5 mM CaCl2 (pH 9.0), loaded onto a 5 mL HiTrap Q HP column (GE Healthcare), and eluted with a linear gradient to 20 mM Tris-HCl, 20 mM CaCl2, 800 mM NaCl (pH 8.5). The highly purified, des-Gla factor VIIa pool at 0.5 mg/mL was mixed with 10 mM of the small S1 pocket binding inhibitor benzamidine, incubated on ice for 1 h, and concentrated by ultrafiltration to 25 mg/mL. This preparation was divided into aliquots, flash frozen, and stored at −80 °C until needed for crystallization. Fragments were selected for crystallographic study on the basis of structural interest and binding affinity and were used to soak the preformed des-Gla factor VIIa crystals grown previously in the presence of benzamidine. Data were collected at APS beamline 17BM. The structures were originally refined with CNX41 and Quanta.42 For deposition, some were rerefined with BUSTER43 and COOT.44 For crystallographic data and refinement statistics for the Xray crystal structures of compounds 6, 7, 15, and 18, see the Supporting Information. Computational Chemistry and Molecular Modeling. Crossdocking P1 Fragments (See Supporting Information). The inhibitor/ factor VIIa crystallographic structures 4JZE and 4JZF from the Protein Data Bank (PDB) were refined using the Protein Prep utility in Maestro,25 and the ligand P1 substructures, 1,6-diamino-isoquinoline and 3-amino-benzamide, respectively, were extracted. Ions, glycerol molecules, and waters were removed except the water above Tyr228. His57 was protonated because this was most consistent with the original crystal structures. The amino acid content and formal charges were kept constant across the ensemble. Grids for docking into each protein S1 were prepared using Glide with defaults and using the P1 groups as reference points.18 Docking itself was similarly done with default settings at the extra-precision level (XP). The scores reported here were the “XP scores”. Assessment of Docking Score Sensitivity to Receptor Structure As a Function of Ligand Complexity and Size (See Supporting Information). Receptors and docking grids for Glide were prepared as described in the section below for “Fragment Virtual Screening”. A series of simple organic fragments were docked into each of these receptors using Glide SP with defaults. To illustrate the trend of increased sensitivity of score on receptor with increased ligand size and complexity, the results for benzene, aniline, and 1,6 diamino isoquinoline were tabulated (see Supporting Informaton). Fragment Virtual Screening. With Pipeline Pilot,16 about 30000 fragments (≤3 rotable bonds and ≤17 heavy atoms) were extracted
(2)
IC50 1+
S Km
(3)
Vmax[S] Km 1 +
(
[I] Ki
(
[I] K is
) + [S]
(4)
Vmax[S] Km 1 +
) + (1 + )[S] [I] K ii
(5)
In eqs 1 to 5, v is the observed velocity of the reaction, Vmax is the maximal velocity, vo is the velocity of the control in the absence of inhibitor, vs is the velocity in the presence of inhibitor, [S] is the concentration of substrate, Km is the Michaelis constant for the substrate, I is the concentration of inhibitor, A is the minimum activity remaining (usually locked at zero), B is the maximum activity remaining (usually locked at 1.0), n is the Hill coefficient (a measure of the number and cooperativity of potential inhibitor binding sites), IC50 is the concentration of inhibitor that produces 50% inhibition, Ki is the dissociation constant of the enzyme/inhibitor complex, and, for a mixed-type noncompetitive inhibitor, Kii and Kis are the dissociation constants of the enzyme/inhibitor and enzymesubstrate/inhibitor complexes, respectively. Although for a few inhibitors slightly better fits could be obtained with eq 5 than with eq 4, the differences in Ki and Kis were small. The Ki values from eq 4 are reported throughout. The factor VII deficient prothrombin time clotting assay (FVII-def PT) was performed as previously described.4a NMR Screening. Binding of the fragments to factor VIIa was detected with the saturation transfer difference (STD) NMR experiment39 using 4 μM factor VIIa protein and 1 mM test compound. (For preparation of factor VIIa for the NMR studies, see the Supporting Information). A frozen stock (310 μM) of factor VIIa was thawed on ice and diluted to 4 μM with NMR buffer (25 mM TrisHCl, pD 7.1, 100 mM NaCl, 0.5 mM EDTA, 100% D2O). Diluted factor VIIa (490 μL) was added to 10 μL of compound (50 mM stock in DMSO), and the sample was mixed and transferred to a Norell 5 mm HT NMR tube. All NMR data was acquired at 20 °C on a Varian INOVA 600 MHz spectrometer equipped with a 5 mm HCN cold probe. For the on-resonance experiments, the protein was saturated by applying the saturating rf pulse at 0.5 ppm (1.0 ppm bandwidth) during the last 4.3 s of a 5 s relaxation delay. Off-resonance spectra were collected in an identical fashion but with the “saturating” RF pulse at −30 ppm. Data were obtained with an interleaved acquisition 2804
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Journal of Medicinal Chemistry from the ACD.15 Chemical filters were then applied to further remove molecules with un-drug-like or otherwise undesired features, yielding the set of 18000 fragments, which were subsequently used for protein ensemble docking. Given the uncertainties of finding and characterizing neutral fragments at the outset of this endeavor, we elected to include cationic fragments in the process as well, which we expected would bind more strongly and enable us to troubleshoot and optimize the overall screening and crystallography process as needed. The selection of the ensemble of factor VIIa structures for docking was done by overlaying in Maestro25 all publicly available and proprietary crystal structures of factor VIIa/ligand complexes with acceptable temperature factors in the active site region and resolution (
