Molecular Basis for Multiple Omapatrilat Binding Sites within the ACE

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Molecular Basis for Multiple Omapatrilat Binding Sites within the ACE C-domain – Implications for Drug Design Gyles E. Cozier, Lauren B. Arendse, Sylva L. Schwager, Edward D. Sturrock, and K. Ravi Acharya J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01309 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 29, 2018

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Journal of Medicinal Chemistry

Binding of omapatrilat to ACE

Molecular Basis for Multiple Omapatrilat Binding Sites within the ACE C-domain – Implications for Drug Design

Gyles E. Cozier+, Lauren B. Arendse#, Sylva L. Schwager#, Edward D. Sturrock# and K. Ravi Acharya*,+

+Department

of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY,

United Kingdom #Department

of Integrative Biomedical Sciences, Institute of Infectious Disease and Molecular

Medicine, University of Cape Town, Observatory 7935, Cape Town, Republic of South Africa Running title: Binding of omapatrilat to ACE *Corresponding Author K. Ravi Acharya, Department of Biology & Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom; [email protected]; Tel. (+44) 1225-386238, Fax. (+44) 1225-386779.

Keywords: Omapatrilat, angiotensin-1 converting enzyme, domain specificity, neutral endopeptidase, crystallography, enzyme kinetics, enzyme structure, metalloprotease

ACS Paragon Plus Environment

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Binding of omapatrilat to ACE ABSTRACT: Omapatrilat was designed as a vasopeptidase inhibitor with dual activity against the zinc metallopeptidases angiotensin-1 converting enzyme (ACE) and neprilysin (NEP). ACE has two homologous catalytic domains (nACE and cACE) which exhibit different substrate specificities. Here we report high-resolution crystal structures of omapatrilat in complex with nACE and cACE, and show omapatrilat has sub-nanomolar affinity for both domains. The structures show nearly identical binding interactions for omapatrilat in each domain, explaining the lack of domain selectivity. The cACE complex structure revealed an omapatrilat dimer occupying the cavity beyond the S2 subsite, and this dimer had low micromolar inhibition of nACE and cACE. These results highlight residues beyond the S2 subsite that could be exploited for domain selective inhibition. In addition, it suggests the possibility of either domain specific allosteric inhibitors that bind exclusively to the non-prime cavity, or the potential for targeting specific substrates rather than completely inhibiting the enzyme. INTRODUCTION The human zinc dipeptidylcarboxypeptidase angiotensin-1 converting enzyme (ACE, EC 3.4.15.1) plays an important role in cardiovascular physiology 1. Not only is it part of the renin-angiotensinaldosterone system (RAAS), where it converts angiotensin I to the vasoactive peptide hormone angiotensin II 2, but in addition it cleaves the vasodilatory peptide bradykinin (BK) 3. Due to this central role in blood pressure regulation it has been a key target for controlling the blood pressure response. Two isoforms of ACE are known, testis (tACE) and somatic (sACE) ACE, which consist of one and two catalytically active domains respectively. In sACE these are known as the N- and Cdomains (nACE and cACE respectively), and although they have similar amino acid sequences and structural topology, they differ in their substrate specificity and inhibitor binding 4-8. Although a range of ACE inhibitors such as captopril, lisinopril, enalapril, ramipril and perindopril have been routinely used for the treatment of hypertension and myocardial infarction as


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Binding of omapatrilat to ACE well as diabetic nephropathy, up to 28% of patients develop side effects after long-term treatment 9, 10. These side effects not only include a persistent dry cough, but also in some cases the more serious condition of angioedema, which indicates diverse involvement of ACE and it’s peptide substrates in multiple pathways 11-13. A characteristic feature of ACE inhibitors currently used clinically is that they bind both nACE and cACE with similar affinities, thereby causing increased plasma BK, which has been a major reason for drug discontinuation in certain patients. Development of domain-selective inhibitors for either nACE or cACE would allow more control for patient treatment depending on their condition, with lower risks of adverse effects. Inhibition of cACE is known to reduce circulating Ang II while plasma BK levels would still be under the control of nACE 14. In contrast, it has been shown in animal models that Ac-SDKP levels are increased by inhibition of nACE alone, and this produces anti-fibrotic and anti-inflammatory effects 15, 16. This suggests that selectively inhibiting nACE would have minimal effects on the RAAS and allow for the targeted treatment of tissue injury and fibrosis without affecting blood pressure. Neutral endopeptidase (neprilysin, NEP) is capable of cleaving a wide variety of peptides relevant to hypertension including BK, substance P, atrial natriuretic peptide, enkephalins and angiotensins

17, 18.

This led to the proposal that inhibition of both ACE and NEP could improve

treatment for conditions such as hypertension 19. NEP and ACE are both zinc metallopeptidases and the similarity between these enzymes, exemplified by their overlapping substrate specificity, enabled the development of single molecules that inhibit both enzymes (i.e., vasopeptidase inhibitors). One such inhibitor, omapatrilat ((4S,7S,10aS)-5-oxo-4-[[(2S)-3-phenyl-2-sulfanylpropanoyl]amino]2,3,4,7,8,9,10,10a-octahydropyrido[2,1-b][1,3]thiazepine-7-carboxylic developed as a dual enzyme inhibitor

20,

acid)

(Figure

1),

was

and indeed showed increased reduction in blood pressure

when compared to inhibition of ACE alone. Omapatrilat has nano-molar (nM) affinities for both NEP



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Binding of omapatrilat to ACE and ACE 20, it is reported to inhibit the cleavage of nACE and cACE specific substrates similarly 21 and inhibits a third bradykinin degrading enzyme aminopeptidase P 22. In addition, the incidence of angioedema was significantly higher and more severe in the subjects treated with omapatrilat (2.17%) than in those receiving the normal ACE inhibitor enalapril (0.68%) 23. The increased incidence of this serious, potentially life-threatening complication was presumed to be related to the accumulation of bradykinin and other related peptides during treatment. Here we describe the high resolution crystal structures of nACE and cACE in complex with omapatrilat and we also report in vitro inhibition data for omapatrilat against the individual ACE domains. Omapatrilat displayed non-selective inhibition, inhibiting both nACE and cACE in the subnanomolar range. The kinetic data is consistent with the structural data, which shows many, and mostly conserved protein-inhibitor interactions for the Zn2+-bound omapatrilat molecule within the active site of each domain. However, the complex with cACE also shows that two additional omapatrilat molecules are able to bind in the binding site cavity, and it is consistent with binding of an omapatrilat dimer. This lends support for the design of an extended molecule exploiting the larger active site groove to provide enhanced specificity for cACE. RESULTS AND DISCUSSION Overall Structure. The nACE and cACE omapatrilat complexes crystallised in P1 and P212121 space groups, and the structures were determined to high resolutions of 1.80 and 1.37 Å respectively (Table 1). There was one molecule of cACE and two molecules of nACE in their respective asymmetric units. As previously shown, the overall fold of both nACE and cACE is mainly α-helical, forming an ellipsoid, with the active site buried in the centre in the form of a two-lobed cavity comprised of the Sʹ and S subsites flanking the catalytic Zn2+ ion. Clear, unambiguous electron density that could be attributed to the inhibitor bound in the S1, S1ʹ and S2ʹ subsites was observed in the mFo-


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Binding of omapatrilat to ACE DFc omit maps for both the nACE and cACE omapatrilat complexes. (Figure 2A and B). Consistent with omapatrilat being a high affinity inhibitor, there are extensive interactions with the protein and active site zinc ion, as described in detail below. Sections of the first 100 residues of both molecules in the asymmetric unit of nACE show high temperature factors (B-factors), and in protein chain B the electron density is poor with some evidence of a secondary conformation of this region. These features are often observed in nACE crystal structures, and this part of the chain has been attributed to a flexible lid-like structure that may open and close to control peptide access into the active site 24. While in some previous structures (e.g. the nACE complex with sampatrilat, PDB code 6F9V) it was possible to model the second conformation 25,

the density for the omapatrilat complex was not sufficient for this purpose. As reported previously for the sampatrilat-cACE complex structure (PDB code 6F9T) 25, Cys-

496 was modified, most likely oxidised to an S-hydroxycysteine. This modification is clearly seen for the current cACE complex with omapatrilat and has been modelled as the same S-hydroxycysteine (Cso) residue. Closer examination of the electron density maps for the cACE/omapatrilat complex indicated electron density that was consistent with two additional omapatrilat molecules bound in the S subsite lobe of the binding cavity (Figure 2C and D). The details of these additional, ‘newly observed’ binding sites are described below. Inhibitor Binding Sites. As described above, in both the nACE and cACE complexes an omapatrilat molecule binds in the S1, S1ʹ and S2ʹ subsites. Listed in Table 2, and depicted in Figures 3 and 4, are the extensive interactions with the inhibitor molecules from the protein chain and Zn2+ ion. Omapatrilat (Figure 1) was designed as a tripeptide mimic 20, having a carboxylic acid ‘C-terminal’, a bicyclic group to occupy the S2ʹ and S1ʹ subsites, and a sulphur containing phenylalanine analogue to



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Binding of omapatrilat to ACE interact with the Zn2+ ion and extend into the S1 subsite. The crystal structures reported here show that the omapatrilat molecule occupies almost identical positions in the two nACE chains and the cACE chain, in a conformation typical of small peptides and peptide mimic inhibitors. The ‘C-terminal’ carboxylic acid is tightly bound, with the O1 atom hydrogen bonding to Gln259, Lys489 and Tyr498 in nACE (Gln281, Lys511 and Tyr520 in cACE), and the O2 atom in a water mediated interaction with Lys489/511 (Figure 3). The atoms of the bicyclic omapatrilat group that mimic a peptide backbone interact with the sidechains of His331 and His491, and the backbone of Ala332 of nACE (His353, His 513 and Ala354 of cACE). The O4 atom of omapatrilat interacts with His361 and Tyr501 of nACE (His383 and Tyr501 of cACE), and along with S2 forms a bidentate interaction with the Zn2+ ion. The strong bidentate interaction with the Zn2+ ion is consistent with the high affinity and slow off-rate observed for omapatrilat in the inhibition assays (see below). There are also a series of hydrophobic interactions in the S2ʹ and S1ʹ subsites with residues Thr358, His361, Phe435, His491 and Tyr501 of nACE (Val380, His383, Phe457, His513 and Tyr523 of cACE). With the phenylalanine sidechain like moiety being the only part of omapatrilat to extend into the S subsites lobe of the binding cavity, the interactions are restricted to being hydrophobic with Ser333 and Phe490 of nACE (Ser355 and Phe512 of cACE). In addition, there is a hydrophobic interaction with an ethylene glycol molecule in chain A of nACE, whereas in cACE the third omapatrilat molecule provides a hydrophobic environment. Despite all these hydrophobic interactions, this ‘phenylalanine side-chain’ is the most flexible part of the omapatrilat molecule highlighted by comparatively less clear electron density and higher B-factors than the rest of the inhibitor. Therefore, the crystal structures show that there is an almost identical set of binding interactions observed for omapatrilat in both nACE and cACE.


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Binding of omapatrilat to ACE Additional Omapatrilat Molecule Binding Sites in cACE. Examination of the experimental electron density maps for the S subsites lobe of the binding cavity in both nACE and cACE usually shows a variety of electron density beyond the bound inhibitor/substrate that can be attributed to water molecules and substituents of the buffers and crystallisation condition used, such as various polyethylene glycol (PEG) molecules. However, in the present cACE omapatrilat complex structure, the density suggested that two extra omapatrilat molecules (omapatrilat 2 and 3) were bound, as shown by the 2mFo-DFc electron density map and the mFo-DFc omit map (Figure 2C and D). The density for these molecules is not complete, and is strongest for the heavier sulphur, oxygen and nitrogen atoms. Therefore, to help confirm that the density does indeed result from omapatrilat molecules, Polder maps were generated which showed almost complete density for omapatrilat 2 and clear density for omapatrilat 3 (Figure 2E and F). The level of electron density observed indicates that these binding sites do not have full occupancy and were refined at 66 and 58% for omapatrilat 2 and 3 respectively. An area of electron density that can be best modelled as an ethylene glycol molecule with 82% occupancy, is located close to the phenylalanine like sidechain of omapatrilat 2, which itself shows poor density even in the Polder map. To avoid steric clashes between the phenylalanine ring and the ethylene glycol molecule, a second conformation of omapatrilat 2 was modelled, although the data indicates that this moiety of the omapatrilat molecule is highly flexible. A view of the binding cavity within the cACE overall structure (Figure 5) shows that these two extra omapatrilat molecules bind on one side, and occupy most of the remaining length of the S subsite lobe not bound by omapatrilat 1. The binding interactions for omapatrilat 2 and 3 are shown in Table 3 and Figures 3 and 4. Omapatrilat 3 has no direct hydrogen bond interactions with the protein, whereas omapatrilat 2 binds



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Binding of omapatrilat to ACE to arginines 124 and 522, as well as an ethylene glycol molecule. Residue Arg124 provides further evidence for presence of the additional omapatrilat molecules. In the cACE complex structure it adopts a dual conformation where orientation B is typical for this enzyme, but this would occupy the same position as omapatrilat 3. So not only is the predominant conformation A (refined at 63% occupancy) allowing room for omapatrilat 3 to bind, it also provides important interactions with omapatrilat 2. Omapatrilat 2 has water mediated interactions involving two water molecules and only two residues (Glu123 and Trp220), whereas six residues (Asn85, Arg124, Asn136, Glu143, Ser516 and Ser517) interact through four water molecules with omapatrilat 3. Omapatrilat 2 has a more extensive set of hydrophobic interactions with four residues (Glu123, Met223, Glu403 and Pro519) and an ethylene glycol molecule, compared to omapatrilat 3 which interacts with only two residues (Asn85 and Val518) and omapatrilat 1. The overall more extensive interactions for omapatrilat 2 compared to omapatrilat 3 is consistent with the density being clearer, and occupancy higher for omapatrilat 2. As stated above both omapatrilat 2 and 3 were refined with an occupancy of above 50%, thereby suggesting that at least some of the time both are bound to cACE. This is of particular note because the distance between the S2 atoms of the molecules when both are bound is 2.01 Å, which suggests a disulphide like bond can form. From the crystal structure alone it is not possible to elucidate whether each molecule of omapatrilat can bind individually, or whether an inhibitor dimer where 2 molecules are linked through their S2 atoms is required for binding. Comparison of Ligand Binding Beyond S2 Subsite. To date the vast majority of substrates and inhibitors that have been co-crystallised with nACE, cACE and AnCE (the Drosophila homologue) have been small, equivalent to a 2-4 residue peptide chain, and therefore bind only in the S2, S1, S1ʹ and S2ʹ subsites at most. Larger molecules that have been tested, were often either hydrolysed by the enzymes such that only a short product remains 26, or were so flexible beyond the S2 subsite that this


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Binding of omapatrilat to ACE region was not visible in the electron density maps

27.

There have been some examples where the

length of a P2 group has facilitated interaction with a longer protein side-chain that would otherwise not be considered part of the S2 subsite, one such example being the nACE complex with RXP407 that shows an interaction with Arg381 28. However, information about interactions with the S3 and more distant subsites, has been observed in only four previous structures. Firstly, both nACE and cACE were co-crystallised with a phosphinic tripeptide (FII), and similar to omapatrilat, the complex with cACE showed an additional inhibitor molecule bound 29. Two further crystal structures of cACE, those in complex with angiotensin II and BPPb 30, contained peptide chains longer than four residues visible in the electron density maps, and in the case of BPPb the full 11-mer was clearly shown. Finally, in the structure of AnCE in complex with BPPb, six residues of the inhibitor were observed

27.

The

complex of cACE with omapatrilat described here provides a fifth structure, and further information about the binding cavity beyond the S2 subsite. Figures 6 and 7 show the position and orientation within the binding cavity, and an overlay of the ligands respectively. This comparison of all five structures shows that the binding within the S2, S1, S1ʹ and S2ʹ subsites is similar, with the ligand structures overlaying closely. This is to be expected, with binding close to the active site Zn2+ ion being closely controlled in a confined space, and the Cterminal binding position being highly conserved. Beyond this region, the binding cavity widens, giving much more space and many more potential binding interactions, and this is highlighted by the wide variety of positions and orientations adopted by the ligands in these subsites. There are some similarities such as BPPb bound to cACE and AnCE closely align up to the S4 subsite, beyond which BPPb is no longer visible in the electron density in the AnCE complex. Angiotensin II also aligns with those BPPb peptides in the S3 subsite, but the P4 residue adopts a different position, and if the final P5 residue had been visible, it would likely also be in a different position to the BPPb molecule. FII has



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Binding of omapatrilat to ACE a very different shape to the peptides, however the C-terminal mimic half of the second molecule that is bound does have some similarity in position to the P3-6 residues of BPPb in cACE, but the second half of its structure does not overlay, and instead is closer in position to the omapatrilat 2 and 3 molecules. This second half of FII, and both omapatrilat molecules bind to the opposite face of the binding cavity to that seen for the other ligands. Therefore the additional omapatrilat molecules in the structure reported here are binding to regions (additional conformational space) of the cACE cavity that have not previously been implicated in ligand binding. Omapatrilat 2 in particular highlights a hydrophobic region comprising of Trp220, Met223 and Pro519 (Trp198, Trp201 and Pro497 in nACE), and the hydrogen bond interactions with Arg124 (Ser100 in nACE) (Figures 3D and 4B). Combining the information from the previous structures with that of omapatrilat-cACE complex indicates that most of the binding cavity can be involved in interactions with ligands. While this makes predicting the interactions for a particular ligand more difficult, it does allow for a significant amount of flexibility in inhibitor design. Kinetic Analysis of nACE and cACE Inhibition by Omapatrilat. Kinetic analysis of nACE and cACE inhibition by omapatrilat was carried out using single domain recombinant ACE proteins and the peptide substrate Cbz-Phe-His-Leu (Z-FHL) in endpoint fluorogenic assays (Figure 8). The Morrison equation, for inhibition under tight binding conditions (where Kiapp ≤ [enzyme]), was fitted to the inhibition data yielding typical active-site titration curves (Figure 8C and D). Under tight binding conditions, the assumptions made by classical inhibition models no longer hold true so Ki values cannot accurately be determined from IC50 plots, as exemplified in Figure 8. Due to the slow binding nature of many protease inhibitors, inhibitors are typically pre-incubated with the enzyme prior to the addition of substrate to ensure that inhibition constants are measured under steady-state conditions. Even though it is well established that omapatrilat is a reversible competitive inhibitor, Kiapp did not


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Binding of omapatrilat to ACE vary with substrate concentration for either domain, indicating that omapatrilat has a very slow off rate and did not dissociate from the enzyme during the time of the experiment, in which case Ki = Kiapp/(1+[S]/Km) does not hold true and Kiapp approximates Ki. There was no significant difference in the affinity of omapatrilat for the two domains, with Ki values in the sub-nanomolar range for both nACE and cACE. This is agreement with the report by Azizi et al., showing that omapatrilat similarly inhibited the somatic ACE-mediated hydrolysis of N- and C-domain site-specific substrates, AcSDKP and hippuryl-histidyl-leucine, at sub-nanomolar concentrations

21.

In addition, the kinetic data is

consistent with the extensive and almost identical set of binding interactions observed for omapatrilat in both nACE and cACE complex structures described above. Kinetic Analysis of nACE and cACE Inhibition by Omapatrilat Dimer. Assessment of omapatrilat dimer inhibitor potential for nACE and cACE was carried out using Z-FHL and a bradykinin-like MCA-RPPGFSAFK(Dnp)-OH substrate (Figure 9). Omapatrilat dimer displayed low micromolar inhibition of cACE with Z-FHL (IC50 2.8 µM) and MCA-RPPGFSAFK(Dnp)-OH (IC50 1.8 µM). Additionally, omapatrilat dimer inhibition of nACE was comparable to cACE for Z-FHL and MCA-RPPGFSAFK(Dnp)-OH (IC50 2.2 µM and IC50 2.1 µM, respectively).

Towards Structure-Based Design of Next Generation ACE Inhibitors: Dual ACE Cdomain Selective/NEP Inhibitors. Dual ACE/NEP inhibitors that selectively inhibit cACE, leaving the nACE free to degrade bradykinin, are predicted to have reduced side-effect profiles while maintaining efficacy, providing a promising new approach to the development of vasopeptidase inhibitors 31. Several C-domain selective inhibitors described to date have been derived from potent non-selective ACE inhibitors, with structural modifications significantly reducing N-domain binding 32, 33.

These structures of nACE and cACE in complex with omapatrilat, together with previously

reported cACE structures in complex with C-selective inhibitors provide a good starting point for the



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Binding of omapatrilat to ACE structure-based design of dual ACE C-domain /NEP inhibitors. Mutagenesis studies have shed light on active-site residues important for conferring domain selectivity 11, 32-34 and exploiting these regions for inhibitor design has proved an effective approach for obtaining domain-selective ACE inhibitors 35.

In addition, crystal structures of the NEP ectodomain in complex with various inhibitors have

provided insight into the specificity of NEP

36-40.

In the absence of a crystal structure of NEP in

complex with omapatrilat, in silico docking was used to predict the binding interactions between omapatrilat and NEP (Figure 10A). Figure 10B shows omapatrilat docked into the active site of NEP aligned to the nACE- and cACE-omapatrilat complexes. The predicted omapatrilat binding mode in NEP differs form that observed in the ACE structures, with the benzyl group occupying the S1ʹ binding site while the 7, 6 fused bicyclic thiazepinone ring binds at the edge of the S2ʹ binding pocket with majority of the ring system positioned within the large open cavity between the two sub-domains of NEP. This is in agreement with the observation that the prime side of NEP is primarily responsible for inhibitor selectivity and potency, with the S1ʹ pocket showing the most stringent specificity and preferentially binding aromatics or large hydrophobic groups

37.

In the predicted NEP-omapatrilat

complex, the thiol group of omapatrilat chelates the zinc ion while the amide carbonyl interacts with Arg717. The carboxylic acid forms a salt bridge with Arg110 and both the carboxylic acid and the thiazepinone carbonyl interact with Asn542. Since the S2’ site is largely unoccupied, the position of flexible residues Trp693 and Phe106, on the boundary of the S1ʹ and S2ʹ binding pockets, which typically adopt different position depending on the bound ligand, do not appear to affect the docking pose of omapatrilat. ACE Inhibitors Targeting Regions Beyond the S2 Subsite. To date, all ACE inhibitor development has concentrated on utilizing the conserved zinc and S2ʹ, S1ʹ and S1 subsite interactions to give potency, and targeted the small differences between nACE and cACE in these and the S2 subsite


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Binding of omapatrilat to ACE to give specificity. While this has produced some success, developing drug-like specific inhibitors has been a challenge. The additional omapatrilat molecules bound to cACE, and the kinetic data showing inhibition by the omapatrilat dimer, suggest regions beyond the S2 subsite could be exploited for drug design. This not only includes the possibility of extended zinc binding inhibitors targeting these regions giving more potential for domain specific inhibition, but also a novel class of non-zinc binding inhibitors that bind only to outer areas of the binding cavity. ACE domains are involved in the hydrolysis of a selection of peptide substrates, and these substrates extend beyond the S2 subsite, in a variety of orientations upon binding. The overlay of the cACE/omapatrilat complex with the ACE structures in complex with BPPb and angiotensin II, and the kinetic data presented in this study, indicate that the omapatrilat dimer binds in the non-prime lobe and acts as a non-competitive inhibitor. In which case, the omapatrilat dimer could be a starting point to develop domain specific, and more potent allosteric inhibitors by targeting unconserved regions of the non-prime lobe. The allosteric binding site of the dimer is proximal to the obligatory substrate binding site, so, it is also conceivable that other peptide substrates may bind in the same region as the omapatrilat dimer, in which case the inhibition could be competitive in these instances. The kinetic data and cACE-omapatrilat crystal structure show that it is possible for small molecules to bind in the non-prime lobe without utilizing interactions with the catalytic zinc ion. Even if no natural ACE substrate binds to the same side of the binding site as the omapatrilat dimer, it is interesting to speculate that inhibitors solely targeted to areas of the binding cavity distant from the catalytic site could take advantage of differences in substrate binding caused by variation in their sequence and length. This approach may produce substrate specific inhibition while leaving the general activity of the enzyme largely unchanged, and like domain-specific inhibition, it has the potential to reduce adverse side effects and may be worth exploring further. It would be of significant benefit in investigating the



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Binding of omapatrilat to ACE possibility of substrate specific inhibitors by knowing which areas of the non-prime lobe to target. Therefore crystal structures of the ACE domains in complex with full-length substrates would be of great benefit. Comparison of omapatrilat dimer binding region in cACE with nACE. In contrast to the high degree of conservation found in the S2, S1, S1ʹ and S2ʹ subsites, the region around the omapatrilat dimer binding site in cACE contains many residues that are not conserved in nACE (Table 4 lists all these residues beyond the S2 subsite). Therefore, this would make it a good region to target for domain specificity. The unconserved residues are located over the whole cavity surface where the omapatrilat dimer binds, this gives a large degree of flexibility in what molecule could be designed to target them. One of the closest to the catalytic site has already been utilised for domain specificity, where nACE Arg381 (Glu403 in cACE) has been attributed to the nACE specificity of RXP 40728. Other unconserved residues of similar distance from the catalytic site include Asn66, Glu143 and Ser516 of cACE (Ser39, Ser119 and Asn494 of nACE), with Glu143 and Ser516 both interacting with the omapatrilat dimer. Targeting residues further from the catalytic site using the conventional P2, P1, P1ʹ and P2ʹ scaffold could prove challenging in producing a drug-like molecule of not too large size, although meeting these challenges maybe necessary in order to obtain the desired domain specificity. An option for potentially overcoming this hurdle could be to design inhibitors that span from interacting with the catalytic zinc ion at one end, to targeting the unconserved residues highlighted by the omapatrilat dimer binding region at the other that is not extending all the way to the S2ʹ subsite. This would reduce the length of molecule required, retain the strong interactions with the zinc ion for high affinity binding, and gain the domain specificity by reaching the less conserved regions of the enzyme.


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Binding of omapatrilat to ACE The binding of an omapatrilat dimer in the cACE complex structure was a fortuitous observation, therefore the molecular structure has not been designed or optimised for this region. However, the dimer already has a micromolar affinity for the ACE domains, which is often the starting point for SAR studies. As mentioned above, when considering designing a molecule that binds in the non-prime cavity away from the catalytic site, there is a wide range of unconserved residues that could be targeted to achieve domain specificity (Table 4). One example is the region where omapatrilat 2 binds in the cACE complex, which has more space than observed in nACE due to sequence differences. Residues Ala208, Ser219 and Met223 of cACE, are replaced by Tyr186, Tyr197 and Trp201 in nACE, and therefore this larger pocket in cACE could be an ideal region to target for domain selective inhibition. CONCLUSIONS In summary, this study has shown that omapatrilat is a non-specific, sub-nanomolar inhibitor of both nACE and cACE, and this can be explained by the near identical binding orientation and almost completely conserved, extensive interactions observed in the high resolution structures of these domains in complex with omapatrilat. Interestingly, two further omapatrilat molecules likely to be a dimer linked by a disulphide like bond between their S2 atoms, were observed in the non-prime lobe of the binding cavity of cACE, but distant from the catalytic site. Kinetic studies showed that the omapatrilat dimer was a micromolar inhibitor of both nACE and cACE using both short and long substrates, thereby suggesting it is a noncompetitive, allosteric inhibitor. These data suggest that not only could more potent and domain specific extended zinc binding inhibitors be designed to additionally target the more distant non-prime subsites, but also new classes of non-zinc binding inhibitors could be developed that only bound in the non-prime lobe. By not



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Binding of omapatrilat to ACE utilizing the strong, conserved zinc and prime subsites binding interactions (which make specificity more difficult), one strategy could use the omapatrilat dimer as a starting point for domain specific, allosteric inhibitors while another could target differences in substrate binding within the large nonprime lobe to develop substrate specific inhibition. Both of these approaches would have the aim of producing drugs with reduced side-effects. EXPERIMENTAL SECTION Enzymes and Omapatrilat. Fully glycosylated (D629 and Δ36NJ) and minimally glycosylated (N389 and G13) N- and C-domain human ACE proteins respectively, were generated by expression in cultured mammalian CHO cells, and purified to homogeneity as described previously 42, 43.

Purity was assessed using SDS-PAGE and shown to be >95% pure. Omapatrilat was obtained from

American Custom Chemicals Corp. and Sigma-Aldrich. X-ray Crystallographic Studies. The minimally glycosylated ACE domains were preincubated with omapatrilat in a 4:1 v/v ratio of protein (8 mg ml-1 cACE and 5 mg ml-1 nACE both in 50 mM Hepes, pH 7.5, 0.1 mM PMSF) and 5 mM inhibitor for 1 hour (cACE on ice, nACE at room temperature). Co-crystals were obtained with 1 µl of the protein-inhibitor complex mixed with an equal volume of reservoir solution (0.1 M MIB buffer pH 4.0, 5% glycerol and 15% PEG 3350 for cACE, and 30% PEG 550 MME/PEG 20000, 0.1 M Tris/Bicine pH 8.5, and 60 mM divalent cations, Molecular Dimensions Morpheus A9 for nACE), which was suspended above the well as a hanging drop. X-ray diffraction data were collected on station I03 at the Diamond Light Source (Didcot, UK). Crystals were kept at constant temperature (100 K) under the liquid nitrogen jet during data collection. Images were collected using a PILATUS3 6M detector (Dectris, Switzerland). Raw data images were


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Binding of omapatrilat to ACE indexed and integrated with DIALS 44, and then scaled using AIMLESS

45

from the CCP4 suite 46.

Initial phases were obtained by molecular replacement with PHASER 47 using PDB code 6F9T 25 for cACE and 6EN5 35 for nACE as the search models. Further refinement was initially carried out using REFMAC5

48

and then Phenix 49, with COOT

50

used for rounds of manual model building. Ligand

and water molecules were added based on electron density in the Fo-Fc Fourier difference map. MolProbity 51 was used to help validate the structures. Crystallographic data statistics are summarized in Table 1. All figures showing the crystal structures were generated using CCP4mg 52 and Pymol 53, and schematic binding interactions are displayed using Ligplot+

54.

Cavity surfaces were produced

using Hollow 55, and the surface electrostatic potential was calculated and displayed using PDB2PQR 56

and APBS 57. ACE Inhibition Assays: Omapatrilat. Fresh 10 mM omapatrilat stocks were prepared in

DMSO on the day of the experiment and immediately diluted in ACE assay buffer (100 mM potassium phosphate buffer, pH 8.3, 300 mM NaCl, 10 µM ZnCl2) to working stock concentrations to limit disulphide bond formation. Omapatrilat integrity was monitored at 50 µM by HPLC to ensure that disulphide bond formation was < 5% during the course of the experiments. Fully glycosylated nACE and cACE proteins were pre-incubated with inhibitor for 15 min at 22⁰C and reactions were initiated by the addition of Cbz-Phe-His-Leu (Z-FHL, Bachem Ltd, nACE Km 600 µM; cACE Km 60 µM). Final concentrations in the 50 µl reactions were as follows: nACE 4 nM, cACE 2nM; omapatrilat 0 – 10 nM; Z-FHL 0.15/0.3/1.2 mM. Each reaction was carried out in triplicate. After incubation at 37 ͦC for 10 minutes, the reactions were stopped by the addition of 165 µl of 0.34 M NaOH containing 2mg/ml of the derivatising agent o-phthalaldyde. Derivatisation was carried out for 10 min at 22⁰C and stopped by the addition of 25 µl of 3 M HCl. Fluorescence intensities were measured at λex= 360 nm and λem= 485 nm using a fluorescence spectrophotometer (Varian Inc.) Graph-Pad Prism software



Page 18 of 52

Binding of omapatrilat to ACE (San Diego, USA) was used to fit inhibition models to the data using non-linear regression. To determine IC50 values, Log(inhibitor) vs response (Vi/Vo) (variable slope) curves were fitted to the data where Vi is initial velocity in the presence of inhibitor and Vo is initial velocity in absence of inhibitor.

To determine Kiapp values, the Morrison equation was fitted to the data:

Y=(1-

((((Et+X+(Kiapp))-(((Et+X+( Kiapp))^2)-4*Et*X)^0.5))/(2*Et))), where Y is Vi/Vo and X is inhibitor concentration. Omapatrilat Dimer. For kinetic analysis of ACE inhibition by the omapatrilat dimer, a 1 mM omapatrilat stock in ACE assay buffer was left at room temperature and dimer formation was monitored by HPLC over ~6 days until complete conversion to the dimer was observed. The sample was analysed using a Poroshell 120 EC-C 18 column on the Agilent 1260 infinity HPLC system. 10 µl of sample was injected onto the column at the indicated time points. Conditions: A: 1% ACN, 0.1% TFA; B: 95 % ACN, 0.1% TFA; 0- 100% B over 10 min; 0.5 mL/min, 214 nm. Omapatrilat dimer was pre-incubated with fully glycosylated nACE and cACE for 15 min at 22⁰C and reactions were initiated with substrate. Reactions were carried out in triplicate and final omapatrilat dimer concentrations were 0 – 20 µM. End-point Z-FHL assays were carried out as described above. For the bradykinin-like FRET peptide, MCA-RPPGFSAFK(Dnp)-OH substrate (R&D Systems), 50 µl of substrate was added to 50 µl enzyme-inhibitor and increase in fluorescent with time was measured at λex= 320 nm and λem= 405 nm. Final concentrations in the 100 µl reactions were as follows: substrate 5 µM, nACE 0.6 nM, cACE 0.3 nM). To determine IC50 values, Log(inhibitor) vs response (Vi/Vo) (variable slope) curves were fitted to the data using Graph-Pad Prism software (San Diego, USA). In silico Docking. The Schrodinger 9.7 Glide docking program (Schrödinger, Inc., New York, NY, USA) was used for the docking experiments. The following NEP X-ray crystal structures were retrieved from the Protein Data Bank (PDB) for docking method validation 1DMT; 1R1H, 1R1I, 1R1J,


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Binding of omapatrilat to ACE 1Y8J, 2QPJ and 2YB9. Water molecules and heteroatoms other than the zinc ion and the bound inhibitor were deleted from the PDB files. Protein structures were prepared for docking using Protein Preparation Wizard, Schrödinger 9.7 (Schrödinger, Inc., New York, NY, USA) to correct bond orders, add hydrogen atoms and optimise their position for optimal hydrogen bond formation, create zeroorder bonds to metals and to minimize heavy atoms to a RMSD threshold of 0.3 Å using OPLS_2005 force fields. The Schrodinger 9.7 Glide docking program (Schrödinger, Inc., New York, NY, USA) was used for the docking experiments. All NEP structures were superimposed and a docking grid box, grid size 30Å X 30Å X 30Å centred on the centroid phosphoramidon ligand (1DMT), was generated using Glide-Receptor Grid Generation with default parameters for van der Waals radius scaling. Docking was performed using the extra precision (XP) Glide docking method.

After method

validation, which involved re-docking the bound inhibitors into their original X-ray crystal structures (cross-docking of inhibitors into the other NEP structures did not typically result in the correct binding pose because of conformational difference between structures depending on the bound ligand in the co-crystal), omapatrilat with the sulfhydryl group in the deprotonated state for zinc co-ordination was docked into the full set of NEP structures and despite the variation in the conformation of active site residues between structures, a similar binding mode, with the benzyl group in the S1′ subsite, was predicted for omapatrilat within all the structures. Images of the inhibitor-enzyme complex were created using PyMOL molecular viewer software.

Supporting Information. Molecular formula strings (CSV) PDB ID Codes The atomic coordinates and structure factors for N- and C-domains ACE bound to omapatrilat



Page 20 of 52

Binding of omapatrilat to ACE complexes have been deposited in the RCSB-Protein Data Bank with the codes 6H5W and 6H5X respectively. Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION Corresponding Author *Phone: +44-1225-386238. Email: [email protected] ORCID K. Ravi Acharya: 0000-0002-3009-4058 Author Contributions G.E.C. performed all the crystallography experiments, analysed the data and wrote the manuscript. L.B.A carried out the in vitro inhibition assays, performed the in silico docking experiments and contributed to the writing of the manuscript. S.L.S. carried out all the protein expression work. E.D.S. supervised the biochemical work and edited the manuscript. K.R.A. supervised the structural study, analysed the data and edited the manuscript. All authors reviewed the manuscript. Notes The authors declare no competing financial interests. ACKNOWLEDGEMENTS We thank the scientists at stations IO3 (Proposal Number mx12342) of Diamond Light Source, Didcot, Oxfordshire (UK), for their support during X-ray diffraction data collection. This work was supported


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Binding of omapatrilat to ACE by the Medical Research Council (U.K.) Project Grant G1001685 (to K.R.A.) and the National Research Foundation (South Africa) CPRR grant 13082029517 (to E.D.S.). K.R.A. and E.D.S. also thank the University of Cape Town (South Africa) and University of Bath (UK) respectively for the Visiting Professorship. ABBREVIATIONS USED ACE, angiotensin-1 converting enzyme; AnCE, Drosophila ACE homologue; BK, bradykinin; BPP, bradykinin potentiating peptide; cACE, ACE C-domain; FII, phosphinic tripeptide; nACE, ACE Ndomain; NEP, neprilysin; RAAS, renin-angiotensin-aldosterone system; sACE, somatic ACE; tACE, testis ACE; Z-FHL, Cbz-Phe-His-Leu



Page 22 of 52

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CCP4mg molecular-graphics software. Acta Crystallogr D Biol Crystallogr 2011, 67, 386-394. 53.

Schrodinger, LLC. The PyMOL molecular graphics system, Version 2.0.

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Laskowski, R. A.; Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams

for drug discovery. J Chem Inf Model 2011, 51, 2778-2786. 55.

Ho, B. K.; Gruswitz, F. HOLLOW: generating accurate representations of channel and

interior surfaces in molecular structures. BMC Struct Biol 2008, 8, 49. 56.

Dolinsky, T. J.; Nielsen, J. E.; McCammon, J. A.; Baker, N. A. PDB2PQR: an automated

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Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Electrostatics of

nanosystems: application to microtubules and the ribosome. Proc Natl Acad Sci U S A 2001, 98, 10037-10041.



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Binding of omapatrilat to ACE FIGURE LEGENDS Figure 1. Structures of omapatrilat, phosphinic tripeptide FII, BPPb and Angiotensin II, with the PDB atom numbering for omapatrilat indicated. Figure 2. Schematic representation of omapatrilat bound to the ACE domains. A-D are overlayed with the final 2mFo-DFc (blue, contoured at 1σ level) electron density map and the mFo-DFc (green, contoured at 3σ level) electron density omit map for (A) Omapatrilat-nACE, (B) Omapatrilat-cACE (molecule 1), (C) Omapatrilat-cACE (molecule 2) and (D) Omapatrilat-cACE (molecule 3) complexes. The zinc ion is shown as a lilac sphere with the coordinating side chains shown as sticks. Alpha-helices and β-strands are shown in rose and dark cyan respectively. Panels (E) and (F) show omapatrilat molecules 2 and 3 respectively overlayed with polder maps. Figure 3. Ligplot representation of the binding site interactions of (A) omapatrilat-nACE chain A, (B) omapatrilat-nACE chain B, (C) omapatrilat-cACE (molecule 1), (D) omapatrilat-cACE (molecule 2 conformation A and molecule 3) and (E) omapatrilat-cACE (molecule 2 conformation B). H-bond/electrostatic interactions are shown in green, hydrophobic interactions in red and water molecules as cyan spheres. Residues solely involved in hydrophobic interactions are represented by red, semi-circular symbols. Figure 4. Schematic representation of (A) omapatrilat-cACE (molecule 1), (B) omapatrilat-cACE (molecule 2) and (C) omapatrilat-cACE (molecule 3) binding sites showing the interactions involved with the 3 omapatrilat molecules. The zinc ion and water molecules are shown as lilac and red spheres respectively, and the coordinating side chains shown as sticks. Alpha-helices and β-strands are shown in rose and dark cyan respectively.


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Binding of omapatrilat to ACE Figure 5. Two views showing the binding cavity (transparent green surface) of Omapatrilat-cACE complex structure indicating how the omapatrilat molecules occupy most of the non-prime cavity. The zinc atom is shown as lilac sphere, omapatrilat molecules as sticks, α-helices as rose cylinders and β-strands in dark cyan. Figure 6. Comparison of the location inside the binding cavity for ligands that interact beyond the S2 subsite. (A) Omapatrilat-cACE, (B) phosphinic tripeptide-cACE, (C) BPPb-cACE, (D) Angiotensin II-cACE (for clarity only one conformation is shown) and (E) BPPb-AnCE. Proteins are depicted as an internal surface coloured by electrostatic potential, ligands and the zinc binding residues are shown as sticks with zinc ions shown as lilac spheres. NB There was no zinc ion bound in the BPPb-cACE crystal structure. Figure 7. Two views of the overlay to show binding orientations of ligands that extend beyond the S2 subsite. Omapatrilat, BPPb, angiotensin II and phosphinic tripeptide from cACE complexes are shown as magenta, light blue, green and lime sticks respectively, and BPPb from AnCE complex is shown as orange sticks. Figure 8. Inhibition of nACE and cACE activity by omapatrilat: (A) + (B) IC50 Log plots (variable slope) and (C) + (D) Morrison plots generated using the same data (Et unconstrained). Enzyme and inhibitor were incubated at 22°C for 15 min and reactions were initiated by the addition of ZFHL substrate (nACE Km 600 µM; cACE Km 60 µM). Reactions were carried out in triplicate and assays were carried out at different substrate concentrations (nACE: final Z-FHL concentrations 0.3 mM and 1.2 mM; cACE: final Z-FHL concentrations 0.15 mM, 0.3 mM and 1.2 mM). The final enzyme concentration in each reaction was 4 nM and 2nM for nACE and cACE respectively, which corresponded well with the enzyme concentration (Et) determined by the Morrison



Page 32 of 52

Binding of omapatrilat to ACE equation. IC50 and Ki values are average values at all substrate concentrations ± standard deviation of the mean. Figure 9. (A) Omapatrilat dimerisation monitored by HPLC. The sample was analysed using a Poroshell 120 EC-C 18 column on the Agilent 1260 infinity HPLC system. 10 µl of sample was injected onto the column at the indicated time points. Conditions: A: 1% ACN, 0.1% TFA; B: 95 % ACN, 0.1% TFA; 0- 100% B over 10 min; 0.5 mL/min, 214 nm. Peaks corresponding to the monomer and dimer were observed at 7.8 and 9.1 minutes, respectively. (B) and (C) Inhibition of nACE and cACE activity by omapatrilat dimer: IC50 Log plots for inhibition of the ACE mediated turnover of (B) Z-FHL (final concentration 500 µM; nACE Km 600 µM; cACE Km 60 µM) and (C) MCA- MCA-RPPGFSAFK(Dnp)-OH (final concentration 5 µM; nACE Km 3.6; cACE Km 2.4 µM) substrates. Enzyme and inhibitor were pre-incubated at 22°C for 15 minutes and reactions were initiated by the addition of substrate. Figure 10. Predicted binding pose for omapatrilat in the active-site of NEP. Omapatrilat (cyan) was docked into NEP PDB structure 1R1H (green) using Schrödinger’s XP Glide. (A) Close up of the omapatrilat binding site with polar contacts indicated by dotted lines and the catalytic Zn2+ ion shown in purple. (B) Predicted binding pose for omapatrilat (green) in the active-site of NEP, aligned to the nACE- and cACE-omapatrilat structures, with omapatrilat shown in yellow and magenta respectively. Domain specific ACE active-site residues important for conferring domain selectivity are shown as sticks with nACE specific residues in orange and cACE specific residues in cyan.


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Binding of omapatrilat to ACE Table 1. X-ray data collection and refinement statistics cACE

nACE

Omapatrilat

Omapatrilat

[85.13-7.50]

[74.00-9.86]

(1.39-1.37)

(1.83-1.80)

Space group

P212121

P1

Cell dimensions (a,b,c)

56.6, 85.1, 134.3 Å

73.16, 76.95, 83.09 Å

angles (α,β,γ)

90.0, 90.0, 90.0°

88.83, 64.22, 75.21°

Molecules/asymmetric unit

1

2

Total / Unique reflections

6,747,076

1,930,117

136,769

141,750

Completeness (%)

[100.0] 100.0 (100.0)

[99.6] 97.4 (100.0)

Rmerge

[0.043] 0.205 (6.916)

[0.031] 0.066 (0.954)

Rpim

[0.006] 0.029 (1.043)

[0.009] 0.018 (0.264)

[13.2] 62.4 (0.8)

[69.5] 20.9 (2.9)

CC1/2

[1.000] 1.000 (0.544)

[1.000] 1.000 (0.903)

Multiplicity

[43.7] 49.3 (44.2)

[13.0] 13.6 (13.9)

Resolution (Å)

Refinement statistics Rwork/Rfree

0.145/0.175

0.164/0.204

Rmsd in bond lengths (Å)

0.007

0.005



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Binding of omapatrilat to ACE Rmsd in bond angles (°)

1.028

0.910

Ramachandran statistics (%) Favoured

98.8

98.4

Allowed

1.0

1.3

Outliers

0.2

0.3

Average B- factors (Å2) Protein

24.2

40.5

Ligand

38.2

64.5

Water

33.5

39.9

Number of atoms Protein

9723

19 711

Ligand

481

695

Water

643

754

PDB code

6H5W

6H5X


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Binding of omapatrilat to ACE Table 2. Comparison of amino acid residues involved in nACE and cACE interactions with omapatrilat. Chains A and B from the asymmetric unit of nACE, and the main omapatrilat (1) binding site for cACE are shown. Conserved interactions are on the same row. nACE (A)

nACE (B)

tACE(1)

Direct hydrogen bond interactions Gln259

Gln259

Gln281

His331

His331

His353

Ala332

Ala332

Ala354

His361

His361

His383

Lys489

Lys489

Lys511

His491

His491

His513

Tyr498

Tyr498

Tyr520

Tyr501

Tyr501

Tyr523

Lys489

Lys511

Ser333

Ser333

Ser355

Thr358

Thr358

Val380

Water-mediated interactions Lys489 Hydrophobic interactions



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Binding of omapatrilat to ACE His361

His361

His383

His365

His365

His387

Phe435

Phe435

Phe457

Phe490

Phe490

Phe512

His491

His491

His513

Tyr501

Tyr501

Tyr523

Edo12

-

-

-

-

Omapatrilat 3


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Binding of omapatrilat to ACE Table 3. Amino acid residues involved in cACE interactions with the two secondary omapatrilat (2 and 3) molecules bound. No interactions are equivalent between the two binding sites. cACE(2)

cACE(3)

Direct hydrogen bond interactions Arg124

-

Arg522

-

Edo2

-

Water-mediated interactions Glu123

Asn85

Trp220

Arg124

-

Asn136

-

Glu143

-

Ser516

-

Ser517

Hydrophobic interactions Glu123

Asn85

Met223

Val518

Glu403

Omapatrilat 1



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Binding of omapatrilat to ACE Pro519

-

Edo6

-


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Binding of omapatrilat to ACE Table 4. Non-conserved residues around the omapatrilat dimer binding site beyond the S2 subsite..

cACE

nACE

Tyr62

Ala35

Asn66

Ser39

Asn70

Asp43

Asn85

Ala58

Ile88

Ser61

Ala89

Gln62

Lys118

Ala94

Asp121

Thr97

Glu123

Gly99

Arg124

Ser100

Glu143

Ser119

Ala208

Tyr186

Tyr213

Phe191

Ser219

Tyr197

Met223

Trp201

Glu403

Arg381

Ser516

Asn494



Page 40 of 52


Figures

Figure 1


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Binding of omapatrilat to ACE Figure 2



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Table of Contents graphic 54x43mm (300 x 300 DPI)


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Molecular Basis for Multiple Omapatrilat Binding Sites within the ACE

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