Browsing by Author "Sturrock, Edward D"
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- ItemMetadata onlyAngiotensin-converting enzyme - new insights into structure, biological significance and prospects for domain-selective inhibitors.(Bentham Science Publishers, 2009) Watermeyer, Jean M; Kröger, Wendy L; Sturrock, Edward D; Ehlers, Mario R WSomatic angiotensin-converting enzyme (ACE) - well known for its role in cardiovascular pathophysiology - has an unusual, two-domain, double active-site structure. The two domains (designated N and C) are 55% identical and each contains a similar active site with overlapping but distinct substrate preferences. While both convert angiotensin I to angiotensin II in vitro, current evidence suggests the C domain site predominates in this role in vivo. The N domain site inactivates a hemoregulatory and antifibrotic peptide, AcSDKP, in vivo, although the significance of this remains unclear. However, differences in the characteristics of the two domains may result in different context-dependent activities, as is the case with other enzymes containing tandem repeats. The N domain may also have a role in modulating C domain activity, through a combination of inter-domain cooperativity and structural stabilization. Comparison of ACE with its structural homologues reveals conservation of peptidase activity and a tendency to hinge about the active-site cleft. Recent work on ACE active-site mutants containing one or more key residues replaced by their cognate residues from the other domain, synthesis of domain-selective inhibitors, and co-crystal structures of each domain with such inhibitors, has led to a better resolution of the basis for domain selectivity and should enable the design of next-generation, domain-selective inhibitors with distinct pharmacological profiles.
- ItemOpen AccessAngiotensin-converting enzyme cleavage of the Alzheimer's beta-amyloid peptide(2015) Larmuth, Kate Morgan; Sturrock, Edward DAngiotensin-1 converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (N and C) with different substrate specificities. ACE is a central component of the intrinsic brain renin angiotensin-aldosterone system (BRAAS), well renowned as the regulator of blood pressure. The BRAAS has alternate functions that extend beyond fluid and blood pressure homeostasis into areas such as neurological function. As a result, it is implicated in many neurodegenerative diseases including Alzheimer's disease (AD). ACE's specific mechanistic role in AD is not entirely clear and is somewhat controversial. However, it has been shown that ACE hydrolyses the amyloid beta (Aβ) peptide, the putative causative agent of AD. This study aimed to investigate the molecular basis of ACE hydrolysis of Aβ by determining : 1) the kinetic parameters of five different forms of human ACE with various N-terminal amyloid beta (Aβ) substrates; 2) the specific active site determinants of Aβ-domain selectivity; and 3) the high-resolution crystal structures of the N-domain of ACE in complex with Aβ(1-16), Aβ(10-16), Aβ(4-10), the FRET Aβ(4-10)Y and Aβ(35-42) peptides. For the physiological Aβ(1-16) peptide, a novel ACE cleavage site was found at His14/Gln15. Furthermore, Aβ(1-16 ) was preferentially cleaved by the truncated N-domain; however, the presence of an inactive C-domain in full-length ACE greatly reduced enzyme activity and affected domain-selectivity. Two fluorogenic substrates, designed specifically to assess ACE's mechanism of Aβ hydrolysis Aβ(4-10)Q and Aβ(4-10)Y, underwent endoproteolytic cleavage at the Asp7/Ser8 bond. The Aβ(4-10)Q peptide was a poor substrate of ACE but was N-selective, with a selectivity driven largely by interactions with the domain-specific residues of the S2 and S2' pockets. The selectivity of the S2' residues were confirmed with a similar, more physiological, fluorogenic Aβ(4-10)Y peptide. This work provides further understanding towards the substrate determinants of N-selectivity, highlighting the importance of the S2' Ser357. ACE C-domain hydrolysed Aβ(4-10)Y with modest efficiency compared to the other substrates, where hydrolysis under the same conditions did not occur. Moreover, Aβ(4-10)Y also displayed N-domain selectivity. In contrast to Aβ(1-16) and Aβ(410)Q, both sACE and the double C-domain (CC-sACE) construct showed positive domain cooperativity towards Aβ(4-10)Y. The high-resolution crystal structures of the N-domain in complex with five Aβ peptide fragments provided an overlapping, conserved, molecular mechanism of peptide binding and evidence of the enzyme's broad exoprotease activity. In addition to the kinetic and structural studies, ACE's signalling response to the N-selective Aβ(1-16) and Aβ(1-42) was investigated using immunodetection and mass spectrometry. Similar to the ACE inhibitor lisinopril, the Aβ peptides elicited ACE signalling by phosphorylation of the cytoplasmic Ser1270 residue and JNK activation. The signalling response of ACE was coupled to increased ACE activity an d expression on treatment with Aβ(1-42). These studies allowed us to rationalise the increased ACE activity and expression found in AD, may arise through direct interactions with Aβ. This work provides a kinetic, structural and mechanistic understanding of the selective cleavage of Aβ by the N and C catalytic sites of ACE. Due to the broad substrate specificity of the two domains of ACE, and the overarching N- selectivity of Aβ hydrolysis, these findings provide rationale for further in vivo pharmacological studies on the mechanism of action C- domain-selective inhibitors, in the context of AD.
- ItemOpen AccessCharacterisation of the ectodomain shedding of angiotensin-converting enzyme(2003) Woodman, Zenda; Sturrock, Edward DBibliography: leaves 240-266.
- ItemOpen AccessCharacterisation of the structural motifs Involved in the cleavage and secretion of human angiotensin-converting enzyme(2014) Conrad, Nailah; Sturrock, Edward D; Schwager, Sylva L UAngiotensin converting enzyme is an ectoprotein prone to regulated proteolytic solubilisation by an as yet unknown protease or sheddase. Proteolytic cleavage of membrane proteins is an essential cellular process that controls their expression and function, and modulates cellular and physiological processes. Testis ACE (tACE) is shed at a higher rate than somatic ACE and it has been proposed that regions in its ectodomain direct its shedding. Discrete secondary structures on the surface of the distal ectodomain of tACE were replaced with their N-domain counterparts to determine their role in the ectodomain shedding of ACE. None of the regions investigated proved to be an absolute requirement for shedding, but the mutant ACE proteins were subject to variations in shedding compared to wild-type tACE. To investigate the role of the proximal ectodomain in shedding the residues H610-L614 were mutated to alanines, causing a decrease in shedding. An extension of this mutation on the N-terminal side to seven alanines resulted in a reduction in ACE activity and, more importantly, it affected the processing of the protein to the membrane, resulting in expression of an underglycosylated form of ACE. When E608-H614 was mutated to the homologous region of the N-domain, processing was normal and shedding only marginally reduced. These data suggest that this region is more crucial for the processing of ACE than is for regulating shedding. Construction of a P628L mutation in tACE showed an increase in shedding. Furthermore, MALDI analysis of a tryptic digest established that the putative glycosylation site N620WT became glycosylated. Further mutagenesis of the P628L mutant to remove the newly formed glycosylation site, resulted in an even greater increase in shedding. Soluble fluorogenic peptides mimicking the ACE stalk were used in a cell-based assay to characterise the contribution of the stalk to ACE shedding. Hydrolysis of the wild-type peptide Abz-NSARSEGPQ-EDDnp was not responsive to phorbol ester or the hydroxamate inhibitor (TAPI), however, it was inhibited by EDTA. The aminopeptidase inhibitor bestatin did not inhibit cleavage or alter the cleavage site. Therefore the protease involved in the cleavage of the ACE stalk peptides is likely different to the sheddase responsible for ACE shedding. Substitution of the P1 and P1' sites of the peptides did not significantly influence the rate of cleavage. All the peptides were cleaved at the E-G bond, which is C-terminal to the physiological R-S cleavage site. Removal of the fluorogenic capping groups resulted in no cleavage of the peptides and lengthening of the peptide did not result in cleavage. This confirms the need for the ACE sheddase and its substrate to be anchored in the membrane and suggests the use of soluble peptide substrates in a cell assay has limited application for investigating the ectodomain shedding of ACE.
- ItemRestrictedCharacterization of domain-selective inhibitor binding in angiotensin-converting enzyme using a novel derivative of lisinopril(Portland Press, 2010) Watermeyer, Jean M; Kröger, Wendy L; O'Neill, Hester G; Sewell, Trevor B; Sturrock, Edward DHuman ACE (angiotensin-converting enzyme) (EC 3.4.15.1) is an important drug target because of its role in the regulation of blood pressure via the renin–angiotensin–aldosterone system. Somatic ACE comprises two homologous domains, the differing substrate preferences of which present a new avenue for domainselective inhibitor design. We have co-crystallized lisW-S, a Cdomain-selective derivative of the drug lisinopril, with human testis ACE and determined a structure using X-ray crystallography to a resolution of 2.30 Å (1 Å = 0.1 nm). In this structure, lisW-S is seen to have a similar binding mode to its parent compound lisinopril, but the P2 tryptophan moiety takes a different conformation to that seen in other inhibitors having a tryptophan residue in this position. We have examined further the domain-specific interactions of this inhibitor by mutating Cdomain-specific active-site residues to their N domain equivalents, then assessing the effect of the mutation on inhibition by lisWS using a fluorescence-based assay. Kinetics analysis shows a 258-fold domain-selectivity that is largely due to the co-operative effect of C-domain-specific residues in the S2 subsite. The high affinity and selectivity of this inhibitor make it a good lead candidate for cardiovascular drug development.
- ItemOpen AccessCharacterization of the physiochemical and biochemical properties of the urinary protein bikunin in South African black and white subjects with respect to calcium oxalate kidney stone formation(2006) Mabizela, Nontobeko; Rodgers, Allen; Sturrock, Edward DApproximately 12 % of males in the Western world are likely to suffer at least one kidney stone in their life. The incidence of kidney stone disease in South Africa is similar in white males; however stone occurrence in the South African black population is extremely rare. The difference between the incidences of kidney stones in the black and white populations in South Africa is unexplained. In general, the role of several urinary proteins in the pathogenesis of this disease has been established. Bikunin is an example. The primary aim of this study was to isolate bikunin from the urine of healthy black and white male subjects and to investigate whether the protein from the black group is a more powerful inhibitor of CaOx crystallization than that from the stone prone white group.
- ItemOpen AccessCrystal structure of protoporphyrinogen oxidase from Myxococcus xanthus and its complex with the inhibitor acifluorfen(American Society for Biochemistry and Molecular Biology, 2006) Corradi, Hazel R; Corrigall, Anne V; Boix, Ester; Mohan, C Gopi; Sturrock, Edward D; Meissner, Peter N; Acharya, K RaviProtoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.
- ItemRestrictedCrystal structure of the N domain of human somatic angiotensin I-converting enzyme provides a structural basis for domain-specific inhibitor design(Elsevier, 2006) Corradi, Hazel R; Schwager, Sylva L U; Nchinda, Aloysius T; Sturrock, Edward D; Acharya, K RaviHuman somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of “domain-specific” second-generation ACE inhibitors.
- ItemRestrictedDeletion of the cytoplasmic domain increases basal shedding of angiotensin-converting enzyme(Elsevier, 2004) Chubb, Anthony J; Schwager, Sylva L U; van der Merwe, Elizabeth; Ehlers, Mario R W; Sturrock, Edward DEctodomain shedding generates soluble isoforms of cell-surface proteins, including angiotensin-converting enzyme (ACE). Increasing evidence suggests that the juxtamembrane stalk of ACE, where proteolytic cleavage-release occurs, is not the major site of sheddase recognition. The role of the cytoplasmic domain has not been completely defined. We deleted the cytoplasmic domain of human testis ACE and found that this truncation mutant (ACE-ΔCYT) was shed constitutively from the surface of transfected CHO-K1 cells. Phorbol ester treatment produced only a slight increase in shedding of ACE-ΔCYT, unlike the marked stimulation seen with wild-type ACE. However, for both wild-type ACE and ACE-ΔCYT, shedding was inhibited by the peptide hydroxamate TAPI and the major cleavage site was identical, indicating the involvement of similar or identical sheddases. Cytochalasin D markedly increased the basal shedding of wild-type ACE but had little effect on the shedding of ACE-ΔCYT. These data suggest that the cytoplasmic domain of ACE interacts with the actin cytoskeleton and that this interaction is a negative regulator of ectodomain shedding.
- ItemOpen AccessDevelopment of N-domain selective Angiotensin-I Converting Enzyme (ACE) inhibitors using Computer Aided Drug Discovery (CADD)(2017) Fienberg, Stephen; Chibale, Kelly; Sturrock, Edward DAngiotensin-I (Ang-I) converting enzyme (ACE) is a zinc metalloprotease that plays a vital role in the Renin Angiotensin Aldosterone System (RAAS) and is a key antihypertensive drug target. In addition to Ang-I, ACE cleaves many other physiological substrates, thus extending its function beyond the regulation of blood pressure. Somatic ACE (sACE) consists of two structurally homologous yet distinct catalytic sites termed the N- and C-domains. The two catalytic domains of ACE have distinct substrate affinities and play different regulatory roles. The antifibrotic tetrapeptide Ac-SDKP is hydrolysed solely by the N-domain and thus is a potential target for interactions between the ligand and unique residues within the active site of the N- and C-domains, which need to be exploited to effect either N- or Cdomain selectivity. N-domain selective ACE inhibition has been demonstrated with peptides while crystallographic studies have shown that the N-domain to C-domain substitution of Arg381 with Glu403 within the S₂ subsite is integral to N-domain selective ACE inhibition. Three computer aided drug discovery (CADD) approaches were pursued to design N-domain selective drug-like ACE inhibitors (ACEi) with an acidic P₂ functional group that would confer N-domain selectivity via an interaction with Arg381 in the S₂ subsite. Firstly, a fragment-based screening protocol was performed by running a set of chemical filters on 16 000 drug fragment compounds (MW < 350), all of which contained a metal chelating group. 60 Ligands capable of binding to both the zinc metal and Arg381 in the S₂ subsite of the N-domain were tested for ACE inhibition against the two domains of ACE. Two of the fragments identified in this screen showed a modest ACE inhibition (IC₅₀ +/- 200 μM), but no domain selectivity. Secondly, a combinatorial library was created to explore the P₂ structure activity relationship (SAR) of a scaffold based on the core structure of the clinical ACEi, Enalaprilat. Over 400 variants were created to generate a combinatorial library. These compounds were docked against the two domains of ACE and a synthetic scheme was developed to synthesise compounds from this library. Using this scheme, one Enalaprilat analogue, SF07 was synthesised as a mixture of diastereomers. SF07 exhibited low micromolar N-domain inhibition with no C-domain inhibition observable below 100 μM. For the third approach, 25 000 compounds containing biological data pertaining to ACE were extracted from the GVK BIO GOSTAR database. These compounds were filtered for drug-like properties and manually inspected for promising P₂ functionality. The N-domain selectivity of these compounds was then assessed via molecular docking against the two domains of ACE. This screen identified a series of diprolyl compounds with varied groups in the P₂ position. These compounds were subsequently synthesised and tested in vitro for inhibition against both domains. The most N-domain selective compound from the series proved to be SG6, a diprolyl compound with an Asp group in the P₂ position. SG6 displayed potent inhibition (Kᵢ = 12 nM) and was 83-fold more selective towards the N-domain than the C-domain. This study has demonstrated the N-domain selective inhibition of ACE by drug-like peptidomimetics. Two promising leads on drug-like N-domain selective ACE inhibitors, SG6 and SF07, have been identified. These two compounds have the potential to pave the way for clinical N-domain selective ACEis and a novel treatment for cardiac and pulmonary fibrosis.
- ItemOpen AccessGenetic and biochemical analysis of ACE inhibitor-induced angioedema in black and coloured South Africans(2011) Moholisa, Retsilisitsoe Raymond; Sturrock, Edward DAngiotensin converting enzyme inhibitors (ACEi) are routinely used as first line treatment for hypertensive patients because of their protective effects against heart and kidney disease. Despite their clinical benefits, ACEi used is associated with adverse side effects such as life threatening angioedema (ACEi-AE) and persistent dry cough (ACEi-cough)
- ItemOpen AccessIdentification and characterisation of proteases in Mycobacterium tuberculosis(1999) Dave, Joel Alex; Ehlers, Mario R W; Sturrock, Edward DVirulence determinants of M. tuberculosis remain largely unknown. Of key interest has been the ability of the bacterium to survive intracellularly within its host cell, the macrophage, and its ability to cause extensive tissue necrosis. Exported proteases are commonly associated with virulence in bacterial pathogens, yet their role in Mycobacterium tuberculosis has virtually not been studied. Preliminary experiments showed M. tuberculosis culture filtrates contained a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca²⁺, features typical of some serine proteases, notably subtilisins, and possibly metalloproteases. Purification attempts were unsuccessful. A family of five genes that encode putative, secreted, serine proteases has recently been described in M. tuberculosis. These proteases share 36-47% sequence identity and are all encoded with putative signal peptides, suggesting that they are translocated across the cytoplasmic membrane. One member, mycP1, was selected for further study. The gene product, mycosin-1, was 30-35% identical to bacterial subtilisin-like serine proteases and contained the classic catalytic triad and oxyanion hole. Mycosin-1 also contained a typical signal peptide, a likely propeptide, and a Cterminal hydrophobic sequence with a high transmembrane potential. Topology analyses predicted mycosin-1 to be a type I ectoprotein. Consistent with this, expression of mycosin-1 in M. tuberculosis and in Mycobacterium smegmatis transformed with mycP1 (M. smegmatis-P1) was limited strictly to the cell envelope, as seen by Western blotting, and immunogold electron microscopy. Only full-length, 50-kDa mycosin-1 was observed by Western blotting in broth-grown M. tuberculosis and M. smegmatis-P1 lysates, whereas a 40-kDa species was detected in 6-week M. tuberculosis culture filtrates. A similar 40-kDa immunoreactive band was also observed in lysates of macrophages infected with M. tuberculosis, consistent with robust transcription of the mycP 1 gene during growth in macrophages. Since putative mature mycosin-1 has a molecular weight of 38.6 kDa, the 40-kDa protein may represent activated mycosin-1 after propeptide cleavage. In conclusion, mycosin-1 is an exported, cell envelopeassociated subtilisin homolog that is expressed during growth of M. tuberculosis in vitro and in macrophages.
- ItemOpen AccessThe importance of N-linked glycosylation on the N-domain of angiotensin-I converting enzyme(2011) Anthony, Colin Scott; Sturrock, Edward DAngiotensin-I converting enzyme (ACE) is an important drug target in the treatment of heart disease due to its role in the regulation of blood pressure. ACE contains two domains, the N- and C-domains, both of which are catalytically active and heavily glycosylated. Glycosylation is one of the most important forms of post-translational modification, having a wide range of functions including protein folding, modulation of the immune response, and providing targeting signals. Glycosylation is required for the expression of active ACE and structural studies of ACE have been fraught with severe difficulties because of surface N-glycosylation of the protein. This problem has been addressed to a large extent with respect to the C-domain, where the role of glycosylation has been extensively characterised and a minimally glycosylated form was able to crystallise reproducibly. As yet, little is known about the degree and importance of N-linked glycosylation on the N-domain. The generation of minimally glycosylated N-domain, however, requires a greater understanding of the relative importance of the individual N-linked glycosylation sites.
- ItemOpen AccessInvestigating domain-selective angiotensin converting enzyme inhibition and oxidative inactivation(2018) Lubbe, Lizelle; Sturrock, Edward D; Sewell B TrevorAngiotensin converting enzyme (ACE) is a zinc metalloprotease comprised of two highly homologous, catalytically active domains (90% active site identity and 60% sequence similarity). The C-domain is responsible for blood pressure regulation via angiotensin I cleavage while the N-domain inactivates an antifibrotic peptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP). Since selective N-domain inhibition will result in AcSDKP accumulation, it shows promise for the treatment of fibrosis without affecting blood pressure. Low bioavailability, however, precludes the use of currently available N-selective ACE inhibitors in a clinical setting. Inhibition of ACE by a phosphinic, peptidomimetic compound, 33RE, was characterized using a continuous assay with quenched fluorogenic substrate. The N-domain displayed nanomolar (Ki = 11.21±0.74nM) and the C-domain micromolar (Ki = 11 278±410nM) inhibition, thus 1000-fold selectivity. Residues predicted to contribute to selectivity based on the N-domain-33RE co-crystal structure were subsequently mutated to their C-domain counterparts. S2 subsite mutation with resulting loss of a hydrogen bond drastically decreased 33RE affinity (Ki = 2794±156nM), yet did not entirely account for the selectivity. Additional substitution of all unique S2’ residues, however, completely abolished N-selectivity (Ki = 10 009±157nM). Interestingly, these residues do not directly bind 33RE. All mutants were therefore subjected to molecular dynamics (MD) simulations in the presence and absence of 33RE in addition to co-crystallization of 33RE with the N-domain mutant having all S2 and S2’ residues mutated. Trajectory analyses highlighted the S2’ residues’ importance in formation of a favourable interface between the ACE subdomains and thus a closed, ligand-bound complex. This was supported by X-ray crystallography and provides a molecular basis for the inter-subsite synergism governing 33RE’s 1000-fold N-domain selectivity. Enzyme kinetics were also used to study the concentration-dependent competitive inhibition and time-dependent irreversible oxidative inactivation of ACE catalysed by the Cu-Gly-GlyHis-lisinopril (CuGGHLis) metallodrug. Although both domains displayed nanomolar affinity for metallodrug binding (N-domain Ki = 44.94±1.84nM and C-domain Ki = 15.57±1.30nM), rapid and complete CuGGHLis-mediated inactivation occurred exclusively in the N-domain upon incubation with ascorbate and H2O2 redox co-reactants (k2 = 59 710 M-1 min-1 ). Michaelis-Menten characterization of the residual activity after partial N-domain inactivation revealed a decreased rate for hydrolysis of a non-domain selective substrate. This suggests that although CuGGHLis binds with similar affinity to both domains, the metal-chelate is optimally orientated in the N- but not the C-domain to catalyze oxidation of residues involved in substrate hydrolysis. The C-domain, in contrast, showed increased susceptibility to oxidative inactivation by diffuse radicals. This is of physiological significance as C-domain inactivation in normotensive individuals could result in accumulation of pro-inflammatory peptides. Since the N-domain is more heavily glycosylated, the potential role of unique glycans in diffuse radical shielding was studied using glycoprotein MD simulations. Unique C-domain solvent tunnels were identified that could increase diffuse radical access and, additionally, the mechanism whereby glycosylation contributes to ACE thermal stability was described for each site. This has implications for future ACE crystallography studies and the design of ACE-modulating agents with potential anti-inflammatory activity. This study demonstrated the utility of combining in vitro and in silico approaches to reveal how subtle amino acid or glycosylation site differences between the highly homologous domains control dynamic behaviour. It furthermore elucidated how two inhibitors with different mechanisms of action selectively target the N-domain active site by exploiting these differences and provided valuable insight for future anti-fibrotic ACE inhibitor design.
- ItemOpen AccessInvestigating the role of the Renin Angiotensin System in cancer(2017) Dunn, Cherise; Leaner, Virna D; Sturrock, Edward DIt has recently been discovered that cancer shares a link with metabolic diseases, including that of cardiovascular disease, diabetes, amongst others, where common sets of genes show similar gene expression. There is thus interest to investigate current therapies for metabolic diseases as possible anti-cancer agents. The renin-angiotensin system (RAS) regulates blood pressure and cardiovascular homeostasis through Angiotensin Converting Enzyme-1 (ACE-1) and its homolog ACE-2. RAS has also been implicated in the progression of various cancers due to the increased action of the vasoconstrictor, angiotensin II, which requires ACE-1 and specifically the Angiotensin Type 1 Receptor (AT1R) for its function. In this study, we investigated the potential association of the endogenous ACE-1 and ACE-2 enzymes in cervical cancer. Our results showed that ACE-1 and AT1R protein expression was elevated in cervical cancer cell lines compared to normal cells and that this correlated with elevated ACE-1 enzyme activity in cancer cells. Treatment with the ACE-1 inhibitors, Captopril and Lisinopril, reduced this activity. We showed that ACE-1 axis stimulation in cancer cells results in increased calcium signaling preferentially via the AT1R and this associates with cancer cell proliferation. Candesartan, an AT1R blocker significantly reduced these effects. ACE-2 expression and activity were decreased in cancer compared to normal cells. Our data shows that ACE2 activators, the natural peptide angiotensin 1-7 and small molecule Diminazene aceturate (DIZE) have anticancer effects with DIZE inducing a G2/M arrest in cancer cells. We also investigated associations between drugs targeting RAS and current chemotherapeutic agents, Cisplatin (CDDP) and Doxorubicin (DOX). Our data shows that ACE-1 axis inhibitors have an antagonistic effect on CDDP, while the ACE-2 activator DIZE associates synergistically with DOX. Taken together, these results suggest that elevated ACE- 1 expression associates with cervical cancer and that the inhibitors of ACE-1 function or activators of ACE-2 function have potential as anticancer therapies as single agents or in combination treatments with current chemotherapeutics.
- ItemOpen AccessMechanisms of chloride modulated activity in the C-domain of angiotensin-converting enzyme(2012) Yates, Christopher John; Sturrock, Edward DThe somatic isoform of angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, primarily cleaves the hypertension-associated angiotensin-I (AngI) and bradykinin peptides, as well as a number of other physiologically relevant peptides in vitro. sACE consists of two homologous and catalytically active N- and C- domains which display marked differences in substrate specificities and chloride activation. To investigate these potential mechanisms, a series of single amino acid substitution mutants (based on analysis of aligned C- and N-domain 3D structures) were generated in a soluble, minimally glycosylated C-domain construct. Evaluation of these constructs was done using AngI and the short synthetic substrates hippuryl-L-histidyl-Lleucine (HHL) and Z-phenylalanyl-L-histidyl-L-leucine (Z-FHL) under differing chloride concentrations. An isothermal titration calorimetry-based assay was developed to determine the effect of chloride concentration on enzyme thermodynamic and kinetic parameters. Chloride binding in the chloride 1 pocket of tACE was found to affect positioning of K511 and potentially alter the conformation of the active site. This would alter C-terminal substrate interactions, which were suggested to affect chloride 2 pocket ion affinity by coordinating Y520 and affect peptide bond rotation and hence substrate interactions. The analysis of the chloride 2 pocket R522Q and R522K mutations revealed a key R522-Y523 Pi-cation interaction that is stabilized via chloride coordination of R522. Substrate interactions in the S2 sub-site were shown to affect positioning of this complex as well as chloride affinity in the chloride 2 pocket. The E403-K118 salt bridge in tACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket, an interaction which is destabilized via substrate interactions within the S2 pocket which results in tighter chloride binding. This work showed that substrate composition to the C-terminal side of the scissile bond, as well as interactions of larger substrates in the S2 sub-site, moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate selective nature of chloride dependence in ACE and how this varies between the N- and C- domains.
- ItemOpen AccessA Molecular Basis for the C-Domain Selectivity of Angiotensin-Converting Enzyme(2009) Kroger, W; Sturrock, Edward DAngiotensin-Converting Enzyme (ACE) plays an essential role in blood pressure regulationand ACE inhibitors are widely used to treat cardiovascular disease. Two isoforms exist,somatic ACE (sACE) consisting of two homologous domains, N- and C-domain, and testisACE (tACE), corresponding to the C-domain of sACE. Despite a high degree of sequenceidentity, these two domains display marked differences in substrate and inhibitor specificity.Furthermore, the C-domain of ACE has been implicated to play a dominant role in bloodpressure control. It has therefore been suggested that development of ACE inhibitortreatments that selectively block the C-domain will result in decreased side-effects comparedto current therapies. Analysis of three-dimensional structures of tACE in complex withdomain-specific inhibitors has enabled the identification of key active-site residues potentiallyplaying a role in domain selectivity. To investigate the contribution of such residues, a seriesof C-domain mutants was generated containing single and multiple N-domain active-sitesubstitutions. These constructs were used to characterise specific interactions using domainselectiveinhibitors and fluorogenic peptides. Mutants tested with the fluorogenic peptidesdisplayed minimal, if any, acquisition of N-domain-like catalytic properties. Of the singlemutations, S2 (F391Y, tACE numbering) and S1 (V518T) pocket substitutions caused thelargest decreases in affinity for the C-selective phosphinic inhibitor RXPA380 (34-fold) andketo-ACE derivatives (14-26 fold), respectively. The V379S mutation caused an unexpectedincrease in affinity (2-10 fold) for C-selective inhibitors containing a P2’ Trp that could beexplained by the formation of a water-mediated hydrogen bond interaction resulting fromrearrangement of inhibitor and protein side-chains within the S2’ pocket. Multiple mutantscontaining an N-domain-like S2’ pocket combined with the S2 F391Y substitution (S2’F)caused the most notable shift in Ki from that of tACE for the highly selective phosphinicinhibitors, RXPA380 (Ki’s tACE = 69 nM; S2’F = 5300 nM) and N-specific RXP407 (Ki’stACE = 2800 nM; S2’F Ki = 16.1 nM). This work identifies key residues contributing to thedomain selectivity of ACE, and highlights the complex combination of effects involved in thisphenomenon. Furthermore, it provides useful insight for the further design of domainselectiveinhibitors.
- ItemOpen AccessA novel angiotensin I-converting enzyme mutation (S333W) impairs N-domain enzymatic cleavage of the anti-fibrotic peptide, AcSDKP(Public Library of Science, 2014) Danilov, Sergei M; Wade, Michael S; Schwager, Sylva L; Douglas, Ross G; Nesterovitch, Andrew B; Popova, Isolda A; Hogarth, Kyle D; Bhardwaj, Nakul; Schwartz, David E; Sturrock, Edward D; Garcia, Joe G NBACKGROUND: Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. RESULTS: We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S 1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. Conclusions and Significance A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis.
- ItemRestrictedProbing the basis of domain-dependent inhibition using novel ketone inhibitors of angiotensin-converting enzyme(ACS Publications, 2008) Watermeyer, Jean M; Kro¨ger, Wendy L; O’Neill, Hester G; Trevor Sewell, B; Sturrock, Edward DHuman angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5S)-5-[(Nbenzoyl)amino]-4-oxo-6-phenylhexanoyl-L-tryptophan (kAW) and (5S)-5-[(N-benzoyl)amino]-4-oxo-6- phenylhexanoyl-L-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4-20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domainselective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties.
- ItemRestrictedProbing the basis of domain-dependent inhibition using novel ketone inhibitors of angiotensin-converting enzyme(American Chemical Society, 2008) Watermeyer, Jean M; Kroger, Wendy L; O'Neill, Hester G; Sewell, B Trevor; Sturrock, Edward DHuman angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5S)-5-[(N-benzoyl)amino]-4-oxo-6-phenylhexanoyl-l-tryptophan (kAW) and (5S)-5-[(N-benzoyl)amino]-4-oxo-6-phenylhexanoyl-l-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4−20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domain-selective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties.