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      Fragment-based design for the development of N-domain-selective angiotensin-1-converting enzyme inhibitors

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          Abstract

          ACE (angiotensin-1-converting enzyme) is a zinc metallopeptidase that plays a prominent role in blood pressure regulation and electrolyte homeostasis. ACE consists of two homologous domains that despite similarities of sequence and topology display differences in substrate processing and inhibitor binding. The design of inhibitors that selectively inhibit the N-domain (N-selective) could be useful in treating conditions of tissue injury and fibrosis due to build-up of N-domain-specific substrate Ac-SDKP ( N-acetyl-Ser–Asp–Lys–Pro). Using a receptor-based SHOP (scaffold hopping) approach with N-selective inhibitor RXP407, a shortlist of scaffolds that consisted of modified RXP407 backbones with novel chemotypes was generated. These scaffolds were selected on the basis of enhanced predicted interaction energies with N-domain residues that differed from their C-domain counterparts. One scaffold was synthesized and inhibitory binding tested using a fluorogenic ACE assay. A molecule incorporating a tetrazole moiety in the P 2 position (compound 33RE) displayed potent inhibition ( K i=11.21±0.74 nM) and was 927-fold more selective for the N-domain than the C-domain. A crystal structure of compound 33RE in complex with the N-domain revealed its mode of binding through aromatic stacking with His 388 and a direct hydrogen bond with the hydroxy group of the N-domain specific Tyr 369. This work further elucidates the molecular basis for N-domainselective inhibition and assists in the design of novel N-selective ACE inhibitors that could be employed in treatment of fibrosis disorders.

          Abstract

          The present paper reports the development of an N-domain selective ACE inhibitor and the molecular basis for the interaction of key active site residues with the ligand.

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          Most cited references43

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          Satisfying hydrogen bonding potential in proteins.

          We have analysed the frequency with which potential hydrogen bond donors and acceptors are satisfied in protein molecules. There are a small percentage of nitrogen or oxygen atoms that do not form hydrogen bonds with either solvent or protein atoms, when standard criteria are used. For high resolution structures 9.5% and 5.1% of buried main-chain nitrogen and oxygen atoms, respectively, fail to hydrogen bond under our standard criteria, representing 5.8% and 2.1% of all main-chain nitrogen and oxygen atoms. We find that as the resolution of the data improves, the percentages fall. If the hydrogen bond criteria are relaxed many of these unsatisfied atoms form weak hydrogen bonds. However, there remain some buried atoms (1.3% NH and 1.8% CO) that fail to hydrogen bond without any immediately obvious compensating interactions.
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            The determination of enzyme inhibitor constants.

            M DIXON (1953)
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              Plasma bradykinin in angio-oedema.

              Bradykinin is believed to be the main mediator of symptoms in hereditary (HA) and acquired (AA) angio-oedema due to C1 esterase inhibitor deficiency, as well as in angio-oedema that complicates treatment with inhibitors of angiotensin-converting enzyme (ACE). Difficulties in the measurement of kinin concentrations, however, have so far precluded the demonstration of an incontrovertible change in plasma bradykinin concentrations in these disorders. By developing a reliable assay we have been able to follow bradykinin concentrations during attacks and during remission in HA and in AA, and also in a patient treated with an ACE-inhibitor. Liquid-phase extraction, high-performance liquid chromatography, and RIA were used for specific measurement of plasma bradykinin concentrations in 22 patients with HA and in 22 healthy volunteers of similar age and sex distribution. Four patients with AA and one hypertensive patient treated with the ACE inhibitor captopril were also studied. Among the healthy volunteers plasma bradykinin concentration was inversely proportional to age. The geometric mean plasma bradykinin concentration in the healthy volunteers was 2.2 fmol/mL (SD 2.2), compared with 3.9 fmol/mL (3.7) among patients with HA during remission (p=0.095). Bradykinin was also high in the patients with AA (10.4 fmol/mL [1.6]). During acute attacks of oedema, in both HA and AA, plasma bradykinin rose to two to 12 times the upper limit of normal. Infusion of C1-esterase inhibitor (the deficient factor in both HA and AA) immediately lowered bradykinin concentrations. In the patient receiving the ACE-inhibitor captopril, bradykinin concentration was very high at 47 fmol/mL during an acute attack of angio-oedema, but normal at 3.2 fmol/mL in remission after withdrawal of the drug. A sensitive method for measurement of plasma bradykinin provided the means to show that concentrations of this peptide decrease with age in healthy people. Although the differences between patients in remission and healthy controls did not reach statistical significance, there were substantial rises in bradykinin during acute attacks of hereditary, acquired, or captopril-induced angio-oedema.
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                Author and article information

                Journal
                Clin Sci (Lond)
                Clin. Sci
                cls
                CS
                Clinical Science (London, England : 1979)
                Portland Press Ltd.
                0143-5221
                1470-8736
                9 September 2013
                22 October 2013
                1 February 2014
                : 126
                : Pt 4
                : 305-313
                Affiliations
                *Institute of Infectious Disease and Molecular Medicine, and Division of Medical Biochemistry, University of Cape Town, Observatory, Cape Town 7935, South Africa
                †Department of Chemistry and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, Cape Town 7701, South Africa
                ‡Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, U.K.
                §Lead Molecular Design, Sant Cugat del Vallès and Pompeu Fabra University, Barcelona, Spain
                Author notes

                1 These authors contributed equally to this work.

                Correspondence: Professor Edward D. Sturrock (email edward.sturrock@ 123456uct.ac.za ), Professor Kelly Chibale (email kelly.chibale@ 123456uct.ac.za ) or Professor K. Ravi Acharya (email K.R.Acharya@ 123456bath.ac.uk ).
                Article
                CS20130403
                10.1042/CS20130403
                3875237
                24015848
                d465c2be-d667-4715-887e-4513aedae773
                © 2014 The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Licence (CC-BY)(http://creativecommons.org/licenses/by/3.0/) which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 19 July 2013
                : 20 August 2013
                : 9 September 2013
                Page count
                Figures: 7, Tables: 5, References: 52, Pages: 9
                Categories
                Original Paper
                S8
                S2

                Medicine
                angiotensin-1-converting enzyme (ace),crystal structure,rxp407,shop, scaffold hopping,in silico screening,inhibitor design,kinetics,ace, angiotensin-1-converting enzyme,ac-sdkp, n-acetyl-ser–asp–lys–pro,n-selective, n-domain selective

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