Substrate-Based Design of the First Class of Angiotensin-Converting Enzyme-Related Carboxypeptidase (ACE2) Inhibitors

Dales, Natalie A.; Gould, Alexandra E.; Brown, James A.; Calderwood, Emily F.; Guan, Bing; Minor, Charles A; Gavin, James M.; Hales, Paul; Kaushik, Virendar K.; Stewart, Michael; Tummino, Peter J.; Vickers, Chad; Ocain, Timothy D.; Patane, Michael A.

doi:10.1021/ja0277226

Cited by 176 publications

(191 citation statements)

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“…Refinement statistics for the inhibitor-bound ACE2 structure are shown in Table II. The bound compound MLN-4760 potently inhibits human ACE2 (IC 50 ϭ 0.44 nM), but weakly inhibits tACE (IC 50 Ͼ 100 M) and carboxypeptidase A (IC 50 ϭ 27 M) (15). The structure of the bound inhibitor is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Similarly, diffraction-quality ACE2 crystals were also grown in the presence of an ACE2 inhibitor, MLN-4760 (ML00106791; (S,S)-2-{1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol-4-yl]-ethylamino}-4-methylpentanoic acid). Compound MLN-4760 corresponds to compound 16 of Dales et al (15). Crystallization trials used 2 l of reservoir solution plus 2 l of ACE2 at 5.9 mg/ml containing 0.1 mM inhibitor.…”

Section: Methodsmentioning

confidence: 99%

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ACE2 X-Ray Structures Reveal a Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis

Towler

Staker²,

Prasad³

et al. 2004

Journal of Biological Chemistry

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669

841

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The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH ؉ E zincbinding consensus sequence. ACE2 has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). To gain further insights into this enzyme, the first crystal structures of the native and inhibitor-bound forms of the ACE2 extracellular domains were solved to 2.2-and 3.0-Å resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other (ϳ16°) that brings important residues into position for catalysis. The potent inhibitor MLN-4760 ((S,S)-2-{1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino}-4-methylpentanoic acid) makes key binding interactions within the active site and offers insights regarding the action of residues involved in catalysis and substrate specificity. A few active site residue substitutions in ACE2 relative to ACE appear to eliminate the S 2 substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2.The angiotensin-converting enzyme (ACE) 1 -related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH ϩ E zinc-binding consensus sequence (1, 2). The catalytic domain of ACE2 is 42% identical to that of its closest homolog, somatic angiotensinconverting enzyme (sACE; EC 3.4.15.1), a peptidyl dipeptidase that plays an important role in the renin angiotensin system for blood pressure homeostasis. The loss of ACE2 in knockout mice has no effect on blood pressure, but reveals ACE2 as an essential regulator of heart function (3). In a recent discovery, ACE2 was identified as a functional receptor for the coronavirus that is linked to the severe acute respiratory syndrome (SARS) (4, 5).The physiological differences observed in the phenotypes of ACE (6, 7) and/or ACE2 (3) knockout mice presumably reflect the significant differences in substrate specificity and reactivity between these enzymes. Many substrates for ACE2 were identified by screening biologically active peptides (8). In all cases, only carboxypeptidase activity was found. Of the seven best in vitro peptide substrates identified (k cat /K m Ͼ 10 5 M Ϫ1 s Ϫ1 ), proline and leucine are the preferred P 1 residues, with a partiality for hydrophobic residues in the P 1 Ј position, although basic residues at P 1 Ј are also cleaved (peptide-binding subsites in proteins are as previously defined (9)). Some of the best in vitro peptide substrates are apelin-13, des-Arg 9 -bradykinin, angiotensin II, and dynorphin A-(1-13). The longest peptide substrate identified is a 36-residue peptide, apelin-36 (8). An examination of the ACE2 and ACE literature may be found in recently published reviews (10 -12).We report here the first crystal ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

ACE2 X-Ray Structures Reveal a Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis

Towler

Staker²,

Prasad³

et al. 2004

Journal of Biological Chemistry

Self Cite

669

841

View full text Add to dashboard Cite

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“…9 This theory would explain the symbiosis between B 0 AT1 and ACE2 in the small intestine, since the carboxy amino acids released by ACE2 cleavage are mostly neutral amino acids, but also cationic. 87 A catalytic dead mutant of ACE2 however still had the same effect on B 0 AT1 function. The slc6a19 knockout model (B 0 AT1) has normal expression of ACE2 and Tmem27 suggesting that transporter ablation has no impact on RAS system members.…”

Section: 11mentioning

confidence: 89%

Collectrin and ACE2 in renal and intestinal amino acid transport

Singer¹,

Camargo²

2011

Channels

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Neutral amino acid transporters of the SLC6 family are expressed at the apical membrane of kidney and/or small intestine, where they (re-)absorb amino acids into the body. In this review we present the results concerning the dependence of their apical expression on their association to partner proteins. We will in particular focus on the situation of B(0)AT1 and B(0)AT3, which associate with members of the renin-angiotensin system (RAS), namely Tmem27 and angiotensin-converting enzyme 2 (ACE2), in a tissue specific manner. The role of this association in relation to the formation of a functional unit related to Na(+) or amino acid transport will be assessed. We will conclude with some remarks concerning the relevance of this association to Hartnup disorder, where some mutations have been shown to differentially interact with the partner proteins

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“…8A). Next, two inhibitors were chosen to test for their effects on renal Ang-(1-7) formation: MLN-4760 (MLN), a specific ACE2 inhibitor (14), and Z-polyl-prolinal (ZPP), a PCP and PEP inhibitor (51,58). At 100 mol/l Ang II and 5 min of incubation time, Ang-(1-7) formation was inhibited by MLN (1 mol/l) to 38 Ϯ 1% but not by ZPP (10 mol/l) (Fig.…”

Section: Ang III and Ang-(1-7) Degradation In Kidneymentioning

confidence: 99%

Mass spectrometry for the molecular imaging of angiotensin metabolism in kidney

Grobe

Elased

Cool

et al. 2012

American Journal of Physiology-Endocrinology and Metabolism

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Grobe N, Elased KM, Cool DR, Morris M. Mass spectrometry for the molecular imaging of angiotensin metabolism in kidney. Am J Physiol Endocrinol Metab 302: E1016 -E1024, 2012. First published February 7, 2012 doi:10.1152/ajpendo.00515.2011.-To better understand the tissue distribution and activity of enzymes involved in angiotensin II (Ang II) processing, we developed a novel molecular imaging method using matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) mass spectrometry. Mouse kidney sections (12 m) were incubated with 10 -1,000 mol/l Ang II for 5-15 min at 37°C. The formed peptides Ang III and Ang-(1-7) were identified by MALDI-TOF/TOF. A third metabolite, Ang-(1-4), was generated from further degradation of Ang-(1-7). Enzymatic processing of Ang II was dose and time dependent and absent in heat-treated kidney sections. Distinct spatial distribution patterns (pseudocolor images) were observed for the peptides. Ang III was localized in renal medulla, whereas Ang-(1-7)/Ang-(1-4) was present in cortex. Regional specific peptide formation was confirmed using microdissected cortical and medullary biopsies. In vitro studies with recombinant enzymes confirmed activity of peptidases known to generate Ang III or Ang-(1-7) from Ang II: aminopeptidase A (APA), Ang-converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP), and prolyl endopeptidase (PEP). Renal medullary Ang III generation was blocked by APA inhibitor glutamate phosphonate. The ACE2 inhibitor MLN-4760 and PCP/PEP inhibitor Z-pro-prolinal reduced cortical Ang-(1-7) formation. Our results establish the power of MALDI imaging as a highly specific and information-rich analytical technique that will further aid our understanding of the role and site of Ang II processing in cardiovascular and renal pathologies. matrix-assisted laser desorption ionization imaging; angiotensin-converting enzyme 2; prolyl carboxypeptidase; prolyl endopeptidase; aminopeptidase A THE POTENT VASOCONSTRICTOR ANGIOTENSIN II (Ang II) is the main effector peptide of the renin-angiotensin system (RAS), controlling renal function and blood pressure. Inhibitors of Ang II synthesis and receptor binding are commonly used in the management of hypertension, congestive heart failure, and renal disease. However, there is growing evidence that therapeutic interventions targeting receptor binding and peptide generation have not provided as much cardiovascular risk reduction as anticipated (1, 56). The search for treatment alternatives continues, particularly since reports have emerged of novel Ang II binding sites as well as reactivation of Ang II synthesis after chronic treatment with Ang-converting enzyme (ACE) inhibitors (4, 32,41,44). Ang II degradation might be a target for potential new treatment strategies against Ang II's vasoconstrictive actions (16,42). However, this requires a reliable method to assess Ang II processing enzyme activities, particularly in pathological states shown to be associated with up/downregulation of RAS enzymes (7,15,59,61). Recognizing the ad...

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Substrate-Based Design of the First Class of Angiotensin-Converting Enzyme-Related Carboxypeptidase (ACE2) Inhibitors

Cited by 176 publications

References 11 publications

ACE2 X-Ray Structures Reveal a Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis

ACE2 X-Ray Structures Reveal a Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis

Collectrin and ACE2 in renal and intestinal amino acid transport

Mass spectrometry for the molecular imaging of angiotensin metabolism in kidney

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