Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased ϳ10-fold by Cl ؊ and F ؊ but is unaffected by Br ؊ . ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatographymass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only.

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 ...

Angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a recently identified zinc metalloprotease with carboxypeptidase activity that was identified using our genomics platform. We implemented a rational design approach to identify potent and selective ACE2 inhibitors. To this end, picomolar inhibitors of ACE2 were designed and synthesized.

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Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.