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Journal ArticleDOI

RXP 407, a phosphinic peptide, is a potent inhibitor of angiotensin I converting enzyme able to differentiate between its two active sites

TL;DR: RXP 407, a highly potent and selective inhibitor of the N-terminal active site of wild ACE, may lead to a new generation of ACE inhibitors able to block in vivo only a subset of the different functions regulated by ACE.
Abstract: The human somatic angiotensin converting enzyme (ACE) contains two homologous domains, each bearing a zinc-dependent active site. All of the synthetic inhibitors of this enzyme used in clinical applications interact with these two active sites to a similar extent. Recently, several lines of evidence have suggested that the N-terminal active site of ACE might be involved in specific hydrolysis of some important physiological substrates, like Acetyl-Seryl-Aspartyl-Lysyl-Proline, a negative regulator of hematopoietic stem cell differentiation and proliferation. These findings have stimulated studies aimed at identifying new ACE inhibitors able to block only one of the two active sites of this enzyme. By screening phosphinic peptide libraries, we discovered a phosphinic peptide Ac-Asp-(L)Pheψ(PO2-CH2)(L)Ala-Ala-NH2, called RXP 407, which is able to differentiate the two ACE active sites, with a dissociation constant three orders of magnitude lower for the N-domain of the enzyme. The usefulness of a combinatorial chemistry approach to develop new lead structures is underscored by the unusual chemical structure of RXP 407, as compared with classical ACE inhibitors. As a highly potent and selective inhibitor of the N-terminal active site of wild ACE (Ki = 12 nM), RXP 407, which is metabolically stable in vivo, may lead to a new generation of ACE inhibitors able to block in vivo only a subset of the different functions regulated by ACE.
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Journal ArticleDOI
TL;DR: In this paper, the authors examined the kinetics of angiotensin peptide cleavage by full-length human ACE, the separate N- and C-domains of ACE, ACE2, and NEP (neprilysin), and the activity of the enzyme preparations was determined by active-site titrations using competitive tight-binding inhibitors and fluorogenic substrates.
Abstract: In the RAS (renin–angiotensin system), Ang I (angiotensin I) is cleaved by ACE (angiotensin-converting enzyme) to form Ang II (angiotensin II), which has effects on blood pressure, fluid and electrolyte homoeostasis. We have examined the kinetics of angiotensin peptide cleavage by full-length human ACE, the separate N- and C-domains of ACE, the homologue of ACE, ACE2, and NEP (neprilysin). The activity of the enzyme preparations was determined by active-site titrations using competitive tight-binding inhibitors and fluorogenic substrates. Ang I was effectively cleaved by NEP to Ang (1–7) (kcat/Km of 6.2×105 M−1·s−1), but was a poor substrate for ACE2 (kcat/Km of 3.3×104 M−1·s−1). Ang (1–9) was a better substrate for NEP than ACE (kcat/Km of 3.7×105 M−1·s−1 compared with kcat/Km of 6.8×104 M−1·s−1). Ang II was cleaved efficiently by ACE2 to Ang (1–7) (kcat/Km of 2.2×106 M−1·s−1) and was cleaved by NEP (kcat/Km of 2.2×105 M−1·s−1) to several degradation products. In contrast with a previous report, Ang (1–7), like Ang I and Ang (1–9), was cleaved with a similar efficiency by both the N- and C-domains of ACE (kcat/Km of 3.6×105 M−1·s−1 compared with kcat/Km of 3.3×105 M−1·s−1). The two active sites of ACE exhibited negative co-operativity when either Ang I or Ang (1–7) was the substrate. In addition, a range of ACE inhibitors failed to inhibit ACE2. These kinetic data highlight that the flux of peptides through the RAS is complex, with the levels of ACE, ACE2 and NEP dictating whether vasoconstriction or vasodilation will predominate.

554 citations

Journal ArticleDOI
TL;DR: The tetrahedral transition state is believed to be specifically stabilized in enzyme active sites, which has inspired numerous studies on their applications in regulating the activity of proteases, including the development of many potent inhibitors of various enzymes, such as the antihypertensive drug fosinopril.
Abstract: R-Aminophosphonic acids are broadly defined as analogues of amino acids in which the carboxylic group is replaced by a phosphonic acid or related group (usually phosphonous or phosphinic acids). This results in the presence of the characteristic N C P scaffold (Scheme 1). The biological activity and natural occurrence of these compounds (often called R-aminophosphonates) were discovered half a century ago. Since then, the chemistry and biology of this class of compounds have been developed into a distinct branch of phosphorus chemistry. It is generally acknowledged that R-aminophosphonates possess a broad capability of influencing physiologic and pathologic processes, with applications ranging from agrochemistry to medicine. In some cases, these compounds have been commercialized. A number of excellent reviews on various aspects of their activity in natural systems have been published. 12 The mode of action of aminophosphonates primarily involves the inhibition of enzymes of different class and origin. Despite its long history, this area of research remains intensively explored and frequently delivers new promising lead compounds in medicinal chemistry. The N C P molecular fragment and its chemistry offer many possibilities for structural modifications, which have resulted in broad biological relevance (Scheme 1). Often, R-aminophosphonic and phosphinic acids are considered simple analogues of their natural counterparts, carboxylic acids. Although carboxylic and phosphonic acid groups differ in shape (tetrahedral at phosphorus versus planar at carbon), acidity (with phosphonic acid being significantly more acidic), and steric bulk (the phosphorus atom has a much larger atomic radius than carbon), they frequently exhibit similar properties, with the phosphonic acid being recognized by enzymes or receptors as false substrates or inhibitors. However, the tetrahedral geometry of substituents around the phosphorus moiety causes it to resemble the high-energy transition state (TS) of ester and amide bond hydrolyses. The tetrahedral transition state is believed to be specifically stabilized in enzyme active sites, which has inspired numerous studies on their applications in regulating the activity of proteases. This approach has been most successful in the case of metalloproteases, which have an organophosphorus moiety in their active sites that facilitates the chelation of metal ions. This approach has resulted in the development of many potent inhibitors of various enzymes, such as the antihypertensive drug fosinopril, an angiotensin I converting enzyme (ACE) inhibitor. Recently, the N C P scaffold has been used to construct extended transition state analogues of amide bond synthesis or hydrolysis to find potent inhibitors of enzymes such as glutamine synthetase or urease. Reactive peptidyl phosphonate diaryl esters have been successfully used to covalently modify members of the serine hydrolase superfamily. This approach exploits their ability to phosphonylate the hydroxyl residue of the active-site serine of these enzymes. They act as competitive, irreversible inhibitors, which, after the formation of an initial enzyme substrate complex, bind to the active site via a transesterification reaction and thus block its catalytic function. The activity and selectivity of the interactions of inhibitors with target enzymes can be adjusted by structural optimization of the S1 residues and/or by the development of an extended peptide chain. Finally, aminomethylenebisphosphonic acids form a separate class of medicinally important compounds bearing the N C P skeleton. They are hydrolytically stable analogues of pyrophosphate characterized by a common P C P fragment in which a carbon phosphorus bond replaces an oxygen phosphorus bond. Their primary medical application is in combating osteoporosis. They exhibit very high affinity to bone tissue, being rapidly adsorbed at the bone surface, and they regulate the bone remodeling process. Because the action of bisphosphonates is limited to osseous tissue, they have also been used to deliver conjugated chemotherapeutic agents to bone. Likely because of their strong chelating properties, bisphosphonates also exhibit inhibitory properties toward a wide variety of metalloenzymes. In this Perspective, we present the key features of theN C P molecular fragment that govern the activity of the molecules that incorporate it. A general overview of known modes of action and target enzyme classes is briefly presented. Recent representative medicinal chemistry projects are described and discussed, including the achievements of our research group on leucine aminopeptidase and urease. Particular attention is given to the molecular aspects of the N C P mechanism of action and to the rational design of new compounds based on threedimensional structures. The potential future applications of this class of compounds are also discussed.

472 citations

Journal ArticleDOI
TL;DR: The structural features of current inhibitors are discussed and how next-generation ACE inhibitors could be designed by using the three-dimensional molecular structure of human testis ACE are outlined.
Abstract: Current-generation angiotensin-converting enzyme (ACE) inhibitors are widely used for cardiovascular diseases, including high blood pressure, heart failure, heart attack and kidney failure, and have combined annual sales in excess of US $6 billion. However, the use of these ACE inhibitors, which were developed in the late 1970s and early 1980s, is hampered by common side effects. Moreover, we now know that ACE actually consists of two parts (called the N- and C-domains) that have different functions. Therefore, the design of specific domain-selective ACE inhibitors is expected to produce next-generation drugs that might be safer and more effective. Here we discuss the structural features of current inhibitors and outline how next-generation ACE inhibitors could be designed by using the three-dimensional molecular structure of human testis ACE. The ACE structure provides a unique opportunity for rational drug design, based on a combination of in silico modelling using existing inhibitors as scaffolds and iterative lead optimization to drive the synthetic chemistry.

290 citations

Journal ArticleDOI
David Coates1
TL;DR: Mouse knockout experiments, and comparative work with invertebrate homologues, suggest that the two domains of angiotensin converting enzyme have clearly distinct roles.

249 citations

Journal ArticleDOI
TL;DR: New therapies in preclinical and early clinical stages of development are focused on, including novel small molecule inhibitors and receptor agonists/antagonists, less conventional strategies such as gene therapy to suppress angiotensinogen at the RNA level, recombinant ACE2 protein, and novel bispecific designer peptides.
Abstract: Despite the success of renin-angiotensin system (RAS) blockade by angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 receptor (AT1R) blockers, current therapies for hypertension and related cardiovascular diseases are still inadequate. Identification of additional components of the RAS and associated vasoactive pathways, as well as new structural and functional insights into established targets, have led to novel therapeutic approaches with the potential to provide improved cardiovascular protection and better blood pressure control and/or reduced adverse side effects. The simultaneous modulation of several neurohumoral mediators in key interconnected blood pressure–regulating pathways has been an attractive approach to improve treatment efficacy, and several novel approaches involve combination therapy or dual-acting agents. In addition, increased understanding of the complexity of the RAS has led to novel approaches aimed at upregulating the ACE2/angiotensin-(1-7)/Mas axis to counter-regulate the harmful effects of the ACE/angiotensin II/angiotensin III/AT1R axis. These advances have opened new avenues for the development of novel drugs targeting the RAS to better treat hypertension and heart failure. Here we focus on new therapies in preclinical and early clinical stages of development, including novel small molecule inhibitors and receptor agonists/antagonists, less conventional strategies such as gene therapy to suppress angiotensinogen at the RNA level, recombinant ACE2 protein, and novel bispecific designer peptides.

214 citations


Cites background from "RXP 407, a phosphinic peptide, is a..."

  • ...N-domain–selective inhibitors may prove useful for indications such as fibrosis, where it would be beneficial to inhibit N-domain–specific Ac-SDKP formation without affecting blood pressure (Dive et al., 1999; Douglas et al., 2014; Fienberg et al., 2018)....

    [...]

References
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Journal ArticleDOI
22 Apr 1977-Science
TL;DR: A hypothetical model of the active site of angiotensin-converting enzyme, based on known chemical and kinetic properties of the enzyme, has enabled a new class of potent and specific inhibitors, carboxyalkanoyl and mercaptoalkanoysl derivatives of proline, to be designed.
Abstract: A hypothetical model of the active site of angiotensin-converting enzyme, based on known chemical and kinetic properties of the enzyme, has enabled us to design a new class of potent and specific inhibitors. These compounds, carboxyalkanoyl and mercaptoalkanoyl derivatives of proline, inhibit the contractile response of guinea pig ileal strip to angiotensin I and augment its response to bradykinin. When administered orally to rats, these agents inhibit the pressor effect of angiotensin I, augment the vasodepressor effect of bradykinin, and lower blood pressure in a model of renovascular hypertension.

1,759 citations

Journal ArticleDOI
TL;DR: A computer program with the code name DYNAFIT was developed for fitting either the initial velocities or the time course of enzyme reactions to an arbitrary molecular mechanism represented symbolically by a set of chemical equations.

1,388 citations

Journal ArticleDOI
TL;DR: In assays of the human matrix metalloproteinases, Mca‐Pro‐ Leu‐Gly‐Leu‐Dpa‐Ala‐Arg‐NH2 is about 50 to 100 times more sensitive than dinitrophenyl‐Pro •Leu •Gly •LeU‐Trp •Ala •d‐Arg •NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates.

739 citations

Journal ArticleDOI
TL;DR: The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication, and is consistent with the presence of a single gene for ACE in the haploid human genome.
Abstract: The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-converting enzyme (ACE; peptidyl-dipeptidase A, kininase II; EC 3.4.15.1) purified from human kidney were used to design oligonucleotide probes. The nucleotide sequence of ACE mRNA was determined by molecular cloning of the DNA complementary to the human vascular endothelial cell ACE mRNA. The complete amino acid sequence deduced from the cDNA contains 1306 residues, beginning with a signal peptide of 29 amino acids. A highly hydrophobic sequence located near the carboxyl-terminal extremity of the molecule most likely constitutes the anchor to the plasma membrane. The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication. Each of these two domains contains short amino acid sequences identical to those located around critical residues of the active site of other metallopeptidases (thermolysin, neutral endopeptidase, and collagenase) and therefore bears a putative active site. Since earlier experiments suggested that a single Zn atom was bound per molecule of ACE, only one of the two domains should be catalytically active. The results of genomic DNA analysis with the cDNA probe are consistent with the presence of a single gene for ACE in the haploid human genome. Whereas the ACE gene is transcribed as a 4.3-kilobase mRNA in vascular endothelial cells, a 3.0-kilobase transcript was detected in the testis, where a shorter form of ACE is synthesized.

723 citations

Journal ArticleDOI
TL;DR: Observations provide strong evidence that ACE possesses two independent catalytic domains and suggest that they may have different functions.

374 citations