RESUMEN
Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I-converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X-ray crystallography and molecular dynamics simulations. Here, we report the first cryo-EM structures of full-length, glycosylated, soluble sACE (sACES1211 ). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N- and C-terminal domains of monomeric sACES1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N-terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.
Asunto(s)
Enfermedades Cardiovasculares , Hipertensión , Microscopía por Crioelectrón , Dimerización , Humanos , Peptidil-Dipeptidasa ARESUMEN
Discovered more than 30 years ago, the angiotensin AT2 receptor (AT2R) has evolved from a binding site with unknown function to a firmly established major effector within the protective arm of the renin-angiotensin system (RAS) and a target for new drugs in development. The AT2R represents an endogenous protective mechanism that can be manipulated in the majority of preclinical models to alleviate lung, renal, cardiovascular, metabolic, cutaneous, and neural diseases as well as cancer. This article is a comprehensive review summarizing our current knowledge of the AT2R, from its discovery to its position within the RAS and its overall functions. This is followed by an in-depth look at the characteristics of the AT2R, including its structure, intracellular signaling, homo- and heterodimerization, and expression. AT2R-selective ligands, from endogenous peptides to synthetic peptides and nonpeptide molecules that are used as research tools, are discussed. Finally, we summarize the known physiological roles of the AT2R and its abundant protective effects in multiple experimental disease models and expound on AT2R ligands that are undergoing development for clinical use. The present review highlights the controversial aspects and gaps in our knowledge of this receptor and illuminates future perspectives for AT2R research. SIGNIFICANCE STATEMENT: The angiotensin AT2 receptor (AT2R) is now regarded as a fully functional and important component of the renin-angiotensin system, with the potential of exerting protective actions in a variety of diseases. This review provides an in-depth view of the AT2R, which has progressed from being an enigma to becoming a therapeutic target.
Asunto(s)
Receptor de Angiotensina Tipo 2 , Sistema Renina-Angiotensina , Angiotensinas/metabolismo , Angiotensinas/farmacología , Sitios de Unión , Humanos , Ligandos , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Receptor de Angiotensina Tipo 1/metabolismo , Receptor de Angiotensina Tipo 2/metabolismoRESUMEN
Angiotensin converting enzyme (ACE) exerts strong modulation of myeloid cell function independently of its cardiovascular arm. The success of the ACE-overexpressing murine macrophage model, ACE 10/10, in treating microbial infections and cancer opens a new avenue into whether ACE overexpression in human macrophages shares these benefits. Additionally, as ACE inhibitors are a widely used antihypertensive medication, their impact on ACE expressing immune cells is of interest and currently understudied. In the present study, we utilized mass spectrometry to characterize and assess global proteomic changes in an ACE-overexpressing human THP-1 cell line. Additionally, proteomic changes and cellular uptake following treatment with an ACE C-domain selective inhibitor, lisinopril-tryptophan, were also assessed. ACE activity was significantly reduced following inhibitor treatment, despite limited uptake within the cell, and both RNA processing and immune pathways were significantly dysregulated with treatment. Also present were upregulated energy and TCA cycle proteins and dysregulated cytokine and interleukin signaling proteins with ACE overexpression. A novel, functionally enriched immune pathway that appeared both with ACE overexpression and inhibitor treatment was neutrophil degranulation. ACE overexpression within human macrophages showed similarities with ACE 10/10 murine macrophages, paving the way for mechanistic studies aimed at understanding the altered immune function.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina , Macrófagos , Peptidil-Dipeptidasa A , Proteómica , Humanos , Macrófagos/metabolismo , Proteómica/métodos , Peptidil-Dipeptidasa A/metabolismo , Peptidil-Dipeptidasa A/genética , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Células THP-1 , Lisinopril/farmacología , Proteoma/metabolismo , Ratones , Animales , Triptófano/metabolismoRESUMEN
The renin-angiotensin system (RAS) is a key regulator of human arterial pressure. Several of its effects are modulated by angiotensin II, an octapeptide originating from the action of angiotensin-I converting enzyme (ACE) on the decapeptide angiotensin-I. ACE possess two active sites (nACE and cACE) that have their own kinetic and substrate specificities. ACE inhibitors are widely used as the first-line treatment for hypertension and other heart-related diseases, but because they inactivate both ACE domains, their use is associated with serious side effects. Thus, the search for domain-specific ACE inhibitors has been the focus of intense research. Angiotensin (1-7), a peptide that also belongs to the RAS, acts as a substrate of nACE and an inhibitor of cACE. We have synthetized 15 derivatives of Ang (1-7), sequentially removing the N-terminal amino acids and modifying peptides extremities, to find molecules with improved selectivity and inhibition properties. Ac-Ang (2-7)-NH2 is a good ACE inhibitor, resistant to cleavage and with improved cACE selectivity. Molecular dynamics simulations provided a model for this peptide's selectivity, due to Val3 and Tyr4 interactions with ACE subsites. Val3 has an important interaction with the S3 subsite, since its removal greatly reduced peptide-enzyme interactions. Taken together, our findings support ongoing studies using insights from the binding of Ac-Ang (2-7)-NH2 to develop effective cACE inhibitors.
Asunto(s)
Angiotensina I , Peptidil-Dipeptidasa A , Humanos , Peptidil-Dipeptidasa A/metabolismo , Angiotensina I/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/química , Péptidos/farmacologíaRESUMEN
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.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Hipertensión/tratamiento farmacológico , Sistema Renina-Angiotensina/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Insuficiencia Cardíaca/fisiopatología , Humanos , Hipertensión/fisiopatología , Terapia Molecular DirigidaRESUMEN
Angiotensin-converting enzyme (ACE) is best known for its formation of the vasopressor angiotensin II that controls blood pressure but is also involved in other physiological functions through the hydrolysis of a variety of peptide substrates. The enzyme contains two catalytic domains (nACE and cACE) that have different affinities for ACE substrates and inhibitors. We investigated whether nACE inhibitor backbones contain a unique property which allows them to take advantage of the hinging of nACE. Kinetic analysis showed that mutation of unique nACE residues, in both the S2 pocket and around the prime subsites (S') to their C-domain counterparts, each resulted in a decrease in the affinity of nACE specific inhibitors (SG6, 33RE and ketoACE-13) but it required the combined S2_S' mutant to abrogate nACE-selectivity. However, this was not observed with the non-domain-selective inhibitors enalaprilat and omapatrilat. High-resolution structures were determined for the minimally glycosylated nACE with the combined S2_S' mutations in complex with the ACE inhibitors 33RE (1.8â Å), omapatrilat (1.8â Å) and SG6 (1.7â Å). These confirmed that the affinities of the nACE-selective SG6, 33RE and ketoACE-13 are not only affected by direct interactions with the immediate environment of the binding site, but also by more distal residues. This study provides evidence for a more general mechanism of ACE inhibition involving synergistic effects of not only the S2, S1' and S2' subsites, but also residues involved in the sub-domain interface that effect the unique ways in which the two domains stabilize active site loops to favour inhibitor binding.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Dominio Catalítico , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Presión Sanguínea/fisiología , Cristalografía por Rayos X , Glicosilación , Humanos , Cinética , Ligandos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Peptidil-Dipeptidasa A/genética , Unión Proteica , Conformación Proteica en Lámina beta/genética , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Angiotensin converting enzyme (ACE) is well-known for its role in blood pressure regulation via the renin-angiotensin aldosterone system (RAAS) but also functions in fertility, immunity, haematopoiesis and diseases such as obesity, fibrosis and Alzheimer's dementia. Like ACE, the human homologue ACE2 is also involved in blood pressure regulation and cleaves a range of substrates involved in different physiological processes. Importantly, it is the functional receptor for severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 responsible for the 2020, coronavirus infectious disease 2019 (COVID-19) pandemic. Understanding the interaction between SARS-CoV-2 and ACE2 is crucial for the design of therapies to combat this disease. This review provides a comparative analysis of methodologies and findings to describe how structural biology techniques like X-ray crystallography and cryo-electron microscopy have enabled remarkable discoveries into the structure-function relationship of ACE and ACE2. This, in turn, has enabled the development of ACE inhibitors for the treatment of cardiovascular disease and candidate therapies for the treatment of COVID-19. However, despite these advances the function of ACE homologues in non-human organisms is not yet fully understood. ACE homologues have been discovered in the tissues, body fluids and venom of species from diverse lineages and are known to have important functions in fertility, envenoming and insect-host defence mechanisms. We, therefore, further highlight the need for structural insight into insect and venom ACE homologues for the potential development of novel anti-venoms and insecticides.
Asunto(s)
Betacoronavirus/patogenicidad , Infecciones por Coronavirus/enzimología , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/enzimología , Receptores Virales/metabolismo , Internalización del Virus , Enzima Convertidora de Angiotensina 2 , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Animales , Antivirales/uso terapéutico , Betacoronavirus/efectos de los fármacos , COVID-19 , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/virología , Interacciones Huésped-Patógeno , Humanos , Pandemias , Peptidil-Dipeptidasa A/química , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/virología , Conformación Proteica , Receptores Virales/química , SARS-CoV-2 , Relación Estructura-Actividad , Tratamiento Farmacológico de COVID-19RESUMEN
Angiotensin-converting enzyme (ACE) is a zinc metalloprotease best known for its role in blood pressure regulation. ACE consists of two homologous catalytic domains, the N- and C-domain, that display distinct but overlapping catalytic functions in vivo owing to subtle differences in substrate specificity. While current generation ACE inhibitors target both ACE domains, domain-selective ACE inhibitors may be clinically advantageous, either reducing side effects or having utility in new indications. Here, we used site-directed mutagenesis, an ACE chimera and X-ray crystallography to unveil the molecular basis for C-domain-selective ACE inhibition by the bradykinin-potentiating peptide b (BPPb), naturally present in Brazilian pit viper venom. We present the BPPb N-domain structure in comparison with the previously reported BPPb C-domain structure and highlight key differences in peptide interactions with the S4 to S9 subsites. This suggests the involvement of these subsites in conferring C-domain-selective BPPb binding, in agreement with the mutagenesis results where unique residues governing differences in active site exposure, lid structure and dynamics between the two domains were the major drivers for C-domain-selective BPPb binding. Mere disruption of BPPb interactions with unique S2 and S4 subsite residues, which synergistically assist in BPPb binding, was insufficient to abolish C-domain selectivity. The combination of unique S9-S4 and S2' subsite C-domain residues was required for the favourable entry, orientation and thus, selective binding of the peptide. This emphasizes the need to consider factors other than direct protein-inhibitor interactions to guide the design of domain-selective ACE inhibitors, especially in the case of larger peptides.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Oligopéptidos/química , Peptidil-Dipeptidasa A/química , Animales , Células CHO , Catálisis , Cricetulus , Cristalografía por Rayos X , Humanos , Mutagénesis Sitio-Dirigida , Peptidil-Dipeptidasa A/genética , Dominios ProteicosRESUMEN
Angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system (RAS), which is primarily responsible for blood pressure homeostasis. Studies have shown that ACE inhibitors yield cardiovascular benefits that cannot be entirely attributed to the inhibition of ACE catalytic activity. It is possible that these benefits are due to interactions between ACE and RAS receptors that mediate the protective arm of the RAS, such as angiotensin II receptor type 2 (AT2R) and the receptor MAS. Therefore, in this study, we investigated the molecular interactions of ACE, including ACE homodimerization and heterodimerization with AT2R and MAS, respectively. Molecular interactions were assessed by fluorescence resonance energy transfer and bimolecular fluorescence complementation in human embryonic kidney 293 cells and Chinese hamster ovary-K1 cells transfected with vectors encoding fluorophore-tagged proteins. The specificity of dimerization was verified by competition experiments using untagged proteins. These techniques were used to study several potential requirements for the germinal isoform of angiotensin-converting enzyme expressed in the testes (tACE) dimerization as well as the effect of ACE inhibitors on both somatic isoforms of angiotensin-converting enzyme expressed in the testes (sACE) and tACE dimerization. We demonstrated constitutive homodimerization of sACE and of both of its domains separately, as well as heterodimerization of both sACE and tACE with AT2R, but not MAS. In addition, we investigated both soluble sACE and the sACE N domain using size-exclusion chromatography-coupled small-angle X-ray scattering and we observed dimers in solution for both forms of the enzyme. Our results suggest that ACE homo- and heterodimerization does occur under physiologic conditions.
Asunto(s)
Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Multimerización de Proteína/fisiología , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Cristalización , Células HEK293 , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Somatic angiotensin converting enzyme (sACE) is comprised of two homologous domains (N and C domains), whereas the smaller germinal isoform (tACE) is identical to the C domain. Both isozymes share an identical stalk, transmembrane and cytoplasmic domain, and undergo ectodomain shedding by an as yet unknown protease. Here we present evidence for the role of regions distal and proximal to the cleavage site in human ACE shedding. First, because of intrinsic differences between the N and C domains, discrete secondary structures (α-helix 7 and 8) on the surface of tACE were replaced with their N domain counterparts. Surprisingly, neither α-helix 7 nor α-helix 8 proved to be an absolute requirement for shedding. In the proximal ectodomain of tACE residues H610-L614 were mutated to alanines and this resulted in a decrease in ACE shedding. An N-terminal extension of this mutation caused a reduction in cellular ACE activity. 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 moderately decreased suggesting that this region is more crucial for the processing of ACE than it is for regulating shedding. Finally, to determine whether glycosylation of the asparagine proximal to the Pro1199-Leu polymorphism in sACE affected shedding, the equivalent P623L mutation in tACE was investigated. The P623L tACE mutant showed an increase in shedding and MALDI MS analysis of a tryptic digest indicated that N620WT was glycosylated. The absence of an N-linked glycan at N620, resulted in an even greater increase in shedding. Thus, the conformational flexibility that the leucine confers to the stalk, is increased by the lack of glycosylation reducing access of the sheddase to the cleavage site.
Asunto(s)
Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células CHO , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Micropartículas Derivadas de Células/ultraestructura , Cricetulus , Activación Enzimática , Humanos , Peptidil-Dipeptidasa A/ultraestructura , Unión Proteica , Conformación Proteica , Dominios Proteicos , Relación Estructura-ActividadRESUMEN
Sampatrilat is a vasopeptidase inhibitor that inhibits both angiotensin I-converting enzyme (ACE) and neutral endopeptidase. ACE is a zinc dipeptidyl carboxypeptidase that contains two extracellular domains (nACE and cACE). In this study the molecular basis for the selectivity of sampatrilat for nACE and cACE was investigated. Enzyme inhibition assays were performed to evaluate the in vitro ACE domain selectivity of sampatrilat. The inhibition of the C-domain (Ki = 13.8 nM) by sampatrilat was 12.4-fold more potent than that for the N-domain (171.9 nM), indicating differences in affinities for the respective ACE domain binding sites. Interestingly, replacement of the P2 group of sampatrilat with an aspartate abrogated its C-selectivity and lowered the potency of the inhibitor to activities in the micromolar range. The molecular basis for this selective profile was evaluated using molecular modeling methods. We found that the C-domain selectivity of sampatrilat is due to occupation of the lysine side chain in the S1 and S2 subsites and interactions with Glu748 and Glu1008, respectively. This study provides new insights into ligand interactions with the nonprime binding site that can be exploited for the design of domain-selective ACE inhibitors.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Mesilatos/farmacología , Peptidil-Dipeptidasa A/metabolismo , Inhibidores de Proteasas/farmacología , Tirosina/análogos & derivados , Inhibidores de la Enzima Convertidora de Angiotensina/química , Humanos , Mesilatos/química , Modelos Moleculares , Peptidil-Dipeptidasa A/química , Inhibidores de Proteasas/química , Dominios Proteicos , Tirosina/química , Tirosina/farmacologíaRESUMEN
Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg(522). Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu(403)-Lys(118) salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite 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.
Asunto(s)
Cloruros/química , Peptidil-Dipeptidasa A/química , Sustitución de Aminoácidos , Sitios de Unión , Cloruros/metabolismo , Cristalografía por Rayos X , Humanos , Mutación Missense , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Somatic angiotensin-I converting enzyme (sACE) has an essential role in the regulation of blood pressure and electrolyte fluid homeostasis. It is a zinc protease that cleaves angiotensin-I (AngI), bradykinin, and a broad range of other signalling peptides. The enzyme activity is provided by two homologous domains (N- and C-), which display clear differences in substrate specificities and chloride activation. The presence of chloride ions in sACE and its unusual role in activity was identified early on in the characterisation of the enzyme. The molecular mechanisms of chloride activation have been investigated thoroughly through mutagenesis studies and shown to be substrate-dependent. Recent results from X-ray crystallography structural analysis have provided the basis for the intricate interactions between ACE, its substrate and chloride ions. Here we describe the role of chloride ions in human ACE and its physiological consequences. Insights into the chloride activation of the N- and C-domains could impact the design of improved domain-specific ACE inhibitors.
Asunto(s)
Cloruros/metabolismo , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos MolecularesRESUMEN
The somatic isozyme of ACE (angiotensin I-converting enzyme) comprises two distinct zinc-dependent catalytic domains with different substrate specificities for angiotensin I (cleaved selectively by the C-domain) and bradykinin (cleaved equally efficiently by both the N- and C-domains). Classical ACEIs (ACE inhibitors) target both domains, with side effects such as cough and angio-oedema being attributed, in part, to N-domain inhibition, probably through bradykinin accumulation. We questioned whether a novel C-domain-selective ACEI (lisW-S) has anti-hypertensive effects without influencing bradykinin status. AngII (angiotensin II)-dependent hypertension was studied in mice that express active human renin in the liver (TtRhRen). Compared with wild-type littermates, TtRhRen mice displayed cardiac hypertrophy and had significantly elevated SBP [systolic BP (blood pressure)] as determined by tail cuff sphygmomanometry (150±3 compared with 112±5 mmHg; P<0.05) and telemetry (163±3 compared with 112±2 mmHg; P<0.01). Treatment with the non-selective ACEI lisinopril (1 mg/kg of body weight per day via an osmotic mini-pump for 2 weeks) reduced SBP (127±3 compared with. 154±6; P<0.05). Similarly, treatment with the C-domain selective ACEI lisW-S (lisinopril-tryptophan; 3.6 mg/kg of body weight per day via an osmotic mini-pump for 2 weeks) reduced BP. Treatment with lisinopril or lisW-S significantly reduced levels of AngII in kidneys (~4-fold; P<0.001). Ang-(2-8) [angiotensin-2-8)] was significantly reduced by lisinopril, but not by lisW-S. Plasma bradykinin levels were significantly increased only in the lisinopril group. These data suggest that C-domain-selective ACEIs reduce BP and AngII levels similarly to classical ACEIs. C-domain-selective ACEIs have the potential to avoid undesirable effects on the bradykinin system common to classic ACEIs and may represent a novel approach to the treatment of hypertension.
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Angiotensina II/fisiología , Inhibidores de la Enzima Convertidora de Angiotensina/uso terapéutico , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Angiotensina II/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Bradiquinina/sangre , Bradiquinina/metabolismo , Cardiomegalia/etiología , Cardiomegalia/prevención & control , Enfermedad Crónica , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Hipertensión/complicaciones , Hipertensión/fisiopatología , Hipertrofia , Riñón/metabolismo , Riñón/patología , Lisinopril/análogos & derivados , Lisinopril/uso terapéutico , Ratones , Ratones TransgénicosRESUMEN
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 P2 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 His388 and a direct hydrogen bond with the hydroxy group of the N-domain specific Tyr369. This work further elucidates the molecular basis for N-domain-selective inhibition and assists in the design of novel N-selective ACE inhibitors that could be employed in treatment of fibrosis disorders.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Presión Sanguínea/efectos de los fármacos , Diseño de Fármacos , Oligopéptidos/química , Peptidil-Dipeptidasa A/química , Ácidos Fosfínicos/química , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Sitios de Unión/fisiología , Conformación ProteicaRESUMEN
Human somatic angiotensin-converting enzyme (ACE) is a key zinc metallopeptidase that plays a pivotal role in the renin-angiotensin-aldosterone system (RAAS) by regulating blood pressure and electrolyte balance. Inhibition of ACE is a cornerstone in the management of hypertension, cardiovascular diseases, and renal disorders. Recent advances in structural biology techniques have provided invaluable insights into the molecular mechanisms underlying ACE inhibition, facilitating the design and development of more effective therapeutic agents. This review focuses on the latest advancements in elucidating the structural basis for ACE inhibition. High-resolution crystallographic studies of minimally glycosylated individual domains of ACE have revealed intricate molecular details of the ACE catalytic N- and C-domains, and their detailed interactions with clinically relevant and newly designed domain-specific inhibitors. In addition, the recently elucidated structure of the glycosylated form of full-length ACE by cryo-electron microscopy (cryo-EM) has shed light on the mechanism of ACE dimerization and revealed continuous conformational changes which occur prior to ligand binding. In addition to these experimental techniques, computational approaches have also played a pivotal role in elucidating the structural basis for ACE inhibition. Molecular dynamics simulations and computational docking studies have provided atomic details of inhibitor binding kinetics and energetics, facilitating the rational design of novel ACE inhibitors with improved potency and selectivity. Furthermore, computational analysis of the motions observed by cryo-EM allowed the identification of allosteric binding sites on ACE. This affords new opportunities for the development of next-generation allosteric inhibitors with enhanced pharmacological properties. Overall, the insights highlighted in this review could enable the rational design of novel ACE inhibitors with improved efficacy and safety profiles, ultimately leading to better therapeutic outcomes for patients with hypertension and cardiovascular diseases.
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Inhibidores de la Enzima Convertidora de Angiotensina , Peptidil-Dipeptidasa A , Humanos , Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Simulación de Dinámica Molecular , Relación Estructura-Actividad , Simulación del Acoplamiento Molecular , Sistema Renina-Angiotensina/efectos de los fármacos , Microscopía por Crioelectrón , Unión Proteica , AnimalesRESUMEN
Human somatic angiotensin-1-converting enzyme (sACE) is composed of a catalytic N-(nACE) and C-domain (cACE) of similar size with different substrate specificities. It is involved in the regulation of blood pressure by converting angiotensin I to the vasoconstrictor angiotensin II and has been a major focus in the development of therapeutics for hypertension. Bioactive peptides from various sources, including milk, have been identified as natural ACE inhibitors. We report the structural basis for the role of two lacototripeptides, Val-Pro-Pro and Ile-Pro-Pro, in domain-specific inhibition of ACE using X-ray crystallography and kinetic analysis. The lactotripeptides have preference for nACE due to altered polar interactions distal to the catalytic zinc ion. Elucidating the mechanism of binding and domain selectivity of these peptides also provides important insights into the functional roles of ACE.
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Inhibidores de la Enzima Convertidora de Angiotensina , Peptidil-Dipeptidasa A , Humanos , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Cinética , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , AngiotensinasRESUMEN
The renin-angiotensin system (RAS) is central to regulation of blood pressure and electrolyte homeostasis. Angiotensin-converting enzyme (ACE), a key protease in the RAS, has a range of substrates, including N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP). The peptide Ac-SDKP is cleared almost exclusively by ACE, and specifically by the N-domain active site of this enzyme. N-Acetyl-Ser-Asp-Lys-Pro is a negative regulator of haematopoietic stem cell differentiation and is a potent antifibrotic agent. In this review, the physiological actions of Ac-SDKP are presented, together with the potential clinical usefulness of raising Ac-SDKP levels. This emphasizes the possible opportunity of N-domain-selective ACE inhibitors or ACE-resistant Ac-SDKP analogues for the treatment of fibrosis.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Antifibrinolíticos/farmacología , Diseño de Fármacos , Oligopéptidos/farmacología , Sistema Renina-Angiotensina/fisiología , Inhibidores de la Enzima Convertidora de Angiotensina/química , Animales , Antifibrinolíticos/química , Oligopéptidos/química , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacosRESUMEN
Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase containing two homologous domains. While the C-domain plays a major role in blood pressure regulation, the N-domain hydrolyzes the antifibrotic agent N-acetyl-Ser-Asp-Lys-Pro. Thus, N-domain selective (N-selective) inhibitors could be useful in the treatment of conditions relating to excessive tissue fibrosis. New keto-ACE analogues were designed that contained functionalities considered important for N-selective inhibitor RXP407 binding, namely, a P(2) Asp, N-acetyl group, and C-terminal amide. Such functionalities were incorporated to assess the structural determinants for N-selective binding in a novel inhibitor template. Inhibitors containing a C-terminal amide and modified P(2)' group were poor inhibitors of the N-domain, with several of these displaying improved inhibition of the C-domain. Molecules with both a C-terminal amide and P(2) Asp were also poor inhibitors and not N-selective. Compounds containing a free C-terminus, a P(2) Asp and protecting group displayed a change of more than 1000-fold N-selectivity compared with the parent molecule. Molecular docking models revealed interaction of these P(2) groups with S(2) residues Tyr369 and Arg381. This study emphasizes the importance of P(2) functionalities in allowing for improved N-selective binding and provides further rationale for the design of N-selective inhibitors, which could be useful in treating tissue fibrosis.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/síntesis química , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Dominio Catalítico , Dipéptidos/síntesis química , Dipéptidos/farmacología , Nitrógeno , Peptidil-Dipeptidasa A/química , Inhibidores de la Enzima Convertidora de Angiotensina/química , Técnicas de Química Sintética , Dipéptidos/química , Evaluación Preclínica de Medicamentos , Modelos Moleculares , Peptidil-Dipeptidasa A/metabolismo , Especificidad por SustratoRESUMEN
Somatic angiotensin I-converting enzyme (ACE)has two homologous active sites (N and C domains) that show differences in various biochemical properties.In a previous study, we described the use of positionals canning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain(Bersanetti et al., 2004). In the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. The peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P 3 to P 1 ' positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz = ortho -aminobenzoic acid; Dnp = 2,4-dinitrophenyl)showed the highest selectivity for ACE N-domain( k cat /K m = 1.76 µ m -1 · s -1) . Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. The binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.