Study of bradykinin metabolism in human and rat plasma by liquid chromatography with inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry.

Bradykinin is a small peptide that acts mainly as a hormone by activating specific receptors that confer protection against the development of hypertension. The efficacy of bradykinin is influenced by the activities of various kininases present in plasma and blood. In this study, both human and rat plasma were incubated with a labelled form of bradykinin (at 4 and 12.5 microM), that will be referred to as bromobradykinin. The metabolic fate of bromobradykinin was monitored by liquid chromatography coupled to an orthogonal acceleration time-of-flight mass spectrometer (oaTOF). Quantification measurements of the bromine-containing metabolites were performed on-line, via flow splitting, by inductively coupled plasma mass spectrometry (ICPMS). The data obtained highlighted that the mechanism(s) of bradykinin metabolism in human and rat plasma are different, with the metabolism of bradykinin in rat plasma being much more aggressive than that observed in human plasma. In addition to the known bradykinin metabolites, e.g. [1,5], [1,7] from ACE, [1,8] from carboxypeptidase and [2,9] from aminopeptidase activity, we have identified the presence of new bradykinin metabolites in both human and rat plasma. These have been identified as fragment [5], the amino acid phenylalanine, which was present in both the human and rat plasma and the fragments [2,8] and [4,8] in rat plasma. To our knowledge it is the first time that these fragments have been recorded in human and rat plasma. The occurrence of these new fragments provides evidence for the presence of potentially new enzymes and mechanisms of bradykinin metabolism. The method described here provides a powerful technique for monitoring the activity of the many kininases involved in bradykinin metabolism such as ACE (angiotensin I converting enzyme), carboxypeptidase N and aminopeptidase P. In addition, this procedure could be used as a screening assay for selecting and monitoring the actions of inhibitors of the enzymes implicated in bradykinin metabolism directly in plasma or serum.     

Carnosine to Combat Novel Coronavirus (nCoV): Molecular Docking and Modeling to Cocrystallized Host Angiotensin-Converting Enzyme 2 (ACE2) and Viral Spike Protein

Aims: Angiotensin-converting enzyme 2 (ACE2) plays an important role in the entry of coronaviruses into host cells. The current paper described how carnosine, a naturally occurring supplement, can be an effective drug candidate for coronavirus disease (COVID-19) on the basis of molecular docking and modeling to host ACE2 cocrystallized with nCoV spike protein. Methods: First, the starting point was ACE2 inhibitors and their structure–activity relationship (SAR). Next, chemical similarity (or diversity) and PubMed searches made it possible to repurpose and assess approved or experimental drugs for COVID-19. Parallel, at all stages, the authors performed bioactivity scoring to assess potential repurposed inhibitors at ACE2. Finally, investigators performed molecular docking and modeling of the identified drug candidate to host ACE2 with nCoV spike protein. Results: Carnosine emerged as the best-known drug candidate to match ACE2 inhibitor structure. Preliminary docking was more optimal to ACE2 than the known typical angiotensin-converting enzyme 1 (ACE1) inhibitor (enalapril) and quite comparable to known or presumed ACE2 inhibitors. Viral spike protein elements binding to ACE2 were retained in the best carnosine pose in SwissDock at 1.75 Angstroms. Out of the three main areas of attachment expected to the protein–protein structure, carnosine bound with higher affinity to two compared to the known ACE2 active site. LibDock score was 92.40 for site 3, 90.88 for site 1, and inside the active site 85.49. Conclusion: Carnosine has promising inhibitory interactions with host ACE2 and nCoV spike protein and hence could offer a potential mitigating effect against the current COVID-19 pandemic.     

Structure-based pharmacophore design and virtual screening for novel angiotensin converting enzyme 2 inhibitors

The metallopeptidase Angiotensin Converting Enzyme (ACE) is an important drug target for the treatment of hypertension, heart, kidney, and lung disease. Recently, a close and unique human ACE homologue termed ACE2 has been identified and found to be an interesting new cardiorenal disease target. With the recently resolved inhibitor-bound ACE2 crystal structure available, we have attempted a structure-based approach to identify novel potent and selective inhibitors. Computational approaches focus on pharmacophore-based virtual screening of large compound databases. Selectivity was ensured by initial screening for ACE inhibitors within an internal database and the Derwent World Drug Index, which could be reduced to zero false positives and 0.1% hit rate, respectively. An average hit reduction of 0.44% was achieved with a five feature hypothesis, searching approximately 3.8 million compounds from various commercial databases. Seventeen compounds were selected based on high fit values as well as diverse structure and subjected to experimental validation in a bioassay. We show that all compounds displayed an inhibitory effect on ACE2 activity, the six most promising candidates exhibiting IC50 values in the range of 62-179 microM.     

Improved multicomponent,one-pot synthesis of functionalized tetrahydropyrans cardiovascular dysfunction regulators

A simple, efficient, and straightforward one-pot three-component synthesis of functionalized tetrahydropyrans ( 6a-l ) is carried-out in the presence of an inexpensive and environmentally benign Eaton's reagent as a catalyst to discover some ACE inhibitors and anti-inflammatory agents. Angiotensin-converting enzyme (ACE) is highly involved in the renin-angiotensin system and often directly associated with cardiovascular dysfunction via inflammation. Thus, the cardiovascular protective effect of newly synthesized tetrahydropyrans ( 6a-l ) was evaluated through ACE inhibition, human red blood cell (HRBC) membrane stabilization, and molecular docking studies. As the results complied, compounds 6e and 6f were identified as effective ACE inhibitors. Docking studies, HRBC based anti-inflammatory assay, and SAR studies revealed that compounds 6e and 6f could be good anti-inflammatory agents apart from ACE inhibitors.     

亲和色谱法筛选中药中血管紧张素转化酶抑制剂

以壳聚糖微球为载体、戊二醛（glutaraldehyde,GA）为交联剂对血管紧张素转化酶（Angiotensin converting enzyme,ACE）进行固定化．用固化的ACE作为亲和介质,利用血管紧张素转化酶抑制剂（Angiotensin convertingenzym einhibitor,ACEI）与ACE之间的亲和作用,结合高效液相色谱对亲和前后的体系进行检测,比较两者各组分色谱峰的差异,以此实现快速筛选复杂体系中的ACE抑制剂．应用赖诺普利（Lisinopril）、九肽抑制剂、依那普利（Enalapril）、培哚普利（Perindopril）、卡托普利（Captopril）等已上市的ACEI对方法进行验证,反映方法具有高度选择性．将方法应用于中药地龙及山楂筛选,发现共有5个组分与ACE有亲和作用,并且都能抑制ACE酶活性,它们对酶活性抑制的IC50值在0．45～4．62μg／mL范围．通过对亲和方法重现性考察,6次测定的相对标准偏差小于1％,说明方法可靠．提出的亲和色谱．色谱指纹差异法非常适合于从中药及天然产物等复杂混合物库中快速筛选靶点活性物质．     

Acidity,lipophilicity, solubility,absorption, and polar surface area of some ACE inhibitors

Computational chemical methods have been used to correlate the molecular properties of the 10 ACE inhibitors (captopril, enalapril, perindopril, lisinopril, ramipril, trandolapril, quinapril, fosinopril, benazepril, and cilazapril) and some of their active metabolites (enalaprilat, perindoprilat, ramiprilat, trandolaprilat, quinaprilat, fosinoprilat, benazeprilat, and cilazaprilat). The computed pK _a values correlate well with the available experimental values. In the dicarboxylic ACE inhibitors, the carboxyalkyl carboxylate group of the ACE inhibitors studied is more acidic than the C-terminal carboxylate. However, at physiological pH = 7.4 both carboxyl groups of ACE inhibitors are completely ionized and the dicarboxyl-containing ACE inhibitors behave as strong acids. The available experimental partition coefficients of these ACE inhibitors investigated are well reproduced by the neural network-based ALOGPs and the fragment-based KoWWiN methods. All parent drugs (and prodrugs), with the exception of fosinopril, are compounds with low lipophilicity. Calculated pK _a, lipophilicity, solubility, absorption, and polar surface area of the most effective ACE inhibitors for the prevention of myocardial infarction, perindopril and ramipril, were found similar. Therefore, it is probable that the experimentally observed differences in the survival benefits in the first year after acute myocardial infarction in patients 65 years of age or older correlate closely to the physicochemical and pharmacokinetic characteristics of the specific ACE inhibitor that is used.     

Metallodrug Profiling against SARS-CoV-2 Target Proteins Identifies Highly Potent Inhibitors of the S/ACE2 interaction and the Papain-like Protease PLpro

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has called for an urgent need for dedicated antiviral therapeutics. Metal complexes are commonly underrepresented in compound libraries that are used for screening in drug discovery campaigns, however, there is growing evidence for their role in medicinal chemistry. Based on previous results, we have selected more than 100 structurally diverse metal complexes for profiling as inhibitors of two relevant SARS-CoV-2 replication mechanisms, namely the interaction of the spike (S) protein with the ACE2 receptor and the papain-like protease PL^pro. In addition to many well-established types of mononuclear experimental metallodrugs, the pool of compounds tested was extended to approved metal-based therapeutics such as silver sulfadiazine and thiomersal, as well as polyoxometalates (POMs). Among the mononuclear metal complexes, only a small number of active inhibitors of the S/ACE2 interaction was identified, with titanocene dichloride as the only strong inhibitor. However, among the gold and silver containing complexes many turned out to be very potent inhibitors of PL^pro activity. Highly promising activity against both targets was noted for many POMs. Selected complexes were evaluated in antiviral SARS-CoV-2 assays confirming activity for gold complexes with N-heterocyclic carbene (NHC) or dithiocarbamato ligands, a silver NHC complex, titanocene dichloride as well as a POM compound. These studies might provide starting points for the design of metal-based SARS-CoV-2 antiviral agents.     

Validated ligand mapping of ACE active site

Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 A, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.     

Virtual Screening of Natural Chemical Databases to Search for Potential ACE2 Inhibitors

The angiotensin-converting enzyme II (ACE2) is a multifunctional protein in both health and disease conditions, which serves as a counterregulatory component of RAS function in a cardioprotective role. ACE2 modulation may also have relevance to ovarian cancer, diabetes, acute lung injury, fibrotic diseases, etc. Furthermore, since the outbreak of the coronavirus disease in 2019 (COVID-19), ACE2 has been recognized as the host receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The receptor binding domain of the SARS-CoV-2 S-protein has a strong interaction with ACE2, so ACE2 may be a potent drug target to prevent the virus from invading host cells for anti-COVID-19 drug discovery. In this study, structure- and property-based virtual screening methods were combined to filter natural product databases from ChemDiv, TargetMol, and InterBioScreen to find potential ACE2 inhibitors. The binding affinity between protein and ligands was predicted using both Glide SP and XP scoring functions and the MM-GBSA method. ADME properties were also calculated to evaluate chemical drug-likeness. Then, molecular dynamics (MD) simulations were performed to further explore the binding modes between the highest-potential compounds and ACE2. Results showed that the compounds 154-23-4 and STOCK1N-07141 possess potential ACE2 inhibition activities and deserve further study.     

Antioxidant activity of peptide-based angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) inhibitors are important for the treatment of hypertension as they can decrease the formation of vasopressor hormone angiotensin II (Ang II) and elevate the levels of vasodilating hormone bradykinin. It is observed that bradykinin contains a Ser-Pro-Phe motif near the site of hydrolysis. The selenium analogues of captopril represent a novel class of ACE inhibitors as they also exhibit significant antioxidant activity. In this study, several di- and tripeptides containing selenocysteine and cysteine residues at the N-terminal were synthesized. Hydrolysis of angiotensin I (Ang I) to Ang II by ACE was studied in the presence of these peptides. It is observed that the introduction of L-Phe to Sec-Pro and Cys-Pro peptides significantly increases the ACE inhibitory activity. On the other hand, the introduction of L-Val or L-Ala decreases the inhibitory potency of the parent compounds. The presence of an L-Pro moiety in captopril analogues appears to be important for ACE inhibition as the replacement of L-Pro by L-piperidine 2-carboxylic acid decreases the ACE inhibition. The synthetic peptides were also tested for their ability to scavenge peroxynitrite (PN) and to exhibit glutathione peroxidase (GPx)-like activity. All the selenium-containing peptides exhibited good PN-scavenging and GPx activities.     

96孔板法用于高通量血管紧张素转化酶抑制剂体外检测

为在体外迅速检测血管紧张素转化酶抑制剂( ACEI)的抑制活性,选用96孔板,以呋喃丙烯酰三肽(FAPGG)为模拟底物,通过检测血管紧张素转化酶(ACE)酶解FAPGG生成N-[3-(呋喃)丙稀醇酰-2-苯丙氨酸( FAP)和双甘氨肽(GG)后340 nm处吸光值的下降衡量ACE的活性,采用加入ACEI前后ACE的活性变化衡量ACEI的活性.考察了不同缓冲体系、Cl-浓度、ACE酶活性(ACE酶浓度)、缓冲体系的pH值等对上述检测模型反应体系的影响,建立了高通量降血压肽活性体外检测方法,本方法最多可同时检测96个样品的ACE抑制活性,上机分析时间仅需10 s.不同批次活性平行测定的相对标准偏差均小于0.001％,p=0.667＞0.05,各测定结果无显著差异,重现性好,精密度较高.采用本方法测定了商品血管紧张素转化酶抑制剂Captopril的IC50值为16.19 nmol/L,与文献报道的测定结果一致.     

Characterization of dipeptidylcarboxypeptidase of <Emphasis Type="Italic">Leishmania donovani</Emphasis>: a molecular model for structure based design of antileishmanials

Leishmania donovani dipeptidylcarboxypeptidsae (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase has been identified and characterized as a putative drug target for antileishmanial chemotherapy. The kinetic parameters for LdDCP with substrate, Hip-His-Leu were determined as, Km, 4 mM and Vmax, 1.173 μmole/ml/min. Inhibition studies revealed that known ACE inhibitors (captopril and bradykinin potentiating peptide; BPP1) were weak inhibitors for LdDCP as compared to human testicular ACE (htACE) with Ki values of 35.8 nM and 3.9 μM, respectively. Three dimensional model of LdDCP was generated based on crystal structure of Escherichia coli DCP (EcDCP) by means of comparative modeling and assessed using PROSAII, PROCHECK and WHATIF. Captopril docking with htACE, LdDCP and EcDCP and analysis of molecular electrostatic potentials (MEP) suggested that the active site domain of three enzymes has several minor but potentially important structural differences. These differences could be exploited for designing selective inhibitor of LdDCP thereby antileishmanial compounds either by denovo drug design or virtual screening of small molecule databases.     

Structure-based design and optimization of antihypertensive peptides to obtain high inhibitory potency against both renin and angiotensin I-converting enzyme

The human renin–angiotensin system (RAS) plays an essential role in regulating blood pressure and systemic vascular resistance. Renin and angiotensin I-converting enzyme (ACE) are two key enzymes in RAS and have long been recognized as attractive antihypertensive targets. Here, a synthetic strategy was proposed integrating quantitative structure–activity relationship (QSAR), molecular dynamics (MD) simulation and binding free energy analysis to discover novel dual renin and ACE peptidic inhibitors. With the strategy a number of candidates were generated virtually, from which eight promising peptides were selected and synthesized for biological assay. Consequently, three peptides (RYLP, YTAWVP and YRAWVL) were successfully identified to have satisfactory inhibitory profile against both renin and ACE with IC₅₀ values of <1 mM and <10 μM, respectively. Structural analysis and energetic dissection revealed different binding modes of peptide to renin and ACE; a peptide only inserts its C-terminus into the active site of ACE, whereas the whole peptide packs tightly against renin. In addition, when limited to structural diversity it is hard to reconcile the renin and ACE inhibitory activities of short peptides such as dipeptides. These findings can be used to guide peptide optimization with improved biological activity.     

Development of non-peptide ACE inhibitors as novel and potent cardiovascular therapeutics: An in silico modelling approach

Angiotensin-converting enzyme (ACE) inhibitors have been acknowledged as first-line agents for the treatment of hypertension and a variety of cardiovascular disorders. In this context, quantitative structure–activity relationship (QSAR) models for a series of non-peptide compounds as ACE inhibitors are developed based on Simplified Molecular Input-Line Entry System (SMILES) notation and local graph invariants. Three random splits into the training and test sets are used. The Monte Carlo method is applied for model development. Molecular docking studies are used for the final assessment of the developed QSAR model and the design of novel inhibitors. The statistical quality of the developed model is good. Molecular fragments responsible for the increase/decrease of the studied activity are calculated. The computer-aided design of new compounds, as potential ACE inhibitors, is presented. The predictive potential of the applied approach is tested, and the robustness of the model is proven using different methods. The results obtained from molecular docking studies are in excellent correlation with the results from QSAR studies. The presented study may be useful in the search for novel cardiovascular therapeutics based on ACE inhibition.     

Liquid chromatographic study of the enzymatic degradation of endomorphins, with identification by electrospray ionization mass spectrometry.

The recently discovered native endomorphins play an important role in opioid analgesia, but their metabolic fate in the organism remains relatively little known. This paper describes the application of high-performance liquid chromatography combined with electrospray ionization mass spectrometry to identify the degradation products resulting from the incubation of endomorphins with proteolytic enzymes. The native endomorphin-1, H-Tyr-Pro-Trp-Phe-NH2 (1), and endomorphin-2, H-Tyr-Pro-Phe-Phe-NH2 (2), and an analog of endomorphin-2, H-Tyr-Pro-Phe-Phe-OH (3), were synthetized, and the levels of their resistance against carboxypeptidase A, carboxypeptidase Y, aminopeptidase M and proteinase A were determined. The patterns of peptide metabolites identified by this method indicated that carboxypeptidase Y first hydrolyzes the C-terminal amide group to a carboxy group, and then splits the peptides at the Trp3-Phe4 or Phe3-Phe4 bond. The remaining fragment peptides are stable against the enzymes investigated. Carboxypeptidase A degrades only analog 3 at the Phe3-Phe4 bond. Aminopeptidase M cleaves the peptides at the Pro2-Trp3 or Pro2-Phe3 bond. The C-terminal fragments hydrolyze further, giving amino acids and Phe-NH2-s while the N-terminal part displays a resistance to further aminopeptidase M digestion. Proteinase A exhibits a similar effect to carboxypeptidase Y: the C-terminal amide group is first converted to a carboxy group, and one amino acid is then split off from the C-terminal side.     

Constrained search of conformational hyperspace

Summary We introduce a new method for determining pharmacophore or active site geometries by analysis of the structures of a series of active compounds. The method, constrained search, and the key concepts on which it is based, is described and illustrated by its application to 28 potent inhibitors of angiotensin-converting enzyme (ACE). The data set is one utilized by Mayer et al. [J. Comput.-Aided Mol. Design, 1 (1987) 3–16] to determine a unique geometry for the active site. Our experiment validated the previously reported results, obtained by a systematic search, while reducing the computer time requirement by more than two orders of magnitude. The experiment also identified a previously unrecognized alternative active site geometry for the ACE series.     

Inhibition of angiotensin-converting enzyme by peptides from the venom of the viperEchis multisquamatus

Angiotensin I-converting enzyme [dipeptidyl carboxypeptidase] (ACE, CE 3.4.15.1) isolated in the pure form from ox kidney, has been characterized physicochemically and kinetically. Inhibition of the enzyme by homogeneous peptides from the venom of the Central Asian viperEchis multisquamatus Ch. has been investigated. Institute of Biochemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 62 32 56. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 821–825, November–December, 1998.     

Microwave-assisted Solid-phase Synthesis,Biological Evaluation and Molecular Docking of Angiotensin I-converting Enzyme Inhibitors

Short peptides based on the tripeptides, Leu-Arg-Pro and Leu-Lys-Pro, were synthesized by microwaveassisted solid-phase synthesis method, in order to make a search for potential inhibitors for angiotensin I-converting enzyme(ACE) with minimum side effects in the treatment of hypertension. One peptide with the sequence Leu-Arg-Pro-Phe-Phe shows the strongest inhibition towards ACE with an IC50 value of 0.26 μmol/L in vitro. The study of structure-activity relationship shows that the introduction of a bulky group into the N-terminal of this series of inhibitors may enlarge steric hindrance, resulting in the poor inhibitory activity towards ACE. The inhibitory activity decreased in turn when L-Pro, D-Pro or Ac6c was at the C-terminal respectively. The binding interaction between each of these inhibitors and testicular ACE(tACE) was performed by molecular docking. The results suggest that Leu-Arg-Pro-Phe-Phe mainly occupied the S1 subsite of tACE, and made contact with tACE via seven H-bonds. It appeared that the site on the peptide that bound with tACE was influenced by the configuration of the amino acid, L- or D-form, at the C-terminal of the peptide.     

Phosphorus-containing derivatives of L-aspartic and L-glutamic acids

Phosphorylureas (including those with the 1,3,2-oxazaphosphinane ring) containing L-aspartate and L-glutamate fragments, as well as 1,3,2-oxazaphosphinane derivatives of phos-phorylacetic acid with an L-aspartate fragment and of phosphamide with an L-glutamate fragment, were obtained in a search for structural analogs of aspartate transcarbamoylase and glutamate carboxypeptidase inhibitors.