Angiotensin-converting enzyme inhibitors: synthesis and biological activity of N-substituted tripeptide inhibitors

A new series of highly potent angiotensin-converting enzyme (ACE) inhibitors, 1-(N2-substituted L-lysyl-gamma-D-glutamyl)octahydro-1H-indole-2-carboxylic acids, was synthesized; various acyl groups were introduced at the alpha-amino group of the N-terminal P1 Lys. The effect of the N2-acyl groups on in vitro inhibitory activity and oral antihypertensive effect was examined. All of the synthesized N-acyl tripeptides were found to have in vitro inhibitory activity at an approximately nanomolar level, and showed antihypertensive potency in renal hypertensive rats at a dose of 10 mg/kg, when administered orally. Among them, compounds 7e, g and 9f, i, m showed potent and long-lasting antihypertensive effects compared with enalapril (2a). Their structure-activity relationships are also discussed.     

Renin inhibitors. I. Synthesis and structure-activity relationships of transition-state inhibitors containing homostatine analogues at the scissile bond.

The synthesis and structure-activity relationships of transition-state renin inhibitors containing the homostatine analogues at the scissile bond are described. These inhibitors incorporate the amino acid side chains corresponding to positions 7-12 (P4-P2') of angiotensinogen. Ethyl, 2-hydroxyethyl and 3-hydroxypropyl groups at position 2 of the homostatine analogues (P1') are more effective for increasing potency than the isopropyl group. A combination of residues at P1, P3 and P4 is important for potency and this result suggests that S1, S3 and S4 form a huge hydrophobic core together in renin.     

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.     

Purification of two low-molecular-mass serine proteinase inhibitors from chicken liver.

Two serine proteinase inhibitors, designated clTI-1 and clTI-2 were purified from livers of chickens to apparent homogeneity by a combination of ethanol-acetone fractionation, gel filtration and ion-exchange chromatography on CM-cellulose and Mono S columns. The inhibitor clTI-1 is a single polypeptide chain, low-molecular-mass protein (Mr about 6200), very stable to heat and ethanol. It inhibits chicken, porcine and bovine trypsins as well as human plasmin. The second protein, clTI-2 of Mr 17,000 was shown to be a very effective inhibitor of both trypsins and human cathepsin G. Since both inhibitors are sensitive to arginine modification with phenylglyoxal it is assumed that this amino acid residue is present at the P1 position of the reactive site peptide bond. The N-terminal amino acid sequence of 28 residues of clTI-2 (SVDVSKYPSTVSKDGRTLVACPRILSPV) revealed a high homology of this protein to the third domain of the chicken ovoinhibitor, whereas, the clTI-1 (APPAAEKYYSLPPGAPRYYSPVV) has some sequence identity to a fragment of the human inter-alpha-trypsin inhibitor.     

Essential of Proline and Valine Residues in the Peptide Derived from Lactoferrin for Angiotensin Converting Enzyme Inhibition

We synthesized Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu, the peptide contained in lactoferrin (Lf), to identify the angiotensin converting enzyme (ACE) inhibition. In an attempt to know the structure‐activity relationship of this peptide, we replaced Pro (the third amino acid residues from N‐terminal) or Val (the fourth amino acid residues from N‐terminal) with Ala (neutral amino acid), Glu (acidic amino acid) or Lys (basic amino acid) to produce six peptides. From the in vitro ACE inhibition (IC₅₀) of these synthesized peptides, the original peptide (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu) showed higher ACE inhibition than the replaced six peptides. Thus, replacement of Pro at the third amino acid residues or Val at the fourth position with Ala, Glu or Lys revealed the ACE inhibition to be lower than the original form of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu. Otherwise, we added one peptide at the C‐terminal of Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu and found both products with an addition of Val (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Val) or Ile (Leu‐Arg‐Pro‐Val‐Ala‐Ala‐Glu‐Ile) showing a lower ACE inhibition than the original one. The ACE inhibitions produced by both replaced peptides were without significance. Also, deletion of the last peptide at the C‐terminal (Leu‐Arg‐Pro‐Val‐Ala‐Ala) failed to produce a marked change of ACE inhibition as compared to the original one. These results suggest that Pro and Val are essential in the peptide for inhibition of ACE activity.     

Synthesis, characterization and antioxidant activity of angiotensin converting enzyme inhibitors

Angiotensin converting enzyme (ACE) catalyzes the conversion of angiotensin I (Ang I) to angiotensin II (Ang II). ACE also cleaves the terminal dipeptide of vasodilating hormone bradykinin (a nonapeptide) to inactivate this hormone. Therefore, inhibition of ACE is generally used as one of the methods for the treatment of hypertension. 'Oxidative stress' is another disease state caused by an imbalance in the production of oxidants and antioxidants. A number of studies suggest that hypertension and oxidative stress are interdependent. Therefore, ACE inhibitors having antioxidant property are considered beneficial for the treatment of hypertension. As selenium compounds are known to exhibit better antioxidant behavior than their sulfur analogues, we have synthesized a number of selenium analogues of captopril, an ACE inhibitor used as an antihypertensive drug. The selenium analogues of captopril not only inhibit ACE activity but also effectively scavenge peroxynitrite, a strong oxidant found in vivo.     

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.     

Analysis method of the angiotensin-I converting enzyme inhibitory activity based on micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.     

Substrate specificity and novel selective inhibitors of TNF-alpha converting enzyme (TACE) from two-dimensional substrate mapping

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.     

Design, synthesis and biological evaluation of thrombin inhibitors based on a pyridine scaffold

A series of 2,4-disubstituted pyridine derivatives has been designed, synthesised and evaluated as thrombin inhibitors. A Grignard exchange reaction was used to introduce various benzoyl substituents in position 4 of the pyridine ring, where they serve as P3 residues in binding to thrombin. In position 2 of the pyridine ring, a para-amidinobenzylamine moiety was incorporated as P1 residue by an SNAr reaction using ammonia as nucleophile followed by a reductive amination. A crystal structure obtained for one of the compounds in the active site of thrombin revealed that the basic amidine group of the inhibitor was anchored to Asp 189 at the bottom of the S1 pocket. A comparison with melagatran, bound in the active site of thrombin, revealed a good shape match but lack of hydrogen bonding possibilities in the S2-S3 region for the thrombin inhibitors reported in this study.     

Discovery of novel indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors by virtual screening

In this study, a combination of virtual screening methods were utilized to identify novel potential indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors. A series of IDO1 potential inhibitors were identified by a combination of following steps: Lipinski's Rule of Five, Veber rules filter, molecular docking, HipHop pharmacophores, 3D-Quantitative structure activity relationship (3D-QSAR) studies and Pan-assay Interference Compounds (PAINS) filter. Three known categories of IDO1 inhibitors were used to constructed pharmacophores and 3D-QSAR models. Four point pharmacophores (RHDA) of IDO1 inhibitors were generated from the training set. The 3D-QSAR models were obtained using partial least squares (PLS) analyze based on the docking conformation alignment from the training set. The leave-one-out correlation (q²) and non-cross-validated correlation coefficient (r²_pred) of the best CoMFA model were 0.601 and 0.546, and the ones from the best CoMSIA model were 0.506 and 0.541, respectively. Six hits from Specs database were identified and analyzed to confirm their binding modes and key interactions to the amino acid residues in the protein. This work may provide novel backbones for new generation of inhibitors of IDO1.     

血红蛋白片段的合成及生物活性

采用多肽固相合成方法, 以Wang 树脂为载体, Fmoc为N-端氨基酸保护基, HOBt-HBTU为缩合试剂, 合成了一系列血红蛋白α链的片段, 产物经RP-HPLC和质谱进行了确定. 生物活性研究结果表明, 该系列多肽具有较高的血管紧张素Ⅰ转换酶抑制活性, 但不具有α-葡萄糖苷酶抑制活性.     

Design and synthesis of sulfur based inhibitors of matrix metalloproteinase-1

Fibroblast collagenase (MMP-1), a member of the matrix metalloproteinases family, is believed to be a pathogenesis of arthritis, by cleaving triple-helical type II collagen in cartilage. From the similarity of the active site zinc binding mode with hydroxamate, we designed and synthesized alpha-mercaptocarbonyl possessing compounds (3-5), which incorporated various peptide sequences as enzyme recognition sites. The P4-P1 peptide incorporating compound (3) exhibited as potent inhibition as the hydroxamate (1) and the carboxylate (2) type inhibitors, with an IC50 of 10(-6) M order against MMP-1. But the inhibitor (3) related compounds (6-8) displayed decreased or no inhibitory potencies. These results suggest that the existence of both the carbonyl and thiol groups might be critical for the inhibition, and the distance between the two functional groups is important for inhibitory potency. For Pn' peptide incorporating compounds (4a-k), except for 4h and 4k, all compounds showed IC50 values under sub-nanomolar. Among them, for potent inhibition, Leu was better than Phe and Val as the P1' amino acid, and the P2' position amino acid was necessary, and preferentially Phe. Insertion of the Pn peptide into 4d or 4k, giving compounds 5a-c, did not increase the activities of 4d and 4k. Substitution of the mercapto group with other functional groups lost the activity of compound 4a. The stereochemical preference at the thiol-attached position was also determined by preparation of both isomers of 4a. It was found that the S configuration compound (36b) is approximately 100 times more potent than the corresponding R-isomer (36a).     

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.     

Hot-spot guided design of macrocyclic inhibitors of the LSD1-CoREST1 interaction

New modalities such as cyclic peptides are attractive structures to inhibit challenging targets. The interaction between LSD1 and CoREST1 is required for histone demethylation and represents an attractive therapeutic target. The large interaction surface between these two proteins was analyzed by virtual alanine scanning using DrugScore PPI and a cluster of hot-spot residues was identified on CoREST1. The cluster was converted into a series of cyclic peptides and the inhibitory potency was optimized by stereochemical inversion at one of the amino acids alpha carbons combined with modification of amino acid side chains. Active peptides were further studied by variable temperature ¹H NMR and docking to evaluate the effect of conformation on binding. Potent inhibitors of the challenging PPI were obtained and will allow future optimization into more druglike structures.     

Substrate-based design of the first class of angiotensin-converting enzyme-related carboxypeptidase (ACE2) inhibitors

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.     

Mass spectral fragmentation reactions of angiotensin-converting enzyme (ACE) inhibitors

A variety of mass spectrometric techniques have been employed in the study of a series of structurally similar compounds used in the treatment of hypertension. The compounds, known collectively as angiotensin-converting enzyme (ACE) inhibitors, all share the amino acid residue proline or some variant thereof, as a common structural element. The gas phase fragmentation behavior of these compounds has been explored systematically using various instruments and techniques. An interesting dissociation process (rearrangement) unique to one of the compounds, lisinopril, has been investigated using isotopic labeling experiments and exact mass measurements. The general nature of the process has been probed through both the positive and negative ion analyses of fourteen related compounds exhibiting structural homology.     

A molecular docking model of SARS-CoV S1 protein in complex with its receptor, human ACE2

The exact residues within severe acute respiratory syndrome coronavirus (SARS-CoV) S1 protein and its receptor, human ACE2, involved in their interaction still remain largely undetermined. Identification of exact amino acid residues that are crucial for the interaction of S1 with ACE2 could provide working hypotheses for experimental studies and might be helpful for the development of antiviral inhibitor. In this paper, a molecular docking model of SARS-CoV S1 protein in complex with human ACE2 was constructed. The interacting residue pairs within this complex model and their contact types were also identified. Our model, supported by significant biochemical evidence, suggested receptor-binding residues were concentrated in two segments of S1 protein. In contrast, the interfacial residues in ACE2, though close to each other in tertiary structure, were found to be widely scattered in the primary sequence. In particular, the S1 residue ARG453 and ACE2 residue LYS341 might be the key residues in the complex formation.     

新型血管紧张素转化酶抑制剂: 氮杂三肽类似物的合成及结构活性关系

设计与合成一系列氮杂三肽类似物, 测定了氮杂三肽类似物对血管紧张素转化酶的体外抑制活性, 探讨了它们的结构与抑制活性之间的关系, 结果表明氮杂丙氨酸是丙氨酸很好的替代物。