Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity

Quantitative structure activity type models were developed in an attempt to predict the key features of peptide sequences having dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The models were then employed to help predict the potential of peptides, which are currently reported in the literature to be present in the intestinal tract of humans following milk/dairy product ingestion, to act as inhibitors of DPP-IV. Two models (z- and v-scale) for short (2–5 amino acid residues) bovine milk peptides, behaving as competitive inhibitors of DPP-IV, were developed. The z- and the v-scale models (p < 0.05, R² of 0.829 and 0.815, respectively) were then applied to 56 milk protein-derived peptides previously reported in the literature to be found in the intestinal tract of humans which possessed a structural feature of DPP-IV inhibitory peptides (P at the N₂ position). Ten of these peptides were synthetized and tested for their in vitro DPP-IV inhibitory properties. There was no agreement between the predicted and experimentally determined DPP-IV half maximal inhibitory concentrations (IC₅₀) for the competitive peptide inhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitors was conserved. Furthermore, potent in vitro DPP-IV inhibitory activity was observed with two peptides, LPVPQ (IC₅₀ = 43.8 ± 8.8 μM) and IPM (IC₅₀ = 69.5 ± 8.7 μM). Peptides present within the gastrointestinal tract of human may have promise for the development of natural DPP-IV inhibitors for the management of serum glucose.     

Comparison of the susceptibility of porcine and human dipeptidyl-peptidase IV to inhibition by protein-derived peptides

The enzyme dipeptidyl-peptidase IV (DPP-IV) is recognized to be a promising target for the management of type 2 diabetes. Over the last decade, numerous synthetic molecules and more recently, peptides from dietary proteins, have been reported to be able to inhibit DPP-IV activity. Most studies that have investigated the in vitro effect of these inhibitors have used porcine or human DPP-IV. Although structurally alike, it is unclear whether these two species display similar inhibition patterns. Therefore, the objective of this study was to compare the effects of protein-derived peptides on the activity of porcine and recombinant human DPP-IV. The two species showed different inhibition susceptibility to 43 of the 62 peptide sequences investigated. While 37 protein-derived peptides were more effective at inhibiting the porcine DPP-IV, only six caused a stronger inhibition of the activity of the human enzyme. Although the peptides WR, IPIQY and WCKDDQNPHS were found to be among the most potent inhibitors of both species, the inhibitory effect was greater on the porcine enzyme than on human DPP-IV (αK_i or K_i = 11.5, 13.4, 13.3 μM and 31.4, 28.2, 75.0 μM for porcine and human DPP-IV, respectively). Investigation into the mode of action of the most effective inhibitory peptides revealed that both species were inhibited in a similar manner by short fragments (≤5 amino acid residues), but that some of the longer peptides acted differently on the enzymes. This study shows that porcine DPP-IV is generally inhibited with greater potency by protein-derived peptides than is the human enzyme.     

Molecular mechanisms of novel peptides from silkworm pupae that inhibit α-glucosidase

The objectives of this study were to identify peptides that inhibit α-glucosidase using a quantitative structure-activity relationship (QSAR) screening method and a database of silkworm peptides. This study compared the docking characteristics of several peptides with high inhibitory activity against α-glucosidase and summarized the molecular mechanisms by which the silkworm peptides affected α-glucosidase. Four peptides that strongly inhibited α-glucosidase were obtained: Gln-Pro-Gly-Arg with IC₅₀ at 65.8 μmol/L, Ser-Gln-Ser-Pro-Ala at 20 μmol/L, Gln-Pro-Pro-Thr at 560 μmol/L and Asn-Ser-Pro-Arg at 205 μmol/L. Studies docking the peptides to the active site of α-glucosidase (PDB ID: 2QMJ) showed that a common characteristic was Lys776 in 2QMJ, which could be a critical target for α-glucosidase trapping of inhibitory peptides. The results revealed that the four peptides, especially Ser-Gln-Ser-Pro-Ala, could be potential drugs for treating diabetes.     

LuxR-dependent quorum sensing: Computer aided discovery of new inhibitors structurally unrelated to N-acylhomoserine lactones

A virtual screening, involving flexible docking sequences within the LuxR, TraR and LasR binding sites, was used as a structural binding sites similarity filter to specifically target conserved residues in the proteins of the LuxR family (namely Tyr62, Trp66, Tyr70, Asp79, Trp94 for LuxR). This docking-based screening, employing a genetic algorithm, was performed on a 2344 chemical compounds library, together with empirical binding free energy (ΔG_bind) calculations. Docking results were analysed, and the compounds detected with reproducible low ΔG_bind values or identified as being in the top 120 for most of the docking sequences, were selected as hits candidates which interact with conserved residues. Biological evaluation with LuxR-dependent quorum sensing led to the discovery of some new inhibitors, namely tamoxifen, sertraline, pimethixene, terfenadine, fendiline and calmidazolium. Notably, calmidazolium was identified as one of the most potent AHL-structurally unrelated inhibitors of LuxR-dependent quorum sensing, with an IC₅₀ value of 7.0 ± 0.2 μM.     

Characterisation of novel angiotensin-I-converting enzyme inhibitory tripeptide,Gly-Val-Arg derived from mycelium of Pleurotus pulmonarius

It has been shown that fraction D₆ from Pleurotus pulmonarius has the potential to inhibit ACE. After this discovery, additional studies were conducted to obtain peptides from that fraction, as ACE inhibitors. By size exclusion chromatography, single peak was resolved and termed as Psec. The IC₅₀ of Psec was assessed to be 4.50 μg/mL, which was 2.5 times lower than that of D₆. When Psec was resolved by SDS-PAGE, three bands with estimated molecular weight of 63 kDa, 55 kDa and 11 kDa were observed. The protein bands were subjected to MALDI-Tof MS/MS for protein identification. By using the BIOPEP database for predicting in silico digestion of gastrointestinal (GI) enzymes, four stable tripeptides with ACE inhibitor potential resulting from GI enzyme digestion were identified, namely GVR, VVR, NPR, and VVL. The IC₅₀ was estimated to be 55 μg/mL, 93 μg/mL, 110 μg/mL and >250 μg/mL individually. Based on a Lineweaver-Burk plot, tripeptide GVR was determined to be a competitive inhibitor and this was confirmed by molecular docking analysis. At 100 mg/kg of body weight (bw), the tripeptide GVR reduced SBP 33.5 mm Hg in SHRs. The results suggested that this tripeptide is potentially beneficial as an antihypertensive agent.     

(2S,4S)-1-[2-(1,1-Dimethyl-3-oxo-3-pyrrolidin-1-yl-propylamino)acetyl]-4-fluoro-pyrrolidine-2-carbonitrile: A potent,selective, and orally bioavailable dipeptide-derived inhibitor of dipeptidyl peptidase IV

A series of 2-[3-[2-[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethylamino]-3-methyl-1-oxobutyl]-based DPP-IV inhibitors with various monocyclic amines were synthesized. The structure–activity relationships (SAR) led to the discovery of potent DPP-IV inhibitors, having IC₅₀ values of <100 nM with excellent selectivity over the closely related enzymes, DPP-II, DPP8, DPP9 and FAP (IC₅₀ > 20 μM). Of these compounds, the analogues 12a, 12h and 12i exhibited a long-lasting ex vivo DPP-IV inhibition in rats.     

Purification and molecular docking study of angiotensin I-converting enzyme (ACE) inhibitory peptides from hydrolysates of marine sponge Stylotella aurantium

Angioteinsin I-converting enzyme (ACE) inhibitory peptide was isolated from marine sponge (Stylotella aurantium) hydrolysate prepared by various hydrolysis enzymes. The peptic hydrolysate exhibited highest ACE inhibitory activity among them and was fractionated into three ranges of molecular weight. The below 5 kDa fraction showed the highest ACE inhibitory activity and was used for subsequent purification steps. The amino acid sequences of the purified peptides were identified to be Tyr-Arg (337.2 Da), and Ile-Arg (287.2 Da). The purified peptides from marine sponge had an IC₅₀ value of 237.2 μM and 306.4 μM, respectively. The molecular docking study revealed that ACE inhibitory activity of the purified peptides was mainly attributed to the hydrogen bond interactions and Pi interaction between the dipeptides and ACE. The results suggest that marine sponge, S. aurantium would be an attractive raw material for the manufacture of anti-hypertensive nutraceutical ingredients.     

The inhibitory activity of HL-7 and HL-10 peptide from scorpion venom (Hemiscorpius lepturus) on angiotensin converting enzyme: Kinetic and docking study

The hypertension is one of the highest risk factors for stroke, myocardial infarction, vascular disease and chronic kidney disease. Angiotensin converting enzyme (ACE) has an important role in the physiological regulation of cardiovascular system. ACE inhibition is a key purpose for hypertension treatment. In this study, two peptides named HL-7 with the sequence of YLYELAR (MW: 927.07 Da) and HL-10 with the sequence of AFPYYGHHLG (MW: 1161.28 Da) were identified from scorpion venom of H. lepturus. The inhibitory activity of HL-7 and HL-10 was examined on rabbit ACE. The inhibition mechanisms were assayed by kinetic and docking studies. The IC₅₀ values for ACE inhibition of HL-7 and HL-10 were 9.37 µM and 17.22 µM, respectively. Lineweaver-Burk plots showed that two peptides inhibited rabbit ACE with competitive manner. The molecular docking conformed experimental results and showed that the two peptides interacted with N-domain and C-domain active sites. Also, docking study revealed that the two peptides can form hydrogen and hydrophobic bonds at their binding sites. Both peptides had higher affinity to N-domain. Our results showed that HL-7 exhibited more strong interactions with amino acids at active site. It seems that HL-10 peptide could occupy more space, thereby inhibiting the substrate entrance to active site.     

Prenylated xanthones from mangosteen as promising cholinesterase inhibitors and their molecular docking studies

Garcinia mangostana is a well-known tropical plant found mostly in South East Asia. The present study investigated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of G. mangostana extract and its chemical constituents using Ellman's colorimetric method. Cholinesterase inhibitory-guided approach led to identification of six bioactive prenylated xanthones showing moderate to potent cholinesterases inhibition with IC₅₀ values of lower than 20.5 μM. The most potent inhibitor of AChE was garcinone C while γ-mangostin was the most potent inhibitor of BChE with IC₅₀ values of 1.24 and 1.78 μM, respectively. Among the xanthones, mangostanol, 3-isomangostin, garcinone C and α-mangostin are AChE selective inhibitors, 8-deoxygartanin is a BChE selective inhibitor while γ-mangostin is a dual inhibitor. Preliminary structure-activity relationship suggests the importance of the C-8 prenyl and C-7 hydroxy groups for good AChE and BChE inhibitory activities. The enzyme kinetic studies indicate that both α-mangostin and garcinone C are mixed-mode inhibitors, while γ-mangostin is a non-competitive inhibitor of AChE. In contrast, both γ-mangostin and garcinone C are uncompetitive inhibitors, while α-mangostin is a mixed-mode inhibitor of BChE. Molecular docking studies revealed that α-mangostin, γ-mangostin and garcinone C interacts differently with the five important regions of AChE and BChE. The nature of protein–ligand interactions is mainly hydrophobic and hydrogen bonding. These bioactive prenylated xanthones are worthy for further investigations.     

Caffeic acid derivatives: A new type of influenza neuraminidase inhibitors

Recently, many natural products, especially some plant-derived polyphenols have been found to exert antiviral effects against influenza virus and show inhibitory activities on neuraminidases (NAs). In our research, we took caffeic acid which contained two phenolic hydroxyl groups as the basic fragment to build a small compound library with various structures. The enzyme inhibition result indicated that some compounds exhibited moderate activities against NA and compound 15d was the best with IC₅₀ = 7.2 μM and 8.5 μM against N2 and N1 NAs, respectively. The 3,4-dihydroxyphenyl group from caffeic acid was important for the activity according to the docking analysis. Besides, compound 15d was found to be a non-competitive inhibitor with K_i = 11.5 ± 0.25 μM by the kinetic study and also presented anti-influenza virus activity in chicken embryo fibroblast cells. It seemed promising to discover more potent NA inhibitors from caffeic acid derivatives to cope with influenza virus.     

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add l-Ala, d-Glu, meso-A₂pm or l-Lys, and d-Ala-d-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute ‘Diversity Set’ on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC₅₀ = 10 μM) and one novel MurF inhibitor (IC₅₀ = 63 μM).     

Cyclic peptides identified by phage display are competitive inhibitors of the tRNA-dependent amidotransferase of Helicobacter pylori

In Helicobacter pylori, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated Glu-tRNA^Gln and Asp-tRNA^Asn into Gln-tRNA^Gln and Asn-tRNA^Asn, respectively. In this study, we used a phage library to identify peptide inhibitors of GatCAB. A library displaying loop-constrained heptapeptides was used to screen for phages binding to the purified GatCAB. To optimize the probability of obtaining competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA^Gln, we used that purified substrate in the biopanning process of the phage-display technique to elute phages bound to GatCAB at the third round of the biopanning process. Among the eluted phages, we identified several that encode cyclic peptides rich in Trp and Pro that inhibit H. pylori GatCAB in vitro. Peptides P10 and P9 were shown to be competitive inhibitors of GatCAB with respect to its substrate Glu-tRNA^Gln, with K_i values of 126 and 392 μM, respectively. The docking models revealed that the Trp residues of these peptides form π-π stacking interactions with Tyr81 of the synthetase active site, as does the 3′-terminal A76 of tRNA, supporting their competitive behavior with respect to Glu-tRNA^Gln in the transamidation reaction. These peptides can be used as scaffolds in the search for novel antibiotics against the pathogenic bacteria that require GatCAB for Gln-tRNA^Gln and/or Asn-tRNA^Asn formation.     

Discovery of non-peptide inhibitors of Plasmepsin II by structure-based virtual screening

Plasmepsin II (PM II) is an attractive target for anti-malaria drug discovery, which involves in host hemoglobin degradation in the acidic food vacuole. In this study, we demonstrated the successful use of structure-based virtual screening to identify inhibitors of PM II from two chemical database. Five novel non-peptide inhibitors were identified and revealed moderate inhibitory potencies with IC₅₀ ranged from 4.62 ± 0.39 to 9.47 ± 0.71 μM. The detailed analysis of binding modes using docking simulations for five inhibitors showed that the inhibitors could be stabilized by forming multiple hydrogen bonds with catalytic residues (Asp 34 and Asp 214) and also with other key residues.     

SAR studies of differently functionalized chalcones based hydrazones and their cyclized derivatives as inhibitors of mammalian cathepsin B and cathepsin H

Cathepsins have emerged as potential drug targets for melanoma therapy and engrossed attention of researchers for development and evaluation of cysteine cathepsin inhibitors as cancer therapeutics. In this direction, we have designed, synthesized, and assayed in vitro a small library of 30 low molecular weight functionalized analogs of chalcone hydrazones for evaluating structure–activity relationship aspects and inhibitory potency against cathepsin B and H. The maximum inhibitory effect was exerted by chalcone hydrazones, which are open chain analogues followed by their cyclized derivatives, pyrazolines and pyrazoles. All the synthesized compounds were established as reversible inhibitors of these enzymes. Cathepsin B was selectively inhibited by the compounds in each series. Compounds 1d, 2d and 4d were recognized as most potent inhibitors of cathepsin B in this study with K_i values of 0.042 μM, 0.053 μM and 0.131 μM whereas 1b (K_i = 1.111 μM), 2b (K_i = 1.174 μM) and 4b (K_i = 1.562 μM) inhibited cathepsin H activity effectively. And, preeminent cathepsin B inhibitors were –NO₂ functionalized however, –Cl substituted moieties were the most persuasive inhibitors for cathepsin H among all the designed compounds. Molecular docking studies performed using iGemdock provided valuable insights.     

Synthesis and α-glucosidase inhibitory activity evaluation of N-substituted aminomethyl-β-d-glucopyranosides

A series of N-substituted 1-aminomethyl-β-d-glucopyranoside derivatives was prepared. These novel synthetic compounds were assessed in vitro for inhibitory activity against yeast α-glucosidase and both rat intestinal α-glucosidases maltase and sucrase. Most of the compounds displayed α-glucosidase inhibitory activity, with IC₅₀ values covering the wide range from 2.3 μM to 2.0 mM. Compounds 19a (IC₅₀ = 2.3 μM) and 19b (IC₅₀ = 5.6 μM) were identified as the most potent inhibitors for yeast α-glucosidase, while compounds 16 (IC₅₀ = 7.7 and 15.6 μM) and 19e (IC₅₀ = 5.1 and 10.4 μM) were the strongest inhibitors of rat intestinal maltase and sucrase. Analysis of the kinetics of enzyme inhibition indicated that 19e inhibited maltase and sucrase in a competitive manner. The results suggest that the aminomethyl-β-d-glucopyranoside moiety can mimic the substrates of α-glucosidase in the enzyme catalytic site, leading to competitive enzyme inhibition. Moreover, the nature of the N-substituent has considerable influence on inhibitory potency.     

Purification,modification and inhibition mechanism of angiotensin I-converting enzyme inhibitory peptide from silkworm pupa (Bombyx mori) protein hydrolysate

Angiotensin I-converting enzyme (ACE) inhibitory peptide from silkworm pupa (Bombyx mori) was purified, modified, as well as inhibition mechanism by using molecular docking analysis. Silkworm pupa protein was hydrolyzed by neutral protease and the obtained hydrolysate was subjected to various types of chromatography to acquire peptide isolate. Then the molecular mass and amino acid sequence of the peptide was determined by MALDI-TOF/TOF MS. Subsequently, thermal and digestive stability of the peptide were explored through a high temperature processing and a simulated gastrointestinal digestion. Finally, the peptide was modified to smaller peptides and investigated their potentiate activities. Results showed that the peptide from silkworm pupa was determined to be Gly-Asn-Pro-Trp-Met (603.7 Da) with IC₅₀ 21.70 μM. Stability testing showed that ACE inhibitory activities were not significantly changed at temperature from 40 to 80 °C as well as during in vitro gastrointestinal digestion. The inhibitory activity of four modified peptides were Trp-Trp > Gly-Asn-Pro-Trp-Trp > Asn-Pro-Trp-Trp > Pro-Trp-Trp, and the IC₅₀ of Trp-Trp was 10.76 μM Docking simulation revealed that the inhibitory activity was closely related to the spatial structure of peptide and zinc ions. The purified peptide and four modified peptides may be beneficial as functional food or drug for treating hypertension.     

Synthesis,biological evaluation and molecular docking studies of chromone hydrazone derivatives as α-glucosidase inhibitors

A series of chromone hydrazone derivatives 4a–4p have been synthesized, characterized by ¹H NMR and ¹³C NMR and evaluated for their in vitro α-glucosidase inhibitory activity. Out of these tested compounds, six (4a, 4b, 4d, 4j, 4o and 4p) displayed potent α-glucosidase inhibitory activity with IC₅₀ values in the range of 20.1 ± 0.19 μM to 45.7 ± 0.23 μM, as compared to the standard drug acarbose (IC₅₀ = 817.38 ± 6.27 μM). Among this series, compound 4d (IC₅₀ = 20.1 ± 0.19 μM) with 4-sulfonamide substitution at phenyl part of hydrazide was found to be the most active compound. Lineweaver-Burk plot analysis indicated that compound 4d is a non-competitive inhibitor of α-glucosidase. The binding interactions of the most active analogs were confirmed through molecular docking studies. Docking studies showed 4d are interacting with the residues Glu-276, Asp-214, Asp-349 and Arg-439 through hydrogen bonds, arene-anion and arene-cation interactions. In summary, our studies shown that these chromone hydrazone derivatives are a new class of α-glucosidase inhibitors.     

Discovery of 3,3-di(indolyl)indolin-2-one as a novel scaffold for α-glucosidase inhibitors: In silico studies and SAR predictions

3,3-Di(indolyl)indolin-2-ones 4a-4n were synthesized and evaluated for their in vitro α-glucosidase inhibitory activity. These newly synthesized compounds showed moderate to potent α-glucosidase inhibitory activity with IC₅₀ range from 5.98 ± 0.11 to 145.95 ± 0.46 μM, when compared to the standard drug acarbose. Among this series of 3,3-di(indolyl)indolin-2-ones, compound 4j (5.98 ± 0.11 μM) having a 2-fluorobenzyl group on the indole ring was found to be the most active compound. Molecular docking studies showed that compound 4j have high binding affinities with the active site of α-glucosidase enzyme through hydrogen bonds, arene-cation, π-π stacking and hydrophobic interactions. This study showed these 3,3-di(indolyl)indolin-2-ones as a new class of α-glucosidase inhibitors.     

Ionic liquid mediated synthesis of mono- and bis-spirooxindole-hexahydropyrrolidines as cholinesterase inhibitors and their molecular docking studies

One pot, three-component reaction of 1-acryloyl-3,5-bisarylmethylidenepiperidin-4-ones with isatin and sarcosine in molar ratios of 1:1:1 and 1:2:2 furnished to mono- and bis-spiropyrrolidine heterocyclic hybrids comprising functionalized piperidine, pyrrolidine and oxindole structural motifs. Both mono and bis-spiropyrrolidines displayed good inhibitory activity against acetylcholinesterase (AChE) with IC₅₀ values of 2.36–9.43 μM. For butyrylcholinesterase (BChE), mono-cycloadducts in series 8 with IC₅₀ values of lower than 10 μM displayed better inhibitory activities than their bis-cycloadduct analogs in series 9 with IC₅₀ values of 7.44–19.12 μM. The cycloadducts 9j and 8e were found to be the most potent AChE and BChE inhibitors with IC₅₀ values of 2.35 and 3.21 μM, respectively. Compound 9j was found to be competitive inhibitor of AChE while compound 8e was a mixed-mode inhibitor of BChE with calculated K_i values of 2.01 and 6.76 μM, respectively. Molecular docking on Torpedo californica AChE and human BChE showed good correlation between IC₅₀ values and free binding energy values of the synthesized compounds docked into the active site of the enzymes.     

Angiotensin I converting enzyme (ACE) inhibitory peptides from salmon byproduct protein hydrolysate by Alcalase hydrolysis

Angiotensin I converting enzyme (ACE) inhibitory peptides were produced from salmon byproduct proteins via enzymatic hydrolysis using Alcalase, flavourzyme, neutrase, pepsin, protamex and trypsin. Among them, Alcalase hydrolysate showed the highest ACE inhibitory activity, thus ACE inhibitory peptides were purified using consecutive chromatography. The purified ACE inhibitory peptides were identified to be Val-Trp-Asp-Pro-Pro-Lys-Phe-Asp (P1), Phe-Glu-Asp-Tyr-Val-Pro-Leu-Ser-Cys-Phe (P2), and Phe-Asn-Val-Pro-Leu-Tyr-Glu (P4) by time of flight-mass spectrometry/mass spectrometry (TOF-MS) analysis. The IC₅₀ values against ACE activity were 9.10 μM (P1), 10.77 μM (P2) and 7.72 μM (P4). The inhibition mode of P1, P2 and P4 was analyzed using the Lineweaver–Burk plots, demonstrating P1 to be a non-competitive inhibitor, P2 and P4 having a mixed inhibition mode. Taken together, the salmon byproduct protein hydrolysate and/or its active peptides can be used in foods for its benefits against hypertension and related diseases.