In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC₅₀ 216 μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC₅₀ values of 3.5 and 44.2 μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.     

Evaluating Khaya senegalensis for Dipeptidyl Peptidase-IV Inhibition Using in Vitro Analysis and Molecular Dynamic Simulation of Identified Bioactive Compounds

The dipeptidyl peptidase-IV (DPP-IV) inhibitory activity of Khaya senegalensis extracts was evaluated. The DPP-IV from a rat kidney was purified to a purification fold of 2.3. Among extracts from K. senegalensis, the hexane extract had the best DPP-IV inhibitory activity, with IC₅₀ value of 1.56±0.61 μg/mL and was fractionated to eleven fractions (A–K). Fraction I had the best DPP-IV inhibition via uncompetitive pattern. GC-MS analysis of fraction I showed that the major bioactive compounds were 3-amino-3-hydroxyimino-N-phenylpropanamide ( 1 ) and 11-(2-cyclopenten-1-yl)undecanoic acid ( 2 ), with good binding affinities toward DPP-IV, based on molecular docking,. They were then subjected to molecular dynamic simulation using WEBGRO and utilizing a GROMACS system for 100 ns. The 3-amino-3-hydroxyimino-N-phenylpropanamide-DPP-IV complex was more stable and compact than the other complex. K. senegalensis contains compounds like 1 that might be used for the design of new DPP-IV inhibitors.     

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.     

(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.     

Identification of Novel Human Dipeptidyl Peptidase-IV Inhibitors of Natural Origin (Part II): In Silico Prediction in Antidiabetic Extracts

Background

Natural extracts play an important role in traditional medicines for the treatment of diabetes mellitus and are also an essential resource for new drug discovery. Dipeptidyl peptidase IV (DPP-IV) inhibitors are potential candidates for the treatment of type 2 diabetes mellitus, and the effectiveness of certain antidiabetic extracts of natural origin could be, at least partially, explained by the inhibition of DPP-IV.

Methodology/Principal Findings

Using an initial set of 29,779 natural products that are annotated with their natural source and an experimentally validated virtual screening procedure previously developed in our lab (Guasch et al.; 2012) [1], we have predicted 12 potential DPP-IV inhibitors from 12 different plant extracts that are known to have antidiabetic activity. Seven of these molecules are identical or similar to molecules with described antidiabetic activity (although their role as DPP-IV inhibitors has not been suggested as an explanation for their bioactivity). Therefore, it is plausible that these 12 molecules could be responsible, at least in part, for the antidiabetic activity of these extracts through their inhibitory effect on DPP-IV. In addition, we also identified as potential DPP-IV inhibitors 6 molecules from 6 different plants with no described antidiabetic activity but that share the same genus as plants with known antidiabetic properties. Moreover, none of the 18 molecules that we predicted as DPP-IV inhibitors exhibits chemical similarity with a group of 2,342 known DPP-IV inhibitors.

Conclusions/Significance

Our study identified 18 potential DPP-IV inhibitors in 18 different plant extracts (12 of these plants have known antidiabetic properties, whereas, for the remaining 6, antidiabetic activity has been reported for other plant species from the same genus). Moreover, none of the 18 molecules exhibits chemical similarity with a large group of known DPP-IV inhibitors.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human α₁-antitrypsin in double immunodiffusion. PI-1 corresponding to α₁ - antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200–300 Kd) than that of PI-1, and did not crossreact with antisera against human α₁-antitrypsin, α₂-macroglobulin, α₁-antichymotrypsin, α₂-plasmin inhibitor, inter-α-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including α₁-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Identification of Novel Human Dipeptidyl Peptidase-IV Inhibitors of Natural Origin (Part I): Virtual Screening and Activity Assays

Background

There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a) to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b) to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c) to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site.

Methodology/Principal Findings

We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity). Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives.

Conclusions/Significance

We have demonstrated that our virtual-screening protocol was successful in identifying novel lead compounds for developing more potent DPP-IV inhibitors.     

Novel trans-2-aryl-cyclopropylamine analogues as potent and selective dipeptidyl peptidase IV inhibitors

A series of trans-2-aryl-cyclopropylamine derived compounds were synthesized and evaluated their biological activities against DPP-IV. The structure-activity relationships (SAR) led to the discovery of novel series of DPP-IV inhibitors, having IC₅₀ values of <100 nM with excellent selectivity over the closely related enzymes, DPP8, DPP-II and FAP. The studies identified a potent and selective DPP-IV inhibitor 24b, which exhibited the ability to both significantly inhibit plasma DPP-IV activity in rats and improve glucose tolerance in lean mice and diet induced obese mice.     

In silico identification of novel ACE and DPP-IV inhibitory peptides derived from buffalo milk proteins and evaluation of their inhibitory mechanisms

The capacity of buffalo milk proteins to release bioactive peptides was evaluated and novel bioactive peptides were identified. The sequential similarity between buffalo milk proteins and their cow counterparts was analysed. Buffalo milk proteins were simulated to yield theoretical peptides via in silico proteolysis. The potential of selected proteins to release specific bioactive peptides was evaluated by the A value obtained from the BIOPEP–UWM database (Minkiewicz et al. in Int J Mol Sci 20(23):5978, 2019). Buffalo milk protein is a suitable precursor to produce bioactive peptides, particularly dipeptidyl peptidase IV (DPP-IV) and angiotensin I-converting enzyme (ACE) inhibitory peptides. Two novel ACE inhibitory peptides (KPW and RGP) and four potential DPP-IV inhibitory peptides (RGP, KPW, FPK and KFTW) derived from in silico proteolysis of buffalo milk proteins were screened using different integrated bioinformatic approaches (PeptideRanker, Innovagen, peptide-cutter and molecular docking). The Lineweaver–Burk plots showed that KPW (IC₅₀?=?136.28?±?10.77 μM) and RGP (104.72?±?8.37 μM) acted as a competitive inhibitor against ACE. Similarly, KFTW (IC₅₀?=?873.92?±?32.89 μM) was also a competitive inhibitor of DPP-IV, while KPW and FPK (82.52?±?10.37 and 126.57?±?8.45 μM, respectively) were mixed-type inhibitors. It should be emphasized that this study does not involve any clinical trial.

     

Discovery of conformationally rigid 3-azabicyclo[3.1.0]hexane-derived dipeptidyl peptidase-IV inhibitors

The induction of conformationally restricted N-(aryl or heteroaryl)-3-azabicyclo[3.1.0]hexane derivatives at P₂ region of compounds of 2-cyanopyrrolidine class was explored to develop novel DPP-IV inhibitors. The synthesis, structure–activity relationship, and selectivity against related proteases are delineated.     

Inhibition of anthrax lethal factor: lability of hydroxamate as a chelating group

The metalloprotease activity of lethal factor (LF) from Bacillus anthracis (B. anthracis) is a main source of toxicity in the lethality of anthrax infection. Thus, the understanding of the enzymatic activity and inhibition of B. anthracis LF is of scientific and clinical interests. We have designed, synthesized, and studied a peptide inhibitor of LF, R9LF-1, with the structure NH₂–(d-Arg)₉–Val–Leu–Arg–CO–NHOH in which the C-terminal hydroxamic acid is commonly used in the inhibitors of metalloproteases to chelate the active-site zinc. This inhibitor was shown to be very stable in solution and effectively inhibited LF in kinetic assays. However, its protection on murine macrophages against lethal toxin’s lysis activity was relatively weak in longer assays. We further observed that the hydroxamic acid group in R9LF-1 was hydrolyzed by LF, and the hydrolytic product of this inhibitor is considerably weaker in inhibition of potency. To resist this unique hydrolytic activity of LF, we further designed a new inhibitor R9LF-2 which contained the same structure as R9LF-1 except replacing the hydroxamic acid group with N,O-dimethyl hydroxamic acid (DMHA), –N(CH₃)–O–CH₃. R9LF-2 was not hydrolyzed by LF in long-term incubation. It has a high inhibitory potency vs. LF with an inhibition constant of 6.4 nM had a better protection of macrophages against LF toxicity than R9LF-1. These results suggest that in the development of new LF inhibitors, the stability of the chelating group should be carefully examined and that DMHA is a potentially useful moiety to be used in new LF inhibitors.     

Pharmacophore modeling and 3D-QSAR analysis of phosphoinositide 3-kinase p110α inhibitors

Pharmacophore modeling studies were undertaken for a series of compounds belonging several groups of phosphoinositide 3-kinase (PI3K) p110α inhibitors: 4-morpholino-2-phenylquinazolines derivatives, pyrido[3′,2′:4,5]furo-[3,2-d]pyrimidine derivatives, imidazo[1,2-a]pyridine derivatives, sulfonylhydrazone substituted imidazo[1,2-a]pyridines, and LY294002. A five-point pharmacophore with three hydrogen bond acceptors (A), one hydrophobic group (H), and one aromatic ring (R) as pharmacophore features was developed. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of R ² = 0.95 for training set compounds. The model generated showed excellent predictive power, with a correlation coefficient of Q ² = 0.88 and r _pret² = 0.95 for a test set of 14 compounds. Furthermore, the structure–activity relationships of PI3K p110α inhibitors were elucidated and the activity differences between them discussed. Docking studies were also carried out wherein active and inactive compounds were docked into the active site of the PI3K p110α crystal structure to analyze PI3K p110α–inhibitor interactions. The results provide insights that will aid optimization of these classes of PI3K p110α inhibitors for better activity, and may prove helpful for further lead optimization and virtual screening.     

Structure based virtual screening of MDPI database: Discovery of structurally diverse and novel DPP-IV inhibitors

Inhibition of dipeptidyl peptidase IV (DPP-IV) has been emerged as a promising approach for the treatment of type 2 diabetes (T2D). Structure based virtual screening (SBVS) of Molecular Diversity Preservation International (MDPI) database was performed using Glide and Gold against DPP-IV enzyme. Six promising hits were identified and tested for DPP-IV inhibition. Three compounds were found to be active at low micromolar concentration. The 3-(1-hydrazinyl-1-(phenylamino)ethyl)-4-hydroxy-1-methylquinolin-2(1H)-one (compound A) was found to be the most potent hit with an IC₅₀ of 0.73 μM. These three compounds (A, B and D) were then assessed for their glucose lowering effects in glucose fed hyperglycemic female Wistar rats. The glucose lowering effects of compounds also confirms their potential as anti-diabetic agents. The present study demonstrates a successful utilization of in silico SBVS tools in identification of novel and potential DPP-IV inhibitor.     

Discovery of β-aminoacyl containing thiazolidine derivatives as potent and selective dipeptidyl peptidase IV inhibitors

A series of β-aminoacyl containing thiazolidine derivatives was synthesized and evaluated for their ability to inhibit DPP-IV. Several thiazolidine derivatives with an acid moiety were found to be potent DPP-IV inhibitors. Among them, compound 2da is the most active in this series with an IC₅₀ value of 1 nM, and it showed excellent selectivity over DPP-IV related enzymes including DPP-2, DPP-8, and DPP-9. Compound 2da is chemically and metabolically stable, and showed no CYP inhibition, hERG binding or cytotoxicity. Compound 2db, an ester prodrug of 2da, showed good in vivo DPP-IV inhibition after oral administration in rat and dog models.     

Gibberellin-ethylene interaction controls radial expansion in citrus roots

The involvement of gibberellins (GAs) and ethylene in the process of root radial expansion was studied in young seedlings of Carrizo citrange [Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.]. The GA inhibitors cycocel, paclobutrazol, and tetcyclacis enhanced radial expansion of the root tip (up to 2.3-fold) as a result of increases in stele diameter and inner cortex width. The GA deficiency increased cell number and width, and changed the polarity of growth, generating wider and shorter cortical cells in the elongation zone. In the presence or absence of GA inhibitors, GA₃ decreased root tip width and reduced all parameters related to radial expansion. The ethylene inhibitors (aminooxyacetic acid; cobalt ions, CoCl₂; silver thiosulfate) suppressed swelling induced by GA deficiency, generating thinner cells just as GA₃ did. In contrast to GA₃, ethylene inhibitors produced longer cells strongly resembling those of the untreated seedlings. Ethylene released by ethephon did not modify root tip width in control plants, while root diameter behind the root tip was increased. In the presence of low and ineffective concentrations of cycocel, the ethylene precursor 1-aminocyclopropane-1-carboxylic acid increased radial expansion of root tips (1.3-fold) and changed the polarity of growth, producing wider and shorter inner cortical cells as GA inhibitors did. These observations imply, first, that ethylene is the hormonal effector of the process of root radial expansion and, second, that the endogenous GAs modulate the promotive response of ethylene. Received: 4 October 1996 / Accepted: 25 December 1996     

In silico QSAR studies of anilinoquinolines as EGFR inhibitors

Members of the epidermal growth factor receptor (EGFR) family of proteins are frequently overactive in solid tumors. A relatively new therapeutic approach to inhibit the kinase activity is the use of ATP-competitive small molecules. In silico techniques were employed to identify the key interactions between inhibitors and their protein receptors. A series of EGFR inhibitory anilinoquinolines was studied within the framework of hologram quantitative structure activity relationship (HQSAR), density functional theory (DFT)-based QSAR, and three-dimensional (3D) QSAR (CoMFA/CoMSIA). The HQSAR analysis implied that substitutions at certain sites on the inhibitors play an important role in EGFR inhibition. DFT-based QSAR results suggested that steric and electronic interactions contributed significantly to the activity. Ligand-based 3D-QSAR and receptor-guided 3D-QSAR analyses such as CoMFA and CoMSIA techniques were carried out, and the results corroborated the previous two approaches. The 3D QSAR models indicated that steric and hydrophobic interactions are dominant, and that substitution patterns are an important factor in determining activity. Molecular docking was helpful in identifying a bioactive conformer as well as a plausible binding mode. The docked geometry-based CoMFA model with steric and electrostatic fields effect gave q ² = 0.66, r ² = = 0.94 with r ² _predictive = 0.72. Similarly, CoMSIA with hydrophobic field gave q ² = 0.59, r ² = 0.85 with r ² _predictive = 0.63. Bulky groups around site 3 of ring “C”, and hydrophilic and bulky groups at position 6 of ring “A” are desirable, with a hydrophobic and electron-donating group at site 7 of ring “A” being helpful. Accordingly, potential EGFR inhibitors may be designed by modification of known inhibitors.     

New Antidiabetic Targets of α-Glucosidase Inhibitory Peptides,SVPA, SEPA,STYV and STY: Inhibitory Effects on Dipeptidyl Peptidase-IV and Lipid Accumulation in 3T3-L1 Differentiated Adipocytes with Scavenging Activities Against Methylglyoxal and Reactive Oxygen Species

Type 2 diabetes mellitus (T2DM) is a multifactorial disease that requires multiple therapeutic strategies for its management. Bioactive peptides with multiple anti-diabetic targets are attractive therapeutic molecules. The present study was conducted to identify additional anti-diabetic targets of α-glucosidase inhibitory peptides, SVPA, SEPA, STYV, and STY. The α-glucosidase inhibitory activity of the peptides was in the order STYV?>?STY?>?SEPA?>?SVPA while molecular docking against human dipeptidyl peptidase IV (DPP-IV) showed that SVPA had the best binding affinity. In contrast, in vitro studies indicated that SEPA had a significantly higher (P?<?0.05) DPP-IV inhibitory activity (IC₅₀?=?5.78?±?0.19 mM) than other peptides. SVPA and SEPA showed mixed inhibition pattern while STYV and STY were uncompetitive inhibitors of the enzyme. IPI (diprotin A), STYV and STY were not cytotoxic while SEPA displayed some cytotoxicity. In differentiated 3T3-L1 adipocytes, SVPA and STYV were found to induce a significant (P?<?0.05) decrease in intracytoplasmic lipid accumulation when added during the differentiation process while STY, GSH and IPI caused significant reduction (P?<?0.05) in the lipid accumulation when added after the differentiation. The SVPA, SEPA and STYV were better scavengers of methylglyoxal than STY but STYV had the best scavenging activities toward reactive oxygen species and nitric oxide. It was concluded that the four α-glucosidase inhibitory peptides including IPI have multiple targets against type T2DM but, overall, of the four peptides evaluated, STYV tends to have better potential for application as a multifunctional anti-diabetic peptide.