Inhibition of Dipeptidyl Peptidase-4 by Flavonoids: Structure–Activity Relationship, Kinetics and Interaction Mechanism

Abstract

With the aim to establish a structure-inhibitory activity relationship of flavonoids against dipeptidyl peptidase-4 (DPP-4) and elucidate the interaction mechanisms between them, a pannel of 70 structurally diverse flavonoids was used to evaluate their inhibitory activities against DPP-4, among which myricetin, hyperoside, narcissoside, cyanidin 3-O-glucoside, and isoliquiritigenin showed higher inhibitory activities in a concentration-dependent manner. Structure-activity relationship analysis revealed that introducing hydroxyl groups to C3', C4', and C6 of the flavonoid structure was beneficial to improving the inhibitory efficacy against DPP-4, whereas the hydroxylation at position 3 of ring C in the flavonoid structure was unfavorable for the inhibition. Besides, the methylation of the hydroxyl groups at C3', C4', and C7 of the flavonoid structure tended to lower the inhibitory activity against DPP-4, and the 2,3-double bond and 4-carbonyl group on ring C of the flavonoid structure was essential for the inhibition. Glycosylation affected the inhibitory activity diversely, depending on the structure of flavonoid aglycone, type of glycoside, as well as the position of substitution. Inhibition kinetic analysis suggested that myricetin reversibly inhibited DPP-4 in a non-competitive mode, whereas hyperoside, narcissoside, cyanidin 3-O-glucoside, and isoliquiritigenin all reversibly inhibited DPP-4 in a mixed type. Moreover, the fluorescence quenching analysis indicated that all the five flavonoid compounds could effectively quench the intrinsic fluorescence of DPP-4 by spontaneously binding with it to form an unstable complex. Hydrogen bonds and van der Waals were the predominant forces to maintain the complex of myricetin with DPP-4, and electrostatic forces might play an important role in stabilizing the complexes of the remaining four flavonoids with DPP-4. The binding of the tested flavonoids to DPP-4 could also induce the conformation change of DPP-4 and thus led to inhibition on the enzyme. Molecular docking simulation further ascertained the binding interactions between DPP-4 and the selected five flavonoids, among which hyperoside, narcissoside, cyaniding 3-O-glucoside, and isoliquiritigenin inserted into the active site cavity of DPP-4 and interacted with the key amino acid residues of the active site, whereas the binding site of myricetin was located in a minor cavity close to the active pockets of DPP-4.

Natural products from plants targeting key enzymes for the future development of antidiabetic agents

“…Representative DPP-IV inhibitory avonoids (1-72) and other aromatic compounds (73-100) are summarized in Table 1. Perhaps, one of the most extensive analyses of the effect of avonoids on DPP-IV was carried out by Pan et al, 44 who analyzed 70 different avonoids of different structural groups using a human recombinant enzyme and monitoring the reaction by measuring the uorescence generated by the substrate Gly-Pro-7-amido-4-methyl coumarin hydrobromide.…”

“…They also demonstrated that the uorometric method is more sensitive than the colorimetric. 39,72 Regrettably, unlike Proença et al, 39 Pan et al, 44 did not include the results for any positive control in the study; therefore, potency comparisons between such control and the tested avonoids cannot be performed. In the study conducted by the former authors, natural avonoids did not reach 50% inhibition at 200 mM and were thus less effective and potent than positive controls assayed (sitagliptin and diprotin A with IC 50 values of 0.064 and 31 mM, respectively).…”

“…Review Natural Product Reports Some of the DPP-IV avonoid inhibitors affect glucose homeostasis through different mechanisms according to the human, animal, and in vitro studies. 7,24,31,37,73 For example, anthocyanins, in general, modify the activities of glucose transporter-4 (GLUT-4), SGLTs, a-glucosidase, protein tyrosine phosphatase 1B (PTP-1B), GLP-1/GLP-1R and peroxisome proliferator-activated receptor-g. 44,74 Catechins decrease blood glucose levels by activating glucose transporters and the insulin receptor. Some avanones and avones, such as hesperidin (20) and genistein (35) control blood glucose levels by acting through different mechanisms.…”

“…Representative DPP-IV inhibitory flavonoids ( 1–72 ) and other aromatic compounds ( 73–100 ) are summarized in Table 1. 42–71 …”

Insight into the Role of Active Site Protonation States and Water Molecules in the Catalytic Inhibition of DPP4 by Vildagliptin

“…These residues are within a 10 Å radius from the catalytic triad and could play a relevant role in controlling the noncovalent binding and catalytic properties of DPP4 due to differential electrostatic effects arising from their possible protonation forms (Figure A). , His126 is part of the S2 recognition domain of DPP4 and has been proposed to play a structuring role by engaging in hydrogen bonding interactions with the substrate anchoring residue Glu205 (Figure B) . His126 has also been proposed as a binding site residue for noncovalent DPP4 inhibitors. − Asp663 also belongs to the S2 recognition subsite and is responsible for the differential substrate specificity and enzyme activity between DPP4 and the homologous fibroblast activation protein α (FAP) . This residue locates nearby the anionic side chain of Glu206 and could form a hydrogen bond that has been associated with the appearance of downfield resonance signals at ∼16 ppm in native and mutant forms of DPP4 in ligand-free and complex states with covalent and noncovalent inhibitors (Figure C) .…”