Pharmacology, physiology, and mechanisms of action of dipeptidyl peptidase-4 inhibitors

Abstract

Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane molecule and a soluble circulating protein. Both membrane-associated and soluble DPP4 exert catalytic activity, cleaving proteins containing a position 2 alanine or proline. DPP4-mediated enzymatic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the molecular mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biology of DPP4 with a focus on: 1) identification of pharmacological vs physiological DPP4 substrates; and 2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chemical reduction of DPP4 activity. We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4 inhibitors in both preclinical and clinical studies. Finally, we highlight experimental pitfalls and technical challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.

Figure 1

Membrane-bound DPP4 contains residues 1–766, whereas sDPP4 contains residues 39–766. sDPP4 is lacking the cytoplasmic domain [residues 1–6], transmembrane domain [residues 7–28], and the flexible stalk [residues 29–39]. Both membrane-bound and circulating sDPP4 share many domains including the glycosylated region [residues 101–535, specific residues 85, 92, 150], ADA binding domain [340–343], fibronectin binding domain [468–479], cysteine-rich domain [351–506, disulfide bonds are formed from 385–394, 444–472, and 649–762], and the catalytic domain [507–766 including residues composing the catalytic active site 630, 708, and 740].

Figure 3

DPP4 cleavage regulates substrate/receptor interactions. A, DPP4 cleaves NPY [1–36] and PYY [1–36]. The intact forms of these peptides signal through Y1R-Y5R. After DPP4 cleavage, NPY [3–36] and PYY [3–36] are generated and preferentially signal through the Y2R and Y5R. B, DPP4 cleaves SP [1–11], which signals through the NK1R receptor to generate SP [5–11], which can signal through (NK1R, -2R, -3R).

Figure 2

Experimental paradigms for identification of DPP4 substrates. A, In vitro pharmacology. Cells expressing DPP4, serum, or recombinant/isolated DPP4 are utilized as an enzyme source and incubated with putative DPP4 substrates in vitro, after which the extent of truncation of the peptide at the penultimate residue can be analyzed. B, In vivo pharmacology. Pharmacological concentrations of peptides of interest are infused into wild-type mice or rats or Dpp4^−/− mice, Fischer 344 rats, or animals or humans treated with a selective DPP4 inhibitor, and quantification of relative levels of intact vs cleaved peptide are monitored. C, In vivo physiology. Identification of endogenous physiological peptide substrates. In this paradigm, intact peptides and DPP4-cleaved peptides are detected at different levels in wild-type vs Dpp4 knockout animals or in animals or humans treated with a selective DPP4 inhibitor.

Figure 4

Endocrine pathways altered during DPP4 inhibition. Inhibition of DPP4 protease activity with selective DPP inhibitors produces multiple biological actions in peripheral tissues. The peptide substrates that have been identified as downstream targets mediating key cardiometabolic actions of DPP4 are indicated.