Crystal Structures of DPP-IV (CD26) from Rat Kidney Exhibit Flexible Accommodation of Peptidase-Selective Inhibitors

Abstract

Dipeptidyl peptidase IV (DPP-IV) belongs to a family of serine peptidases, and due to its indirect regulatory role in plasma glucose modulation, DPP-IV has become an attractive pharmaceutical target for diabetes therapy. DPP-IV inactivates the glucagon-like peptide (GLP-1) and several other naturally produced bioactive peptides that contain preferentially a proline or alanine residue in the second amino acid sequence position by cleaving the N-terminal dipeptide. To elucidate the details of the active site for structure-based drug design, we crystallized a natural source preparation of DPP-IV isolated from rat kidney and determined its three-dimensional structure using X-ray diffraction techniques. With a high degree of similarity to structures of human DPP-IV, the active site architecture provides important details for the design of inhibitory compounds, and structures of inhibitor-protein complexes offer detailed insight into three-dimensional structure-activity relationships that include a conformational change of Tyr548. Such accommodation is exemplified by the response to chemical substitution on 2-cyanopyrrolidine inhibitors at the 5 position, which conveys inhibitory selectivity for DPP-IV over closely related homologues. A similar conformational change is also observed in the complex with an unrelated synthetic inhibitor containing a xanthine core that is also selective for DPP-IV. These results suggest the conformational flexibility of Tyr548 is unique among protein family members and may be utilized in drug design to achieve peptidase selectivity.

Dipeptidyl Aminopeptidase IV fromStenotrophomonas maltophiliaExhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

“…4. The position of the catalytic triad, Ser610, Asp685, and His717, was found to be conserved in enzymes belonging to the POP family (14,18,21,35,47), and the active-site serine was also found to have the disallowed conformation in the Ramachandran plot. The Tyr524 residue corresponds to Tyr547 in porcine and human DPIVs, and this residue functions as an oxyanion hole (6).…”

“…Glu206 and Glu207 residues were found on the ␣ 2 helix. These glutamate residues function in the recognition of the N-terminal amino group of a substrate (14,21,35,47). The ␤-propeller domain forms a funnel shape with a solvent-filled tunnel at the center of eight blades.…”

“…DPIV has also been identified as CD26 antigen, a surface differentiation marker involved in the transduction of mitogenic signals in thymocytes and T lymphocytes in mammals (7,16,55) and in cell matrix adhesion through specific interactions with fibronectin and collagen (4,46). The crystal structures of human, porcine, and rat DPIV enzymes have been determined (14,21,35,47). Although DPIV enzymes from bacteria and fungi, including Flavobacterium meningosepticum (56), Bacteroides fragilis (32), Prevotella intermedia (50), Porphyromonas gingivalis (11), Trichophyton rubrum (41), Lactobacillus helveticus (37), Aspergillus fumigatus (5), Aspergillus oryzae (52), etc., have been identified, no structural information has been reported regarding microbial DPIV.…”

Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase

“…Type A (Fig. 7(a)) contains all enzymes with a tyrosine OH contributing to the oxyanion site, even though the amino acid sequences of the four-residue motifs are different: YAGP for dipeptidyl-peptidase IV (Engel et al, 2003;Hiramatsu et al, 2003;Longenecker et al, 2006), YGGF for prolyl-oligopeptidase (Fü löp et al, 1998;Shan et al, 2005), and YGGP for fibroblast activation protein a (Aertgeerts et al, 2005) and prolyl tripeptidyl aminopeptidase (Ito et al, 2006). In these structures a b-turn following the four-residue motif helps to position the tyrosine.…”

Structural Chemistry and Molecular Modeling in the Design of DPP4 Inhibitors

“…Thus, potential for off-target toxicity remained an issue that needed to be addressed in the development of suitable back-up compounds. This point was addressed by utilizing information from a combination of internal and external sources: modeling [15], 3D-QSAR approaches [11] and enzymes structure/activity analysis [16][17][18]. This resulted in several back-ups molecules being synthesized and tested, up to Sitagliptin approval.…”