A comparative study of the binding modes of recently launched dipeptidyl peptidase IV inhibitors in the active site

https://doi.org/10.1016/j.bbrc.2013.03.010Get rights and content

Highlights

  • We analyze the co-crystal structures of the six DPP-4 inhibitors in parallel.

  • We show three binding patterns on the basis of the inhibitor’s binding subsites.

  • Binding to the S1′, S2′ or S2 extensive subsites increases inhibitory activity.

Abstract

In recent years, various dipeptidyl peptidase IV (DPP-4) inhibitors have been released as therapeutic drugs for type 2 diabetes in many countries. In spite of their diverse chemical structures, no comparative studies of their binding modes in the active site of DPP-4 have been disclosed. We determined the co-crystal structure of vildagliptin with DPP-4 by X-ray crystallography and compared the binding modes of six launched inhibitors in DPP-4. The inhibitors were categorized into three classes on the basis of their binding subsites: (i) vildagliptin and saxagliptin (Class 1) form interactions with the core S1 and S2 subsites and a covalent bond with Ser630 in the catalytic triad; (ii) alogliptin and linagliptin (Class 2) form interactions with the S1′ and/or S2′ subsites in addition to the S1 and S2 subsites; and (iii) sitagliptin and teneligliptin (Class 3) form interactions with the S1, S2 and S2 extensive subsites. The present study revealed that the additional interactions with the S1′, S2′ or S2 extensive subsite may increase DPP-4 inhibition beyond the level afforded by the fundamental interactions with the S1 and S2 subsites and are more effective than forming a covalent bond with Ser630.

Introduction

Dipeptidyl peptidase IV (DPP-4, EC 3.4.14.5) inhibitors are a new class of oral anti-hyperglycemic agents for the treatment of type 2 diabetes. The glucose lowering effect of DPP-4 inhibitors is mediated by suppressing the degradation of the incretin hormone glucagon-like peptide-1 and stimulating insulin secretion in response to increased blood glucose levels [1]. Prescriptions for recently launched DPP-4 inhibitors for type 2 diabetes have been expanding because of their high effectiveness and safety.

Among the recently marketed DPP-4 inhibitors (Table 1), vildagliptin [2], saxagliptin [3] and teneligliptin [4] are peptide mimetic compounds, which have been discovered by replacing segments of peptide-based substrates [5]. In contrast, sitagliptin [6], alogliptin [7] and linagliptin [8] are non-peptide mimetic compounds, which have been discovered by optimization of the initial lead compounds identified by random screening [5]. Therefore, their chemical structures are diverse, suggesting that each of their binding modes in DPP-4 would be unique.

DPP-4 is a highly specific serine protease that recognizes an amino acid sequence having proline or alanine at the N-terminal penultimate (P1) position and inactivates or generates biologically active peptides [9]. The amino acid sequence and three-dimensional structure of DPP-4 are well known [10], [11]. The structure comprises a β-propeller domain and a catalytic domain, which together embrace an internal cavity housing the active center. This cavity is connected to the bulk solvent by a “propeller opening” and a “side opening” [12]. The conventional hypothesis suggests that substrates and inhibitors enter or leave the active site via the side opening [12], [13].

While some comparative studies on the pharmacological effects of DPP-4 inhibitors have been reported [14], there have been no reports comparing their binding modes in DPP-4. X-ray co-crystal structures of five inhibitors, sitagliptin [6], saxagliptin [15], alogliptin [16], linagliptin [8] and teneligliptin [4], with DPP-4 were determined by each originator except vildagliptin. Because these inhibitors have diverse chemical structures, a comparative study of their binding modes in DPP-4 is of considerable interest. Although it is well known that all DPP-4 inhibitors bind to the S1 and S2 subsites in common, it has not been systematically understood whether other subsites exist and whether each inhibitor binds to these in a distinct manner. In this study, we determined the co-crystal structure of vildagliptin with DPP-4, analyzed those of the six inhibitors in parallel and studied the relationships between their binding interactions with DPP-4 and their inhibitory activity.

Section snippets

Synthesis of vildagliptin

Vildagliptin was prepared according to the method described by Villhauer et al. [2].

X-ray crystallographic studies

The protein of human DPP-4 (33-766) secreted from insect cells was purified and crystallized according to the method reported by Hiramatsu et al. [17] The protein–inhibitor complex was obtained by soaking a preformed DPP-4 crystal in the presence of vildagliptin and preserving it in liquid nitrogen for data collection at 100 K. X-ray diffraction data were collected at the High Energy Accelerator Research

Definition of subsites in the active site of DPP-4

In the active site of a protease, subsites are generally defined by the binding site of the substrate peptide [20]. The amino acids in the substrate peptide are numbered from the point of cleavage (P2, P1, P1′, P2′ …), and the protein subsites occupied by the respective amino acids are also numbered in the same fashion (S2, S1, S1′, S2′…). In the case of DPP-4, the N-terminus of the substrate peptide is recognized by Glu205 and Glu206, and Ser630 cleaves at the N-terminus penultimate position (P

Acknowledgments

The authors thank Dr. Hideo Kubodera, Dr. Okimasa Okada and Dr. Kunitomo Adachi for their helpful discussion.

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