Crystal Structures of DPP-IV (CD26) from Rat Kidney Exhibit Flexible Accommodation of Peptidase-Selective Inhibitors Kenton L. Longenecker,* ,‡ Kent D. Stewart, ‡ David J. Madar, § Clarissa G. Jakob, ‡ Elizabeth H. Fry, ‡ Sherwin Wilk, | Chun W. Lin, § Stephen J. Ballaron, § Michael A. Stashko, § Thomas H. Lubben, § Hong Yong, § Daisy Pireh, § Zhonghua Pei, § Fatima Basha, § Paul E. Wiedeman, § Thomas W. von Geldern, § James M. Trevillyan, § and Vincent S. Stoll ‡ Department of Structural Biology and Department of Metabolic Disease, Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, Abbott Park, Illinois 60064-6098, and Department of Pharmacology, Mount Sinai School of Medicine, New York, New York 10029 ReceiVed January 27, 2006; ReVised Manuscript ReceiVed April 28, 2006 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. Inhibition of the proteolytic enzyme dipeptidyl peptidase IV (DPP-IV, 1 EC 3.4.15.5) is increasingly appreciated as a potential therapeutic strategy for non-insulin-dependent (type 2) diabetes, a major world health concern (1, 2). DPP-IV is believed to be responsible for the rapid inactivation of glucagon-like peptide 1 (GLP-1), which is a naturally produced bioactive peptide that enhances insulin secretion and inhibits glucagon release upon oral ingestion of nutrients (3). The bioactivity of GLP-1 influences multiple aspects of glucose homeostasis and importantly promotes normalization of blood glucose levels in diabetic patients. The therapeutic rationale for DPP-IV inhibition is to extend the half-life of GLP-1 bioactivity and thereby improve the impaired glucose tolerance of the diabetic patient. DPP-IV may also mediate additional effects by processing other bioactive peptides such as gastric inhibitory peptide, substance P, bradykinin, neu- ropeptide Y, and various chemokines and cytokines, so the consequences of DPP-IV inhibition are of special interest in ongoing in vivo investigations (4). In addition to possessing peptidase activity, DPP-IV may impact functions of the immune system, potentially acting as a costimulatory protein on the surface of T-cells through interactions with other proteins (5). For some time, the physiological binding partner of adenosine deaminase (ADA) was ascribed to a protein of unknown sequence named CD26, but its identity with DPP-IV is now recognized. However, the functional consequences of ADA binding are uncertain and continue to be an area of ongoing research. In contrast to many mammalian species, rat DPP-IV lacks ADA binding activity and provides an important comparison to its relatives. Recent structural results on complexes of DPP-IV and ADA have defined the interacting surfaces and suggest structural differences for the functional variation among species (6, 7). Many physical characteristics underlying the function of DPP-IV (CD26) are known (8). DPP-IV is found in two forms in the body, consisting of a membrane-bound form that is tethered to extracellular surfaces and a soluble form that circulates in the plasma. The immobilized form is tethered by an N-terminal peptide sequence of ∼35 residues that upon cleavage yields the soluble form. DPP-IV belongs * To whom correspondence should be addressed: Department of Structural Biology, R46Y, Bldg. AP10, 100 Abbott Park Rd., Abbott Park, IL 60064. Phone: (847) 938-1428. Fax: (847) 937-2625. E-mail: Kenton.Longenecker@Abbott.com. ‡ Department of Structural Biology, Global Pharmaceutical Research and Development, Abbott Laboratories. § Department of Metabolic Disease, Global Pharmaceutical Research and Development, Abbott Laboratories. | Mount Sinai School of Medicine. 1 Abbreviations: GLP-1, glucagon-like peptide 1; DPP-IV, dipeptidyl peptidase IV; ADA, adenosine deaminase; POP, prolyl oligopeptidase; rmsd, root-mean-square deviation. 7474 Biochemistry 2006, 45, 7474-7482 10.1021/bi060184f CCC: $33.50 © 2006 American Chemical Society Published on Web 05/26/2006