Dipeptidyl Peptidase Inhibitors, an Emerging Drug Class for Inﬂammatory Disease?

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provided by CiteSeerX Review Dipeptidyl peptidase inhibitors, an emerging drug class for inﬂammatory disease?

Roger Yazbeck1,2, Gordon S. Howarth1,2,3 and Catherine A. Abbott1

1 School of Biological Sciences, Flinders University, Adelaide, South Australia, Australia 2 Centre for Paediatric and Adolescent Gastroenterology, Women’s and Children’s Hospital, Adelaide, South Australia, Australia 3 Discipline of Agricultural and Animal Science, School of Agriculture, Food and Wine, The University of Adelaide, South Australia, Australia

Dipeptidyl peptidase (DPP)-4 is a member of the S9b activity of DPP4, thus prolonging the insulinotrophic serine protease family, which also includes DPP8 and effects of GLP1 [5]. Altered DPP activity has been reported DPP9. DPP4 cleaves a number of regulatory factors, in a number of disease pathologies (Table 2). In vivo and in including chemokines and growth factors. DPP4 inhibi- vitro studies have described the anti-inflammatory proper- tors have recently emerged as an effective treatment ties of non-selective DPP inhibitors, including val-pyrr, Ile- option for type 2 diabetes. Early in vitro studies demon- Thia Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrroli- strated that DPP4 inhibitors inhibit T-cell proliferation dide, suggesting pharmacological application of this novel and cytokine production, leading to their investigation in drug class for inflammatory disease. This review summar- numerous pre-clinical models of inflammatory diseases, izes the application of DPP inhibitors to a range of disease including arthritis, multiple sclerosis and inflammatory settings, including the clinical success of DPP4 inhibitors bowel disease. Recent data suggest that the early DPP4- for type 2 diabetes, as well as pre-clinical studies in animal specific inhibitors might also bind DPP8 and DPP9, thus models of arthritis, multiple sclerosis (MS) and inflamma- exerting their effects through non-specific binding. This tory bowel disease (IBD). Recent insights into the selectiv- review highlights recent insights into the applicability of ity of DPP inhibitors and potential off-target inhibition are DPP inhibitors as novel pharmacological agents for discussed. inflammatory disease. The DPP family Introduction DPP4 The dipeptidyl peptidase (DPP)-4 family, also known as the DPP4 is the archetypal member of the DPP4 family. It was S9b family, contains four structurally homologous first identified in 1966 by Hopsu-Havu and Glenner as enzymes, DPP4, fibroblast activation protein (FAP), glycylproline naphthylamidase [6] and was purified from DPP8 and DPP9 [1]. Enzymes within this family have a rat liver and pig kidney in 1967 and 1968, respectively rare substrate specificity, cleaving N-terminal dipeptides [7,8]. DPP4 is a 110-kDa type-II integral membrane gly- from regulatory factors containing proline or alanine in the coprotein with ubiquitous expression. DPP4 enzyme penultimate position [2]. The most extensively studied and activity has been recorded in rats, mice and humans in well-characterized member of the DPP family is DPP4, a epithelial cells of the intestine, kidney, liver, lung, thymus, ubiquitously expressed transmembrane glycoprotein with lymph node, spleen, prostate and recently in adipocytes, as known substrates that include several growth factors, well as on activated lymphocytes and monocytes [3]. There neuropeptides and chemokines (Table 1). DPP4 is also is also a soluble form of DPP4 that is thought to result from known as the cell surface antigen CD26 and has a co- proteolytic cleavage of the membrane-bound form in the stimulatory function in the immune response [3]. There circulation [1]. DPP4 is also known as the cell surface is increased awareness of the importance of the physio- antigen CD26 and it can associate with the lymphocyte logical functions of DPP8 and DPP9 [1]. DPP2, which cell-surface molecules CD45 and adenosine deaminase to belongs to the S28 serine protease family, lacks structural have a co-stimulatory function in the immune response [3]. homology to members of the S9b family; however, it is able Resting B cells and natural killer (NK) cells express low to bind some DPP inhibitors and has been reviewed in levels of CD26 on their cell surface; however, DPP4 is detail by Maes et al. [4]. significantly upregulated in these cell types after antigenic DPP4 was initially identified as a therapeutic target for stimulation [9]. type 2 diabetes owing to its degradation of the incretin, DPP4 is homodimeric, and possesses catalytic activity glucagon-like peptide (GLP)-1. Since then, several phar- only in its dimer form. The unique crystal structure of maceutical companies have released novel inhibitors, com- DPP4 consists of two domains, an a/b-hydrolase domain monly referred to as the gliptins, that target the enzyme and an eight-blade b-propeller domain [3] (Figure 1). The b-propeller encloses a large cavity that is characterized by 630 708 740 Corresponding author: Abbott, C.A. ([email protected]). the catalytic triad, Ser , Asp and His , which resides

Table 1. Known DPP4 substrates and biological significance after N-terminal cleavage Substrate Significance N-Terminus Reference Regulatory peptides Gastric inhibitory peptide (GIP) Inactivation Tyr-Ala-Asp- [63] Gastrin-releasing peptide (GRP) Inactivation Val-Pro-Leu- [63] GLP1 Inactivation His-Ala-Glu- [62] GLP2 Inactivation His-Ala-Asp- [64] Growth-hormone-releasing factor (GHRF) Inactivation Tyr-Ala-Glu- [65] Pituitary adenylate-cyclase-activating polypeptide (PACAP)-(1–38) Inactivation His-Ser-Asp- [66] Peptide YY(1–36) Altered receptor specificity Tyr-Pro-Ile- [67] Neuropeptides Neuropeptide-Y (NPY) Altered receptor specificity Tyr-Pro-Ser- [24] Substance P Inactivation Arg-Pro-Lys- [68] Chemokines Eotaxin (CCL11) Altered receptor specificity Gly-Pro-Gly- [69] IP10 (CXCL10) Altered receptor specificity Val-Pro-Leu- [18] ITAC (CXCL11) Altered receptor specificity Phe-Pro Met- [18] Macrophage-derived chemokine (MDC, CCL22) Altered receptor specificity Gly-Pro-Tyr- [70] Monokine induced by IFN-g (Mig, CXCL9) Reduced activity Thr-Pro-Val- [71] RANTES (CCL5) Altered receptor specificity Ser-Pro-Tyr- [72] Stromal-cell-derived factor (SDF-1, CXCL12) Altered receptor specificity Lys-Pro-Val [18] within the a/b-hydrolase fold [3]. DPP4 is responsible for confined to the cytoplasm [2,12]. Homology modeling of the hydrolysis of a number of regulatory factors, including DPP8 and DPP9 has led to a greater understanding of the regulatory peptides, neuropeptides and chemokines, as potential and catalytic mechanism of these enzymes outlined in Table 1. [13,14]. Recently, many laboratories have developed inhibitors that distinguish between DPP4 and DPP8/9 to FAP, DPP8 and DPP9 further elucidate the function of this interesting family of DPP4, FAP, DPP8 and DPP9 are homologous enzymes enzymes [15,16]. Unfortunately, DPP8/9 inhibitors bind with a conserved glutamic acid motif and a Ser-Asp-His DPP8 and DPP9 with equal efficiency; thus, the aim of catalytic triad [10] (Figure 1). FAP, also known as seprase, current research in our laboratory is to express and purify is a 95-kDa type II integral membrane glycoprotein that is recombinant DPP8 and DPP9 to determine their crystal only expressed at sites of tissue remodelling, such as structures. This will aid the development of inhibitors that wound healing and liver cirrhosis [3]. FAP is structurally can discriminate between the two closely related enzymes. homologous to DPP4, sharing 48% amino acid sequence Although the physiological functions of DPP8 and DPP9 identity. In addition to its exopeptidase activity, FAP also remain to be determined, in vitro assays have shown that has endopeptidase activity, and is capable of degrading they cleave some DPP4 substrates, including GLP1, GLP2, gelatin and type I collagen [3]. Although FAP was pre- neuropeptide Y (NPY), and peptide YY [17]. DP8 also viously considered to exist only in the membrane-bound cleaves the chemotactic factors stromal cell-derived factor form, recent findings have revealed low concentrations of (SDF)-1a and -1b, inflammatory protein-10 and interferon- circulating soluble FAP in human plasma [11]. inducible T-cell chemoattractant [18]. However, these are Recently, DPP8 and DPP9 have been the focus of atten- unlikely in vivo substrates because of the intracellular tion because of their immunoregulatory and therapeutic localization of DPP8 and DPP9. Investigations to identify potential. DPP8 and DPP9 have structural homology to in vivo substrates for these enzymes are ongoing. Cells DPP4 and both possess the unique ability to cleave the overexpressing DPP8 and DPP9 exhibit increased spon- prolyl bond [2,12]. Both DPP8 and DPP9 share significant taneous apoptosis, impaired cell migration, impaired cell amino acid identity to DPP4. In contrast to DPP4 and FAP, adhesion and impaired wound healing, implicating a role these enzymes lack a transmembrane domain and are at sites of tissue damage and remodeling for these enzymes

Table 2. Altered circulating DP activity and membrane DPP4 expression in both neurological and inﬂammatory diseases Membranea Circulating Reference Psychoneuroendocrine disease Anxiety ND # [55] Anorexia and bulemia "#[56] Depression ND # [57] Inﬂammatory disease Arthritis ND # [40,42] Asthma ND # [58] Atopic dermatitis # ND [27] Atherosclerosis " ND [59] Chronic tonsillitis ND " [60] IBD #"[29,48] Multiple sclerosis " ND [28] Systemic lupus erythematosus # ND [61] aND, not determined; ", increased DP activity; #, decreased DP activity.

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CN)*TFA, are non-speciﬁc and inhibit more than one DPP [23], as demonstrated in a number of studies, casting doubt over the molecular targets of these inhibitors [23–25]. Altered levels of DPP4 expression and DPP enzyme activity have been demonstrated in a number of human diseases (Table 2). It has been reported that the signaling capacity of DPP4 mediates immune function and disease pathogenesis in these immune-driven conditions, including anorexia [26], atopic dermatitis [27],MS[28] and IBD [29]. Changes in circulating DPP enzyme activity, originally attributed to DPP4, have also been reported (Table 2); however, the substrates used to assess enzyme activity, predominantly H-Ala-Pro and H-Gly-Pro, can also be cleaved by DPP8 and DPP9. Therefore, it is possible that the changes in enzyme activity observed are due to members of the DPP4 family other than DPP4. The possible involvement of DPPs in driving diverse disease pathologies has resulted in investigations into the effect of DPP inhibitors in preclinical models of inﬂammatory disorders.

Type 2 diabetes mellitus Early in vivo studies demonstrated that DPP4 inhibitors can prolong the in vivo half-life of the incretin GLP1 in different animal models of type 2 diabetes [30]. Several DPP4 selective inhibitors have now been developed by various pharmaceutical companies as treatment options for type 2 diabetes (Table 3). In 2006, the DPP4 inhibitor sitagliptin (Januvia1) was granted FDA approval as a monotherapy for type 2 diabetes. It has since gained approval for therapeutic use in more than 40 countries worldwide, including the USA, Europe and Australia. A second DPP4 inhibitor, NVP-LAF237 (vildagliptin), has

Figure 1. Structural homology between members of the DPP family. (a) The DPP4 been used in several large-scale clinical trials to success- structure (PDB i.d. 1R9N; blue) is overlaid on our DPP8 model (pink) and depicted fully determine its efficacy in the management of type 2 as a ribbon diagram. The catalytic site lies between the b-propeller and the a/b- diabetes [31–33]. Recently, the European Medicines Agen- hydrolase domains. The significant structural homology (35% amino acid identity and 55% amino acid similarity) between the two enzymes, particularly at the active cy has approved the combination treatment of vildagliptin 1 site, indicates why so many so-called DPP4 selective inhibitors also bind other (under the trade name Galvus ) and the anti-diabetic drug members of the DPP family. The DPP8 model was created using Modeller 9 v. 2 metformin for clinical use, although FDA approval for use [73] and the DPP4 (1R9N), FAP (1Z68) and DPP6 (1XFD) templates. Human protein sequences were obtained from SwissProt and accession numbers are AAA51943 in the USA requires further clinical trials. Two recent for DPP4 and AAG29766 for DPP8. Figure prepared using Pymol [54].(b) Schematic meta-analyses revealed that type 2 diabetes patients being representation of some members of the DPP family. The conserved glutamic acid treated with vildagliptin or sitagliptin had a slightly motif and catalytic triad residues essential for enzyme activity are highlighted. Not to scale. increased risk of developing an infection, specifically naso- pharyngitis and urinary tract infection [34,35]. However, no adverse events have been reported to date, confirming [19]. Studies have identified DPP8 and DPP9 enzyme the safety and tolerability of DPP4 inhibitors. activity in human leukocytes [3,20,21], suggesting that these cells contain novel substrates for DPP8 and DPP9 Arthritis and indicating an immunoregulatory function of the In some of the earliest in vitro studies, DPP4 inhibitors enzymes. suppressed mitogen- and antigen-stimulated T-cell activation and proliferation, possibly by suppression of inter- DPP inhibitors and therapeutic potential: preclinical leukin (IL)-2 and interferon (IFN)-g production [36]. Later models studies revealed that DPP4 is also expressed on B cells and The emergence of DPP4 and its involvement in many NK cells [1]. The action of DPP4 inhibitors suggests that it disease conditions have led to the development of several is the enzyme activity of DPP4 that mediates its immu- potent inhibitors of DPP4 activity [22]. These inhibitors are nostimulatory effects and not its signaling function [37]. often small-molecule compounds that competitively and However, it has also been demonstrated that the enzyme reversibly bind to the active site of the enzyme. However, activity of DPP4 is not required for its co-stimulatory owing to structural similarities at the catalytic site of activity of T cells; instead, CD26 can mediate its action members of the DPP4 family, the early DPP4-specific through direct interactions with the cell-surface molecule inhibitors, such as val-pyrr, Ile-Thia and Ile-Pyrr-(2- CD45 [1]. Interestingly, Dpp4 gene knockout (Dpp4/)

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Table 3. DPP4 inhibitors as therapies for type 2 diabetes Company Inhibitor Trade name Current status Merck Sitagliptin Januvia1 Approved in more than 40 countries, including USA, Europe and Australia Novartis Vildagliptin Galvus1 Approved in Europe. Awaiting FDA approval following further Phase III trials Bristol-Myers Squibb and Saxagliptin Onglyza1 Awaiting FDA approval Astra Zeneca Takeda Alogliptin Awaiting FDA approval mice develop normally and have no obvious immune phe- undergoes N-terminal truncation by DPP4, so that it is notype (Box 1) [38,39]. However, the absence of an inflam- no longer able to bind to its receptor. In this study, SDF1 matory stimulant might have masked the potential concentrations were higher in Dpp4/ mice with exper- involvement of DPP4 in immune cell activation and pro- imental arthritis. Busso et al. proposed that in DPP4 liferation in these mice. deficient mice, SDF1 levels are not regulated and this Altered DPP4 activity was first noted in mice with may contribute to the increase in disease severity observed rheumatoid arthritis [40]. Soluble DPP4 enzyme activity [42]. In vitro T-cell proliferation assays using concanavalin was significantly decreased, whereas cell-surface expres- A (conA) revealed no difference between wild-type and sion of CD26 was increased, indicating a role in auto- Dpp4/ mice; however, IFN-g production was signifi- immune diseases. Tanaka et al. determined the in vivo cantly higher in Dpp4/ compared to wild-type mice. efficacy of DPP inhibitors in normal and DPP4-deficient By contrast, Fan et al. [43] and Yan et al. [44] observed a Fischer rats using a type II collagen-induced or alkyldia- decrease in cell proliferation in Dpp4/ mouse spleen mine-induced rat model of arthritis [41]. The inhibitors lymphocytes (MSL) stimulated with conA or pokeweed alanylpyrrolidine-boronic acid (Ala-boroPro), Lys- mitogen (PWM) compared to their wild-type counterparts. [Z(NO2)]-thiazolidide and the irreversible Ala-Pro-nitro- However, IL-4 and IFN-g concentrations were greater in benzoylhydroxylamine were dissolved in saline and admi- MSL supernatants from Dpp4/ mice compared to wild- nistered orally or subcutaneously using prophylactic and type controls [44], supporting the findings of Busso et al. therapeutic dosage regimes. Treatment with any of the Interestingly, Dpp4/ mice exhibited delayed secretion of three inhibitors reduced hind paw swelling, an indicator of IFN-g and had lower IL-4 levels following stimulation with / disease severity. When Lys-[Z(NO2)]-thiazolidide was PWM [44]. These data imply that Dpp4 mice have an added to spleen and lymph node cell isolates from normal altered immune response; however, more research is and DPP4-deficient Fischer rats, the inhibitor suppressed required to delineate the mechanisms involved. Taken in vitro antigen- and mitogen-induced lymphocyte prolifer- together, results from these studies and those with DPP ation in both rat strains. This finding suggests that Lys- inhibitors suggest that inhibition of DPP activity has [Z(NO2)]-thiazolidide has broader specificity and could potential as an anti-inflammatory treatment modality have been targeting other members of the DPP4 family for arthritis, although the mechanisms by which DPP such as DPP8 and DPP9. inhibitors exert their actions are still unclear. The enzyme Using Dpp4/ mice, Busso et al. [42] uncovered poten- activity of DPP4 is required in part for its immunomodu- tial molecular targets for DPP4 in antigen- and collagen- latory effects; however, the signaling function of DPP4 also induced models of arthritis. They demonstrated a decrease seems to play a significant role. Furthermore, confor- in plasma DPP activity during arthritis that could be mational changes arising from interactions with specific attributed solely to DPP4 [42]. Furthermore, damage inhibitors at the active site might induce conformational severity, as indicated by synovial thickness, knee-joint changes in the enzyme, leading to anti-inflammatory inflammation and histological grading, was more severe effects (Figure 2) [43]. Moreover, it will be important to in Dpp4/ mice than in wild-type controls. SDF1 is a determine whether targeted inhibition of DPP4, DPP8 or proinflammatory chemotactic factor that binds to its re- DPP9, or a combination thereof, is required to gain thera- ceptor CXCR4 to stimulate cytokine production. SDF1 peutic benefit. Further work in this area is required to answer these compelling questions.

Box 1. Dpp4 knockout mice Multiple sclerosis Dpp4 knockout (Dpp4/) mice were first described by Marguet et al. DPP4 inhibitors have been extensively studied in blood and were used to demonstrate the essential role of DPP4 in the samples from patients with MS and in experimental aller- inactivation of glucagon-line peptide GLP1 [38]. GLP1 is a potent gic encephalomyelitis (EAE), an animal model of MS that is insulinotrophic hormone capable of stimulating glucose-dependent insulin secretion. Dpp4/ mice exhibited a healthy phenotype, but induced by inoculation with central nervous system anti- insulin secretion and glucose tolerance increased on presentation of gens to illicit an immune response and paralytic disease. T- a glucose challenge. Despite the lack of DPP4 protein in these mice, cell clones from MS patients express signiﬁcantly higher DPP enzyme activity is still detected, indicating that other members levels of DPP4 compared to resting peripheral blood T cells / oftheDPP4familyareactive[20,37,38]. Dpp4 mice have [45]. Addition of the DPP4 inhibitor Lys[Z(NO )]-thiazoli- inherently lower bodyweight than their wild-type counterparts, with 2 reduced food intake and increased energy expenditure, contributing dide or Lys[Z(NO2)]-pyrrolidide to myelin basic protein- to a resistance to obesity and improved metabolic control [39]. stimulated T-cell clones inhibited production of IL-4, IFN-g Dpp/ mice have also been used to investigate the involvement of and tumor necrosis factor (TNF)-a, suggesting that inhi- DPP4 and other family members in animal models of disease, bition of DP activity in vivo may suppress the inﬂamma- including DSS-induced colitis, arthritis and MS. tory response associated with MS.

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Figure 2. Proposed working model of the potential anti-inflammatory mechanism of DPP inhibitors. After binding of the inhibitor to the active site of membrane-bound DPP4, we propose a conformational change in the intracellular domain of the enzyme. Alternatively, the inhibitor could pass across the cell membrane and bind to DPP8 or DPP9. This might then lead to initiation of an intracellular pathway, resulting in a reduction in pro-inflammatory cytokine secretion, suppressed T-lymphocyte proliferation and increased production of the pro-inflammatory cytokine TGF-b. Figure adapted from data published by Fan et al. [43] and Preller et al. [46].

A reduction in pro-inflammatory cytokine production is greater in patients with Crohn’s disease, but not ulcerative evident in mice with EAE, together with an increase in colitis, compared to normal human controls. This indicated transforming growth factor (TGF)-b and reduced T-cell that changes in GLP2 secretion during IBD are indepen- proliferation after treatment with Lys[Z(NO2)]-thiazoli- dent of DPP4 activity [48]. This is supported by higher dide [45]. Preller et al. addressed the role of DPP4 in the membrane expression of DPP4 on T lymphocytes isolated / EAE TH1 immune response using Dpp4 mice [46]. from IBD patients compared to healthy controls [29,47], Similar to the results obtained by Busso et al. [42], EAE indicating that DPP4 might play a significant role in disease severity was significantly greater in Dpp4/ perpetuating the inflammatory response associated with compared to wild-type mice. Concentrations of IFN-g IBD. and TNF-a were also elevated in Dpp4/ mice and latent We previously investigated the effect of DPP4 deficiency TGF-b was lower in comparison to wild-type controls, in mice during experimental colitis. We hypothesized that implicating a direct role in the production of these cyto- Dpp4/ mice would have higher concentrations of the kines for DPP4. The authors proposed that synthetic and intestinotrophic growth factor GLP2 and therefore natural ligand inhibitors of DPP4 could bind to the active increased protection and repair during dextran sulfate site of the enzyme, leading to a reduction in pro-inflam- sodium (DSS)-induced colitis [51]. Disease severity was matory cytokines, an increase in TGF-b and decreased T- greater on day 14 and crypt area was reduced on day 9 of cell proliferation, whereas this regulatory pathway might DSS-induced colitis in Dpp4/ mice. Our study supported not have been present in Dpp4/ mice [46]. However, the the hypothesis advocated by Preller et al. that the absence authors did not address the potential involvement of DPP8, of DPP4 on the cell surface might exaggerate inflammatory DPP9 and DPP2, which are all active in Dpp4/ mice and responses [46]. However, there were no differences in might also play a role in regulating inflammatory markers of intestinal growth, including cell proliferation, responses. crypt depth and colon weight, in Dpp4/ compared to wild- type mice, suggesting that the biological activity of GLP2 IBD was not increased in these animals. Assessment of circu- Although the effect of DPP4 inhibition in IBD has not been lating DP enzyme activity indicated that there was no studied extensively, much evidence indicates that DPP4 difference between wild-type and Dpp4/ mice, has a role in IBD pathogenesis. Serum from IBD patients suggesting that the residual DPP enzyme activity might contains lower levels of circulating DPP4 activity [32,47– have been responsible for GLP2 cleavage and explaining 49]. In vivo inhibition of DPP4 activity using val-pyrr in why no differences in these parameters were observed [51]. healthy rats and mice enhances the intestinotrophic effects The effects of synthetic inhibitors of DPP4 in an animal of GLP2 [50] and it has been proposed that the reduction in model of IBD have been reported by Bank et al. [52]. Using soluble DPP4 activity in IBD patients might be a compen- the DSS colitis model, BALB/c mice were treated with satory mechanism to preserve biologically active GLP21–33 Lys[Z(NO2)]-pyrrolidide and an aminopeptidase N inhibi- [48]. However, Xiao et al. demonstrated that concen- tor, either alone or in combination [52]. Significant trations of biologically active GLP21–33 were significantly decreases in disease activity and increased concentrations

604 Review Trends in Pharmacological Sciences Vol.30 No.11 of TGF-b and FOXP3 were recorded in mice receiving the of DPP activity has provided a novel therapeutic option for combination treatment compared to vehicle treatment. several human diseases, including type 2 diabetes, MS, However, treatment with Lys[Z(NO2)]-pyrrolidide alone arthritis and IBD. The approval of the DPP4 selective also seemed to reduce disease severity. In our laboratory, inhibitors Januvia1 and Galvus1 for therapeutic use in we recently demonstrated the efficacy of two DPP inhibi- patients with type 2 diabetes highlights the safety, toler- tors, Ile-Thia and Ile-Pyrr-(2-CN)*TFA, in wild-type mice ability and efficacy of this class of drugs. With more se- with DSS-induced colitis. Both these inhibitors signifi- lective inhibitors for DPP8 and DPP9 being developed, cantly reduced disease activity after 6 days of DSS inges- there is now an opportunity for researchers to delineate tion [53]. Furthermore, histological indicators such as the individual role of these enzymes in both innate and crypt depth and area were improved in mice treated with adaptive immune responses. The pre-clinical data avail- either Ile-Thia or Ile-Pyrr-(2-CN)*TFA. These studies pro- able indicate that non-selective DPP inhibitors possess vide further evidence of the anti-inflammatory effects of anti-inflammatory properties. However, further studies DPP inhibitors. In recent work we used inhibitors on are required to assess whether selective or non-selective DPP4-deficient mice and found they offered no protective inhibition of DPPs is required to potentiate any anti- effects (Yazbeck et al., unpublished data). This is further inflammatory effects. In conclusion, DPP inhibitors evidence that the presence of DPP4 on the cell surface is represent an exciting and novel drug class for the treat- critical for the immunomodulatory effects of DPP4 inhibi- ment of inflammatory disease; however, it is essential to tors and that the interaction between the inhibitor and define and optimize the molecular mechanisms of this drug DPP4 is essential for a reduced inflammatory response. class before advocating any clinical use. Although the role of other members of the DPP family was not specifically addressed in these studies, data from our Acknowledgements laboratory implicate DPP8 and DPP2 in experimental The authors would like to thank Dr Melissa Pitman for constructing the colitis (Yazbeck et al., unpublished data). However, further DPP4/DP8 overlay image used in Figure 1. studies are required to better characterize the specific role References of these enzymes during the course of colitis. 1 Pitman, M.R. et al. (2009) Dipeptidyl peptidase 8 and 9 – guilty by association? Front. Biosci. 14, 3619–3633 DPPs and immune regulation 2 Abbott, C.A. et al. (2000) Cloning, expression and chromosomal Lankas et al. demonstrated that the early DPP4 selective localization of a novel human dipeptidyl peptidase (DPP) IV inhibitors Lys[Z(NO )]-thiazolidide and Lys[Z(NO )]-pyr- homolog, DPP8. Eur. J. Biochem. 267, 6140–6150 2 2 3 Gorrell, M.D. (2005) Dipeptidyl peptidase IV and related enzymes in rolidide also inhibit DPP8, DPP9 and DPP2 [23]. This cell biology and liver disorders. Clin. Sci. (Lond.) 108, 277–292 finding suggests that off-target effects of the inhibitors 4 Maes, M.B. et al. (2007) Dipeptidyl peptidase II (DPPII), a review. Clin. might have been, in part, responsible for their immuno- Chim. Acta 380, 31–49 suppressive actions, including suppression of T-lympho- 5 Ahren, B. (2007) DPP-4 inhibitors. Best Pract. Res. Clin. Endocrinol. cyte proliferation and pro-inflammatory cytokine Metab. 21, 517–533 6 Hopsu-Havu, V.K. and Glenner, G.G. (1966) A new dipeptide production, as well as an increase in TGF-b production. naphthylamidase hydrolyzing glycyl-prolyl-b-naphthylamide. In a recent study, Reinhold et al. addressed the specific role Histochemie 7, 197–201 of DPP4, DPP8, DPP9 and DPP2 in mitogen-stimulated 7 Hopsu-Havu, V.K. et al. (1968) A hog kidney aminopeptidase liberating MSL from wild-type and Dpp4/ mice [37]. The authors N-terminal dipeptides. Partial purification and characteristics. Acta demonstrated that Lys[Z(NO )]-thiazolidide and Chem. Scand. 22, 299–308 2 8 Hopsu-Havu, V.K. and Sarimo, S.R. (1967) Purification and Lys[Z(NO2)]-pyrrolidide and a specific DPP8/9 inhibitor charcterization of an aminopeptidase hydrolyzing glycyl-proline- all suppressed DNA synthesis in PWM-stimulated MSL naphthylamide. Hoppe-Seyler’s Z. Physiol. Chem. 348, 1540–1550 from wild-type and Dpp4/ mice [37]. The results of this 9 Bauvois, B. et al. (1999) Constitutive expression of CD26/ study are the first to implicate a direct role in T-lymphocyte dipeptidylpeptidase IV on peripheral blood B lymphocytes of patients with B chronic lymphocytic leukaemia. Br. J. Cancer 79, regulation for these enzymes and suggest that they are a 1042–1048 novel target for immunosuppressive drug therapies. How- 10 Abbott, C.A. et al. (1999) Two highly conserved glutamic acid residues ever, the authors acknowledge that DP8/9 may have been in the predicted b propeller domain of dipeptidyl peptidase IV are compensating for the loss of DPP4 in Dpp4/ mice [37]. required for its enzyme activity. FEBS Lett. 458, 278–284 We propose a working model, hypothesizing potential 11 Lee, K.N. et al. (2004) A novel plasma proteinase potentiates a2- antiplasmin inhibition of fibrin digestion. Blood 103, 3783–3788 pathways for the anti-inflammatory effects of DP inhibi- 12 Ajami, K. et al. (2004) Dipeptidyl peptidase 9 has two forms, a broad tors (Figure 2). Further studies using more potent and tissue distribution, cytoplasmic localization and DPIV-like peptidase selective inhibitors of each member of the DPP family are activity. Biochim. Biophys. Acta 1679, 18–28 required to characterize their role in immune regulation. 13 Pitman, M.R. et al. (2006) Prediction of dipeptidyl peptidase (DP) 8 structure by homology modelling. Adv. Exp. Med. Biol. 575, 33–42 Summary 14 Rummey, C. and Metz, G. (2007) Homology models of dipeptidyl DPP4, DPP8 and DPP9 are expressed in immune cells and peptidases 8 and 9 with a focus on loop predictions near the active represent novel pharmacological targets for inflammatory site. Proteins 66, 160–171 diseases. DPP4, the principal member of this enzyme 15 Van der Veken, P. et al. (2008) Inhibitors of dipeptidyl peptidase 8 and family, has a number of regulatory functions, including dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads. Bioorg. Med. Chem. Lett. 18, 4154–4158 the hydrolysis of several peptide hormones and chemo- 16 Van Goethem, S. et al. (2008) Inhibitors of dipeptidyl peptidase 8 and kines and a co-stimulatory effect on T-cell activation via its dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors. signaling function. The emergence of synthetic inhibitors Bioorg. Med. Chem. Lett. 18, 4159–4162

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17 Bjelke, J.R. et al. (2006) Dipeptidyl peptidase 8 and 9 specificity and 41 Tanaka, S. et al. (1997) Suppression of arthritis by the inhibitors of molecular characterization compared to dipeptidyl peptidase IV. dipeptidyl peptidase IV. Int. J. Immunopharmacol. 19, 15–24 Biochem. J. 396, 391–399 42 Busso, N. et al. (2005) Circulating CD26 is negatively associated with 18 Ajami, K. et al. (2008) Stromal cell-derived factors 1a and 1b, inflammation in human and experimental arthritis. Am. J. Pathol. 166, inflammatory protein-10 and interferon-inducible T cell chemo- 433–442 attractant are novel substrates of dipeptidyl peptidase 8. FEBS Lett. 43 Fan, H. et al. (2003) Dipeptidyl peptidase IV/CD26 in T cell activation, 582, 819–825 cytokine secretion and immunoglobulin production. Adv. Exp. Med. 19 Yu, D.M. et al. (2006) Extraenzymatic functions of the dipeptidyl Biol. 524, 165–174 peptidase IV-related proteins DP8 and DP9 in cell adhesion, 44 Yan, S. et al. (2003) Deficiency of CD26 results in a change of cytokine migration and apoptosis. FEBS J. 273, 2447–2460 and immunoglobulin secretion after stimulation by pokeweed mitogen. 20 Yu, D.M. et al. (2009) The in vivo expression of dipeptidyl peptidases 8 Eur. J. Immunol. 33, 1519–1527 and 9. J. Histochem. Cytochem. (in press) doi:10.1369/jhc.2009.953760. 45 Reinhold, D. et al. (2006) Dipeptidyl peptidase IV (DP IV, CD26) and 21 Maes, M.B. et al. (2007) Dipeptidyl peptidase 8/9-like activity in human aminopeptidase N (APN, CD13) as regulators of T cell function leukocytes. J. Leukoc. Biol. 81, 1252–1257 and targets of immunotherapy in CNS inflammation. Int. 22 Hoffmann, T. and Demuth, H.U. (2002) Therapeutic strategies Immunopharmacol. 6, 1935–1942 exploiting DP IV inhibition – target disease: type 2 diabetes. In 46 Preller, V. et al. (2007) TGF-b1-mediated control of central nervous Ectopeptidases: CD13/Aminopeptidase N and CD26/ system inflammation and autoimmunity through the inhibitory Dipeptidylpeptidase IV in Medicine and Biology (Langner, J. and receptor CD26. J. Immunol. 178, 4632–4640 Ansorge, S., eds), pp. 259–278, Kluwer Academic/Plenum 47 Rose, M. et al. (2002) T-cell immune parameters and depression in 23 Lankas, G.R. et al. (2005) Dipeptidyl peptidase IV inhibition for the patients with Crohn’s disease. J. Clin. Gastroenterol. 34, 40–48 treatment of type 2 diabetes: potential importance of selectivity over 48 Xiao, Q. et al. (2000) Circulating levels of glucagon-like peptide-2 in dipeptidyl peptidases 8 and 9. Diabetes 54, 2988–2994 human subjects with inflammatory bowel disease. Am. J. Physiol. 24 Frerker, N. et al. (2007) Neuropeptide Y (NPY) cleaving enzymes: Regul. Integr. Comp. Physiol. 278, R1057–1063 structural and functional homologues of dipeptidyl peptidase 4. 49 Varljen, J. et al. (2005) Clinical relevance of the serum dipeptidyl Peptides 28, 257–268 peptidase IV (DPP IV/CD26) activity in adult patients with Crohn’s 25 Bjelke, J.R. et al. (2006) Dipeptidyl peptidases 8 and 9: specificity and disease and ulcerative colitis. Croat. Chem. Acta 78, 427–432 molecular characterization compared with dipeptidyl peptidase IV. 50 Hartmann, B. et al. (2000) Dipeptidyl peptidase IV inhibition enhances Biochem. J. 396, 391–399 the intestinotrophic effect of glucagon-like peptide-2 in rats and mice. 26 Hildebrandt, M. et al. (2001) Eating disorders: a role for dipeptidyl Endocrinology 141, 4013–4020 peptidase IV in nutritional control. Nutrition 17, 451–454 51 Geier, M.S. et al. (2005) Development and resolution of experimental 27 Jarmolowska, B. et al. (2007) Serum activity of dipeptidyl peptidase IV colitis in mice with targeted deletion of dipeptidyl peptidase IV. J. Cell. (DPPIV; EC 3.4.14.5) in breast-fed infants with symptoms of allergy. Physiol. 204, 687–692 Peptides 28, 678–682 52 Bank, U. et al. (2008) Inflammatory bowel diseases: multiple benefits 28 Reinhold, D. et al. (2008) DP IV/CD26, APN/CD13 and related from therapy with dipeptidyl- and alanyl-aminopeptidase inhibitors. enzymes as regulators of T cell immunity: implications for Front. Biosci. 13, 3699–3713 experimental encephalomyelitis and multiple sclerosis. Front. 53 Yazbeck, R. et al. (2008) Inhibiting dipeptidyl peptidase activity Biosci. 13, 2356–2363 partially ameliorates colitis in mice. Front. Biosci. 13, 6850–6858 29 Hildebrandt, M. et al. (2001) Dipeptidyl peptidase IV (DP IV, CD26) in 54 Delano, W.L. (2002) The PyMOL Molecular Graphics System, DeLano patients with inflammatory bowel disease. Scand. J. Gastroenterol. 36, Scientific 1067–1072 55 Emanuele, E. et al. (2006) Circulating levels of soluble CD26 30 Demuth, H.U. et al. (2005) Type 2 diabetes – therapy with dipeptidyl are associated with phobic anxiety in women. Prog. peptidase IV inhibitors. Biochim. Biophys. Acta 1751, 33–44 Neuropsychopharmacol. Biol. Psychiatry 30, 1334–1336 31 Ahren, B. et al. (2004) Twelve- and 52-week efficacy of the dipeptidyl 56 van West, D. et al. (2000) Lowered serum dipeptidyl peptidase IV peptidase IV inhibitor LAF237 in metformin-treated patients with activity in patients with anorexia and bulimia nervosa. Eur. Arch. type 2 diabetes. Diabetes Care 27, 2874–2880 Psychiatry Clin. Neurosci. 250, 86–92 32 Pratley, R.E. et al. (2006) Twelve-week monotherapy with the DPP-4 57 Maes, M. et al. (1997) Lower serum dipeptidyl peptidase IV activity in inhibitor vildagliptin improves glycemic control in subjects with type 2 treatment resistant major depression: relationships with immune- diabetes. Horm. Metab. Res. 38, 423–428 inflammatory markers. Psychoneuroendocrinology 22, 65–78 33 Ristic, S. and Bates, P.C. (2006) Vildagliptin: a novel DPP-4 inhibitor 58 van der Velden, V.H. et al. (1998) Expression of aminopeptidase N and with pancreatic islet enhancement activity for treatment of patients dipeptidyl peptidase IV in the healthy and asthmatic bronchus. Clin. with type 2 diabetes. Drugs Today (Barc.) 42, 519–531 Exp. Allergy 28, 110–120 34 Richter, B. et al. (2008) Emerging role of dipeptidyl peptidase-4 59 Hosono, M. et al. (2003) Increased expression of T cell activation inhibitors in the management of type 2 diabetes. Vasc. Health Risk markers (CD25, CD26, CD40L and CD69) in atherectomy specimens Manage. 4, 753–768 of patients with unstable angina and acute myocardial infarction. 35 Amori, R.E. et al. (2007) Efficacy and safety of incretin therapy in type 2 Atherosclerosis 168, 73–80 diabetes: systematic review and meta-analysis. J. Am. Med. Assoc. 298, 60 Vlahovic, P. et al. (2007) Elevated serum dipeptidyl peptidase IV 194–206 activity in patients with chronic tonsillitis. Ann. Clin. Biochem. 44, 36 Schon, E. et al. (1989) Dipeptidyl peptidase IV in human T 70–74 lymphocytes. Impaired induction of interleukin 2 and g interferon 61 Kobayashi, H. et al. (2002) Reduction of serum soluble CD26/ due to specific inhibition of dipeptidyl peptidase IV. Scand. J. dipeptidyl peptidase IV enzyme activity and its correlation with Immunol. 29, 127–132 disease activity in systemic lupus erythematosus. J. Rheumatol. 37 Reinhold, D. et al. (2009) Role of dipeptidyl peptidase IV (DP IV)-like 29, 1858–1866 enzymes in T lymphocyte activation: investigations in DP IV/CD26- 62 Baggio, L.L. and Drucker, D.J. (2007) Biology of incretins: GLP-1 and knockout mice. Clin. Chem. Lab. Med. 47, 268–274 GIP. Gastroenterology 132, 2131–2157 38 Marguet, D. et al. (2000) Enhanced insulin secretion and improved 63 Ahren, B. and Hughes, T.E. (2005) Inhibition of dipeptidyl peptidase- glucose tolerance in mice lacking CD26. Proc. Natl. Acad. Sci. U. S. A. 4 augments insulin secretion in response to exogenously 97, 6874–6879 administered glucagon-like peptide-1, glucose-dependent 39 Conarello, S.L. et al. (2003) Mice lacking dipeptidyl peptidase IV are insulinotropic polypeptide, pituitary adenylate cyclase-activating protected against obesity and insulin resistance. Proc. Natl. Acad. Sci. polypeptide, and gastrin-releasing peptide in mice. Endocrinology U. S. A. 100, 6825–6830 146, 2055–2059 40 Gotoh, H. et al. (1989) Activities of dipeptidyl peptidase II and 64 Yazbeck, R. et al. (2009) Growth factor based therapies and intestinal dipeptidyl peptidase IV in synovial fluid from patients with disease: is glucagon-like peptide-2 the new way forward? Cytokine rheumatoid arthritis and osteoarthritis. Clin. Chem. 35, 1016–1018 Growth Factor Rev. 20, 175–184

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65 Mentlein, R. et al. (1993) Dipeptidyl-peptidase IV hydrolyses gastric 70 Proost, P. et al. (1999) Truncation of macrophage-derived chemokine by inhibitory polypeptide, glucagon-like peptide-1(7-36) amide, peptide CD26/dipeptidyl-peptidase IV beyond its predicted cleavage site affects histidine methionine and is responsible for their degradation in human chemotactic activity and CC chemokine receptor 4 interaction. J. Biol. serum. Eur. J. Biochem. 214, 829–835 Chem. 274, 3988–3993 66 Lambeir, A.M. et al. (2001) Kinetic study of the processing by 71 Proost, P. et al. (2001) Amino-terminal truncation of CXCR3 agonists dipeptidyl-peptidase IV/CD26 of neuropeptides involved in impairs receptor signaling and lymphocyte chemotaxis, while pancreatic insulin secretion. FEBS Lett. 507, 327–330 preserving antiangiogenic properties. Blood 98, 3554–3561 67 Unniappan, S. et al. (2006) Effects of dipeptidyl peptidase IV on the 72 Oravecz, T. et al. (1997) Regulation of the receptor speciﬁcity and satiety actions of peptide YY. Diabetologia 49, 1915–1923 function of the chemokine RANTES (regulated on activation, normal 68 Guieu, R. et al. (2006) CD26 modulates nociception in mice via its T cell expressed and secreted) by dipeptidyl peptidase IV (CD26)- dipeptidyl-peptidase IV activity. Behav. Brain Res. 166, 230–235 mediated cleavage. J. Exp. Med. 186, 1865–1872 69 Forssmann, U. et al. (2008) Inhibition of CD26/dipeptidyl peptidase IV 73 Sali, A. and Overington, J.P. (1994) Derivation of rules for comparative enhances CCL11/eotaxin-mediated recruitment of eosinophils in vivo. protein modeling from a database of protein structure alignments. J. Immunol. 181, 1120–1127 Protein Sci. 3, 1582–1596

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