Biochemical and Biophysical Research Communications 434 (2013) 191–196
`
`Contents lists available at SciVerse ScienceDirect
`
`Biochemical and Biophysical Research Communications
`
`j o u r n a l h o m e p a g e : w w w . e l s ev i e r . c o m / l o c a t e / y b b r c
`
`A comparative study of the binding modes of recently launched dipeptidyl
`peptidase IV inhibitors in the active site
`Mika Nabeno a,⇑, Fumihiko Akahoshi a, Hiroyuki Kishida a, Ikuko Miyaguchi a, Yoshihito Tanaka a,
`
`Shinichi Ishii b, Takashi Kadowaki c
`a Medicinal Chemistry Research Laboratories II, Mitsubishi Tanabe Pharma Corporation, Saitama, Japan
`b Pharmacology Research Laboratories II, Mitsubishi Tanabe Pharma Corporation, Saitama, Japan
`c Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 28 February 2013
`Available online 15 March 2013
`
`Keywords:
`Type 2 diabetes
`Dipeptidyl peptidase IV inhibitor
`X-ray co-crystal structure
`Binding subsite
`
`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 sub-
`sites and a covalent bond with Ser630 in the catalytic triad; (ii) alogliptin and linagliptin (Class 2) form
`
`
`2 subsites in addition to the S1 and S2 subsites; and (iii) sitagliptin andinteractions with the S01 and/or S0
`teneligliptin (Class 3) form interactions with the S1, S2 and S2 extensive subsites. The present study
`
`
`revealed that the additional interactions with the S01, S02 or S2 extensive subsite may increase DPP-4 inhi-
`bition 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.
`
`Ó 2013 Elsevier Inc. All rights reserved.
`
`1. 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 hor-
`mone 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), vil-
`dagliptin [2], saxagliptin [3] and teneligliptin [4] are peptide mi-
`metic 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 com-
`pounds, 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.
`
`⇑ Corresponding author. Address: Medicinal Chemistry Research Laboratories II,
`
`Research Division, Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawagishi,
`Toda-shi, Saitama 335-8505, Japan. Fax: +81 48 433 2610.
`E-mail address: nabeno.mika@mv.mt-pharma.co.jp (M. Nabeno).
`
`0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
`http://dx.doi.org/10.1016/j.bbrc.2013.03.010
`
`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-dimen-
`sional structure of DPP-4 are well known [10,11]. The structure
`comprises a b-propeller domain and a catalytic domain, which to-
`gether 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 re-
`ports comparing their binding modes in DPP-4. X-ray co-crystal
`structures of five inhibitors, sitagliptin [6], saxagliptin [15], aloglip-
`tin [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 under-
`stood 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
`
`AstraZeneca Exhibit 2176
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`Page 1 of 6
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`M. Nabeno et al. / Biochemical and Biophysical Research Communications 434 (2013) 191–196
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`Table 1
`Recently launched DPP-4 inhibitors.
`
`Compound name
`
`Sitagliptin
`
`Chemical structure
`
`Release year
`
`2006
`
`Originator
`
`Merck & co.
`
`Vildagliptin
`
`Saxagliptin
`
`Alogliptin
`
`Linagliptin
`
`2007
`
`2009
`
`2010
`
`Novartis
`
`Astrazeneca and bristol-myers squibb
`
`Takeda
`
`2011
`
`Boehringer ingelheim
`
`Teneligliptin
`
`2012
`
`Mitsubishi tanabe pharma
`
`Data were collected in Thomson Reuters Integrity on February 4, 2013.
`
`of the six inhibitors in parallel and studied the relationships be-
`tween their binding interactions with DPP-4 and their inhibitory
`activity.
`
`2. Materials and methods
`
`2.1. Synthesis of vildagliptin
`
`Vildagliptin was prepared according to the method described by
`Villhauer et al. [2].
`
`2.2. 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 vildaglip-
`tin and preserving it in liquid nitrogen for data collection at 100 K.
`X-ray diffraction data were collected at the High Energy Accelera-
`tor Research Organization (KEK) beam line BL5 and processed
`using the program HKL2000 [18]. The structure of the DPP-4–
`inhibitor complex was solved by molecular replacement with the
`program PHASER [19], utilizing the previously determined coordi-
`nates of DPP-4 with the Protein Data Bank (PDB) accession code
`3VJK. Data collection and model refinement statistics are summa-
`rized in Table 2.
`
`2.3. Comparison of X-ray complex structures
`
`The co-crystal structures of five inhibitors with human DPP-4
`have been reported [PDB: 1X70 (sitagliptin), 3BJM (saxagliptin),
`3G0B (alogliptin), 2RGU (linagliptin), and 3VJK (teneligliptin)].
`They were superimposed on the co-crystal structure of a substrate
`peptide, diprotin A with DPP-4 (PDB: 1NU8) to analyze the binding
`subsites. The molecular modeling software Molecular Operating
`Environment version 2011.10 (Chemical Computing Group, Inc.,
`Montreal, Canada) was used for analysis and graphical visualiza-
`tion of the X-ray co-crystal structures.
`The contact area between the inhibitor and DPP-4 was calcu-
`lated using the molecular modeling software Discovery Studio ver-
`sion 3.5 (Accelrys,
`Inc., San Diego, USA). For each co-crystal
`structure, the molecular surface area of the inhibitor, and its sol-
`vent-exposed surface area in DPP-4 were calculated. The difference
`between these areas was defined as the contact area.
`
`3. Results and discussion
`
`3.1. X-ray co-crystal structures of six inhibitors with DPP-4
`
`3.1.1. 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
`
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`
`Table 2
`Data collection and refinement statistics.
`
`PDB entry code
`
`Crystal
`Space group
`Unit cell parameters: a (Å)
`b (Å)
`c (Å)
`
`Data
`Resolution (Å)
`Unique reflections
`Redundancy
`Completeness (%)
`a
`Rmerge
`I/r (I)
`
`Refinement
`Resolution (Å)
`Unique reflections
`Completeness (%)
`Data in the test set
`R-work
`R-free
`R.m.s.d. bond lengths (Å)
`R.m.s.d. bond angles (°)
`
`Ramachandran plot
`Favored regions (%)
`Allowed regions (%)
`No. of non-H atoms/average B (Å2)
`Protein
`Ligand
`Water
`
`Vildagliptin
`
`3W2T
`
`P212121
`118.22
`126.24
`138.09
`
`50.00–2.36 (2.44–2.36)
`82418 (7691)
`5.0 (4.6)
`97.1 (91.7)
`0.080 (0.234)
`16.3 (6.96)
`
`30.00–2.36 (2.42–2.36)
`78,227 (5407)
`97.3 (92.1)
`4103 (265)
`0.180 (0.206)
`0.231 (0.287)
`0.011
`1.319
`
`96.1
`3.9
`
`12,228/32.7
`44/23.7
`1027/34.7
`
`P
`P
`Values in parentheses are for the highest-resolution shell.
`jðI hIiÞj=
`ðIÞ, where I is the observed intensity.
`a Rmerge =
`
`(P2, P1, P01, P02 . . .), and the protein subsites occupied by the respec-
`
`
`tive amino acids are also numbered in the same fashion (S2, S1, S0
`1,
`S0
`2. . .). 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 (P1). Although, in principle, no sub-
`sites are defined after S2 in DPP-4, our recent study has shown that
`not the substrates but the inhibitors can bind well beyond the S2
`subsite to increase their inhibitory activity [4,21]. We therefore de-
`fined the site beyond S2 as the S2 extensive subsite, which is com-
`posed of Val207, Ser209, Phe357 and Arg358.
`
`3.1.2. Binding mode of vildagliptin
`The co-crystal structure of vildagliptin with DPP-4 is shown
`in Fig. 1(A). The cyanopyrrolidine binds to the S1 subsite, with
`the nitrile forming a covalent imidate adduct with the hydroxyl
`of Ser630 in the catalytic triad. The imidate nitrogen forms a
`hydrogen bond with the side-chain hydroxyl of Tyr547. The
`remaining part including the adamantane binds to the S2 subsite,
`where the carbonyl group forms a hydrogen bond with Asn710
`and the amino group forms salt bridges with Glu205 and
`Glu206. The hydroxyl group on the adamantyl moiety forms
`hydrogen bonds with His126 and Ser209 via the water
`molecules.
`
`3.1.3. Categorization of the six inhibitors on the basis of their binding
`subsites
`The co-crystal structures of the six inhibitors with DPP-4 super-
`imposed on that of the substrate peptide (diprotin A, Ile-Pro-Ile)
`are shown in Fig. 1(B)–(H). We categorized the six inhibitors into
`three classes on the basis of their binding subsites. (i) Vildagliptin
`and saxagliptin have the most basic binding modes, binding to only
`the S1 and S2 subsites (Class 1). (ii) Alogliptin and linagliptin bind
`
`to the S1, S2 and S0
`1 subsites. Moreover, only linagliptin additionally
`binds to the S0
`2 subsite (Class 2). (iii) Sitagliptin and teneligliptin
`bind to the S1, S2 and S2 extensive subsites (Class 3). Fig. 2 shows
`the concept of this categorization.
`
`3.2. Relationship between the inhibitory activity and the binding mode
`of each class
`
`We focus on the characteristic binding interactions with DPP-4
`because other details have been described in previous studies
`[4,6,8,15,16]. It is well known that all the DPP-4 inhibitors form
`salt bridges with Glu205 and Glu206 in the S2 subsite, which have
`vital roles in the inhibitory activity. The potency of the six DPP-4
`inhibitors is shown in Table 3 [22].
`
`3.2.1. Class 1: vildagliptin and saxagliptin
`Because vildagliptin and saxagliptin were designed as peptide
`mimetics, they overlap with the P1 and P2 residues of the substrate
`peptide. As described above, their cyanopyrrolidine moieties bind
`to the S1 subsite, forming a covalent bond between the nitrile
`group and Ser630, and their hydroxy adamantyl groups bind to
`the S2 subsite. While they bind in almost the same mode, one of
`the reasons why saxagliptin has 5-fold higher activity than vildag-
`liptin is attributed to the cyclopropanated cyanopyrrolidine of sax-
`agliptin. Although it was originally intended to enhance the
`chemical stability of the cyanopyrrolidine [3], introduction of the
`cyclopropane moiety afforded an additional hydrophobic interac-
`tion with the side chain of Tyr666 in the S1 subsite. Moreover,
`the direct hydrogen bond between the hydroxyl group of saxaglip-
`tin and the side chain of Tyr547 may also contribute to its higher
`potency.
`
`3.2.2. Class 2: alogliptin and linagliptin
`The chemical structures of alogliptin and linagliptin are far dif-
`ferent from those of the substrate peptides. The cyanobenzyl group
`of alogliptin and the butynyl group of linagliptin bind to the S1 sub-
`site. Their uracil rings form p–p interactions with Tyr547, which
`undergoes a conformational change in the S0
`1 subsite. One of the
`reasons why linagliptin has 8-fold higher activity than alogliptin
`may be because only linagliptin binds to the S0
`2 subsite. The phenyl
`component of the quinazoline substituent forms a p–p interaction
`with Trp629 located in the S0
`2 subsite [23]. Eckhardt et al. reported
`that the introduction of the quinazoline moiety improved its po-
`tency 88-fold [8].
`
`3.2.3. Class 3: sitagliptin and teneligliptin
`The trifluorophenyl moiety of sitagliptin and the thiazoline
`moiety of teneligliptin bind to the S1 subsite. The triazolopyr-
`azine moiety and trifluoromethyl substituent of sitagliptin and
`the (1-phenylpyrazol-5-yl) piperazine moiety, referred to here
`as the ‘‘anchor lock domain,’’ of teneligliptin bind to the S2
`extensive subsite. Although both inhibitors appear to bind to
`the subsites in the same manner, teneligliptin has 5-fold higher
`activity. We suggest three potential reasons for the difference.
`The first reason may be related to their chemical structures. Be-
`cause teneligliptin consists of a considerably rigid ‘‘J-shaped’’
`structure formed by five rings, four of which are directly con-
`nected, the loss in entropy is small upon binding to DPP-4.
`The second reason may be related to the binding interactions
`with the S2 subsite. The carbonyl group of teneligliptin, derived
`from the peptide mimetics, forms a hydrogen bond with the
`side chain of Asn710. The third reason may be related to the
`binding to the S2 extensive subsite. As shown in Fig. 3, for ten-
`eligliptin, introduction of the ‘‘anchor lock domain’’, which binds
`to the S2 extensive subsite, increased the activity by 1500-fold
`over the corresponding fragment that binds to S1 and S2 only
`
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`(a) vildagliptin–DPP4 complex
`
`(b) peptide (diprotin A, Ile-Pro-Ile)
`
`(c) vildagliptin
`
`(d) saxagliptin
`
`(e) alogliptin
`
`(f) linagliptin
`
`(g) sitagliptin
`
`(h) teneligliptin
`
`Fig. 1. Binding modes of each inhibitor in the active site of DPP-4. (A) Co-crystal structure of vildagliptin (cyan) bound to DPP-4 (orange) (PDB: 3W2T). (B) Co-crystal
`structure of the substrate peptide, diproin A (magenta) bound to DPP-4 (orange). (C)–(H) Co-crystal structures of each inhibitor (cyan) bound to DPP-4 (orange) superimposed
`on the substrate peptide (magenta). The active site of DPP-4 is shown as a gray-colored surface. Blue, red, yellow and green colors indicate nitrogen, oxygen, sulfur and
`fluorine atoms respectively, and others indicate carbon atoms. Interactions between inhibitors and water molecules are not shown in (B)–(H). PDB codes are noted in Section 2.
`
`[4,24]. On the other hand, for sitagliptin, previous studies re-
`vealed that the introduction of the substituent binding to the
`S2 extensive subsite increased the activity by 7-fold [6,25]. To
`investigate the reason for the difference in increased activity,
`we applied the estimation method (see Section 2) to the calcu-
`lation of contact areas in the S2 extensive subsite. The results
`showed that teneligliptin has a contact area of 0.92 nm2 (total
`contact area, 2.08 nm2), while sitagliptin has a contact area of
`0.71 nm2 (total contact area, 1.90 nm2). This result indicates that
`teneligliptin may bind more tightly to the S2 extensive subsite
`as a result of stronger hydrophobic interactions mediated by
`
`the ‘‘anchor lock domain’’. Binding of the anchor lock domain
`may relate to the residence time of the inhibitor in DPP-4 and
`the long in vivo duration of action.
`
`3.3. Particularity of the S2 extensive subsite
`
`As mentioned above, the S2 extensive subsite, which is not in-
`volved in substrate binding, contributes to increase the inhibitory
`activity for some DPP-4 inhibitors, but the particularity of the S2
`extensive subsite has not been well known. In other related prolyl
`peptidases,
`including DPP-8, DPP-9 and fibroblast activation
`
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`
`can increase not only their inhibitory activity but also their selec-
`tivity against other related prolyl peptidases.
`In conclusion, we comparatively present X-ray co-crystal struc-
`tures of six inhibitors with DPP-4 and categorized them into three
`classes on the basis of their binding subsites. As a result of the
`comparative study of the three classes, it is suggested that DPP-4
`inhibition tended to increase with an increase in the number of
`binding subsites. Furthermore, the additional interactions with
`
`
`the S01, S02 or S2 extensive subsite may increase DPP-4 inhibition be-
`yond the level afforded by the fundamental interactions with the
`S1 and S2 subsites and are more effective than forming a covalent
`bond with Ser630.
`
`Acknowledgments
`
`The authors thank Dr. Hideo Kubodera, Dr. Okimasa Okada and
`Dr. Kunitomo Adachi for their helpful discussion.
`
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`Table 3
`The DPP-4 inhibitory activity [22].
`
`Compound
`
`DPP-4 inhibition, IC50
`(nmol/L)
`
`Vildagliptin
`Saxagliptin
`Alogliptin
`Linagliptin
`Sitagliptin
`Teneligliptin
`
`29.2
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`4.9
`0.6
`10.3
`1.9
`
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`
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`assayed in the same system. The activity of compound 22t and sitagliptin is obtained from [25] and [6], respectively.
`
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