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Feb 5, 2019 - ABSTRACT: Vildagliptin (VIL) and saxagliptin (SAX) are two covalent drugs for the treatment of type 2 diabetes mellitus. The principal ...
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Research Article Cite This: ACS Catal. 2019, 9, 2292−2302

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Covalent Inhibition Mechanism of Antidiabetic DrugsVildagliptin vs Saxagliptin Yong-Heng Wang,†,‡ Fan Zhang,† Hongjuan Diao,† and Ruibo Wu*,† †

School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, P. R. China Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, P. R. China

‡

ACS Catal. Downloaded from pubs.acs.org by UNIV OF NEW ENGLAND on 02/11/19. For personal use only.

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ABSTRACT: Vildagliptin (VIL) and saxagliptin (SAX) are two covalent drugs for the treatment of type 2 diabetes mellitus. The principal pharmacological effects of VIL and SAX are known to arise from their biochemical reactions at the active site of dipeptidyl peptidase-4 (DPP-4), a serine protease that rapidly inactivates incretin hormones in plasma. However, the details of the catalytic mechanisms and the origin of the different pharmacokinetics behavior for the two scaffold-similar drugs are less clear. By employing quantum mechanical/molecular mechanical molecular dynamics simulations in this work, it is illuminated that the catalytic process involves two major steps: reversible covalent bonding which covalently modifies the antidiabetic target DPP-4 and irreversible hydrolysis reaction which converts the drugs into inactive metabolites. The reaction free energy profiles indicate that VIL is dissociated from DPP-4 mainly through the hydrolysis pathway, while SAX overwhelmingly through the reverse process of covalent bonding. Therefore, the inhibition is pseudoirreversible for VIL, while reversible for SAX. Further comparative studies reveal that the 4,5-methylene substituent of pyrrolidine ring in SAX is responsible for the different dissociation kinetics features and its higher inhibitory activity compared to the VIL. All these findings are in agreement with the previously reported experimental results and guidable for further covalent drug design toward DPP-4. KEYWORDS: enzyme catalysis, QM/MM, DPP-4, vildagliptin, saxagliptin

1. INTRODUCTION Type 2 diabetes mellitus (T2DM) is a global epidemic with many complications such as cardiovascular disease, kidney disease, eye disease, nerve damage, pregnancy complications, and so on, estimated to affect approximately 425 million people in 2017. 1 Traditional medications for T2DM include biguanides, sulfonylureas, meglitinides, thiazolidinediones, αglucosidase inhibitors, etc.2 Unfortunately, most of these agents have undesired side effects, like weight gain, edema, digestive problems, and importantly, hypoglycemic episodes.3,4 Vildagliptin (VIL) and saxagliptin (SAX) are two relatively new oral drugs for the treatment of T2DM with good efficacy, benign side-effect profile, and low hypoglycemia risk.3−7 Like many other gliptins, VIL and SAX are usually prescribed for people with T2DM who have not had good response to the traditional antidiabetic medications such as sulfonylureas. It is well-known that their common drug target is dipeptidyl peptidase-4 (DPP-4), a highly specific serine protease that cleaves N-terminal dipeptides from polypeptides with L-proline or L-alanine at the penultimate position.8−11 In contrast to the other noncovalent gliptins, the biochemical mechanism of therapeutic action for VIL and SAX is unusual: they covalently modify the DPP-4 enzyme through formation of imidate with Ser630 using the nitrile group (enzyme−drug covalent complex, © XXXX American Chemical Society

Scheme 1), which prevents the degradation of the incretin hormones (GLP-1: glucagon-like peptide-1; GIP: glucosedependent insulinotropic polypeptide). Therefore, VIL and SAX’s inhibitory activities toward DPP-4 increase the circulating levels of these incretin hormones and thus potentiate insulin synthesis and secretion, inhibit glucagon release, and reduce glucose production by the liver.3−7 The structures of VIL and SAX are highly similar (Scheme 1), and both are designed to mimic the native substrates, with the nitrile in the position of the scissile bond of the substrate.3−7 However, they have different pharmacokinetics which leads to the distinct dissociation pathways: VIL is dissociated from the enzyme−drug covalent complex through enzymatic hydrolysis reaction probably catalyzed by DPP-4 itself, which gives the free enzyme and an inactive carboxylic acid, LAY151,12 while SAX would escape through the reverse procedure of covalent bonding and then mainly be hydroxylated by cytochrome P450 into 5-hydroxysaxagliptin (see Scheme 1), which is an active metabolite with ∼50% pharmacological activity of SAX.13,14 In view of this experimental evidence, VIL is not Received: December 19, 2018 Revised: January 25, 2019 Published: February 5, 2019 2292

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VIL and SAX in DPP-4, including covalent bonding and hydrolysis reaction, in an effort to disclose the origin of the different dissociation kinetics.24,25

Scheme 1. Presumed Biochemical Processes of Vildagliptin (VIL) and Saxagliptin (SAX)

2. METHODS 2.1. Preparation of Enzyme−Drug Noncovalent Complexes. Due to its transient nature, no structure of DPP-4-VIL or DPP-4-SAX noncovalent complex has been determined in spite of extensive available structures of DPP-4 enzymes. Instead, the covalent complexes for both VIL and SAX have been reported (PDB codes: 3W2T26 and 3BJM27 respectively). Considering the crystal waters, which form hydrogen bonds with the drug and protein residues and may participate in the hydrolysis reaction, in the active pocket of 3W2T are more complete than those in 3BJM, we chose chain A of 3W2T as the initial structure for protein. The protonation states of titratable side chains were determined via H++ program,28and their individual local hydrogen bond networks were carefully examined. Specially, Asp708 is deprotonated; His740 is singly protonated at δ position to form hydrogen bond with Asp 708; and the amino groups at the acyl side chain of VIL and SAX are protonated. For VIL, the enzyme−drug noncovalent complex was directly built from crystal structure 3W2T by breaking the covalent bond between inhibitor and residue Ser630, while for SAX, the structure of inhibitor was abstracted from 3BJM and then put into the active site of protein prepared from 3W2T by maintaining the hydrogen bond networks and relative orientation as in 3BJM. 2.2. Classical MD Simulations. All simulations were performed with AMBER12;29 the AMBER99SB30 force field was employed for the protein, and the TIP3P31 model was used for solvent water molecules. The force field parameters of VIL and SAX were generated from the AMBER GAFF force field.32 The partial atomic charges of VIL and SAX were obtained from the restrained electrostatic potential (RESP)33 charge based on HF/6-31G* calculation with the Gaussian 09 package.34 The initial coordinates and topology files were generated by the tleap program with neutralization and solvation. Both models (VIL and SAX) were treated with the same MD protocols via employing the periodic boundary condition with cubic models. First, minimizations were carried out to relax the solvent and optimize the system. After that, each system was heated from 0 to 300 K gradually under the NVT ensemble for 100 ps, and then another 100 ps of NPT ensemble MD simulations at 300 K and the target pressure of 1.0 atm were carried out. In the NPT ensemble, the system temperature was controlled by the Langevin thermostat method.35 Afterward, a 30 ns MD simulation under the NVT ensemble at 300 K was performed for each model to obtain the equilibrious enzyme−drug complexes. All hydrogen-containing bonds during the MD simulations were constrained using the SHAKE algorithm.36 A cutoff of 12 Å was set for van der Waals and no cutoff for electrostatic interactions. Finally, snapshots of each system from the stable trajectories were used as the initial structures for the next QM/MM simulations. 2.3. Setup for Born−Oppenheimer QM(DFT)/MM MD Simulations. All QM/MM calculations were performed with the modified QChem37 and Tinker38 programs developed by Prof. Yingkai Zhang’s group (http://www.nyu.edu/projects/ yzhang/). Both QM/MM models (VIL and SAX) were prepared by deleting the solvent molecules beyond 30 Å from the reaction center (the nitrile carbon atom) of the inhibitor VIL and SAX. The inhibitor (VIL or SAX), Ser630, Asp708, His740, and

only a covalent inhibitor but also a non-native substrate of DPP4, whereas SAX is likely to be a reversible covalent inhibitor without hydrolysis reactivity. The different dissociation kinetics between VIL and SAX should be originated from the subtle structural discrepancies of the acyl side chain and substituent group on the pyrrolidine ring (Scheme 1). The structural discrepancy of the acyl side chain, i.e., the different positions of the adamantane group which is located at the N atom in VIL while at the α-C atom in SAX, is expected to affect the binding affinity with DPP-4 but less likely to determine the chemical reaction kinetics characteristics since the adamantane group is distal from the reactive center, the nitrile group. In contrast, the 4,5-methylene group on the pyrrolidine ring in SAX, which is absent in VIL, is close to the reactive center nitrile group. Thus, the introduction of the methylene group in SAX may be responsible for the loss of hydrolysis reactivity in DPP-4. Nevertheless, if the hydrolysis reaction is blocked by the steric hindrance of the methylene group, it is very perplexing as to why the covalent bonding/ modification is not blocked by the methylene group. In spite of significant efforts being devoted to investigate the mechanisms of action of VIL and SAX in clinical trials,3−7,13 there has been little understanding regarding the different biochemical reactions of these two structure-similar covalent drugs in DPP-4, while the decipherment of their covalent modification and possible catalytic reaction in DPP-4 is essential for understanding their pharmacokinetics/pharmacodynamics characteristics and useful for covalent drug design, which has recently attracted much attention in pharmacology due to its potential benefits, including high potency, low dose, extended duration of action, and general applicability to target proteins with shallow binding sites.15−20 Herein we have employed Born−Oppenheimer quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations20−23 to systematically investigate the possible biochemical process of 2293

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Figure 1. (a) Plausible covalent bonding mechanism; (b) free energy profiles for covalent bonding; (c) evolutions of key distances along A-to-B process for VIL; (d) evolutions of key distances along A-to-B process for SAX.

reaction coordinates defined as shown in Figure 1a and 3a. After that the reaction paths were divided into a number of windows, and the MM subsystems were further equilibrated by 500 ps classical MD simulations with the frozen QM subsystem for each window. Finally, the resulting snapshots were used as the initial structures for umbrella sampling at the QM/MM level. The system temperature was controlled by the Langevin thermostat method at 300 K,35 and the Newton equations of motion were integrated by the Beeman algorithm.45 After biased-potentialbased QM/MM MD umbrella sampling,46 the WHAM47,48 program was employed to map out the free energy profile with data collected from each window. Each window was simulated for 20 ps with 1 fs time step. The first 10 ps were used to equilibrate the system, and the last 10 ps were used to collect sampling structure and data for free energy analysis. The last 10 ps for each umbrella window has been split into three parts to estimate the free energy error bar, 10−15, 15−20, and 10−20 ps, respectively (Figures S1−S4). This free energy calculation

Tyr547 were considered as the QM area in covalent bonding process and an additional water molecule was considered in the hydrolysis process, while other atoms were considered in the MM area. All of these atoms in the QM subsystem were described with M06-2X39,40 in combination with the 6-31G(d) basis set, and the 6-31G(d,p) basis set was used for the migrated hydrogen. The QM/MM boundary was treated by the improved pseudo-bond approach.41−43The same molecular mechanical force field as in the previous classical MD simulations was used for all of the remaining atoms. The spherical boundary condition was employed, and atoms more than 24 Å away from the spherical center were fixed. The 12 and 18 Å cutoffs were employed for van der Waals and electrostatic interactions, respectively. There was no cutoff for electrostatic interactions between QM and MM regions. The QM/MM systems were minimized again for several iterations, and more than 5 ps QM/ MM MD simulations were performed. The resulting conformations were used to map out the minimum energy path with the reaction coordinate (RC) driving method44 according to the 2294

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Figure 2. Key intermediate structures (A, B, and C) of covalent bonding for VIL and SAX; the average values and the standard deviations of key distances are given in Å. The key residues and water molecules are noted; more details for the water hydrogen bond network in the pocket is shown in Figures S6−S9.

distance between the nitrile carbon atom and the oxygen atom of Ser630 is 2.50 and 2.63 Å for VIL and SAX, respectively, indicating the nitrile group is ready to be attacked by Ser630. The nucleophilic addition of Ser630 to the nitrile groups of VIL and SAX is accompanied by a proton transfer from the hydroxyl of Ser630 to the ε-N of His740 and a proton transfer from the δN of His740 to Asp708.54−56 Thus, the D−H dyad (Asp708− His740) is directly involved in the catalytic reaction as a general base to promote the deprotonation of Ser630, consistent with the reported mutation experiments that the mutation of either Asp708 or His740 leads to the loss of inhibition or catalysis activity in DPP-4.9−11,27Our QM/MM MD simulations indicate that these processes are highly concerted. As shown in Figure 1c and 1d, along the reaction from A to B state, the distances r1, r3, and r5 are decreased while r2 and r4 are increased synchronously. This step is rate-determining for the covalent bonding, and its free energy barrier is only 8.5 and 10.4 kcal/mol for VIL and SAX (Figure 1b), respectively, indicating that the covalent modification of Ser630 in DPP-4 is kinetically feasible both for VIL and SAX. Via the concerted C−O bond formation and proton transfers, imidate anion intermediate B is formed, and the negative charge

protocols are successfully utilized and validated in our previous studies.49−53

3. RESULTS AND DISCUSSION 3.1. Covalent Bonding. The mechanism details of covalent bonding are shown in Figure 1, and the key intermediate structures are shown in Figure 2. In the noncovalent drug− enzyme complex A (see Figure 2), VIL is well accommodated in the active pocket of DPP-4 by supplying the secondary ammonium group to form one salt bridge hydrogen bond with each of Glu205 and Glu206 and forming a very stable hydrogen bond between the nitrile N and Tyr547; SAX also exists in a optimal near-covalent-bonding pose with similar hydrogen bond network except for an additional hydrogen bond between the adamantane hydroxyl group and Tyr547 and an additional salt bridge hydrogen bond between the primary ammonium group and Glu206. This subtle difference in hydrogen bond network, which is thought to be associated with substrate recognition,9−11 mostly stems from the structural discrepancies in the acyl side chain between VIL and SAX. With the assist of the steady hydrogen bond network, the electrophilic nitrile groups of VIL and SAX are close to the nucleophilic residue Ser630. The 2295

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Figure 3. Plausible hydrolysis reaction mechanism; (b) free energy profiles for hydrolysis reaction; (c) evolutions of key distances along C*-to-D process for VIL; (d) evolutions of key distances along C*-to-D process for SAX. Considering intermediate C is not a reactive state for the hydrolysis reaction, the intermediate C* is employed herein as the starting point of the hydrolysis reaction. Due to the limitation of computational strategy on studying proton exchange between solvent water network and amino acid residues, we cannot predict the free energy profile for the conversion of C-toC* process, which is known to be very feasible and likely spontaneous.

deprotonated Tyr547. Therefore, the additional hydrogen bond of the hydroxyl oxygen of Tyr547 with the adamantane hydroxyl hydrogen, which results from the structural discrepancies in the acyl side chain between VIL and SAX, may not significantly affect the covalent bonding in energy, as it will be replaced by the hydrogen bond with a molecule of solvent water when it is absent. The whole C−O bond formation and series of proton transfer processes are exothermic by 10.1 and 5.8 kcal/mol for VIL and SAX, respectively (Figure 1b), leading to the effective covalent inhibition of VIL/SAX toward DPP-4. In view of the free energy barrier for the inverse procedure of covalent bonding for VIL and SAX, 18.6 and 16.2 kcal/mol, respectively (Figure 1b), the dissociation of SAX from the target DPP-4 is more facile than that of VIL if both of them are reversible covalent inhibitor. This is obviously conflicted with the experimental investigation that SAX presents higher pharmacological activity than VIL.3,12−14

is transferred from Asp708 to N7 atom of VIL or SAX (Figure 1a). As shown in Figure 2, this anion hole would be stabilized by a new hydrogen bond between N7 atom and Tyr631, as well as an additional hydrogen bond between N7 atom and a molecule of solvent water, which are spontaneously rearranged and formed. Nevertheless, (see Figure 1a and 1b), intermediate B is assessed as a metastable state since its nitrogen anion will be quenched immediately by the proton transfer from Tyr547 almost without barrier (only 0.6 and 0.4 kcal/mol for VIL and SAX, respectively), leading to the formation of imidate intermediate C. In intermediate C (see Figure 2), the oxygen anion of the deprotonated Tyr547 is stabilized by the as-formed imine group, the adamantane hydroxyl group, and two molecules of water through hydrogen bond interaction for SAX, and the hydrogen network for VIL is similar except that the adamantane hydroxyl group is replaced by a molecule of solvent water to form hydrogen bond with the oxygen anion of the 2296

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Figure 4. Key intermediate structures (C*, D, and E) of hydrolysis reaction for VIL and SAX; the average values and the standard deviations of key distances are given in Å. The key residues and water molecules are noted; more details for the water hydrogen bond network in the pocket are shown in Figures S6−S9.

Similar to the forementioned nucleophilic addition of Ser630 with the nitrile group (namely A-to-B process), the nucleophilic addition of water with the imidate group (namely C*-to-D process) is also involved the C−O bond formation and serial of proton transfer. The C−O bond formation is accompanied by a proton transfer from the water to the ε-N of His740 and a proton transfer from the δ-N of His740 to Asp708 (Figure 3a). In contrast to the covalent bonding (section 3.1), these processes are concerted but asynchronous, and this feature is even more obvious for SAX (Figure 3c and 3d). The proton transfer from the δ-N of His740 to Asp708 takes place first, leading to the deprotonation of His740 which enhances its basicity, and then the proton transfer from the water to the ε-N of His740, leading to the formation of hydroxide with stronger nucleophilicity, and finally the C−O bond formation. This difference between the two nucleophilic additions may be attributed to two aspects. On the one hand, the acidity of water is weaker than the hydroxyl of Ser630, which means a stronger base is needed for facilitating deprotonation; on the other hand, the amidate group is less electrophilic than the nitrile group, which means a stronger nucleophile is needed for addition. The nucleophilic addition of water is the rate-determining step for hydrolysis reaction, and its

Therefore, the inhibitory mechanism of VIL and SAX is probably different and related to the potential hydrolysis reactivity of intermediate C (see section 3.2). 3.2. Hydrolysis Reaction. Since the active pocket of DPP-4 is hydrophilic and there are many solvent waters around the catalytic residues (see Figures S6−S9), the protonation states of Tyr547, His740, and Asp708 are ready to be rescued to form a reactive intermediate C*, which is employed as the starting point to investigate the hydrolysis reaction in this work (Figures 3 and 4). During our MD simulations of C* state, including MM MD and QM/MM MD simulations, we observed that a water molecule was trapped by hydrogen bond interactions with the εN of His740 and the acyl O of VIL or SAX (Figures 3 and 4), which were also observed in X-ray crystal structures (PDB codes: 3W2T and 6B1E).26 This hydrogen bond network makes the nucleophilic O atom of the water close to the electrophilic C atom of the imidate, and the distance is 2.80 and 2.97 Å for VIL and SAX, respectively. Hence, this water is considered as a plausible nucleophile to hydrolyze the covalent complex. The hydrolysis reaction includes two steps: the nucleophilic addition of water to form tetrahedral intermediate D and the departure of Ser630 to form imidic acid E (Figure 3a). 2297

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Figure 5. Distribution of dihedral angle of N1−C2−C6−N7, denoted as ϕ (down), and angle of C6−C2−(midpoint of C4 and C5), denoted as θ (up), along the reaction from A state to E state in our QM(DFT)/MM MD simulations. The ϕ value reflects the orientation of the −CN(H) group; the difference of θ values between VIL and SAX reflects the deformation of pyrrolidine ring in SAX, i.e., the larger the difference of θ values is, the more notablethe distortion of pyrrolidine ring is.

free energy barrier is 17.6 and 25.8 kcal/mol for VIL and SAX (Figure 3b), respectively, suggesting that the hydrolysis is kinetically feasible for VIL but difficult for SAX. Via the concerted but asynchronous C−O bond formation and proton transfers, tetrahedral intermediate D is formed, and the negative charge is transferred from Asp708 to N7 atom of VIL or SAX and is stabilized by the hydrogen bond interactions with Tyr547 and Tyr631 (Figure 4). The free energy of D state relative to C* state is 5.3 and 19.4 kcal/mol for VIL and SAX, respectively. By overcoming a free energy barrier of 6.2 (VIL) or 4.2 kcal/mol (SAX) from D state, the C−O bond between the drug molecular and Ser630 of enzyme is broken, accompanying a proton transfer from the ε-N of His740 to the hydroxyl of Ser630 and a proton transfer from Asp708 to the δ-N of His740 (Figure S5). By undergoing these processes, a final catalytic product imidic acid E is formed, and the enzyme is rescued to the initial catalytic state. The whole hydrolysis process is highly endothermic by 8.1 kcal/mol for SAX, suggesting the hydrolysis of SAX is thermodynamically unfavorable, while it is thermoneutral for VIL (Figure 3b). Considering the imidic acid E would be further hydrolyzed into carboxylic acid at the active site of DPP-4 or in solvent as detected experimentally,12 the exothermicity would facilitate the drug release and make the hydrolysis for VIL thermodynamically favorable, which is consistent with the observation in the metabolism experiments.12 For VIL, the free energy barrier for the hydrolysis reaction is a little lower than the reverse procedure of covalent bonding (17.6 vs 18.6 kcal/mol, Figure 3b and 1b). Thus, the dissociation of VIL from the enzyme−drug covalent complex is more likely through the hydrolysis pathway. For SAX, the free energy for the hydrolysis reaction is much higher than the reverse procedure of covalent bonding (25.8 vs 16.2 kcal/mol, Figures 3b and 1b). Thus, the hydrolysis reaction of SAX is prohibited, and it will escape from the DPP-4 pocket overwhelmingly through the reverse procedure of the covalent binding. Since the hydrolysis reaction is irreversible (Scheme 1), VIL will lose its pharmacological activity once it is hydrolyzed into the carboxylic acid LAY151, which has no inhibitory activity toward DPP-4.

On the contrary, the covalent bonding of SAX is reversible, and its enzyme−drug covalent complex is thermodynamically very stable; thus, the dynamic biochemical equilibrium is maintained for the dissociated SAX and SAX-DPP-4 covalent complex. The elimination rate of SAX from plasma may depend on the rate of renal clearance4 and cytochrome P450-mediated metabolism which converts approximately 50% of administered dose of SAX into 5-hydroxysaxagliptin with approximately 50% potency of SAX,13,14 while the elimination rate of VIL mainly depends on the rate of hydrolysis reaction catalyzed by DPP-4 itself. That may be the reason why the plasma half-life of SAX (∼27 h) is much longer than that of VIL (∼2 h) and thus SAX shows higher potent than VIL: therapeutic dosage of SAX is 5 mg once a day, while VIL is 50 mg twice a day.3,12−14 Therefore, the different pharmacological activities of the two covalent drugs VIL and SAX are likely to be determined by the different dissociation kinetics features in DPP-4. 3.3. Origin of Different Dissociation Kinetics. As assumed in the Introduction section, the different dissociation kinetics between VIL and SAX may be originated from the structural discrepancies of the acyl side chain and substituent group on the pyrrolidine ring (Scheme 1). According to the intermediate structures obtained by our QM/MM MD simulation (Figures 2 and 4), the structural discrepancy of the acyl side chain leads to three subtle differences: (a) The primary ammonium in SAX forms three salt bridge hydrogen bonds with Glu205 and Glu206, while the secondary ammonium in VIL forms two salt bridge hydrogen bonds with Glu205 and Glu206 (Figure 2 and 4); these hydrogen bonds play an important role in anchoring substrate or inhibitor for enzyme catalysis or inhibition as proposed by previous studies.9−11 However, they are so far from the reactive center nitrile group that it would not directly affect chemical reaction kinetics. (b) The adamantane hydroxyl group in SAX forms hydrogen bond with Tyr547, while this hydrogen bond is absent for VIL. This hydrogen bond may stabilize the oxygen anion of the deprotonated Tyr547 in intermediate C state (Figure 2). However, it is compensated by the hydrogen 2298

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SAX (Figure 5). The increase of φ, i.e., clockwise rotation of the −CNH group around C2−C6 bond, which results from the attack of the nucleophilic water, may lead to the increase of steric clash between the −CNH group and the methylene group in SAX; thus, it is hysteretic in SAX relative to VIL (Figure 5). Moreover, the steric repulsion in SAX is so harsh after TS3 state that a remarkable distortion of the pyrrolidine ring occurs (Figure 5), and the rotation of the −CNH group is blocked around 60°. This may be responsible for the large free energy difference between SAX and VIL for TS3 state (25.7 − 17.6 = 8.1 kcal/mol) and D state (19.4 − 5.3 = 14.1 kcal/mol, Figure 3b). Accordingly, it is the introduction of the methylene to the pyrrolidine ring, which leads to the rotation forbidden of the −CNH group, that distinguishes the hydrolysis reaction from the covalent bonding through the distinct steric interactions and selectively blocks the hydrolysis reaction by raising its reaction energy barrier. Essentially, the nature behind the above phenomenon is the different hybridization evolution of the reactive C6 atom between the two processes (covalent bonding vs hydrolysis reaction). For the rate-determining step of covalent bonding (Figure 1), the hybridization style of C6 atom changes from sp to sp2, while for the rate-determining step of hydrolysis reaction (Figure 3), the hybridization style of C6 atom changes from sp2 to sp3. Although the sp-to-sp2 covalent bonding process bends the CN(H) group and makes it point to the inside of the pyrrolidine ring, its steric hindrance with the 4,5-methylene group at 54° direction is small since the sp2-hybridization intermediate B or C adopts an eclipsed conformation (Figure 5) with average φ value of ∼10°, which is the optimal conformation for sp2-hybridization carbon.57,58 In contrast, since the sp3hybridization carbon has a tetrahedral structure, the rotation of the −CNH group is required for the sp2-to-sp3 hydrolysis reaction process, and the sp3-hybridization intermediate D adopted a staggered conformation (Figure 5) with average φ value of ∼50°, which is the optimal conformation for sp3hybridization carbon.58 This leads to the harsh steric repulsion between the −CNH group and the methylene group (at 54° direction) and selectively forbids the sp2-to-sp3 hydrolysis process for SAX. 3.4. Drug−Enzyme Interactions. As shown in Scheme 2, according to whether a covalent bond between an enzyme and

bond with solvent water for VIL as discussed in section 3.1, and thus it may have little contribution to the energy difference between VIL and SAX. (c) The structure of VIL is more stretched than that of SAX (Figure 2 and 4). This difference makes the electrophilic nitrile group of VIL closer to the nucleophile, Ser630 or water, than that of SAX (2.50 vs 2.63 Å in A state, 2.80 vs 2.79 Å in C* state, and 2.55 vs 2.63 Å in E state), which makes the nucleophilic addition of VIL a little easier (lower reaction barrier) than SAX. However, this effect is always existing and cannot distinguish the hydrolysis reaction from the covalent bonding in reaction kinetics. Accordingly, the structural discrepancy of the acyl side chain between SAX and VIL is not the determinant factor for their different dissociation kinetics in DPP-4, and it most likely originated from the substituent effect of pyrrolidine ring. As shown in Figures 2 and 4, the 4,5-methylene group on the pyrrolidine ring in SAX is spatially close to the reactive nitrile group, and thus it may affect the reaction kinetics through steric interaction. Nevertheless, how could it distinguish the hydrolysis reaction from the covalent bonding and selectively block the hydrolysis reaction? To answer this question, we monitor the distribution of dihedral angle of N1−C2−C6−N7, denoted as ϕ, and the angle of C6−C2−(midpoint of C4 and C5), denoted as θ, along the reaction pathways (Figure 5). The ϕ value reflects the relative orientation of the −CN(H) group, which may have steric hindrance with substituents on pyrrolidine ring; the difference of θ values between VIL and SAX reflects the distortion of pyrrolidine ring in SAX due to the steric hindrance between the −CN(H) group and the 4,5-methylene group. For the covalent bonding process, as shown in Figure 5, the ϕ value fluctuated around an average of ∼10°, and the range of the fluctuation gradually decreased from A to C state for both VIL and SAX. Though the fluctuation of ϕ near A state is considerable (from −40° to 60°), the steric hindrance between the CN group and the substituent on pyrrolidine ring is negligible since the linear CN group points to the outside of the pydrrolidine ring (Figure 2). In the TS1 state, the C N group is bended by the attack of Ser630 and points partly to the inside of the pyrrolidine ring (Figures S6 and S8), and the fluctuation of φ ranges from −15° to 35°. Since the methylene group on the pyrrolidine ring of SAX is located at about 54° direction (Figure 5), its steric hindrance with −CN group is tiny and results in a small free energy difference of TS1 between VIL and SAX (10.4 − 8.5 = 1.9 kcal/mol, Figure 1b). In the B state (Figure 2), the CN group is even more bended due to the formation of C−O bond, which may increase the steric hindrance in SAX, leading to the increase of the free energy difference between VIL and SAX (3.3 − 0.3 = 3.0 kcal/mol, Figure 1b). In C state (Figure 2), the bulkier CNH group, with both N and H atom interacting with the methylene (Figure S8 and S9), may further increase the steric hindrance in SAX, leading to the further enhancement of the free energy difference between VIL and SAX (10.1 − 5.8) = 4.3 kcal/mol, Figure 1b). Nevertheless, the overall steric hindrance between the −CN(H) group and the methylene in the covalent bonding process is small, for the distortion of pyrrolidine ring of SAX relative to that of VIL is small (corresponding to the small difference of θ values between VIL and SAX, Figure 5). For the hydrolysis process, the average ϕ value first maintains around 10° and gradually increases to 90° for VIL and to 60° for

Scheme 2. Interaction Modes between Enzyme and Inhibitor

an inhibitor is formed or not, the inhibitory interaction can be classified into noncovalent and covalent inhibition, and the covalent inhibition can be further classified into reversible and irreversible based on the reversibility of the as-formed covalent bond;15−20 particularly, if the covalent inhibitor would be further metabolized by the enzyme, i.e., the inhibitor is a substrate of the enzyme, this interaction is termed as “pseudoirreversible covalent inhibition”.59−62 Among these interactions, the pseudoirreversible covalent inhibition has the 2299

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ACS Catalysis most complicated dissociation kinetics, which is associated with the reverse reaction rate constant k−2 and the metabolism rate constant k3 for the E′−I′ complex, and it will turn into reversible inhibition when k3 is much smaller than k−2, i.e., the metabolism pathway is blocked. For VIL, since the barrier for metabolism is lower than that for the reverse process of covalent bonding (17.6 vs 18.6, Figure 1b and 3b), i.e., k3 > k−2, the drug−enzyme interaction is pseudoirreversible, while for SAX, since the barrier for metabolism is much higher than that for the reverse process of covalent bonding (25.8 vs 16.2, Figure 1b and 3b), i.e., k3 ≪ k−2, the drug−enzyme interaction is reversible. Thus, the current system provides a strategy for the transformation from pseudoirreversible to reversible covalent inhibition by substituent steric effect, which enhances the pharmacological activity (see more examples from Chart S1),26,63,64 and this strategy may be useful for further rational covalent drug design not only for the current enzyme system but also for other serine proteases54−56,65−67 which have similar catalytic mechanisms as DPP-4.

Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB)

4. CONCLUSIONS We have illuminated the covalent inhibition mechanisms of two structure-similar antidiabetic drugs, VIL and SAX, from the view of enzyme catalysis using elaborative QM(DFT)/MM MD simulations. The drug−target interaction process involves two major steps: reversible covalent bonding which covalently modifies the Ser630 of DPP-4 and irreversible hydrolysis reaction which converts the drugs into inactive metabolites. VIL involves a pseudoirreversible covalent inhibition and dissociates from the target mainly through the hydrolysis pathway, whose reaction barrier is a little lower than the inverse process of covalent bonding, while SAX abides by a reversible covalent inhibition and escapes from the pocket through the inverse procedure of covalent-bonding pathway, whose barrier is much lower than the hydrolysis reaction. The previously experimentally reported difference in pharmacological activity of the two drugs is attributed to the distinguishable dissociation kinetics features of VIL and SAX as clarified herein, originating from the substituent steric effect of pyrrolidine ring, which distinguishes the sp2-to-sp3 hydrolysis reaction process from the sp-to-sp2 covalent bonding process and selectively blocks the hydrolysis reaction and thus enhances the pharmacological activity of SAX. The understanding on the basic principles of the pseudoirreversible covalent inhibition in DPP-4 would be guidable for rational covalent drug design targeting serine proteases (including DPP4) in the future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscatal.8b05051. Figures S1−S9; Chart S1 (PDF) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB) Representative snapshots for reactants, transition states, and intermediates from QM/MM MD simulations (PDB)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Ruibo Wu: 0000-0002-1984-046X Notes

The authors declare no competing financial interest. 2300

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ACS Catalysis

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ACKNOWLEDGMENTS

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REFERENCES

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This work was supported by the National Natural Science Foundation of China (21773313 and 21803080), Guangdong Natural Science Founds for Distinguished Young Scholars (2016A030306038), and the National Key R&D Program of China (2017YFB0202600). We thank the Guangzhou and Shenzhen Supercomputer Center for providing computational source, and we also thank the Three Big ConstructionsSupercomputing Application Cultivation Projects from SYSU.

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