Thermodynamic and Structural Characterization of Halogen Bonding

Brief Article pubs.acs.org/jmc

Thermodynamic and Structural Characterization of Halogen Bonding in Protein−Ligand Interactions: A Case Study of PDE5 and Its Inhibitors Jing Ren, Yang He, Wuyan Chen, Tiantian Chen, Guan Wang, Zhen Wang, Zhijian Xu, Xiaomin Luo, Weiliang Zhu, Hualiang Jiang, Jingshan Shen,* and Yechun Xu* CAS Key Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (CAS), 555 Zuchongzhi Road, Shanghai 201203, China S Supporting Information *

ABSTRACT: The significance of halogen bonding in protein−ligand interactions has been recognized recently. We present here the first comprehensive thermodynamic and structural characterization of halogen bonding in PDE5−inhibitor interactions. ITC studies reveal that binding strength of the halogen bonding between chlorine, bromine, and iodine of inhibitor and the protein is −1.57, −3.09, and −5.59 kJ/mol, respectively. The halogens interact with the designed residue Y612 and an unexpected buried water molecule.

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diseases associated with low cGMP level.23 For example, sildenafil, the prototypical member of PDE5 inhibitors, was approved by FDA as the first oral medicine for the treatment of male erectile dysfunction (ED) in 199824 and was also launched for the treatment for pulmonary arterial hypertension (PAH) in 2005.25 Other inhibitors, vardenafil, tadalafil, and avanafil, are also used to treat ED and PAH.26−28 Additionally, the medical application of PDE5 inhibitors in other diseases such as stroke, Raynaud’s disease, overactive bladder, and premature ejaculation also has been indicated.29,30 We have previously described the design, synthesis, and pharmacological evaluation of monocyclic pyrimidinones as novel inhibitors of PDE5.11,31,32 To introduce halogen bonding between halogen atom of inhibitors and the hydroxyl oxygen atom of residue Y612 at the active-site of PDE5, the 5-position of the pyrimidinone ring was substituted by halogens (Figure 1A).11 On the basis of our previous work, we here present a comprehensive thermodynamic study on the binding of PDE5 with five inhibitors which share an identical structure scaffold but have different substituents, H, F, Cl, Br, and I, at the 5position of the pyrimidinone ring (Figure 1A). Isothermal titration calorimetry (ITC) experiments were carried out to measure the strength of halogen bonding between chlorinated, brominated, or iodinated inhibitor and PDE5, providing the thermodynamic properties of halogen bonding in protein− ligand interactions for the first time. In addition to the available

INTRODUCTION Halogenation of organic compounds has been widely used in processes of hit-to-lead or lead-to-drug conversions, and many drugs and drug candidates in clinical development contain halogen.1,2 Recently, halogen bonding, noncovalent intermolecular interaction occurring between Lewis bases (O, N, and S) and Lewis acids (Cl, Br, and I), has been recognized as prevalent interaction between halogenated ligand and target protein.3−10 A few examples have been reported in which SAR studies, and X-ray crystallographic data have been used in concert as evidence for incorporation of the halogen bonding in biological activities.10−17 However, the occurrence of halogen bonding in protein−ligand complexes is largely the result of database surveys rather than rational design. So far the solution thermodynamic data is only available for halogen bonding appearing in the system of small molecules or polymers but not for those involved in protein−ligand interactions.7 Although several systematic computational studies aiming at an improved understanding of structure−energy relationships of halogen bonding for rational drug discovery have been reported,4,18−21 a thorough experimental characterization of halogen bonding in protein−ligand recognition is still lacking. Phosphodiesterase type 5 (PDE5) is a cGMP-specific enzyme and mostly expressed in smooth muscle tissue of corpus cavernosum, heart, lung, platelets, prostate, urethra, bladder, liver, brain, and stomach.22 Because of the significant role of cGMP in multiple signaling pathways, inhibitors of PDE5, which prevent the hydrolysis of cGMP thus maintaining the desired level of cGMP, become effective treatment to © 2014 American Chemical Society

Received: February 12, 2014 Published: April 5, 2014 3588

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residue Q817, π−π stacking interactions with the phenyl ring of F820, and hydrophobic interactions with residues L765, V782, A783, and F786. Actually, the binding modes of inhibitors 1−5 to PDE5 are similar as structures of five inhibitors overlapped well except for the methylpiperazine group (Figures 1D). Because this group hardly has interaction with neighboring residues, its orientation is affected by the crystal packing. For example, in the crystal packing interface of the PDE5-4 complex with space group of P212121, two H-bonds, with distances of 2.4 and 2.9 Å, respectively, were formed between the methylpiperazine nitrogen in one copy and two carboxyl oxygen of residue E858 in the other copy of the protein (Figure S1 in the Supporting Information), maintaining the orientation of the methylpiperazine group of 4. Such H-bonds was not found in the other four structures with the same space group of P3121. Therefore, the methylpiperazine group adopted a different orientation in the complex of PDE5-4 compared with the others. On the basis of the five crystal structures of PDE5 in complex with inhibitors, the distance between the 5-substituent (X) and the hydroxyl oxygen atom (OH) of residue Y612 and the angle of the C−X···OH were measured (Table 1). In PDE5-1, dH···OH is 0.44 nm, demonstrating that there is no interaction between the two atoms. A distance of 0.39 nm for dF···OH in PDE5-2 indicates that a weak H-bond might be formed between F and the hydroxyl group of Y612. The distances between OH and Cl, Br, and I are 0.36, 0.34, and 0.37 nm, respectively. The angle of C−Cl···OH, C−Br···OH, and C−I···OH is 141, 149, and 122°, respectively. The values of dX···OH as well as the angle suggest that the binding strength of bromide to OH might be the strongest among three interactions between Y612 and inhibitors 3, 4, and 5. To get deep insight into halogen bonding occurring in PDE5−inhibitor interactions, the thermodynamics behavior of five inhibitors binding to the catalytic domain of PDE5 in solution were investigated by ITC. The averaged dissociation constant (Kd), binding free energy (ΔG), enthalpy (ΔH), and entropy term (−TΔS) resulted from three independent ITC measurements are listed in Table 1. Figure S2 in the Supporting Information displays the representative ITC results and fitting curve of each inhibitor binding to PDE5. The correlation coefficient between Kd and IC50 of five compounds is 0.96, although there is difference in the values of IC50 and Kd for a single compound. The IC50 was obtained by a tritium scintillation proximity assay with using the full-length of rabbit PDE5,11 while in ITC measurement the recombinant catalytic domain of human PDE5 was used, which might explain the difference between two values. The binding free energy of PDE5 interacting with the inhibitor (ΔG) revealed that the

Figure 1. Structures of five inhibitors and their complexes with the catalytic domain of PDE5. (A) Chemical structures of the five inhibitors. (B) The (F0 − Fc) difference electron-density maps contoured at 3.0 σ for inhibitor 5 bound in PDE5. (C) The detailed interactions between 5 and PDE5. (D) The superimposition of 1 (green), 2 (cyan), 3 (magenta), 4 (yellow), and 5 (salmon) by fitting the coordinates of proteins of five complex structures.

complex structures of PDE5-2, PDE5-3, and PDE5-4, we determined two more crystal structures in which the catalytic domain of PDE5 is bound with inhibitors 1 or 5. The binding strength in combination with the geometry of the halogen bonds shown in the crystal structures, a structure−energy relationship of halogen bonding between halogen and oxygen is obtained. Moreover, the freshly determined complex structure of PDE5-5 unexpectedly reveals that the iodine of inhibitor 5 forms halogen bonding to not only the designed the hydroxyl oxygen of residue Y612 but also the oxygen of a buried water molecule which is simultaneously H-bonded with three residues. A particular thermodynamic behavior of fluorinated ligand binding to protein is also discussed.

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RESULTS Complex structures of the catalytic domain of PDE5 bound with 1 and 5 were solved by soaking the inhibitors into the apo crystals of PDE5 with space group of P3121. The crystallographic data of these two structures are summarized in Table S1 in the Supporting Information. Figure 1B,C show the binding of inhibitor 5 to PDE5. The major interactions of 5 with the protein are the classical bidentate H-bonds with

Table 1. Geometry of Halogen Bonding, Thermodynamic Property and IC50 of Inhibitors Binding to PDE5 compd 1 2 3 4 5

(H) (F) (Cl) (Br) (I)

PDB code 4OEX 3SHY 3SHZ 3SIE 4OEW

d1a (nm) c

0.45 0.39 0.36 0.34d 0.37

σ1 a (deg) c

153 151 141 149d 122

σ2a (nm) 0.5 N/A 0.41 0.42 0.34

θ2a (deg) 153 N/A 164 164 166

ΔG (kJ/mol)

Kd (M) (1.45 (1.73 (7.67 (4.16 (1.52

± ± ± ± ±

0.09) 0.11) 1.41) 0.32) 0.19)

× × × × ×

−6

10 10−6 10−7 10−7 10−7

−33.33 −32.89 −34.90 −36.42 −38.92

ΔH (kJ/mol) −31.07 −14.74 −41.38 −49.02 −54.48

± ± ± ± ±

0.17 0.44 0.17 2.9 1.96

−TΔS (kJ/mol) −2.26 −18.15 6.48 12.60 15.56

IC50b (nM) 51.8 90.9 35.9 13.3 7.2

± ± ± ± ±

12.1 22.4 11.5 4.5 2.1

The distance between the halogen or hydrogen atom (X) and the oxygen atom of Y612 (d1) or water molecule (d2), the σ-hole angle of C−X··· OH(Y612)(σ1) or C−X···OW(water molecule)(σ2) . bThe values were cited from our previous study.11 cHydrogen atoms were added to the compound 1 before determining the distance as well as the angle. dA mean value of the one resulted from the chain A and chain B in an asymmetric unit. a

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replacing of 5-H by 5-Cl, -Br, and -I substituent gained stronger binding to the enzyme, while the substitution of F at the same position resulted in a weaker binding of 2 to PDE5 (Table 1 and Figure 2). Further exploration of the effect of enthalpy and

Figure 2. The binding free energy (ΔG), enthalpy (ΔH), and entropy term (−TΔS) of five inhibitors binding to the catalytic domain of PDE5 in solution (A) and ΔΔG, ΔΔH, and Δ(−TΔS) between 1 and the other four inhibitors (B).

Figure 3. The microenvironment of 5-substituent of inhibitors. (A) Superimposition of inhibitors 1 (green), 2 (cyan), 3 (magenta), 4 (yellow), and 5 (salmon), residues Y612, A767, and D764, and a structural water molecule by fitting the coordinates of proteins. (B−F) View of each inhibitor and its neighboring residues and structural water, 1 (B), 2 (C), 3 (D), 4 (E), and 5 (F). Distances (in Å) are labeled.

entropy on protein−ligand interactions indicated that the binding of 1, 3, 4, and 5 to PDE5 is mainly driven by the enthalpic term, while the influence of entropy on the binding is negative (Figure 2A). However, in the case of 2 binding to the enzyme, the positive entropic contribution to the binding affinity is even higher than enthalpy.

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enzyme is strong and it could act like a residue. The distance between 5-I and oxygen (OW) of the water molecule, dI···OW, is 0.34, and the angle of C−I···OW is 165.6°, suggesting perfect halogen bonding occurs between 5 and this structural water. In the complexes of PDE5-3 and PDE5-4, the angle of C−Cl··· OW and C−Br···OW is 163.6 and 164.4°, respectively. Although distances of 5-Cl and 5-Br to OW are over 0.4 nm, halogen bonding might occur between Cl or Br and the water molecule too. Quantum mechanics (QM) calculations were performed on the simplified model (Figure S3 in the Supporting Information) to calculate the halogen bonding interaction energies between 5-Cl, 5-Br, and 5-I and Y612 as well as the water molecule. The coordinates of the heavy atoms in the model which were directly extracted from the crystal structures were fixed and only the hydrogens were free during the energy optimization. The optimized orientation of hydrogens is shown in Figure S3 in the Supporting Information. Table S2 in the Supporting Information shows that the 5-substituent of three inhibitors have halogen bonding interactions with both the residue Y612 and the water molecule. The interaction energy between the 5Cl or 5-Br and Y612 is stronger than the energy between this substituent and the water molecule, while it is opposite in the case of PDE5-5 complex. This is consistent with the geometry shown in three complex structures. It is well-known that halogen substituents typically form multiple interactions with proteins beside halogen bonding such as hydrophobic interactions.33 The distances between the halogen and neighboring carbon atoms were measured based on the complex structures. It revealed that distances of Cl···C, Br···C, and I···C are over 0.4 nm, which is used as a cutoff to measure the hydrophobic-interacted atom pair in LIGPLOT.34 Only the distance between 5-Br of 4 and Cζ of Y612 is 0.38 nm. Therefore, the hydrophobic interactions between 5-Cl, 5-Br, or 5-I and neighboring residues can be ignored as compared to halogen bonding. Eventually, we conclude that the contribution of halogen bonding to the binding affinity of inhibitors to PDE5 or the binding strength of halogen bonding is about −1.57, −3.09, and

DISCUSSION Halogen Bonding of C−X···O. We have determined the thermodynamic properties of five inhibitors binding to PDE5 by ITC measurements. Although the replacement of H by F at the 5-position of the pyrimidinone ring resulted in a loss in binding affinity, inclusion of the 5-Cl substituent increased the binding affinity of 3 with PDE5 as compared to 1. Further increases in the affinity were obtained by replacing the 5-Cl substituent by a bromo (4) and iodo (5) group. Five inhibitors are identical in structure scaffold except the for 5-position substituent. Moreover, the crystal structures revealed that the binding modes of five inhibitors to PDE5 are quite similar and there is no interaction between the 5-H and protein. Therefore, the energy difference of 2, 3, 4, and 5 to 1, ΔΔG, ΔΔH, and Δ(−TΔS) could refer to the thermodynamic data of the interactions between the 5-substituent and PDE5. The ΔΔG of 5-Cl, 5-Br, and 5-I binding to PDE5 is about −1.57, −3.09, and −5.59 kJ/mol, respectively. In addition, the enthalpy term (ΔΔH) for the 5-Cl, 5-Br, and 5-I binding to PDE5 is about −10.31, −17.95, and −23.41 kJ/mol, respectively, while the contribution of entropy to the binding is always negative (Figure 2B). It suggests that the formation of interactions between 5-Cl, 5-Br, or 5-I and PDE5 is predominantly enthalpy-driven. The purpose of introducing halogen at 5-position of the pyrimidinone ring is to generate halogen bonding between 5substituent and the hydroxyl oxygen atom of residue Y612. In light of the geometry data shown in Table 1, a halogen bond is formed between 5-Br and OH of Y612. We noticed that the distance of I···OH and the σ-hole angle of C−I···OH do not show a perfect halogen bond occurring between 5-I and Y612. However, the ΔΔG of 5-I binding to PDE5 is −5.59 kJ/mol, much higher than the other two. A closer inspection of crystal structures revealed that there is a buried water molecule simultaneously H-bonding with the main chain of D764 and A767 and the side-chain of Y612 (Figure 3). This structural water is also found in most of the other PDE5 structures, demonstrating that the binding of the water molecule to the 3590

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−5.59 kJ/mol in the complex of PDE5-3, PDE5-4, and PDE55, respectively. Not only the designed halogen bonding between the 5-X and Y612 but also the one with the unexpected structure water molecule occurred. As an example, the energy of 5.59 kJ/mol in the complex of PDE5-5 is ascribed to the halogen bonding of C−I···OW as well as C−I···OH. However, the binding strength of the two types of halogen bonding in the three complexes is different. The strength of C− X···OW is stronger than the one of C−X···OH in the complex of PDE5-5 while it is opposite in the other two cases, which is correlated well with the geometry of the halogen bond. For example, the distance of 5-Cl to the OH of Y612 is above the sum of the van der Waals radii of chloride and oxygen, and the bond of Cl···OH tilts far away from the axis of C−Cl, so the halogen bonding of C−Cl···OH is weak. In the case of C−Br··· OH and C−I···OW, the dX···O is below the sum of the van der Waals radii of halogen and oxygen, meanwhile the angle of C− X···O is closer to 180°, the binding strength of these two halogen bonds is thereby stronger than that of C−Cl···OH (Table S2 in the Supporting Information). In conclusion, the combination of thermodynamic, X-ray crystallographic data, and QM calculation not only shows evidence for incorporation of the halogen bonding in binding affinity but also reveals the first structure−energy relationship of halogen bonding. In particular, the discovery and characterization of the halogen bonding between iodine and the buried water molecule in the present study provide a representative case and shed light on the significance of halogen bonding to structural water for rational drug design. Thermodynamic Behavior of Fluorinated Inhibitor Binding to Protein. The unique chemical property of fluorine leads to the thermodynamic data of 2 binding to PDE5 quite differently from those of the others (Figure 2A). The contribution of enthalpy to the binding free energy is significant low in the complex of PDE5-2. The crystal structure suggests that a H-bond might be formed between the 5-F and the hydroxyl group of Y612 because the distance of F···OH is 0.39 nm. Although the H-bonding has a beneficial effect on the binding of 2 to the enzyme, the close distance of F···OH and high electronegativity of both oxygen and fluorine are harmful to formation of interaction. In addition, fluorine can change the basicity of the compound.35 The 5-F substituent reduces the basicity of the nearby NH group, and the typical H-bonding of NH to Q817 is thereby weakened. It is also possible that fluorine acts as a good electron withdrawing group to bring the electron density from the monocyclic pyrimidinone ring and thus reduces the key interactions between this aromatic ring and residues of PDE5 such as F820 and V782. These factors together lead to a lowest enthalpy contribution to the binding free energy between 2 and PDE5. We have mentioned above that in the case of 2 binding to PDE5, the positive entropic contribution to the binding affinity is even higher than enthalpy, while the binding of inhibitors such as 3, 4, and 5 to the enzyme is driven by enthalpy and prevented by entropic effect. In general, the substitution of a hydrogen atom by fluorine increases the lipophilicity. In contrast, it has been mentioned in the literature that the lipophilicity of the fluorinated compound is decreased if there is an oxygen atom in close vicinity to the substituted fluorine, like our inhibitor 2.35 The explanation for such an effect of fluorination is not clear. Lower lipophilicity makes 2 to gain more solvation energy by forming more interactions with water molecules than the other four inhibitors. The binding of 2 to

the enzyme eliminates the interactions and releases the water molecules, leading to a positive contribution of entropy to the formation of PDE5-2 complex. In addition, Figure 3 shows that the structure water molecule simultaneously H-bonding with D764 and A767 is only missed in the complex of PDE5-2, the releasing of this water molecule also gaining positive contribution of entropy to the binding energy. However, the resolution of the crystal structure of PDE5-2 is 2.65 Å (PDB code 3SHY). A higher resolution structure is required to validate whether the missed structure water is caused by the low resolution or the binding of 2. Nonetheless, our study provides a first comprehensive view on the complicated effects of the fluorine substitution on the binding of ligand to protein.

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EXPERIMENTAL SECTION

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ASSOCIATED CONTENT

X-ray Protein Structure Determination. Expression, purification, and crystallization of the catalytic domain of recombinant human PDE5A1, and the synthesis of the five compounds are the same as that described in our previous studies.11,31,32 The purity of all five compounds was >95% as confirmed by HPLC. Inhibitors 1 and 5 were dissolved in the precipitant solution to reach a final concentration of 0.5 mM for soaking. The apo crystal was transferred into the soaking drop and left for 24 h prior to data collection. The protocol for solving the two complex structures are also similar to our previous studies.11,31 The details were described in the Supporting Information. The complete statistics, as well as the quality of the solved structures, are shown in Table S1 in the Supporting Information. The coordinates of two structures and structural factors have been deposited into the PDB with codes 4OEX and 4OEW (Table S1 in the Supporting Information). Isothermal Tritation Calorimetry (ITC). The ITC measurements were conducted at 25 °C with an iTC200 instrument (Microcal, GE Healthcare), and the resulting data were processed by the supplied MicroCal Origin software package. The details of each titration are described in the Supporting Information. Titrations were run in triplicate to ensure reproducibility. In all the cases, a single binding site mode was employed and a nonlinear least-squares algorithm was used to obtain best-fit values of the stoichiometry (n), change in enthalpy (ΔH), and binding constant (K). Thermodynamic parameters were subsequently calculated with the formula ΔG = ΔH − TΔS = −RT ln K, where T, R, ΔG, and ΔS are the experimental temperature, the gas constant, the changes in free energy, and entropy of binding, respectively.

S Supporting Information *

Crystal packing interface in the structure of PDE5 in complex with the inhibitor 4; representative ITC results and fitting curve for each of the five inhibitors binding to PDE5 in solution; models used for interaction energy calculations for the complex structures of PDE5-3, PDE5-4, and PDE5-5; data collection and refinement statistics for two crystal structures of PDE5-1 and PDE5-5; interaction energies among the 5-sustituent of inhibitors 3, 4, and 5, the structure water molecule (W), and residues Y612; determination of crystal structures, isothermal tritation calorimetry (ITC) measurements, and quantum mechanics (QM) calculations. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*For Y.X.: phone, +86-21-50801267; E-mail, [email protected]. *For J.S.: phone, +86-21- 20231962; E-mail, jsshen@mail. shcnc.ac.cn. 3591

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Author Contributions

human cathepsin L and MEK1 kinase: efficient interactions in different environments. ChemMedChem 2011, 6, 2048−2054. (13) Llinas-Brunet, M.; Bailey, M. D.; Goudreau, N.; Bhardwaj, P. K.; Bordeleau, J.; Bos, M.; Bousquet, Y.; Cordingley, M. G.; Duan, J.; Forgione, P.; Garneau, M.; Ghiro, E.; Gorys, V.; Goulet, S.; Halmos, T.; Kawai, S. H.; Naud, J.; Poupart, M. A.; White, P. W. Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335). J. Med. Chem. 2010, 53, 6466−6476. (14) Lemke, C. T.; Goudreau, N.; Zhao, S.; Hucke, O.; Thibeault, D.; Llinas-Brunet, M.; White, P. W. Combined X-ray, NMR, and kinetic analyses reveal uncommon binding characteristics of the hepatitis C virus NS3-NS4A protease inhibitor BI 201335. J. Biol. Chem. 2011, 286, 11434−11443. (15) Jiang, Y.; Alcaraz, A. A.; Chen, J. M.; Kobayashi, H.; Lu, Y. J.; Snyder, J. P. Diastereomers of dibromo-7-epi-10-deacetylcephalomannine: crowded and cytotoxic taxanes exhibit halogen bonds. J. Med. Chem. 2006, 49, 1891−1899. (16) Carter, M.; Voth, A. R.; Scholfield, M. R.; Rummel, B.; Sowers, L. C.; Ho, P. S. Enthalpy−entropy compensation in biomolecular halogen bonds measured in DNA junctions. Biochemistry 2013, 52, 4891−903. (17) Wilcken, R.; Liu, X.; Zimmermann, M. O.; Rutherford, T. J.; Fersht, A. R.; Joerger, A. C.; Boeckler, F. M. Halogen-enriched fragment libraries as leads for drug rescue of mutant p53. J. Am. Chem. Soc. 2012, 134, 6810−6818. (18) Wilcken, R.; Zimmermann, M. O.; Lange, A.; Zahn, S.; Kirchner, B.; Boeckler, F. M. Addressing Methionine in Molecular Design through Directed Sulfur-Halogen Bonds. J. Chem. Theory Comput. 2011, 7, 2307−2315. (19) Wilcken, R.; Zimmermann, M. O.; Lange, A.; Zahn, S.; Boeckler, F. M. Using halogen bonds to address the protein backbone: a systematic evaluation. J. Comput.-Aided Mol. Des. 2012, 26, 935−945. (20) Lange, A.; Zirnmermann, M. O.; Wilcken, R.; Zahn, S.; Boeckler, F. M. Targeting Histidine Side Chains in Molecular Design through Nitrogen−Halogen Bonds. J. Chem. Inf. Model. 2013, 53, 3178−3189. (21) Xu, Z.; Yang, Z.; Liu, Y.; Lu, Y.; Chen, K.; Zhu, W. Halogen Bond: Its Role beyond Drug-Target Binding Affinity for Drug Discovery and Development. J. Chem. Inf Model 2014, 54, 69−78. (22) Giordano, D.; De Stefano, M. E.; Citro, G.; Modica, A.; Giorgi, M. Expression of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in mouse tissues and cell lines using an antibody against the enzyme amino-terminal domain. Biochim. Biophys. Acta 2001, 1539, 16−27. (23) Rawson, D. J.; Ballard, S.; Barber, C.; Barker, L.; Beaumont, K.; Bunnage, M.; Cole, S.; Corless, M.; Denton, S.; Ellis, D.; Floc’h, M.; Foster, L.; Gosset, J.; Holmwood, F.; Lane, C.; Leahy, D.; Mathias, J.; Maw, G.; Million, W.; Poinsard, C.; Price, J.; Russel, R.; Street, S.; Watson, L. The discovery of UK-369003, a novel PDE5 inhibitor with the potential for oral bioavailability and dose-proportional pharmacokinetics. Bioorg. Med. Chem. 2012, 20, 498−509. (24) Boolell, M.; Allen, M. J.; Ballard, S. A.; Gepi-Attee, S.; Muirhead, G. J.; Naylor, A. M.; Osterloh, I. H.; Gingell, C. Sildenafil: an orally active type 5 cyclic GMP-specific phosphodiesterase inhibitor for the treatment of penile erectile dysfunction. Int. J. Impotence Res. 1996, 8, 47−52. (25) Richalet, J. P.; Gratadour, P.; Robach, P.; Pham, I.; Dechaux, M.; Joncquiert-Latarjet, A.; Mollard, P.; Brugniaux, J.; Cornolo, J. Sildenafil inhibits altitude-induced hypoxemia and pulmonary hypertension. Am. J. Respir. Crit. Care Med. 2005, 171, 275−281. (26) Schwartz, B. G.; Kloner, R. A. Drug interactions with phosphodiesterase-5 inhibitors used for the treatment of erectile dysfunction or pulmonary hypertension. Circulation 2010, 122, 88−95. (27) Mohamed, H. A.; Girgis, N. M.; Wilcken, R.; Bauer, M. R.; Tinsley, H. N.; Gary, B. D.; Piazza, G. A.; Boeckler, F. M.; Abadi, A. H. Synthesis and molecular modeling of novel tetrahydro-beta-carboline derivatives with phosphodiesterase 5 inhibitory and anticancer properties. J. Med. Chem. 2011, 54, 495−509.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. J.R. and Y.H. contributed equally. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding was provided by the “100 Talents Project” of CAS to Y.X., the National Natural Science Foundation of China (grant nos. 91013010, 21172233, and 81273435), and the National Science & Technology Major Project (grant no. 2012ZX09301001-005).

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ABBREVIATIONS USED PDE5, phosphodiesterase type 5; ITC, isothermal titration calorimetric; cGMP, cyclic guanosine monophosphate; cAMP, cyclic adenosine monophosphate; ED, erectile dysfunction; PAH, pulmonary arterial hypertension; FDA, U.S. Food and Drug Administration; SAR, structure−activity relationship; Hbond, hydrogen bond

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REFERENCES

(1) Hernandes, M. Z.; Cavalcanti, S. M.; Moreira, D. R.; de Azevedo, W. F., Jr.; Leite, A. C. Halogen atoms in the modern medicinal chemistry: hints for the drug design. Curr. Drug Targets 2010, 11, 303−314. (2) Mirza, A.; Desai, R.; Reynisson, J. Known drug space as a metric in exploring the boundaries of drug-like chemical space. Eur. J. Med. Chem. 2009, 44, 5006−5011. (3) Auffinger, P.; Hays, F. A.; Westhof, E.; Ho, P. S. Halogen bonds in biological molecules. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 16789−16794. (4) Lu, Y.; Shi, T.; Wang, Y.; Yang, H.; Yan, X.; Luo, X.; Jiang, H.; Zhu, W. Halogen bondinga novel interaction for rational drug design? J. Med. Chem. 2009, 52, 2854−2862. (5) Hardegger, L. A.; Kuhn, B.; Spinnler, B.; Anselm, L.; Ecabert, R.; Stihle, M.; Gsell, B.; Thoma, R.; Diez, J.; Benz, J.; Plancher, J. M.; Hartmann, G.; Banner, D. W.; Haap, W.; Diederich, F. Systematic investigation of halogen bonding in protein−ligand interactions. Angew. Chem., Int. Ed. Engl. 2011, 50, 314−318. (6) Parisini, E.; Metrangolo, P.; Pilati, T.; Resnati, G.; Terraneo, G. Halogen bonding in halocarbon−protein complexes: a structural survey. Chem. Soc. Rev. 2011, 40, 2267−2278. (7) Beale, T. M.; Chudzinski, M. G.; Sarwar, M. G.; Taylor, M. S. Halogen bonding in solution: thermodynamics and applications. Chem. Soc. Rev. 2013, 42, 1667−1680. (8) Lu, Y.; Liu, Y.; Xu, Z.; Li, H.; Liu, H.; Zhu, W. Halogen bonding for rational drug design and new drug discovery. Expert Opin. Drug Discovery 2012, 7, 375−383. (9) Voth, A. R.; Ho, P. S. The role of halogen bonding in inhibitor recognition and binding by protein kinases. Curr. Top. Med. Chem. 2007, 7, 1336−1348. (10) Wilcken, R.; Zimmermann, M. O.; Lange, A.; Joerger, A. C.; Boeckler, F. M. Principles and applications of halogen bonding in medicinal chemistry and chemical biology. J. Med. Chem. 2013, 56, 1363−1388. (11) Xu, Z.; Liu, Z.; Chen, T.; Wang, Z.; Tian, G.; Shi, J.; Wang, X.; Lu, Y.; Yan, X.; Wang, G.; Jiang, H.; Chen, K.; Wang, S.; Xu, Y.; Shen, J.; Zhu, W. Utilization of halogen bond in lead optimization: a case study of rational design of potent phosphodiesterase type 5 (PDE5) inhibitors. J. Med. Chem. 2011, 54, 5607−5611. (12) Hardegger, L. A.; Kuhn, B.; Spinnler, B.; Anselm, L.; Ecabert, R.; Stihle, M.; Gsell, B.; Thoma, R.; Diez, J.; Benz, J.; Plancher, J. M.; Hartmann, G.; Isshiki, Y.; Morikami, K.; Shimma, N.; Haap, W.; Banner, D. W.; Diederich, F. Halogen bonding at the active sites of 3592

dx.doi.org/10.1021/jm5002315 | J. Med. Chem. 2014, 57, 3588−3593

Journal of Medicinal Chemistry

Brief Article

(28) Mans, D. J.; Callahan, R. J.; Dunn, J. D.; Gryniewicz-Ruzicka, C. M. Rapid-screening detection of acetildenafils, sildenafils and avanafil by ion mobility spectrometry. J. Pharm. Biomed. Anal. 2013, 75, 153− 157. (29) Sandner, P.; Hutter, J.; Tinel, H.; Ziegelbauer, K.; Bischoff, E. PDE5 inhibitors beyond erectile dysfunction. Int. J. Impotence Res. 2007, 19, 533−543. (30) Kouvelas, D.; Goulas, A.; Papazisis, G.; Sardeli, C.; Pourzitaki, C. PDE5 inhibitors: in vitro and in vivo pharmacological profile. Curr. Pharm. Des. 2009, 15, 3464−3475. (31) Wang, G.; Liu, Z.; Chen, T.; Wang, Z.; Yang, H.; Zheng, M.; Ren, J.; Tian, G.; Yang, X.; Li, L.; Li, J.; Suo, J.; Zhang, R.; Jiang, X.; Terrett, N. K.; Shen, J.; Xu, Y.; Jiang, H. Design, synthesis, and pharmacological evaluation of monocyclic pyrimidinones as novel inhibitors of PDE5. J. Med. Chem. 2012, 55, 10540−10550. (32) Gong, X.; Wang, G.; Ren, J.; Liu, Z.; Wang, Z.; Chen, T.; Yang, X.; Jiang, X.; Shen, J.; Jiang, H.; Asia, H. A.; Xu, Y.; Li, J. Exploration of the 5-bromopyrimidin-4(3H)-ones as potent inhibitors of PDE5. Bioorg. Med. Chem. Lett. 2013, 23, 4944−4947. (33) Bissantz, C.; Kuhn, B.; Stahl, M. A medicinal chemist’s guide to molecular interactions. J. Med. Chem. 2010, 53, 5061−84. (34) Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. LIGPLOT: a program to generate schematic diagrams of protein−ligand interactions. Protein Eng. 1995, 8, 127−134. (35) Bohm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Muller, K.; Obst-Sander, U.; Stahl, M. Fluorine in medicinal chemistry. ChemBioChem 2004, 5, 637−43.

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