The Underestimated Halogen Bonds Forming with Protein Side

Article pubs.acs.org/jcim

The Underestimated Halogen Bonds Forming with Protein Side Chains in Drug Discovery and Design Qian Zhang,† Zhijian Xu,*,‡ and Weiliang Zhu*,‡ †

Department of Computer Science and Technology, East China Normal University, Shanghai 200241, China CAS Key Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

‡

S Supporting Information *

ABSTRACT: Halogen bonds (XBs) have been attracting increasing attention in biological systems, especially in drug discovery and design, for their advantages of both improving drug−target binding affinity and tuning ADME/T properties. After a comprehensive literature survey in drug discovery and design, we found that most of the studies on XBs between ligands and proteins have focused on the protein backbone. Meanwhile, we also noticed that the proportion of side-chain XBs to overall XBs decreases as structural resolution becomes lower and lower. We postulated that protein side chains are more flexible in comparison with backbone structures, leading to more unclear electron density and lower resolution of the side chains. As the classic force field used to refine protein structures from diffraction data cannot handle XBs correctly, some of the interactions are lost during the refinement. On the contrary, there is no change in the corresponding ratio of hydrogen bonds (HBs) during structural resolution because HBs can be handled well with the classic force field. Further analysis revealed that Thr and Gln account for a large part of the decreasing XB trend, which could be partly attributed to the misidentified N, C, or O atoms. In addition, the lost XBs might be recovered after the atoms are reassigned, e.g., by flipping Thr side chains. In summary, formation of XBs with protein side chains is underestimated, and more attention should be paid to the potential formation of XBs between organohalogens and protein side chains during X-ray crystallography studies.

1. INTRODUCTION The halogen bond (XB),1−4 the attractive interaction between the σ hole (positive electrostatic potential)5,6 of a halogen atom (XB donor) and a nucleophile (XB acceptor), has been attracting increasing attention in biological systems,7−15 especially in drug discovery and design,16−21 for its advantages of improving drug−target binding affinity and tuning ADME/T properties.22−25 There are two kinds of XBs (Figure 1): C−X··· Y (Y = O, N, S) and C−X···π (π = aromatic system). Most of the XBs in drug discovery and design are of the C−X···Y type. Therefore, we focus on C−X···Y XBs in this study.

Y is either from the protein backbone or from a protein side chain. After a thorough review of the XB cases in drug discovery and design, we found that XBs with protein side chains are rare. Therefore, it is natural to ask whether the XBs with protein side chains are underestimated. In a survey of the April 2013 release of the Protein Data Bank (PDB), we found that 64.6% of the C−X···Y XBs are formed with protein backbone and 35.4% with side chains.22 In a survey of the September 2013 release of the PDB, Boeckler and co-workers showed that there are twice as many XBs with protein backbone carbonyls as XBs with protein side chains involving O, N, and S combined.17 With quantum-chemical calculations to explore XBs targeting histidine side chains, Boeckler and coworkers showed that nitrogen−halogen bonds involving histidine side chains are underexploited in lead structures, patent applications, and clinical candidate selection.26 On the basis of these results, they stated that XBs with polar protein side chains or aromatic rings are rather underrepresented in the PDB.27 Although the overall backbone/side chain ratio gives a hint of the underestimated side-chain XBs, we need more solid evidence to explore this hypothesis. The force field in the software to determine protein structures by X-ray crystallography cannot handle XBs properly. In general, the protein backbone is rigid and the protein side

Figure 1. Two types of XB in drug discovery and design.

Received: October 17, 2016 Published: December 5, 2016

© 2016 American Chemical Society

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Journal of Chemical Information and Modeling chain is flexible. When the resolution becomes lower, the electron density of the protein side chain becomes more unclear than that of protein backbone. Therefore, we postulated that the proportion of side-chain XBs to total XBs might decrease as the resolution becomes lower. Hence, the proportion could be a good indicator to estimate whether XBs with side chains are underestimated. Accordingly, a PDB survey was performed in this study, and a significant decrease in the percentage of side-chain XBs with respecto to total XBs was indeed observed. On the contrary, there is no change in the corresponding ratio for hydrogen bonds (HBs) because HBs are handled well in the software. Furthermore, we found that XBs with side chains were recovered after flipping of Thr side chains. Thus, the side-chain XBs are most probably underestimated in the PDB, and in the future, attention should be paid to XBs during the refinement of protein structures by Xray crystallography. Side-chain XBs should be routinely considered in drug discovery and design.

considered in the HB survey. A PyMOL script to detect HBs is provided in the Supporting Information.

3. RESULTS AND DISCUSSION 3.1. XBs with Protein Backbones in Drug Discovery and Design. A literature survey indicated that most of the studies concerning XBs in drug discovery and design have focused on the protein backbone. For example, a strong XB is formed between Br of MK204 and the Cys299 backbone carbonyl O atom of human enzyme aldo-keto reductase family member 1B10 (d(Br···O) = 2.89 Å, θ(C−Br···O) = 169.4° (PDB ID 5LIY); the calculated interaction energy is −4.4 kcal/ mol).28 In a systematic study, XBs were found to form between halogenated compounds and the Gly61 backbone carbonyl O atom of human cathepsin L (hCatL) (d(Cl···O) = 3.08 ± 0.11 Å, θ(C−Cl···O) = 173.6 ± 1.1° (PDB ID 2XU1); d(Br···O) = 3.12 Å, θ(C−Br···O) = 176.0° (PDB ID 2YJ2); d(I···O) = 3.12 Å, θ(C−I···O) = 174.6° (PDB ID 2YJ8)).24,29 In MEK1 kinase, the backbone carbonyl O atom of Val127 forms an XB with the I atom of the inhibitor (d(I···O) = 3.35 Å, θ(C−I···O) = 177.8° (PDB ID 3DY7)).29 In cell division protein kinase 2 (CDK2), the Cl atom of 5,6-dichloro-1-β-D-ribofuranosyl-1H-benzimidazole forms an XB with the Glu81 backbone carbonyl O atom (d(Cl···O) = 2.80 ± 0.01 Å, θ(C−Cl···O) = 169.0 ± 0.4° (PDB ID 3MY5)).30 In cell division protein kinase 9 (CDK9), Cl atoms of 5,6-dichloro-1-β-D-ribofuranosyl-1H-benzimidazole form XBs with the backbone carbonyl O atoms of Asp104 (d(Cl···O) = 2.78 Å, θ(C−Cl···O) = 176.5°) and Cys106 (d(Cl···O) = 3.09 Å, θ(C−Cl···O) = 158.9°) (PDB ID 3MY1).30 In 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, chloroplastic (IspD), the Br atom of pseudilin, a marine natural product, forms an XB with the Val239 backbone carbonyl O atom (d(Br···O) = 3.26 Å, θ(C−Br···O) = 164.0° (PDB ID 4NAK)).31 In proteasome subunit β type 5, the Br atom of (2E,3aR,14aS)-9-bromo-2-imino-1,2,3,5,6,14a-hexahydro-4H,8H-imidazo[4′,5′:5,6]pyrrolo[1′,2′:4,5]pyrazino[1,2a]indol-8-one forms an XB with the backbone carbonyl O atom of Thr21 (d(Br···O) = 3.69 ± 0.03 Å, θ(C−Br···O) = 166.1 ± 3.7° (PDB ID 4RUR)).32 In P53 cancer mutant Y220C, an I atom of the small molecule PhiKan784 forms an XB with the backbone carbonyl O atom of Leu145 (d(I···O) = 3.00 ± 0.02 Å, θ(C−I···O) = 171.8 ± 0.3° (PDB ID 4AGL)).33 In dual specificity protein kinase CLK1, a Cl atom of ethyl 3-[(E)-2amino-1-cyanoethenyl]-6,7-dichloro-1-methyl-1H-indole-2-carboxylate forms an XB with the backbone carbonyl O atom of Glu242 (d(Cl···O) = 2.94 Å, θ(C−Cl···O) = 171.2° (PDB ID 2VAG)).34 3.2. XBs with Protein Side Chains in Drug Discovery and Design. Compared with protein backbones, the cases of XBs with protein side chains are rare in drug discovery and design. For example, in c-Jun N-terminal kinase 3 (JNK3), the S atom of Met146 forms an XB with the I atom of N-ethyl-4{[4-(1H-indol-3-yl)-5-iodopyrimidin-2-yl]amino}piperidine-1carboxamide (d(I···S) = 3.10 ± 0.07 Å, θ(C−I···S) = 169.4 ± 1.3° (PDB ID 4X21)).35 In phosphodiesterase type 5 (PDE5), utilizing computational studies, medicinal chemistry, and X-ray crystal structures, we designed XBs to target the Tyr612 phenolate O atom (d(Br···O) = 3.35 ± 0.04 Å, θ(C−Br···O) = 149.2 ± 0.9° (PDB ID 3SIE)).23 Very recently, we repositioned two organohalogen drugs as potent B-Raf V600E inhibitors using the molecular docking software D3DOCKxb36 developed in our lab, which can handle XBs. The predicted XBs with the

2. MATERIALS AND METHODS PDB Survey. The current PDB (April 2016 release) was explored in this study. There are two types of XBs in the PDB: C−X···Y (Y = O, N, S) and C−X···π (Figure 1). We focus on C−X···Y XBs in this study. The criteria are set as an X···Y distance (d) shorter than the sum of the van der Waals radii (∑vdW) and a C−X···Y angle larger than 140° (Table 1).22 Table 1. Geometrical Parameters for XBs in This Study X

Y

d (Å)

θ (deg)

Cl Cl Cl Br Br Br I I I

O N S O N S O N S

140

The XBs in PDB structures with resolution worse than 3.0 Å are too sparse to achieve statistical significance. In addition, the PDB structures with resolution worse than 3.0 Å may have many errors. Therefore, only the structures with resolution equal to or better than 3.0 Å were considered in this study. The geometrical data in the study are the mean ± standard deviation (SD) in the asymmetric unit. For HBs, the criteria are set as a D···A distance (d) less than or equal to 3.2 Å and a H−D···A angle (ω) no larger than 55°, where D stands for the HB donor and A stands for the HB acceptor (Figure 2). The structures of sequences with >90% sequence identity were culled down, and the molecular weight of the organic ligands was restricted to between 150 and 1500 to mimic the druglike properties. Similar to the case of XBs, only X-ray structures with resolution no worse than 3.0 Å were

Figure 2. Geometrical definition of HBs in this study. 23

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S2). Taking these results together, we can state with confidence that formation of XBs with protein side chains are underestimated in PDB. 3.4. XBs with Different Amino Acids. There are 338 sidechain XBs (Table S1) and 784 backbone XBs (Table S2) in the PDB with resolution no worse than 3.0 Å. In 20 amino acids, only Tyr, Trp, His, Met, Cys, Ser, Thr, Asn, Gln, Asp, Glu, Lys, and Arg can form side-chain XBs (Figure 4), while Lys and Arg

side-chain hydroxyl O atoms of Thr508 and Ser602 were confirmed by single-point mutagenesis experiments.37 3.3. Percentages of XBs and HBs with Protein Side Chains at Different Resolution Ranges. There are 105 315 X-ray structures in the April 2016 release of the PDB: 10.1% in the ≤1.5 Å resolution range, 37.8% in the 1.5−2.0 Å resolution range, 30.2% in the 2.0−2.5 Å resolution range, 16.0% in the 2.5−3.0 Å resolution range, and 5.9% in the >3.0 Å resolution range. Statistical analysis of side-chain XBs at different resolution ranges would shed some light on the question of whether the XBs with protein side chains are underestimated. Hence, we defined the percentage of side-chain XBs (side-chain XB percentage) as (side-chain XBs)/(side-chain XBs + backbone XBs) × 100%. For comparison, we also defined the side-chain HB percentage as (side-chain HBs)/(side-chain HBs + backbone HBs) (Figure 3). The side-chain XB percentages

Figure 4. Frequencies of side-chain XBs at different resolution ranges. The percentages of XBs from individual amino acid side chains with respect to that from the total side chains are shown at the top.

are still in question because of their intrinsic positive charge. Met is the most abundant amino acid (20%) in the 338 sidechain XBs, Thr the second (14%), Tyr the third (13%), Ser the fourth (11%), Asn and Asp tied for fifth (8%), Glu the seventh (7%), Gln the eighth (6%), His the ninth (5%), Cys the 10th (4%), Arg the 11th (3%), and Trp and Lys tied for 12th (1%). The side-chain XB percentages for Met are 100%, 81%, 80%, and 86% for the resolution ranges of ≤1.5, 1.5−2.0, 2.0−2.5, and 2.5−3.0 Å, respectively (Figure 5). The percentage is stable

Figure 3. Side-chain XB and HB percentages at different resolution ranges.

are 41%, 39%, 26%, and 21% for the resolution ranges of ≤1.5, 1.5−2.0, 2.0−2.5, and 2.5−3.0 Å, respectively (Figure 3). In addition, to elucidate the role of the distance cutoff on XBs, the cutoff was set as ∑vdW + 0.1 Å, ∑vdW + 0.2 Å, ∑vdW + 0.3 Å, ∑vdW + 0.4 Å, or ∑vdW + 0.5 Å. The side-chain XB percentage showed an apparent decreasing trend at different cutoff values (Figure S1). Considering the structural resolution, the XBs and HBs in the structures with ≤1.5 Å resolution should be most reliable because the electron density is very clear. Figure 3 reveals that as the resolution becomes worse and worse, the side-chain XB percentage becomes lower and lower. Because XBs are considered as clashes in the refinement progress using the classic force field in structure determination, the atoms of backbone XBs with clear electron density would not move too far away from each other. However, the atoms of side-chain XBs with unclear electron density would be forced away from each other during the structural refinement, leading to the disappearance of side-chain XBs. Therefore, the apparent decreasing trend in the side-chain XB percentage suggests that formation of XBs with protein side chains is underestimated in the PDB. Impressively, we observed that the side-chain HB percentages are 56%, 55%, 55%, and 55% for the structures with resolution ranges of ≤1.5, 1.5−2.0, 2.0−2.5, and 2.5−3.0 Å, respectively, which are extremely stable. Furthermore, to elucidate the role of the distance cutoff on HBs, PDB surveys were performed with cutoff values of 2.8, 2.9, 3.0, and 3.1 Å. The side-chain HB percentages are extremely stable for different cutoff values, varying by no more than 2% (Figure

Figure 5. Percentages of side-chain XBs for individual amino acids at different resolution ranges.

at 1.5−3.0 Å resolution range for Met (varies from 80% to 86%). There are only two side-chain XBs for Met at ≤1.5 Å resolution range, which does not show statistical significance. Therefore, Met should not be taken into account for the study of the underestimated side-chain XBs. For Thr, the side-chain XB percentages are 85%, 64%, 36%, and 33% for the resolution range of ≤1.5, 1.5−2.0, 2.0−2.5 and 2.5−3.0 Å, respectively (Figure 5). An overall decreasing trend is observed for Thr that could partly account for the underestimated side-chain XBs. 24

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Journal of Chemical Information and Modeling The most significant amino acid is Gln, with side-chain XB percentages of 100%, 90%, 67%, and 0% for the resolution ranges of ≤1.5, 1.5−2.0, 2.0−2.5, and 2.5−3.0 Å, respectively (Figure 5). Considering that there are 45 backbone XBs for Gln in the 2.5−3.0 Å resolution range, 90 XBs might be overlooked in the Gln case. For Glu, the side-chain XB percentages are 25%, 50%, 35%, and 40% for the resolution ranges of ≤1.5, 1.5−2.0, 2.0−2.5, and 2.5−3.0 Å, respectively (Figure 5). Compared with Asp, with the corresponding percentages of 50%, 53%, 73%, and 75% (Figure 5), the side-chain XBs in the case of Glu might also be overlooked. In addition, the sidechain XBs with His and Cys might be underestimated (Figure 5). 3.5. Recovery of Side-Chain XBs. In the PDB survey, we showed that Thr might partly account for the underestimated side-chain XBs. The electron density could not distinguish between side-chain O and side-chain C, possibly resulting in misassignment of these two atoms. This misassignment could avoid the predicted clash by the classic force field but lead to the disappearance of a true XB. Therefore, if Thr side chains are flipped, some XBs would be recovered. For instance, in the binding site of cytochrome P-450CAM (PDB ID 1PHA, resolution 1.63 Å), the side-chain C of Thr101 forms a close contact with the Cl atom of the inhibitor (d(Cl···C) = 3.26 Å; Figure 6A). If the side chain flips, an XB forms (d(Cl···O) = 3.28 Å, θ(C−Cl···O) = 141.6°; Figure 6B). At the same time, the HB is not disturbed much, with the HB between Thr101 and a water molecule (d(O···O) = 3.36 Å; Figure 6A) changing

to the HB between Thr101 and the heme (d(O···O) = 3.41 Å; Figure 6B).

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CONCLUSIONS A comprehensive literature survey in this study revealed that most of the XBs in drug discovery and design are formed with the protein backbone. The PDB database survey showed an apparent decreasing trend in the percentage of side-chain XBs, suggesting that formation of XBs with protein side chains might be underestimated. On the other side, the percentage of sidechain HBs is extremely stable. Thr and Gln most likely account for the overlooked side-chain XBs. If the side chain flips, some XBs could be recovered. Taking these results together, we can claim with confidence that the formation of XBs with protein side chains is underestimated. Therefore, in the future more attention should be paid to the side-chain XBs during protein structure refinement with X-ray crystallography, and side-chain XBs should be utilized in drug discovery and design.

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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/acs.jcim.6b00628. PyMOL script to detect HBs; 338 side-chain XBs and 784 backbone XBs in the PDB; percentages of side-chain XBs with distance cutoff values equal to the sum of the vdW radii or ∑vdW + 0.1, 0.2, 0.3, 0.4, or 0.5 Å; and percentages of side-chain HBs with distance cutoff values of 2.8, 2.9, 3.0, 3.1, and 3.2 Å (PDF) The HB data set containing 227 199 HBs (ZIP)

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AUTHOR INFORMATION

Corresponding Authors

*Z.X.: Phone: +86-21-50806600-1201. E-mail: [email protected]. cn. *W.Z.: Phone: +86-21-50805020. Fax: +86-21-50807088. Email: [email protected]. ORCID

Zhijian Xu: 0000-0002-3063-8473 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (41301472, 81302699, and 81273435) and the “Personalized MedicinesMolecular Signature-Based Drug Discovery and Development” Strategic Priority Research Program of the Chinese Academy of Sciences (Grant XDA12020309).

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REFERENCES

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Figure 6. Flip of a Thr side chain to form an XB. (A) Original binding site of cytochrome P-450CAM (PDB ID 1PHA). (B) Flip of Thr101 to form an XB. Cytochrome P-450CAM is shown in gray cartoon, the inhibitor in yellow sticks, Thr101 and heme in gray sticks, and a water molecule as a red ball. HBs are shown as red dashed lines and XBs as cyan dashed lines. 25

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DOI: 10.1021/acs.jcim.6b00628 J. Chem. Inf. Model. 2017, 57, 22−26