Molecular Dynamics Simulation of Cyclooxygenase-2 Complexes with

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Molecular dynamics simulation of cyclooxygenase-2 complexes with indomethacin closo-carborane analogs Menyhárt-Botond Sárosi, and Terry P. Lybrand J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00275 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Journal of Chemical Information and Modeling

Molecular dynamics simulation of cyclooxygenase-2 complexes with indomethacin closo-carborane analogs Menyhárt-Botond Sárosi,a,* Terry P. Lybrand,b,* a

Institute of Inorganic Chemistry, Faculty of Chemistry and Mineralogy, Leipzig University, Johannisallee 29, D-04103 Leipzig, Germany b

Departments of Chemistry and Pharmacology, Center for Structural Biology, Vanderbilt University, Nashville, TN 37235-1822, United States

KEYWORDS: carborane-containing pharmacophores, indomethacin derivatives, cyclooxygenase, density functional theory, molecular mechanics, molecular dynamics.

ABSTRACT: Molecular dynamics simulation of carborane-containing ligands in complex with target enzymes is a challenging task due to the unique structure and properties of the carborane substituents, and relative lack of appropriate experimental data to help assess the quality of carborane force field parameters. Here, we report results from energy minimization calculations for a series of carborane-amino acid complexes using carborane force field parameters published previously in the literature and adapted for use with the AMBER ff99SB and ff14SB potential functions. These molecular mechanics results agree well with quantum mechanical geometry optimization calculations obtained using dispersion-corrected density functional theory methods, suggesting that the carborane force field parameters should be suitable for more detailed 1 ACS Paragon Plus Environment

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calculations. We then performed molecular dynamics simulations for the 1,2-, 1,7-, and 1,12dicarba-closo-dodecaborane(12) derivatives of indomethacin methyl ester bound with COX-2. The simulation results suggest that only the ortho-carborane derivative forms a stable complex, in agreement with experimental findings, and provide insight into the possible molecular basis for isomer binding selectivity.

INTRODUCTION Cyclooxygenase (COX) plays a key role in the biosynthesis of prostanoids and occurs in two isoforms with similar three-dimensional structures and high sequence identity.1 COX-1 is constitutively expressed and primarily involved in normal physiological functions. COX-2 is induced mainly by pathological processes such as inflammation and tumorigenesis. COX-2 selective inhibitors are promising antitumor drugs and generally exhibit fewer side effects than nonselective COX inhibitors. COX-2 selectivity can be achieved by exploiting modest differences in size and flexibility of the binding sites of the two COX isoforms.2,3 The COX enzymes are homodimers that behave as heterodimers during inhibition and cross-talk between the COX monomers has been observed.4,5 The communication between the monomers may involve the mobile 123–129 loop of one monomer located at the dimer interface, which interacts with a loop containing residues 541-543 in the partner monomer, and it is hypothesized that ligands which modify this inter-subunit communication could represent a novel class of nonsteroidal anti-inflammatory (NSAID) drugs. The widely-used NSAID indomethacin (Figure 1) is a relatively nonselective COX inhibitor.6,7 COX-2 selectivity can be achieved through conversion of this aryl acetic acid into the neutral methyl ester (1, Figure 1).3,6 A crystal structure is available for the murine COX-2/indomethacin complex,8 but no crystal structure for COX-2 complexed with compound 1 has been published to date. Site2 ACS Paragon Plus Environment

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directed mutagenesis studies suggest that many COX inhibitors form important interactions with a cluster of residues at the entrance of the enzyme active site, denoted as the “constriction site” zone. Inhibitors with free carboxylate groups, such as indomethacin, form interactions with constriction site residues R120 and Y355, while neutral NSAID derivatives, such as compound 1, interact primarily with constriction site residues Y355 and E524.3 Notably, mutation of R120 dramatically diminishes binding of inhibitors with free carboxylate groups but has little or no impact on COX-2 inhibition by indomethacin amides and esters. The larger indomethacin amides and esters are predicted to extend through the constriction site zone and form signiﬁcant interactions with residues that line a large cavity below the constriction site, called the “lobby” region (V89, I112, Y115 and S119), and mutagenesis studies support this prediction.9 These mutagenesis and enzymology studies for neutral indomethacin amide derivatives, combined with earlier computational and structural studies for neutral indomethacin derivatives bound to the COX-1 isoform,10,11 suggest that compound 1 likely binds to COX-2 with a similar binding mode as observed for indomethacin.8 However, docking studies followed by molecular dynamics (MD) simulations suggested two additional possible binding modes in mCOX-2 for compound 1.12 The strategy of incorporating carborane clusters and their derivatives into drug molecules as phenyl mimetics has recently become the focus of intense research in medicinal chemistry and drug design.13,14 The 1,2-, 1,7-, and 1,12-dicarba-closo-dodecaborane(12) isomers (termed ortho, meta, and para isomer, respectively) can serve as three-dimensional analogs of aromatic substituents. Inspired by derivatizations of the 4-chlorophenyl substituent in indomethacin that generated COX-2 selective inhibitors,3,6 Hey-Hawkins and co-workers have recently synthesized indomethacin analogs where the 4-chorophenyl substituent is replaced with a carborane cluster.15 The replacement of the 4-chlorophenyl ring in 1 with an ortho-carborane cluster generates a highly potent and 3 ACS Paragon Plus Environment


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selective COX-2 inhibitor (2o in Figure 1).16 In contrast, substitution of the meta- or para-carborane isomers for the 4-chlorophenyl substituent (2m and 2p in Figure 1, respectively) results in loss of both COX-1 and COX-2 inhibitory activity.16 The inhibition studies were conducted with ovine COX-1 (oCOX-1) and murine COX-2 (mCOX-2).15,16 The carbonyl group adjacent to the ortho-carborane in 2o increases the lability of the cluster for deboronation.17 Decapping of the closo cluster in 2o to an anionic 7,8-nido-dicarbaborate yields a carborane-containing inhibitor 3 (Figure 1) with high COX-2 selectivity and improved solubility and stability.15 The mCOX-2:3 crystal structure (PDB ID: 4Z0L) revealed a different binding mode, compared to the parent compound indomethacin in complex with mCOX-2. The side chain of L531 displays a different rotamer, which exposes a nonpolar side pocket utilized for the binding of NSAIDs of the oxicam family.18,19 The L531 side chain flexibility might also help explain the ability of COX-2 to bind a broad variety of substrates.20 Typical carborane–protein interactions include: B–H···H–X (X = N, C and S) dihydrogen bonds, B–H···Na+ bridging interactions, and B2H···π and C–H···π hydrogen bonds.21-27 The acidic C–H moiety in the carborane cluster also forms C–H···X hydrogen bonds, 13 and may interact with water through weak C–H···OH2 hydrogen bonds.28 The B–H···H–X (X = N, C and S) dihydrogen bonds observed for boranes exhibit particularly short hydrogen–hydrogen contacts in the range of 1.7–2.2 Å.29

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Figure 1. Indomethacin, the methyl ester 1 and carborane-containing derivatives.

Previous molecular docking calculations using the mCOX-2:3 crystal structure (PDB ID: 4Z0L) as a target template suggest that the most probable binding mode for all three isomers of 2 is similar to the experimental binding mode observed for their nido-carborane analog 3 (Figure 2).30 The ester carbonyl in 2o,m,p interacts with S530, the amide carbonyl in 2o,m,p forms polar interactions with R120, and the closo-carborane cluster inserts into the nonpolar pocket exposed by the L531 side chain rotation.15,18,19 Docking calculations using the mCOX-2:celecoxib crystal structure (PDB ID: 3LN1) confirmed the same binding modes for all three isomers of 2, so it does not appear that the



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previous molecular docking studies were biased by the particular COX-2 crystal structure used as a target.30 It is often difficult to correctly rank ligand binding poses in molecular docking calculations, especially when static target structures are used. Molecular dynamics (MD) simulations have been shown to be useful for assessing the viability of binding modes observed in molecular docking calculations, and MD simulations may help clarify the basis for the dramatic activity profile differences for isomer 2o versus 2m and 2p. The isomer-dependent activity profile of 2o,m,p might be attributed to the different position of the mildly acidic carborane C–H protons in the three closo-carborane isomers (Figure 2). The acidic CH group of 2o is oriented away from the nonpolar pocket that accommodates the carborane cluster in the docked mCOX-2:2o complexes. In contrast, the acidic CH groups of 2m and 2p are positioned inside the nonpolar pocket in their docked complexes, and this unfavorable interaction may be sufficient to significantly destabilize the binding of these two derivatives, resulting in negligible COX inhibitory activity.30 A similar observation was made for carborane derivatives of the local anesthetic lidocaine. Those studies revealed that analgesic activity of the lidocaine carborane derivatives decreases with increased exposure of the acidic C-H group to nonpolar protein residues.31


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Figure 2. Stereo view of the docked orientations of 2o (orange) 2m (blue) and 2p (green) inside the mCOX-2 active site from PDB 4Z0L. The carborane CH group carbon atoms are shown as ball and stick representation. mCOX-2 C: gray; N: blue; O: red. COX-2 residues 344 through 354 and hydrogen atoms are not shown for clarity. Water molecules from PDB ID: 4Z0L that are within 5 Å of the ligand are represented as red dots.

In our previous docking studies, we did not perform detailed MD simulations for docked poses of 2o,m,p in the COX-2 binding cavity because it is not clear that there are any appropriate carborane force field parameters that can be used in conjunction with available protein force fields. However, there are some published reports of carborane parameters that reproduce experimental structures and properties for isolated carboranes. Allinger et al. have developed force field parameters that successfully reproduced the experimental structures of substituted 12-vertex carboranes.32 Wimperis, et al., used the Allinger parameters, together with a carborane Lennard-Jones potential 7 ACS Paragon Plus Environment


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developed by Gamba and Powell,33 to perform detailed MD simulations.34 The structure and dynamics of dodecaborate cluster (B12H122−) derivatives in water was studied by Roccatano and coworkers using MD simulations.35 Tjarks et al. have also reported carborane parameters for the AutoDock scoring function that correctly predict the binding modes for a series of drug molecules with closo- and nido-carboranyl substituents.36 In this current study, we have adapted these published carborane force field parameters for use with the AMBER ff99SB and ff14SB potential functions. We first performed energy minimization calculations for a set of carborane-amino acid complexes and compared the results with quantum mechanical geometry optimization calculations for these complexes, to verify that the AMBER plus carborane force field parameters yield reasonable structures. We used dispersion-corrected density functional theory (DFT-D) for the geometry optimization calculations. DFT-D can reliably describe protein–ligand interactions and has been applied successfully to study carborane–biomolecule complexes.22,24,37 We then used the AMBER-carborane force field parameters to perform a series of MD simulations for the COX-2:2o,m,p complexes, to explore the relative stabilities of the ligand binding poses predicted by our previous molecular docking calculations.30

RESULTS AND DISCUSSION Carborane-amino acid complexes We calculated optimized geometries for complexes of all three closo-carborane isomers with each amino acid residue shown in Figure 3 (left), using both the AMBER ff99SB-carborane force field and dispersion-corrected DFT methods. These specific amino acids were chosen because they form the primary carborane contacts in the various docking poses generated previously.30


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Figure 3. Left: closo-carborane (CB) and amino acid structures used for constructing the model systems. Right: optimized structures of the oCB-amino acid complexes and the corresponding rootmean-square deviations (RMSD, in Å) between DFT-D (blue) and MM (orange) minimized structures. Carborane cluster carbon atoms are shown as ball and stick representation. Selected DFTD intermolecular interactions are shown as dashed lines (distances in Å). R: arginine, H0: neutral histidine, H1: protonated histidine, Y: tyrosine, S: serine, L: leucine and F: phenylalanine.

Optimized structures for the ortho-carborane (oCB) complex with each amino acid, using both molecular mechanics (MM, i.e., the force field parameters) and DFT calculations, are superposed in Figure 3 (right). The overall agreement between the two optimization methods is excellent, as 9 ACS Paragon Plus Environment


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Figure 3 illustrates. The oCB-arginine complex is stabilized primarily through B−H···H−N dihydrogen bonds. The oCB-neutral histidine complex is stabilized through a C−H···N hydrogen bond, while a B−H···H−N dihydrogen bond is the main stabilizing interaction in the oCB-protonated histidine complex. The oCB-tyrosine and oCB-phenylalanine complexes are stabilized primarily by a C−H···π interaction. A C−H···O hydrogen bond is the main stabilizing interaction in the oCB-serine complex. The oCB-leucine complex is stabilized by weak (i.e., long) B−H···H−C dihydrogen bonds. The optimized structures for all meta-carborane and para-carborane complexes with each amino acid residue exhibit comparably good agreements between MM and DFT calculations (see Supporting Information for details). The excellent agreement between the MM and DFT geometry optimization calculations suggests that these carborane force field parameters are reasonable and merit further evaluation in detailed MD simulations. We also performed identical calculations for the carborane force field parameters combined with the AMBER ff14SB potential function. The results are essentially identical to those obtained with the ff99SB potential function, so these carborane parameters should be appropriate for use with either AMBER potential function. We used the ff99SB potential function for all protein-carborane MD simulations reported here, to facilitate comparison with previous COX calculations performed with the ff99SB force field.

COX-2:2o,m,p systems We first performed 5 ns of MD simulation for mCOX-2:ligand complexes of each isomer 2o,m,p to relieve any residual unfavorable interactions, using the most probable binding pose predicted in the previous molecular docking calculations as the starting structures (Figure 2).30 We then extended each simulation for ~500 ns to explore the stability of each docking pose. Figure 4 displays ligand 10 ACS Paragon Plus Environment

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RMSD values for all three isomers over the full simulations. All three isomers undergo modest shifts over the first 100-200 ns; these initial docking adjustments appear to be associated primarily with relief of minor unfavorable ligand-protein steric interactions. After this initial relaxation phase, the isomers display notably different fluctuations. Isomer 2o quickly adopts a stable binding mode and exhibits relatively small, infrequent fluctuations for the duration of the MD trajectory. In contrast, both isomers 2m and 2p display substantial fluctuations during most of the MD trajectory, never attaining a truly stable binding mode. Fluctuations for isomer 2p are not as pronounced until ~360 ns, but the corresponding trajectory becomes increasingly unstable after that (Figure 4). The RMSD fluctuations for the protein backbone atoms during the ~500 ns MD simulations are stable around 2.0 Å and the protein fluctuations are similar for all three mCOX-2:2o,m,p complexes (see Supporting Information).

Figure 4. Ligand RMSD for configurations taken at 200 ps intervals from the COX-2:2o,m,p MD simulations relative to the starting structure.



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While there are dramatic differences in the apparent “stability” of the docking poses for the three isomers, based on the structural fluctuations, all three isomers do retain a general binding pose that is consistent with the previous molecular docking calculation results. The mCOX-2:2o,m,p complex final snapshots of each MD trajectory are shown in Figure 5. Visual analysis of the MD trajectories reveals that isomer 2o maintains most protein contacts observed in the initial molecular docking pose, while isomers 2m and 2p display frequent, significant disruptions in ligand-protein contacts over the trajectories and deviate more noticeably from their initial docking poses after ~500 ns (Figure 5 and Supporting Information Figure S5). None of the isomers retained the hydrogen bond with R120 that was observed in the previous molecular docking calculations. The closo-carborane clusters of all three isomers interact primarily with the nonpolar pocket formed by residues M113, V116, L117, I345, V349, L359, L531, L534 and M535. The most dramatic difference in the observed binding interactions for the three isomers concerns the orientation and local chemical environment of the acidic C-H bond in the carborane cluster. This acidic C-H bond is buried in the nonpolar pocket described above in both the 2m and 2p isomer complexes, but fully exposed for potential interactions with solvent and/or polar protein residues in the 2o complex. This apparent inverse correlation between inhibitor activity and exposure of the acidic C-H bond to nonpolar protein residues is reminiscent of the trend observed for lidocaine carborane analogs discussed above.31 We then used the final 400 ns of each MD trajectory to perform MM/PBSA binding free energy calculations, and results are shown in Table 1. The binding free energy preference for isomer 2o versus 2m is strongly influenced by more favorable ligand-enzyme interactions (∆EMM in Table 1), while the interaction energy difference for 2o versus 2p is negligible when averaged over the full 400ns. We also performed MM/PBSA calculations for 100 ns segments of each trajectory (e.g.. sub12 ACS Paragon Plus Environment

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block averaging) to assess the variation in computed results over the course of each trajectory (Table 1). These results parallel the RMSD structural fluctuations closely; the isomer 2o interaction energies are reasonably consistent over the full trajectory, but the interaction energies for isomers 2m and 2p vary more dramatically during different segments of the trajectory, consistent with the much larger structural fluctuations we observe for these isomers. The computed solvation contribution for each isomer is somewhat puzzling, as it seems plausible that all three isomers should have similar desolvation energies. This result might be due to inadequate configurational sampling in these MD trajectories. However, we should emphasize that we have used our standard carborane parameters, including the atomic radii, for these MM/PBSA calculations, but these parameters have not been tested for their suitability in MM/PBSA calculations (the atomic radii set recommended for MM/PBSA calculations with the AMBER force fields does not contain necessary parameters for carborane atoms). This is likely a major source of error for the solvation energy calculations, since MM/PBSA methods can be extremely sensitive to the atomic radii parameter set.38,39,40 While the isomer binding poses predicted from our MD trajectories are in generally good agreement with the binding poses observed in our previous molecular docking calculations, the relative binding free energies computed from this MD-MM/PBSA protocol reflect better agreement with available experimental data, compared to the previous molecular docking calculations.30 In our previous study, neither the docking scoring function results, nor subsequent QM/MM geometry optimization calculations for the docked complexes, reproduced experimental isomer binding preferences accurately and consistently. Our current results suggest that MD simulations combined with MM/PBSA calculations as the “docking scoring function” do provide a more robust and reliable assessment of the preferred ligand binding poses. However, it should be noted that the 13 ACS Paragon Plus Environment


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standard deviations for these MM/PBSA calculations are generally larger than the ∆∆G values computed for each isomer pair, so we cannot use these results for rigorous, quantitative assessment of the relative isomer binding free energies.


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Figure 5. Stereo view of the final snapshots of COX-2:2o, (A) COX-2:2m, (B) and COX-2:2p (C) from 520 ns MD simulations. COX-2: gray; ligand: orange. The relative starting orientation of each ligand is shown in blue. The carborane CH group carbon atoms are shown as ball and stick representation. COX-2 residues 344 through 354 and hydrogen atoms are not shown for clarity. Selected hydrogen bonds are shown as black dashed lines.

Table 1. Binding free energies (∆Gbind) in kcal mol–1 from the last 400 ns simulations. Standard deviations are listed in brackets.

2o

Trajectory sub-block (ns)

∆EMM

∆Gsol

∆Gbind

120-520

–67.71 [5.30]

90.10 [4.37]

22.39 [4.58]

120-220

–63.48 [3.86]

87.53 [4.67]

24.06 [4.59]

220-320

–71.86 [4.12]

92.28 [3.43]

20.42 [3.97]

320-420

–69.08 [4.42]

90.44 [3.38]

21.37 [4.54]

420-520

–66.44 [4.72]

90.14 [4.48]

23.70 [4.12]



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2m 120-520

–65.00 [4.57]

86.76 [4.82]

21.76 [3.95]

120-220

–64.31 [4.66]

84.91 [4.78]

20.60 [3.49]

220-320

–64.33 [4.36]

85.84 [4.79]

21.51 [4.17]

320-420

–66.65 [4.13]

88.28 [4.18]

21.63 [3.90]

420-520

–64.71 [4.70]

88.00 [4.66]

23.29 [3.74]

120-520

–68.41 [4.78]

87.71 [5.45]

19.30 [4.48]

120-220

–69.66 [3.68]

89.60 [3.16]

19.93 [3.86]

220-320

–70.17 [3.41]

88.88 [3.37]

18.71 [3.84]

320-420

–70.42 [3.38]

89.35 [4.54]

18.94 [4.31]

420-520

–63.42 [4.60]

83.04 [6.88]

19.62 [5.57]

2p

∆EMM: gas phase molecular mechanical energy change. ∆Gsol: desolvation free energy change. ∆Gbind = ∆EMM + ∆Gsol

In our previous molecular docking calculations, we observed two alternate ligand binding poses with small populations (i.e., low-probability poses) and generally lower docking scores. One alternate binding pose involved a 180˚ rotation of the indole ring around the amide bond, retaining the carborane cluster in the nonpolar side pocket but reorienting the indole ring system such that the methoxy substituent was positioned near Y385, compared to the major binding pose that places the methyl-ester substituent near Y385 (see Supporting Information Figure S6). A second, alternate binding pose involved a flip of the ligand around the long axis of the indole ring system, resulting in a complete reorientation of the ligands in the binding pocket (see Supporting Information Figure S7). This alternate binding pose was intriguing because it exhibits a favorable interaction between the ligand methyl-ester substituent and R513 at the entrance of the enzyme active site. R513 is replaced by the smaller H513 in the COX-1 isoform, so this alternate binding pose interaction represents a potential (partial) explanation for the COX-2 selectivity observed for these 16 ACS Paragon Plus Environment

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indomethacin carborane derivatives. We performed MD simulations for all three ligands in each of the alternate binding poses as described above. However, the positional fluctuations for all three isomers are large in each alternate binding pose, and none of the three isomers ever attained a stable binding mode in either alternate pose over the full duration of the MD simulations (see Supporting Information Figures S3 & S4). The inhibitor-R513 interaction observed in the second alternate binding pose broke early in the MD trajectories and never reformed, suggesting that R513 is probably not an important determinant for COX-2 selectivity. This observation is consistent with previous experimental studies that provide no compelling evidence for the role of R513 in COX-2 inhibitor selectivity.20,41 Overall, these results are completely consistent with the original molecular docking calculations that indicated both alternate binding poses represent low-probability binding modes. Since none of these alternate binding pose trajectories ever produced stable ligand binding modes, MM/PBSA calculations based on these trajectories are unlikely to yield useful results. Our previous experience, as well as the results listed above particularly for isomers 2m and 2p, show that MM/PBSA calculations based on MD trajectories with large ligand binding position fluctuations yield results with standard deviations that are often larger than the relative binding free energy differences we wish to compute.42

CONCLUSIONS The excellent agreement between MM and DFT-D minimized structures for the carborane–amino acid complexes suggests that the available carborane force field parameters can be adapted for use with the AMBER potential functions in MD simulations. While additional carborane parameter refinement will no doubt be needed as more experimental data are obtained, the current parameter set does appear to produce physically reasonable results for carborane-amino acid interactions and 17 ACS Paragon Plus Environment


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should serve as a useful starting point for further parameter refinement. It will be particularly important to develop better carborane parameters for use in MM/PBSA calculations, since it is extremely unlikely that the current atomic radii set are optimal for this purpose. Additional experimental data will be needed to help refine and improve the carborane parameters, and these data should become available soon as interest in drug analogs with carborane substituents is increasing. The MD simulations indicate that isomer 2o assumes a stable binding mode that is consistent with our previous molecular docking calculations and quite similar to the crystal structure for mCOX-2 with the nido-carborane analog 3.15 By contrast, neither isomer 2m or 2p formed a stable binding mode even after 500 ns, and these results are in good qualitative agreement with the experimental isomer binding trends. We cannot use the MM/PBSA calculations to make quantitative assessment of isomer relative binding free energies because of the large computed standard deviations, due in part to the large ligand RMSD fluctuations for isomers 2m and 2p. The simulations suggest that isomer 2o forms more stable ligand-protein interactions over the 500 ns trajectory compared to isomers 2m and 2p, and this may be due at least in part to the relative orientation of the acidic C-H bond of the carborane cluster in each isomer complex. The acidic C-H bond is accessible to solvent and/or polar protein residues in the 2o complex, but buried in a nonpolar pocket in the complexes with 2m and 2p. Interestingly, this explanation is consistent with the trend observed previously for carborane analogs of the local anesthetic lidocaine.31 The placement of the carborane cluster in the nonpolar pocket exposed by the L531 side chain rotation provides a logical explanation for COX-2 selectivity. The analogous pocket in the COX-1 isoform is smaller, and apparently unable to accommodate large substituents such as the carborane cluster.15 The MD simulations also indicate that the alternate binding poses observed in the molecular docking calculations are unstable, 18 ACS Paragon Plus Environment

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consistent with our previous molecular docking results that suggested those poses were lowprobability binding modes. In summary, the MD simulations reported here correctly reproduce experimental binding trends for a series of carborane isomer analogs of indomethacin methyl ester, helping resolve somewhat ambiguous results obtained in our previous molecular docking calculations. These current results also help us to better understand the important molecular details that may play a primary role in determining carborane isomer binding preferences. It is also clear that the current carborane potential function parameters need further optimization to facilitate more rigorous, quantitative calculations.

COMPUTATIONAL METHODS: Model systems: The small model complexes as well as the individual closo-carboranes and amino acids were optimized at the RI-revPBE-D3bj/def2-TZVP level of theory.43-49 These calculations were carried out with the ORCA software package.50 Dispersion corrected density functional theory (DFT-D) can reliably describe protein–ligand interactions.37 revPBE, together with dispersion correction, is one of the best performing GGA functionals for describing noncovalent interactions.51,52

The DFT-D optimized coordinates were used as starting points for MM

minimizations. The partial atomic charges for the individual closo-carboranes and amino acids were derived according to the RESP procedure53,54,55 at the HF/6-31G* level of theory using Gaussian 0956 and the antechamber program of AmberTools16.57 The amino acid residues were described using the ff99SB force field.58 The carborane bonding interactions were described using the force field developed by Allinger and co-workers.32 Non-covalent interactions with the carborane cluster boron, carbon and hydrogen atoms were described using the LJ parameters of 19 ACS Paragon Plus Environment


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Gamba and Powell.33 MM minimizations were carried out until the root-mean-square of the Cartesian elements of the gradient was less than 0.01 kcal mol–1 Å–1. Minor differences between the ff99SB and ff14SB59 force field results were only observed for the isolated arginine residue (R in Figure 3). However, no differences were observed between the ff99SB and ff14SB minimized oCB-arginine, mCB-arginine and pCB-arginine complexes (see Supporting Information), thus the ff99SB parameters have been used throughout to facilitate comparison with previous COX calculations. MM and MD simulations: The coordinates of the COX-2:2o,m,p docked poses published previously were used for MD simulations.30 The COX-2 structures were prepared using the leap module in AmberTools.57 The crystallographic water molecules found in the 4Z0L PDB file were retained. The ff99SB force field58 was assigned to the COX-2 residues. Ligand hydrogen atoms were automatically added with the UCSF Chimera package60 and manually with GaussView 5.61 The General AMBER Force Field for organic molecules (GAFF) was used to describe the ligands.62 The missing carborane bonding interactions were described using the force field developed by Allinger and co-workers.32 The LJ parameters of Gamba and Powell33 were assigned to the carborane cluster boron, carbon and hydrogen atoms. The bond stretching, angle bending and torsion terms between the carborane cluster and the core indomethacin structure were approximated from HF/6-31G* optimizations and GAFF values either with the parmchk2 module of AmberTools or manually. Partial atomic charges for the ligands were derived in the same fashion as the partial charges of the small model complexes. The heme force field parameters were taken from the work of Furse et al.63 A weak harmonic restraint of 20 kcal mol–1 Å–1 was defined between the heme iron atom and the HIS-388 NE2 atom in both COX-2 monomers.10 Each system was placed into a preequilibrated SPC/E truncated octahedral water box.64 The SPC/E solvent boxes contained the 20 ACS Paragon Plus Environment

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crystallographic water molecules and extended at least 9 Å from the COX-2:2o,m,p complexes. Minimizations and MD simulations were carried out with the sander module.57 During the threestep minimizations, first solvent atoms were held fixed while solute molecules were relaxed, then solute atoms were held fixed while solvent molecules were relaxed and finally both solvent and solute were optimized without any restraints. Each step consisted of 300 steps of steepest descent and 700 steps of conjugate gradient minimization. Subsequently, two 50 ps MD simulations were carried out on all COX-2:2o,m,p complexes with a 2 kcal mol–1 Å–2 restraint on the solute atoms. First, each system was heated from 0 to 300 K in the canonical (NVT) ensemble. Second, each system was relaxed in the isothermic-isobaric (NPT) ensemble (T = 300 K and P = 1 atm). Finally, a 5 ns MD simulation without any restraints was run in the NPT ensemble in order to relieve minor unfavorable ligand-protein steric interactions that might have been still present. Force calculations were performed with periodic boundary conditions and a 9 Å cutoff was used for nonbonded interactions. Covalent bonds connecting hydrogen atoms were constrained with SHAKE.65 A collision frequency of 3 ps–1 was used in the Langevin thermostat to maintain the system temperature.66 A time step of 2 fs was used in all MD simulations. The ~500 ns production MD simulations (~400 ns for the “rotated” binding poses) were carried out with GPU accelerated pmemd.57,67-69 Atom coordinates were saved every 200 ps (5 frames/ns). Each restart of the MD simulations altered the random number generator seed.70 Further numerical analysis was performed with cpptraj.71 Graphical analyses of energy-minimized structures and individual MD snapshots were performed using UCSF Chimera60 and the Molecular Operating Environment (MOE) package.72 Molecular Mechanics/Poisson−Boltzmann Surface Area (MM/PBSA) calculations: MM/PBSA binding free energies were calculated with the MMPBSA.py program.73 The MM/PBSA calculations 21 ACS Paragon Plus Environment


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used the same atomic radii as the MM and MD simulations. The total non-polar solvation free energy was modeled as two terms throughout: the cavity term and the dispersion term.74,75 Entropy contributions were not considered.

SUPPORTING INFORMATION Optimized structures of the mCB- and pCB-amino acid complexes; the bond stretching, angle bending and torsion terms used for the carborane clusters; COX-2 backbone atom RMSD of the reference binding poses; ligand RMSD from the COX-2:2o,m,p MD simulations of the “rotated” and “flipped” binding poses; stereo views of the final COX-2:2o,m,p coordinates from ~400 and ~500 ns MD simulations started from the “rotated” and “flipped” binding poses, respectively. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] ORCID Menyhárt-Botond Sárosi: 0000-0003-4222-0717 Terry P. Lybrand: 0000-0002-2248-104X

ACKNOWLEDGEMENTS


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This work was supported by the German Research Foundation (DFG, SA 2902/2-1). Some calculations were performed using resources at Vanderbilt University Advanced Computing Center for Research and Education. These computational resources are supported in part by National Institutes of Health grants NIH S10 OD020154 and NIH S10 RR031634. M.B.S. is grateful to Prof. Dr. Evamarie Hey-Hawkins for her support.

ABBREVIATIONS COX, cyclooxygenase; LJ, Lennard-Jones; MM, molecular mechanics; MD, molecular dynamics; DFT-D, dispersion-corrected density functional theory; RMS, root-mean-square; RMSD, root-meansquare deviation; RI, resolution-of-identity; GGA, generalized gradient approximation.

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For Table of Contents Use Only

Molecular dynamics simulation of cyclooxygenase-2 complexes with indomethacin closo-carborane analogs

Menyhárt-Botond Sárosi* and Terry P. Lybrand*


Molecular Dynamics Simulation of Cyclooxygenase-2 Complexes with

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