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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Mol Recognit. Author manuscript; available in PMC 2010 October 5.
Published in final edited form as:
J Mol Recognit. 2010 Mar-Apr; 23(2): 173–182.
doi:  10.1002/jmr.989
PMCID: PMC2950069
NIHMSID: NIHMS235730

Including Receptor Flexibility and Induced Fit Effects into the Design of MMP-2 Inhibitors

Abstract

Matrix metalloproteinases (MMPs) comprise a class of flexible proteins required for normal tissue remodeling. Overexpression of MMPs is associated with a wide range of pathophysiological processes, including vascular disease, multiple sclerosis, Alzheimer’s disease, and cancer. Nearly all MMP inhibitors have failed in clinical trials, in part due to lack of specificity. Due to the highly dynamic molecular motions of the MMP-2 binding pockets, the rational drug design of MMP inhibitors has been very challenging. To address these challenges, in the current study we combine computer docking with molecular dynamics (MD) simulations in order to incorporate receptor-flexibility and induced-fit effects into the drug-design process. Our strategy identifies molecular fragments predicted to target multiple MMP-2 binding pockets.

Introduction

Matrix metalloproteinases (MMPs) comprise a class of flexible proteins (Moy, Pisano et al. 1997; Moy, Chanda et al. 1998; Lovejoy, Welch et al. 1999; Moy, Chanda et al. 2000; Zhang, Gonnella et al. 2000) for which computational drug design has been very challenging (Matter and Schudok 2004). There are over 20 MMP enzymes (Chang and Werb 2001) capable of degrading nearly all the components of the extracellular matrix, as well as many other extracellular proteins (Ray and Stetler-Stevenson 1995; Chambers and Matrisian 1997; Kahari and Saarialho-Kere 1999; Kleiner and Stetler-Stevenson 1999; Coussens, Fingleton et al. 2002). MMPs are required for normal tissue remodeling and are involved in many physiological processes, including embryonic growth, signal regulation, inflammation, angiogenesis (Chang and Werb 2001; Hu, Van den Steen et al. 2007), uterine involution, bone resorption, wound healing (Ray and Stetler-Stevenson 1995; Chambers and Matrisian 1997; Kahari and Saarialho-Kere 1999; Kleiner and Stetler-Stevenson 1999), CNS regeneration (Yong 2005), and ovulation (Whittaker, Floyd et al. 1999).

Overexpression of MMPs has been associated with a wide range of pathophysiological processes as well, including vascular disease (Hu, Van den Steen et al. 2007), asthma (Mautino, Oliver et al. 1997; Cataldo, Munaut et al. 2001; Prikk, Maisi et al. 2002), arthritis, multiple sclerosis, Alzheimer’s disease (Coussens and Werb 1996), and cancer (Denis and Verweij 1997; Wojtowicz-Praga, Dickson et al. 1997; Brown 1999), which requires degradation of the extracellular matrix for tumor progression and metastasis. The development of MMP inhibitors for the prevention of cancer metastasis has been a particularly active field of study (Hidalgo and Eckhardt 2001). MMP-2 (gelatinase A) and other gelatinases have received special attention because they are most consistently detected in malignant tissues and are associated with tumor aggressiveness, metastatic potential, and poor prognosis (Hidalgo and Eckhardt 2001; Mook, Frederiks et al. 2004). Nearly all MMP inhibitors have failed in clinical trials, in part due to lack of specificity (Pirard 2007).

Any rational design of MMP-2 inhibitors must take into account the general shape and characteristics of the MMP-2 active site (Pirard 2007). MMP inhibitors are typically comprised of a zinc-binding group (ZBG) with attached moieties that interact with surrounding binding pockets. A number of zinc-chelating groups have been proposed in the literature, including hydroxamates (Whittaker, Floyd et al. 1999), thiols (Skiles, Gonnella et al. 2004), pyrone derivatives (Puerta and Cohen 2003; Puerta, Lewis et al. 2004; Puerta, Griffin et al. 2006), carbamoylphosphonates (Farkas, Katz et al. 2004; Reich, Katz et al. 2005), hydroxyureas (Michaelides, Dellaria et al. 2001; Campestre, Agamennone et al. 2006), hydrazines (Auge, Hornebeck et al. 2003), β-lactams and squaric acids (Cainelli, Galletti et al. 2003; Onaran, Comeau et al. 2005), and nitrogenous ligands (dipicolylamine) (Lim, Schuster et al. 2005; Jacobsen, Lewis et al. 2006). Among these chelators, hydroxamic acid is by far the most frequently used (Whittaker, Floyd et al. 1999; Skiles, Gonnella et al. 2004).

Cohen et al. recently used a bioinorganic approach to discover new classes of zinc-chelating compounds that show improved potency and novel selectivity relative to similar hydroxamate-based ZBGs (Puerta, Lewis et al. 2004; Puerta, Mongan et al. 2005; Jacobsen, Lewis et al. 2006). These ZBGs were crystallized bound to a hydrotris(3,5-phenylmethylpyrazolyl)borate bioinorganic model complex whose zinc-coordinating nitrogen atoms are geometrically positioned in a way that is analogous to the three zinc-coordinating histidine nitrogen atoms of the MMPs (Puerta and Cohen 2003), allowing the ZBG to be modeled within any MMP-2 active site in silico by aligning analogous model and protein atoms.

One identified ZBG, 3-hydroxy-2-methyl-4-pyrone (maltol), is amenable to the addition of moieties that simultaneously interact with multiple MMP binding pockets. This is especially true when maltol is modified to 3-hydroxy-N-methyl-4-oxo-4H-pyran-2-carboxamide. Molecular modeling suggests that two of the hydrogen atoms on the terminal methyl group of this modified maltol point towards the S1′ and S2′ pockets, respectively (Figure 1). S1′ is a cavernous, flexible, hydrophobic pocket that is important for substrate selectivity (Pirard 2007). The S2′ pocket has the greatest geometric variability of all sub-pockets, as judged by force-field interaction energies used to compare the structures of 24 MMPs (nine X-ray structures and 15 homology models). As the shape of the S2′ pocket is not well conserved across the many different MMPs, targeting this pocket may lead to better therapeutic specificity (Lukacova, Zhang et al. 2004).

Figure 1
Superposition of equally spaced snapshots extracted from the MD trajectory of the apo protein. Fitting was performed by aligning the catalytic zinc cation and histidine residues of each structure. The zinc cation (grey) and coordinating nitrogen atoms ...

Due to the high flexibility of the MMP binding pockets (Moy, Pisano et al. 1997; Moy, Chanda et al. 1998; Lovejoy, Welch et al. 1999; Moy, Chanda et al. 2000; Zhang, Gonnella et al. 2000), predicting the binding affinity of MMP inhibitors in silico is particularly difficult. Although structural information extracted from the several available X-ray structures can be used, in principle, to design and improve the selectivity of a given inhibitor towards a specific MMP, the high flexibility of the MMP binding pockets drastically limits the accurate modeling and prediction of inhibitor-protein complexes (Yuan, Marshall et al. 1999; Cuniasse, Devel et al. 2005).

In order to assist future drug design, our group recently studied the flexibility of the apo MMP-2 binding pockets via a molecular dynamics (MD) simulation (de Oliveira, Zissen et al. 2007). The MD simulation revealed that two states, characterized as open and closed, are preferentially sampled. By analyzing the population of those states along the trajectory, we observed that while the closed state is heavily sampled, the open state, in which the S1′ pocket adopts a tunnel-like shape, appears only rarely. The S1′ loop, which has the highest sequence variability among different MMPs, is among the most mobile segments of MMP tertiary structure. Loops S2′ and S3′, which encompass solvent-exposed pockets S2′ and S3′, are also highly flexible (Figure 1).

Previously, our group presented a novel virtual-screening approach called the “relaxed-complex” scheme that accounts for receptor flexibility (Lin, Perryman et al. 2002). In this scheme, protein flexibility is taken into account by docking candidate inhibitors into an ensemble of protein configurations extracted from MD simulations of either the apo or holo receptor (Lin, Perryman et al. 2003; Amaro, Baron et al. 2008). This approach has been applied to several proteins including HIV-1 integrase (Schames, Henchman et al. 2004; Adcock and McCammon 2006), Trypanosoma brucei RNA editing ligase 1 (Amaro, Schnaufer et al. 2008), and influenza neuraminidase (Cheng, Amaro et al. 2008).

Given the dynamics of the MMP-2 binding pockets observed previously, a relaxed-complex technique was employed in the current study. Protein configurations were extracted from the apo MMP-2 simulation and the ZBG was positioned into each frame using in silico modeling based on the Cohen bioinorganic model (Puerta, Lewis et al. 2004; Puerta, Mongan et al. 2005; Jacobsen, Lewis et al. 2006). LUDI, a fragment-based de novo drug-design algorithm (Bohm 1994), was then used to dock fragments into the MMP-2 active site such that the fragments could be easily merged with the ZBG while maintaining interactions with the S1′, S2′, and S3′ pockets across multiple protein configurations extracted from the apo MD simulation.

Next, we extended the relaxed-complex scheme by performing secondary MD simulations of the holo receptor (receptor in complex with each of the thirteen best LUDI-predicted ligands) in order to account for induced-fit effects. These thirteen compounds were then re-ranked according to their ensemble-average predicted binding energy. Re-ranking by the ensemble-average score, which accounts in part for the open-closed dynamics of the active site, significantly reordered the compounds, suggesting that induced-fit effects can have a substantial impact on the computational identification of potential enzyme inhibitors.

Computational Details

Initial molecular dynamics simulations were performed on a 2.80 Å resolution MMP-2 crystal structure (Dhanaraj, Williams et al. 1999) (PDBID: 1QIB). Crystal waters and inhibitors were removed from the structure. Basic residues, i.e. Arg and Lys, were protonated, and acidic residues, i.e. Asp and Glu, were deprotonated. Except for one histidine residue located in the structural domain (His 179), which was protonated in the epsilon position, all histidine residues were protonated in the delta position. To maintain the correct orientation of all zinc-chelating histidine residues and to prevent the escape of zinc atoms to the solvent, harmonic restraints were applied on the distances and bending angles defined by the zinc and the nitrogen atoms of the appropriate histidine residues. The partial charges of the histidine imidazole rings and zinc atoms were calculated using the RESP program. The molecular electrostatic potential was calculated at the HF/6-31G* level.

Following the initial system preparation, we applied 500 steps of steepest descent followed by 1000 steps of conjugate gradient minimization. The relaxed structure was then solvated in a cubic box of pre-equilibrated TIP3P water molecules (Jorgensen, Chandrasekhar et al. 1983), which extended 10 Å further than the protein atoms themselves. To neutralize the system, sodium counterions (Na+) were added to balance the charge of the MMP-2 protein. To relax the waters, the system was then again minimized for 500 of steepest descent followed by 2000 steps of conjugate gradient minimization using constant volume periodic boundaries, with the protein and counterion atoms fixed. To bring the system to the correct density, we carried out an additional 50 ps MD simulation in which only water molecules were allowed to move and NPT ensemble (T = 298K, P=1 bar) was applied. To relax the protein in the solvent environment, all coordinates were then optimized by employing 500 steps of steepest descent followed by 1000 steps of conjugate gradient minimization. We then performed a 500 ps MD simulation to heat the system from 0K to 300K using the NVT ensemble (T = 298K). All subsequent simulations were carried out using the NVT ensemble (T = 298K) and, except for the internal restraints mentioned before and SHAKE constraints on bonds to hydrogen atoms (Ryckaert, Ciccotti et al. 1977), the coordinates of all atoms were allowed to move freely. A final MD simulation of 30 ns was performed. The first 15 ns were used to equilibrate the system, and the last 15 ns were used as the productive run.

Three thousand and one frames were next extracted at regularly spaced intervals from the 15 ns productive portion of the MD simulation. For each frame, the complex formed between the ZBG and the receptor was built by aligning each frame with the crystal-structure coordinates of maltol (3-hydroxy-2-methyl-4-pyrone) bound to [(TpPh,Me)ZnOH] (TpPh,Me = hydrotris(3,5-phenylmethylpyrazolyl)borate), a complex that models the zinc(II) ion coordination within the MMP active site (Puerta and Cohen 2002; Puerta, Schames et al. 2003). Maltol was positioned within the protein active site by minimizing the RMSD distance between the appropriate zinc and the coordinating nitrogen atoms of the protein active site and those of the bioinorganic model (Figures 2A and 2B).

Figure 2
A and B) A molecular dynamics simulation of the apo MMP-2 protein was analyzed in order to account for protein flexibility. Three thousand and one regularly spaced frames were extracted from the simulation, and the ZBG was placed into each protein configuration ...

LUDI was then used to dock potential interacting fragments into the many receptor configurations extracted from the apo MD simulation. The terminal methyl hydrogen atoms of the ZBG were selected as the link sites (Figure 1). Initial docking used the default Discovery-Studio LUDI link fragment library (Accelrys) with the following parameters: maximum alignment angle 20°; maximum alignment RMSD 0.6 Å; search radius 11 Å; rotate bonds two at a time; preselect 4.0; minimum separation 3.0; lipophilic density 40; polar density 40; minimum surface 0; link weight 1.0; lipophilic weight 1.0; H-bond weight 1.0; aliphatic aromatic off; reject bifurcated off; no unpaired polar off; electrostatic check off; minimum score 0; maximum fits 8000; maximum hits all; maximum unfilled cavity 0; energy estimate 1 scoring function (Bohm 1994); and best fit (Puerta, Mongan et al. 2005).

Following the LUDI-guided identification of interacting fragments, the fragments were combined with the ZBG and additional MD simulations of 5 ns were carried out on the resulting protein-ligand complexes comprised of the best LUDI-generated ligand candidates and the MMP-2 receptor. An MD protocol similar to that described above was used. Ligand force-field parameters were determined using the antechamber program inside the AMBER package. Ligand partial charges were calculated using the AM1-BCC charge scheme (Jakalian, Bush et al. 2000). In order to guarantee that the zinc-chelating group of each candidate ligand remain bound to the zinc cation of the active site, harmonic restraints were applied to the distances between the two ligand chelating oxygen atoms and the catalytic zinc cation.

For all MD simulations, the equations of motion were integrated every 2 fs using the Verlet Leapfrog algorithm (Hockney 1968). During MD, temperature and pressure were controlled via a weak coupling to external bath with a coupling constant of 0.5 and 1 ps, respectively (Berendsen, Postma et al. 1984). The center-of-mass motion was removed at regular intervals of 5 ps. The PME summation method was used to treat the long-range electrostatic interactions in the minimization and simulation steps of the solvated systems (Essmann, Perera et al. 1995). The short-range nonbonded interactions were truncated using an 8 Å cutoff and the nonbonded pair list was updated every 10 steps. All calculations were performed in the AMBER package (Case, Darden et al. 2006) with the ff99 force field (Wang, Cieplak et al. 2000).

In order to estimate the ensemble-average binding energy of each protein-ligand complex, seventy frames were extracted at regular intervals from the last 3.5 ns of each 5 ns holo simulation. The AutoDock 4.0 scoring function was used to estimate the ligand-protein binding energy of each frame. AutoDockTools (ADT) version 1.5.2 (Sanner 2005) was used to remove non-polar receptor and ligand hydrogen atoms and to assign Gasteiger charges (Gasteiger and Marsili 1980) to all the atoms of each frame. The MMP-2 zinc cations were manually assigned charges of +2.0 e. AutoGrid 4.0 was used to generate affinity grids centered on the active site. Each grid enclosed a volume of 48.00 Å × 48.00 Å × 48.00 Å with 0.375 Å spacing. For each protein-ligand system, all corresponding AutoDock-predicted binding energies were averaged to give the ensemble-average predicted binding energy (Scoreholo).

For all controls, a procedure similar to the above was used, except that the ZBG 3-hydroxy-4-oxo-4H-pyran-2-carboxamide was used instead of 3-hydroxy-N-methyl-4-oxo-4H-pyran-2-carboxamide, and an extra methyl group was placed on the docked fragments to compensate.

Results and Discussions

Accounting for protein flexibility

The methodology presented here identifies potential inhibitors in a way that accounts for protein flexibility. The approach presented in this work, illustrated in Figure 2, aims to identify ligand candidates that not only interact favorably with conformations typical of the apo form of the protein, but also with potential novel configurations induced by ligand binding.

Based on the ideas behind the relaxed-complex scheme (Lin, Perryman et al. 2002; Lin, Perryman et al. 2003; Amaro, Baron et al. 2008), we first performed a molecular dynamics simulation of the apo protein in order to account for protein flexibility. Three thousand and one regularly spaced frames sampling both open and closed MMP-2 conformations were extracted from the last 15 ns of the 30 ns simulation, and the zinc-binding group (ZBG, 3-hydroxy-N-methyl-4-oxo-4H-pyran-2-carboxamide) was manually placed into each protein configuration via structural alignment of the protein structure with a crystal-structure bioinorganic model of the MMP-2 active site (Puerta, Lewis et al. 2004; Puerta, Mongan et al. 2005; Jacobsen, Lewis et al. 2006) (Figures 2A and 2B).

Next, we selected two strategically positioned ZBG hydrogen atoms as fragment linking points (Figure 1, inset picture). The first hydrogen atom is generally oriented toward the S1′ pocket and the second is located in the vicinity of the S2′ and S3′ pockets. By targeting different binding pockets, we aim to identify fragments that simultaneously interact favorably and specifically with the protein. This approach can be easily extended to enable future combinatorial drug design that achieves high affinity by targeting two or more pockets concurrently.

LUDI docking calculations were then performed on the 3001 snapshots of the apo MD trajectory using both linking hydrogen atoms (Figure 2C). Because the best candidates obtained from LUDI docking calculations may change according to the conformation of the receptor, two strategies were applied to select the “best” interacting fragments. The best fragments were considered to be those that I) were frequently selected as binders across multiple protein configurations regardless of LUDI score or II) had the highest ensemble-wide maximum LUDI score regardless of frequency (Figure 2D). Candidates were considered “frequent binders” if they were selected and docked into at least 60% of the protein configurations, regardless of score. Candidates were considered “strong binders” if their ensemble-maximum LUDI scores fell within 100 units of the highest score of any receptor/fragment combination. Finally, we discarded from our list any fragments that failed to bind in the targeted pockets or that contained long linear aliphatic chains, as compounds with long linear aliphatic chains contain many rotatable bonds and, consequently, large entropic penalties upon binding.

Including Induced-Fit Effects: Rescoring Candidate Ligands

In order to account for the induced-fit changes in protein flexibility that occur upon ligand binding, we performed additional MD simulations of each of the LUDI-generated ligands selected previously in complex with the MMP-2 protein. To model each ligand, we iterated through both the frequent and strong binding fragments and in each case identified the fragment and receptor conformations associated with the ensemble maximum LUDI score. Each candidate fragment, posed in its optimal configuration, was subsequently positioned within the corresponding optimal receptor conformation from the apo simulation and merged with the corresponding ZBG.

A 5 ns MD simulation of each protein-ligand system was then performed to simulate induced-fit effects (Figure 2E). For each system, 100 regularly spaced frames were extracted from the MD trajectory. The AutoDock 4.0 scoring function was used to estimate the ligand energy of binding associated with each frame; visualization of the data revealed that the score converged sufficiently after the first 1.5 ns of each simulation. To predict the binding affinity of each ligand, the AutoDock scores associated with the seventy frames of the last 3.5 ns of each of MD simulations were averaged, yielding an ensemble-average predicted binding energy for each ligand (Figure 2F). Candidate ligands were then re-ranked by this ensemble average. Comparing the ensemble-average AutoDock-predicted binding affinity of each system (Scoreholo) with the predicted binding affinity of the ligand within the apo active site following 5000 steps of steepest-descent minimization (Scoreapo) reveals that accounting for induced-fit conformational changes significantly alters the predicted binding energies of some compounds (Figure 4).

Figure 4
Black) Induced-fit energy contribution. The induced-fit energy contribution is given by Scoreapo – Scoreholo. Grey) Standard deviation of the ensemble-average score. Contributions from induced-fit effects are considered significant if (Scoreapo ...

Table 1 shows the potential ligands obtained by applying the procedure depicted in Figure 2. We identified nine fragments predicted to bind in the S1′ pocket and three predicted to bind in the S2′ or S3′ pockets, hereafter referred to jointly as S2′/S3′. As expected, fragments targeting the S1′ pocket were generally elongated and hydrophobic. Due to the less hydrophobic and more solvent-exposed characteristics of the S2′/S3′ sites, two of the three candidates targeting these pockets were polar and charged. The third, T12 (uncharged), is also a S1′ binder; in fact, ensemble-average predicted binding energies suggest T12 prefers the S1′ pocket to the S2′/S3′ pockets. The predicted binding energies calculated from the apo protein (Scoreapo) were only loosely correlated with the ensemble-average predicted binding energies (Scoreholo) (R2 less than 0.3), indicating that altered receptor flexibility due to induced-fit effects has a significant impact on the predicted energy of binding.

Table 1
MMP-2 inhibitor candidates obtained by applying the procedure illustrated in Figure 2.

For ligands such as those associated with fragments UD2b, UE8, and U97, significant changes in the predicted binding energy occur when the subtle changes in protein configuration that occur upon binding are considered. For instance, the ligand associated with fragment UE8, which was selected as a reliable and strong binder due to its high LUDI frequency and its maximum LUDI score, was among the compounds with the highest binding affinity when only conformations of the apo protein were considered (Table 1, see Scoreapo). However, when induced-fit effects were included in our scoring procedure, the ligand associated with fragment UE8 became the second worst candidate.

Interesting results were also obtained for the ligands associated with the fragments UD2a and UD2b. The high LUDI scores of these tautomers qualified both as strong binders. Nevertheless, after re-scoring the compounds using the AutoDock 4.0 scoring function, the ligand associated with fragment UD2b became the least potent candidate on the list (Scoreapo = −4.40 kcal/mol), though the ligand corresponding to fragment UD2a still scored well. As expected, after equilibrating each ligand-protein complex, the ensemble-average scores (Scoreholo) of the tautomers (UD2a and UD2b) converged to nearly the same value.

To measure the subtle changes in the active-site configuration that yield such drastic changes in the predicted binding energy (i.e. the “induced-fit effects”), we further analyzed the top four predicted inhibitors, those associated with fragments UD2, T12, UB9, and UG1. For each of these inhibitors, the frame from the initial apo MD simulation that gave the highest LUDI score, the same frame following minimization with ligand present, and the corresponding 3.5 ns productive portion of each associated holo trajectory were all aligned by minimizing the RMSD between the active-site non-hydrogen protein atoms of 26 residues lining the active site of the MMP-2 crystal structure (PDB: 1QIB, Table 2). The results suggest that even small changes in the active-site configuration, as judged by the small changes in the RMSD to the crystal-structure active site, can lead to substantial changes in predicted binding affinity, as judged by Scoreapo – Scoreholo. Clearly, even limited protein flexibility can have a major impact on the predicted energy of binding.

Table 2
Analysis of active-site structural changes that occur upon ligand binding. The frame from the initial MD simulation that gave the highest LUDI score (“Frame”), the same frame following minimization (“Min”), and the corresponding ...

An analysis of the holo trajectories of the top four compounds also reveals a number of common ligand binding modes. The H201 side chain plays an important role in the binding of all these compounds. All four form critical pi-pi interactions with H201; additionally, UD2, UB9, and UG1 also form hydrogen bonds with the H201 side chain. Protein backbone carbonyl oxygen atoms are also critical for binding, as they accept hydrogen bonds from three of the top four ligands; UD2, UB9, and UG1 all donate hydrogen bonds to the backbone carbonyl oxygen atoms of residues I222 and A220. Additionally, UD2 and UG1 also form hydrogen bonds with the backbone carbonyl oxygen atoms of residues L218 and L197. Finally, the backbone carbonyl oxygen atom of P221 and the backbone amine of I222 form hydrogen bonds with UD2 and UG1, respectively.

Controls

In order to validate our methodology, we included three controls, AM-2, AM-5, and AM-6, which are known to inhibit MMP-2 with IC50 values of 9.3 μM, 0.61 μM, and > 50 μM, respectively (Puerta, Mongan et al. 2005). A procedure similar to that used above was repeated with these three controls. Our two positive controls, AM-2 and AM-5, had predicted binding energies of 9.18 ± 0.58 and −9.86 ± 0.56 kcal/mol, respectively. Given that AutoDock has a residual standard error of 2.177 kcal mol−1 (Morris, Goodsell et al. 1998), these positive controls have predicted binding energies comparable to those of the best novel compounds identified (Table 1).

AM-6, included as a negative control, was incorrectly identified as a strong binder (Table 1). In order to understand why the current methodology led to this false positive result, we examined the LUDI dockings of the AM-6 fragment into the protein configurations of the apo trajectory. Those dockings with the highest LUDI scores placed the AM-6 fragment deep within the hydrophobic S1′ pocket; one of these dockings was subsequently used to construct the initial frame of the holo AM-6 MD simulation. However, there were very few of these high-scoring dockings, suggesting that MMP-2 can only rarely accommodate the large AM-6 fragment. This observation is supported in part by the fact that LUDI was only able to dock AM-6 into the MMP-2 active sites of 24% of the frames extracted from the apo MD trajectory (Table 1). Even when docking was successful, docking of the AM-6 fragment into a well defined S1′ pocket was rare.

After generating a histogram of all AM-6 LUDI scores, we noted that the most populated cluster corresponded to scores around 450. We therefore repeated the holo AM-6 MD simulation, this time using ligand and protein configurations associated with a LUDI score of 450 to create the initial frame. After 1.5 ns of equilibration, we let the simulation run another 1.35 ns and analyzed the AutoDock-predicted binding energy of the productive ensemble, as described above. As expected, the average AutoDock-predicted binding affinity was substantially lower when more representative protein and AM-6 ligand conformations were considered. In cases where the protein configurations associated with the best LUDI scores are rare, we therefore suggest using more representative protein and ligand configurations for subsequent holo simulations instead.

Generating Composite Compounds

The current scheme is amenable to the design of composite compounds in which optimum fragments targeting both the S1′ and the S2′/S3′ pockets are simultaneously added to the same ZBG (described as targeting “both” pockets in Table 1). In this study, a S1′-binding fragment and a S2′/S3′-binding fragment were selected to create a high-affinity composite compound. Fragment UD2a, which had the highest ensemble-average AutoDock binding affinity (Scoreholo) of all novel hits, was selected as the S1′-binding fragment. As fragment U97 merged with the ZBG was the only S2′/S3′-binding ligand with a potential linker hydrogen pointed in the direction of the S1′ pocket, U97 was selected as the S2′/S3′ fragment. The composite compound was created by simultaneously replacing the two hydrogen-atom linkers (Figure 1, inset figure) with the UD2a and U97 fragments, respectively, producing a chiral compound in the S configuration. The initial orientation of each fragment corresponded to the ensemble-maximum LUDI score for each. Two reasonable receptor configurations were envisioned for the ligand-protein complex: the optimal apo conformation corresponding to fragments U97 and UD2a, respectively (Figures 3A and 3B). To predict the binding affinity of our composite ligand, two sets of MD and AutoDock-rescoring calculations were performed, as described above, each starting from one of these initial protein structures. Figure 3C displays the final configuration of the MD simulation started from the optimal apo conformation for the UD2a fragment. A similar configuration was obtained when the optimal apo conformation for the U97 fragment was used.

Figure 3
LUDI-docked fragments added to a ZBG. A) The ligand associated with fragment UD2a. B) The ligand associated with fragment U97. C) The equilibrated structure of the complex formed between the composite compound (UD2a + U97) and the last MMP-2 configuration ...

After equilibrating each of the two MD simulations of the composite compound (UD2a + U97), the ensemble-average scores (Scoreholo) of both converged to nearly the same value. Accounting for induced-fit effects clearly has a substantial impact on the predicted binding energy by removing dependency on a single, often unrepresentative receptor conformation.

Conclusion

The rational design of MMP inhibitors is challenging due to the flexibility of the MMP active site. In this work, we combined computer docking with MD simulations in order to account for receptor flexibility, conformational selection mechanisms, and induced-fit effects. Our strategy identified fragments that bind the S1′ pocket as well as fragments that bind elsewhere. Subsequently combining both types of fragments with an experimentally validated MMP ZBG group improved predicted potency. Our results indicate the importance of taking into account conformational selection and induced-fit effects when dealing with highly flexible proteins like the MMPs, as these factors can have a significant impact on the predicted binding energies of candidate compounds.

Acknowledgments

This work was supported in part by grants from the UCSD School of Medicine, NSF, NIH, the Center for Theoretical Biological Physics, the National Biomedical Computation Resource, San Diego Supercomputing Center, and Accelrys, Inc. We thank Dr. Seth Cohen and his group for helpful discussions.

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