Ligand Docking and Design in a Flexible Receptor SiteIan L. Alberts*, Nicolay P. Todorov, Per K�llblad+ and Philip M. Dean

De Novo Pharmaceuticals, Compass House, Vision Park, Histon, Cambridge CB4 9ZR, UK, Phone: þ44 1223238054,Fax: þ441223 238088, E-mail: ian.alberts@denovopharma.com

Keywords: Drug design, Conformation, Protein-ligand interaction, Flexibility, Structure generation,Side-chain

Received: November 9, 2004; Accepted: January 6, 2005

AbstractIntrinsic protein flexibility is often ignored in the drug discovery process, however, thereis increasing evidence suggesting that many therapeutic targets are flexible. In this work,we describe a method for incorporating receptor flexibility, based on protein side-chainrearrangements, in ligand docking and design. The approach is applied to the docking ofa highly potent inhibitor in the acetylcholinesterase binding site and to the generation ofligands in the S1’ cavity of human collagenase. Simulations are conducted for both staticand flexible binding sites and from the results we assess the impact of receptor flexibilityin drug discovery procedures.

1 Introduction

Protein flexibility is often ignored in drug discovery. How-ever, designing ligands to modified receptor binding sitestructures may enhance the diversity of the designed mole-cules and extensive evidence in the literature is now avail-able suggesting that many therapeutic targets are flexible[1]. For example, peptidic inhibitors often bind to the na-tive form of renin, whereas, piperidine-based inhibitorsbind to an alternative modified receptor structure, involv-ing rearrangement of three side-chains (Tyr75, Leu73 andTrp39) and opening of the active site flap [2]. Examinationof modified configurations of protein binding sites is,therefore, likely to contribute to the discovery of novelchemically diverse inhibitors that bind with high affinity.

Prediction of the structure of the protein-ligand com-plex is one of the crucial requisites of drug discovery. As aconsequence of the huge number of degrees of freedomavailable to the system, the structure of the target proteinis usually kept rigid and a single, low energy snapshot con-figuration is utilized in the drug discovery process. Howev-er, recently, molecular docking schemes have been devel-oped that incorporate different aspects of protein flexibili-ty. These include the receptor ensemble approach, inwhich a set of protein structural models are identified,from for example, crystal structures, NMR, molecular dy-namics or homology modelling, and ligands are docked ei-ther to all members of the set individually or to a single,average, composite structure [3 – 7]. An alternative ap-proach involves allowing limited conformational freedom

of the protein during the molecular docking simulation,for example, permitting amino-acid side-chain rearrange-ments for specific residues, while keeping the proteinbackbone fixed [8, 9], either by recognizing low-energyside-chain orientations during the docking procedure [10,11] or exploiting libraries of rotamers to represent fav-oured side-chain conformations [12, 13]. A further processused for incorporating receptor structural changes involvescomplex minimization from different initial positions ofthe ligand in the binding site [14].

Despite the well-documented importance of proteinstructural flexibility in drug design [15, 16], most computa-tional approaches utilize a rigid protein structure, whendesigning novel active ligand candidates within the bindingsite [17 – 20]. Analysis of protein crystal structure data hasshown that side-chains have more inherent mobility thanbackbones [9] and, thus, inclusion of side-chain flexibilityrepresents an important initial approach towards the han-dling of fully flexible protein conformations. In this paper,we describe a method for integrating side-chain rearrange-ments into both ligand docking and design processes. Themethod is used to investigate the influence of receptorflexibility in the binding sites of acetylcholinesterase(AChE) and human collagenase (matrix metalloprotei-nase-1 (MMP-1)). Acetylcholinesterase is a serine hydro-lase that is involved in several disease areas, includingmyasthenia gravis and Alzeimer�s disease [21]. Liganddocking experiments are conducted in the AChE bindingsite, involving rotating a single side-chain, Phe330, whichis known to rearrange upon binding of diverse ligands.MMPs have been the focus of rigorous investigation as aresult of their involvement in several pathogenic states, in-cluding cancer [22] and arteriosclerosis [23]. From theanalysis of known MMP-1 crystal structures, it is clear that

QSAR Comb. Sci. 2005, 24 DOI: 10.1002/qsar.200430924 � 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 503

+ Current address: Medivin AB, Lunastigen 7, SE-14144 Hud-dinge, Sweden.

Full Papers

the binding of different ligands induces significant confor-mational changes in the S1’ pocket [24, 25]. In our earlierwork, we conducted a reagent screening protocol withinthe flexible MMP-1 S1’ cavity and identified potent inhibi-tors that would not �fit� into a static site, but bind stronglyin a modified site [26]. This paper extends the previouswork and involves structure design in the MMP-1 S1’pocket connected to a common scaffold that was anchoredin the binding site. In all cases docking and structure gen-eration is compared using a rigid crystal structure and aflexible receptor.

2 Methods

2.1 Receptor model preparation

The AChE and MMP-1 receptor models used in this workare shown in Table 1. They are all ligand-bound com-plexes. Hydrogen atoms were added to the structures usingthe molecular modelling package InsightII (Accelrys Inc.)and their atomic positions were minimized within Discov-

er3 to an energy gradient of 0.001 kcalmol�1��1 using theconjugate gradients algorithm and the CFF force field[27], with the non-hydrogen atoms fixed in their crystalstructure positions. All ligands, non-Zn metal atoms andwater molecules were removed from our dataset of struc-tures. The two AChE models were superposed by align-ment of the Ca position of the backbones structures.

2.2 Algorithm for handling receptor flexibility

The de novo design algorithm Skelgen [20] was extendedto integrate dynamic receptor flexibility by allowing an ad-ditional transition type corresponding to side-chain mobili-ty. Side-chain conformers are represented by c torsion an-gles, and these are randomly modified to give the best in-duced �fit� between the evolving ligand and the flexiblesite. In this work, Skelgen was used both in structure gen-eration and molecular docking modes, involving a simulat-ed annealing protocol to dynamically minimize estimatesof the receptor-ligand binding energy, with the constraintthat all bumps are removed. Further details of the Skelgenalgorithm and practical applications have been describedin detail previously [20].

3 Results

3.1 Acetylcholinesterase – ligand docking with side-chainflexibility

In the catalytic mechanism of AChE, residue Phe330 be-haves like a gateway targeting acetylcholine towards thereaction centre. Significantly different side-chain orienta-tions of Phe330 have been observed in AChE crystal struc-ture complexes, as illustrated in Figure 1 for the overlay oftwo such complexes, 1vot and 1eve. We conducted dockingsimulations of the ligand donepezil, a highly potent mar-keted antocholinergic drug, into the non-cocrystal recep-tor 1vot allowing side-chain rotations of Phe330. For thesake of comparison, reference-docking simulations werealso conducted into the co-crystal structure, 1eve, fromwhich the ligand was extracted (�self� docking [28]). Bind-ing energy and rmsd data for these simulations are pre-sented in Table 2.

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Table 1. Protein structures used for ligand docking and genera-tion

Receptor PDBcode

Resolu-tion/ �

Ligand Reference

AChE 1eve 2.5 E2020 (donepezil) [21]AChE 1vot 2.5 Huperzine A [29]MMP-1 2tcl 2.2 RO 31 – 4724 [30]

Figure 1. Overlay of the crystal structure of 1eve complexed todonepezil (dark grey) and 1vot complexed to huperzine (lightgrey). The side chain conformation of the mobile residuePhe330 is also shown for each protein.

Table 2. Binding energies and binding mode for docking of do-nepezil into its co-crystal receptor 1eve and the non-native re-ceptor 1vot.

Receptor Minimum energydocking solution

Best rmsd(rank)

Rigid 1eve �38.9 1.1 (1)Rigid 1vot �35.1 4.2 (1)Flexible 1eve �39.0 1.1 (1)Flexible 1vot �39.5 1.4 (1)

Full Papers L. L. Alberts et al.

Docking simulations into the static X-ray form of 1evewere able to reproduce the crystal structure of the ligandto within 1 �, however, analogous docking runs into therigid 1vot non-native site were unable to yield a solutioncloser than 4 � to the crystal due to clashes with side-chainatoms of Phe330. Docking into the co-crystal receptor al-lowing side-chain movements by random c angle changesrecovered the correct ligand structure, to within 1.0 �, andcorrect side-chain angles for Phe330. The side-chain is ini-tially assigned random torsion angles, and, thus, recoveryof the correct side-chain conformation, even for the co-crystal receptor, is an important result for validation of themethod. Similarly, allowing rearrangements of Phe330 indocking to the alternative 1vot receptor yielded, as thetop-ranking solution, the correct ligand form to within1.0 � of the crystal in a binding site that is modified fromthe original 1vot structure and resembles that of 1eve (Fig-ure 2). The side-chain of Phe330 has swung away to allowaccommodation of the ligand. The score of this docking so-lution (�39.5) was similar to that of the top-ranking solu-tions obtained by docking into the rigid (�38.9) and flexi-ble (�39.0) native receptor, 1eve. This docking study dem-onstrates the validity of the methodology. With a staticprotein structure of 1vot, the ligand donepezil, which is astrong AChE inhibitor, would not be identified as a �hit�,however, when side-chain mobility is incorporated the ex-perimentally observed ligand binding mode is retrieved asthe top-ranked solution.

3.2 MMP-1 – structure generation in a flexible site

The configuration of the S1’ cavity plays a key role in de-termining the binding characteristics of MMP-1 [24]. We

previously explored structural rearrangements in the S1�pocket in a reagent screening experiment [26]. In thiswork, the earlier studies are extended by executing liganddesign simulations in the flexible S1� cavity of the MMP-1receptor 2tcl, to explore the influence of protein structuralchanges on the diversity of the generated ligands. ManyMMP-1 inhibitors include a hydroxamate or carboxylategroup that interacts with the catalytic zinc ion. In thiswork, we did not attempt to construct the metal-bindinggroup, and, instead the simulations involved connectingthe �growing� ligand to a substituted hydroxamate anchorat substitution position R, shown in Figure 3. The scaffoldwas allowed flexibility in the site during the structure gen-eration, but was anchored through experimentally ob-served distances and H bonding interactions (pharmaco-phoric constraints) with the site (Figure 3).

The six residues Leu181, Arg214, Val215, Ser239,Tyr240 and Phe242 are allowed to rearrange. These are thesame residues that were allowed mobility in our previous

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Figure 2. Overlay of the crystal structures of 1eve (dark grey) and 1vot (light grey), showing the conformation of the mobile residuePhe330. From left-to-right: A) donepezil, the top-ranked docking solution in B) the non-native rigid 1vot site (light grey), and C) theflexible 1vot site (dark grey).

Figure 3. Scaffold and pharmacophore constraints used in theMMP-1 structure generation. The attachment point is at R.

Ligand docking and design in a flexible receptor site

reagent screening work [26]. Ligand generation was per-formed in both the static and flexible 2tcl binding sites andthe designed structures as well as their binding energies inboth sets of experiments are depicted in Figure 4.

The structures generated in the rigid 2tcl receptor con-sisted of a single cyclic group with few substituents. Thering substituents include alkyl, alkoxy, amide and aminogroups. Figure 4A shows nine of the top ranking ligandswith binding energies ranging from �38 to �48 kJ/mol.These small ligand moieties connected to the substitutedhydroxamate scaffold can �fit� in the static S1’ cavity struc-ture. Including receptor flexibility opens the S1’ pocketand, thus, larger ligand groups connected to the hydroxa-mate scaffold can be generated. The larger fragments con-structed typically comprise two ring systems including aro-matic species, aliphatic/aromatic ring pairs and fused ringsystems, with an assortment of substituent groups includ-

ing, hydroxyl, amino, carbonyl and carboxylate groups.Figure 4B shows nine of the top-ranking ligands formedwith binding energies ranging from �48 to �58 kJ/mol.The structures formed in the S1’ cavity are similar to someof the species that were identified as �hits� in the previousvirtual screening study [26]. These structures were judgedto resemble sets of known MMP-1 inhibitors with nM ac-tivities, which were found in the Derwent World Drug In-dex (WDI) (Derwent Information Ltd,. London, U.K.). Asa consequence of side-chain rearrangements, particularlyArg114, Tyr140 and Phe142, ligands of different size andshape, which would not be generated in the original 2tclstructure due to steric clashes with the site, can �fit� into aconformationally modified S1’ pocket. The larger ligandsconstructed in the flexible cavity, which were missed in therigid receptor, were found to have the best binding ener-gies, and resemble some of the most highly active known

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Figure 4. A selection of the top-scoring ligands generated in A) the rigid MMP-1 site and B) the flexible MMP-1 site. The attach-ment point to the anchored scaffold is given by the *. Ligand binding energies are given beneath each structure.

Full Papers L. L. Alberts et al.

MMP-1 inhibitors from the WDI. These results suggestthat the integration of binding site mobility in a drug de-sign process may lead to the recognition of more diverse,active molecules that would be prohibited with a rigid pro-tein structure.

4 Conclusions

In this work, methodology for integrating protein side-chain structural flexibility in molecular docking and designwas described. We used a simulated annealing protocol tooptimise the ligand binding energy [20] and included side-chain rearrangements by permitting c dihedral angle mod-ifications during the simulation. The flexible receptor pro-cedure involving side-chain c angle rotations was shown tobe successful in the ligand docking and design studies de-scribed in this work. The process was validated in thedocking of the potent inhibitor donepezil into AChE.Docking simulations in the rigid non-native protein targetwere unable to identify the experimentally observed bind-ing mode, whereas, the mobile side-chain approach meth-od was able to reproduce the correct binding mode as thebest energy solution. In the ligand design experiments, in-volving connection of the growing molecules to a substitut-ed hydroxamate scaffold in the MMP-1 S1’ cavity, the rigid2tcl receptor leads to the generation of relatively smallunits in the S1’ pocket, consisting of a single cyclic group.Allowing protein side-chain mobility in the generationsimulations exposed the cavity and larger ligands wereconstructed, typically involving two or more cyclic groups,that are not accessible in the static site. These moleculesare strongly binding and show similarities with some of thehighly active inhibitors that were identified in the previousreagent screening study [26]. This work has demonstratedthat incorporating protein structural flexibility is impor-tant for both binding mode predictions and ligand genera-tion. The method described herein represents a valuableapproach for including receptor flexibility into the drugdiscovery process, which may result in the identification ofhighly binding, diverse ligands that would not be recog-nised in a static receptor snapshot.

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Ligand docking and design in a flexible receptor site