Binding Of Sugar Sulfamates To Cas

This study concentrates on the observed binding modes of topiramate and RWJ-37947, tries to develop a theoretical rational for this observation and investigates whether current docking algorithms will reproduce these experimental findings. This should indicate (1) whether at all virtual screening or docking attempts will result in a selection of potential inhibitors and (2) whether virtually selected inhibitors will be correctly ranked according to their potency.

Two x-ray crystal structures of CAIs with a sugar sulfamate moiety in complex with CA II have recently been solved (Casini et al. 2003; Recacha et al. 2002). Topiramate (1) and RWJ-37947 (2; Figure 3.1) inhibit CA II in the nanomolar range (Masereel et al. 2002; Recacha et al. 2002). Topiramate is clinically used as an antiepileptic drug and RWJ-37947 possesses anticonvulsant activity. The difference between the two compounds is substitution of an isopropylidene moiety in topiramate by a cyclic sulfate group in RWJ-37947 (Figure 3.1). Both sugar sulfamate derivatives 1 and 2 bind with their sulfamate moiety to zinc, resulting in a tetrahedral coordination (Figure 3.2). As generally known from NMR spectroscopy, CA inhibitors coordinate with a deprotonated anchoring group to the zinc ion (Liljas et al. 1994). In addition, both compounds make hydrogen bonds with the side chain oxygen atom of Thr-199 and the backbone nitrogen atom of the same residue. The hydroxy group of Thr-199 forms an additional hydrogen bond with Glu-106, such that the Thr-199 hydroxyl acts as a hydrogen-bond acceptor for inhibitor binding.

Despite the similarity in anchoring to the zinc according to a well-known interaction pattern (Liljas et al. 1994), a surprising difference is observed in the binding mode of the two inhibitors with respect to the attached ring system. Topiramate forms hydrogen bonds with amino acid side chains in a hydrophilic binding pocket (Asn-67, Gln-92) and to a water molecule that donates a hydrogen bond to Thr-200 (Figure 3.2A). In addition, this water interacts with the oxygen atom of the ligand's six-membered ring. RWJ-37947, instead, shows a different binding mode in which the ring system is rotated by ca. 180° (mirrored), although an orientation as observed for topiramate would appear feasible (Figure 3.3). Therefore, surprisingly, the cyclic sulfate group points to a more hydrophobic pocket (Leu-198, Pro-202, Phe-131), and except for the sulfamate anchoring group, no further hydrogen bonds are observed (Figure 3.2B).

Given the apparent topological similarity of the two compounds and the surprising difference in their binding modes, the question arises as to whether such a behavior is predictable, and, in particular, whether computational tools can highlight this difference.

Here, the grid-based automated docking program AutoDock (Goodsell and Olson 1990; Morris et al. 1996) was used to address this question and to predict the binding mode of topiramate, RWJ-37947 and three additional commonly used CA inhibitors (see Figure 3.1: acetazolamide 3, brinzolamide 4 and dorzolamide 5). For four compounds, x-ray crystal structures were extracted from the Protein Data Bank (PDB codes 1a42, 1azm, 1cil, 1eou). The crystal structure of CA II in complex with topiramate was kindly provided by Dr. C.T. Supuran, University of Florence. All five protein structures were superimposed with Sybyl® 6.8 (Tripos, Inc., St. Louis, MO), based on all atoms of 20 conserved amino acids in the binding pocket (Ser-29, Pro-30, Asn-61, Gly-63, Gln-92, His-94, His-96, Trp-97, Glu-106, Glu-117, His-119, His-122, Ala-142, Val-143, Ser-197, Thr-199, Pro-201, Glu-205, Trp-209, Asn-244). Comparison of the amino acids in the binding pocket of the superimposed structures indicates slight conformational flexibility for His-64 only (Figure 3.4). This is further corroborated by superpositioning 55 complex structures of hCA II with Relibase (Gunther et al. 2003; Hendlich et al. 2003), which reveals that CA II has a fairly invariant binding site, except for the amino acid at position 64 (Klebe 2003). The biochemical reason for this observation is most likely related to the functional role of His-64 in catalysis, wherein it acts as a proton shuttle in regenerating the zinc-bound hydroxide (Duda et al. 2001, 2003). In general, two main conformations of His-64 are observed: the in conformation, in which His-64 points to the active site, and the out conformation, in which His-64 points to the solvent. In the five complexes mentioned, His-64 adopts both the in (1azm, 1eou) and the out (1a42, 1cil, crystal structure of topiramate) conformations.

FIGURE 3.3 (See color insert following page 148.) The binding mode of RWJ-37947 observed in the crystal structure (A) and manually rotated by 220° around the single bond connecting the ring system with the sulfamate anchor (B). (B) reveals that a binding mode as observed in the topiramate complex is sterically possible. The solvent accessible surface of the binding pocket of CAII hosting topiramate is shown (A, B).

FIGURE 3.3 (See color insert following page 148.) The binding mode of RWJ-37947 observed in the crystal structure (A) and manually rotated by 220° around the single bond connecting the ring system with the sulfamate anchor (B). (B) reveals that a binding mode as observed in the topiramate complex is sterically possible. The solvent accessible surface of the binding pocket of CAII hosting topiramate is shown (A, B).

To allow as much space as possible for ligand placement, 1cil with His-64 in the out conformation was used to dock all five ligands. The structure was constructed for docking by removing all water and ligand atoms, adding polar hydrogens and assigning Amber atomic charges and solvation parameters as required by the

Glu-106 Thr"199 Pro-202

His-96

His-64

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