Protein Surface-Assisted Enhancement in the Binding Affinity of an Inhibitor for Recombinant Human Carbonic Anhydrase-II Abir L. Banerjee, Michael Swanson, Bidhan C. Roy, Xiao Jia, Manas K. Haldar, Sanku Mallik,* and D. K. Srivastava* Contribution from the Department of Chemistry and Molecular Biology, North Dakota State UniVersity, Fargo, North Dakota 58105 Received April 27, 2004; E-mail: dk.srivastava@ndsu.nodak.edu; sanku.mallik@ndsu.nodak.edu Abstract: We elaborate on a novel strategy for enhancing the binding affinity of an active-site directed inhibitor by attaching a tether group, designed to interact with the surface-exposed histidine residue(s) of enzymes. In this approach, we have utilized the recombinant form of human carbonic anhydrase-II (hCA- II) as the enzyme source and benzenesulfonamide and its derivatives as inhibitors. The steady-state kinetic and the ligand binding data revealed that the attachment of iminodiacetate (IDA)-Cu 2+ to benzenesulfonamide (via a triethylene glycol spacer) enhanced its binding affinity for hCA-II by about 40-fold. No energetic contribution of either IDA or triethylene glycol spacer was found (at least in the ground state of the enzyme- inhibitor complex) when Cu 2+ was stripped off from the tether group-conjugated sulfonamide derivative. Arguments are presented that the overall strategy of enhancing the binding affinities of known inhibitors by attaching the IDA-Cu 2+ groups to interact with the surface-exposed histidine residues will find a general application in designing the isozyme-specific inhibitors as potential drugs. Introduction Following the completion of the human genome project, there has been a growing awareness of the potential drug targets for treating a variety of human diseases. 1 The overall strategy in the area of drug discovery has been greatly facilitated by the availability of modern combinatorial and high-throughput facilities. 2 The lead drug compounds, identified by these approaches, 2d-e are often subject to optimizations by the aids of the structural data of the target macromolecules (mostly enzymes and proteins). However, there is an intrinsic limitation in the structure-based drug design, primarily due to inherent flexibility in the protein structure, as well as the limited geometry of the ligand binding pockets (active sites in the case of enzymes) to accommodate extensive variations in the inhibitor structures. 4 Besides, on the basis of the structural coordinates of the selected enzyme-ligand complexes, it is difficult to predict, a priori, whether the desired changes in the inhibitor structure would be better accommodated within the active site pockets. 3 However, such limitations could be overcome, at least in principle, by designing enzyme inhibitors, which would not only bind to their cognate enzyme’s active site pockets, but also bind (by looping around) to the surface-exposed amino acid residues (Figure 1). In pursuit of identifying targetable surface-exposed amino acid residues in the vicinity of the enzyme’s active site pockets, it was realized that various transition-metal ions (Cu 2+ , Zn 2+ , Ni 2+ ) bind to the imidazole group of the histidine residues. 5 The ability of iminodiacetic acid-conjugated Cu 2+ (IDA-Cu 2+ ) to preferentially interact with the histidine residues of proteins has been utilized to perform the two-dimensional crystallization of proteins at lipid bilayers, 6 purification of peptides and proteins, 7 probing the accessibility of histidine residues, 8 map- (1) (a) Lindsay M, A. Nat. ReV. Drug DiscoVery 2003, 2, 831-838. (b) Hemmila, I. A.; Hurskainen, P. Drug DiscoVery Today 2002, 7, 150-156. (C) Stumm, G.; Russ, A.; Nehls, M. Am. J. PharmacoGenomics 2002, 2, 263-271. (d) Guzey, C.; Spigset, O. Drug Saf. 2002, 25, 553-560. (2) (a) Dolle, R. E. J. Comb. Chem. 2003 5, 693-753. (b) Lee, A.; Breitenbucher, J. G. Curr. Opin. Drug DiscoVery DeV. 2003 6, 494-508. 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Inorg. Biochem. 1996, 35, 4490-4496. Figure 1. Surface-assisted enhancement in the binding affinity of an inhibitor. The inhibitor is shown to bind at both the active site pocket as well as a surface-exposed residue of an enzyme. Published on Web 08/07/2004 10.1021/ja047557p CCC: $27.50 © 2004 American Chemical Society J. AM. CHEM. SOC. 2004, 126, 10875-10883 9 10875