By optimizing binding to a selected target protein, modern drug research strives to develop safe and efficacious agents for the treatment of disease. Selective drug action is intended to minimize undesirable side effects from scatter pharmacology. Celecoxib (Celebrex), valdecoxib (Bextra), and rofecoxib (Vioxx) are nonsteroidal antiinflammatory drugs (NSAIDs) due to selective inhibition of inducible cyclooxygenase COX-2 while sparing inhibition of constitutive COX-1. While rofecoxib contains a methyl sulfone constituent, celecoxib and valdecoxib possess an unsubstituted arylsulfonamide moiety. The latter group is common to many carbonic anhydrase (CA) inhibitors. Using enzyme kinetics and X-ray crystallography, we demonstrate an unexpected nanomolar affinity of the COX-2 specific arylsulfonamide-type celecoxib and valdecoxib for isoenzymes of the totally unrelated carbonic anhydrase (CA) family, such as CA I, II, IV, and IX, whereas the rofecoxib methyl sulfone-type has no effect. When administered orally to glaucomatous rabbits, celecoxib and valdecoxib lowered intraocular pressure, suggesting that these agents may have utility in the treatment of this disorder. The crystal structure of celecoxib in complex with CA II reveals part of this inhibition to be mediated via binding of the sulfonamide group to the catalytic zinc of CA II. To investigate the structural basis for cross-reactivity of these compounds between COX-2 and CA II, we compared the molecular recognition properties of both protein binding pockets in terms of local physicochemical similarities among binding site-exposed amino acids accommodating different portions of the drug molecules. Our approach Cavbase, implemented into Relibase, detects similarities between the sites, suggesting some potential to predict unexpected cross-reactivity of drugs among functionally unrelated target proteins. The observed cross-reactivity with CAs may also contribute to differences in the pharmacological profiles, in particular with respect to glaucoma and anticancer therapy and may suggest new opportunities of these COX-2 selective NSAIDs.

In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant d-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and (1)H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.

Structural and mechanistic studies show that when the selection criteria of the immune system are changed, catalytic antibodies that have the efficiency of natural enzymes evolve, but the catalytic antibodies are much more accepting of a wide range of substrates. The catalytic antibodies were prepared by reactive immunization, a process whereby the selection criteria of the immune system are changed from simple binding to chemical reactivity. This process yielded aldolase catalytic antibodies that approximated the rate acceleration of the natural enzyme used in glycolysis. Unlike the natural enzyme, however, the antibody aldolases catalyzed a variety of aldol reactions and decarboxylations. The crystal structure of one of these antibodies identified the reactive lysine residue that was selected in the immunization process. This lysine is deeply buried in a hydrophobic pocket at the base of the binding site, thereby accounting for its perturbed pKa.

Hydrogen bonds are key interactions determining protein-ligand binding affinity and therefore fundamental to any biological process. Unfortunately, explicit structural information about hydrogen positions and thus H-bonds in protein-ligand complexes is extremely rare and similarly the important role of water during binding remains poorly understood. Here, we report on neutron structures of trypsin determined at very high resolutions ≤1.5 Å in uncomplexed and inhibited state complemented by X-ray and thermodynamic data and computer simulations. Our structures show the precise geometry of H-bonds between protein and the inhibitors N-amidinopiperidine and benzamidine along with the dynamics of the residual solvation pattern. Prior to binding, the ligand-free binding pocket is occupied by water molecules characterized by a paucity of H-bonds and high mobility resulting in an imperfect hydration of the critical residue Asp189. This phenomenon likely constitutes a key factor fueling ligand binding via water displacement and helps improving our current view on water influencing protein–ligand recognition.

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