An Experimental Approach to Mapping the Binding Surfaces of Crystalline Proteins

Abstract

Porcine pancreatic elastase has been used as the model enzyme in the design and development of a crystallographic method that allows mapping of the binding surface of a protein by solving its crystal structure in a variety of organic solvents. The ultimate goal of this method is to aid in the process of drug design, where each of the chosen organic molecules represents a given functional group in a larger inhibitor molecule. This method of multiple solvent crystal structures (MSCS) has a theoretical counterpart in the method of multiply copy simultaneous search (MCSS) (Miranker, A.; Karplus, M. Proteins: Struct., Funct., Genet. 1991, 11, 29-34) and is the first experimental method that can be used as a check to the theory. The MSCS method is presented here with acetonitrile as the probe organic solvent. The procedure involved does not cause significant changes in the structure of elastase as compared to the structure in aqueous solution, and the positions found for the acetonitrile molecules in the active site are compared to those of similar functional groups belonging to known inhibitors bound to elastase.

Fragment-guided design of subnanomolar β-lactamase inhibitors active in vivo

“…In themselves, these inhibitors hold promise as leads to overcome a pervasive and growing threat to public health. More generally, whereas fragments are widely used to nucleate early discovery (13)(14)(15)(16)(17)(18)(19)(20)(21)(22), this study suggests that they also may be used to guide late-stage optimization into chemotypes and geometries that would be hard to systematically sample by other methods.…”

“…Whereas fragments have been used previously for new chemotype discovery (13)(14)(15)(16)(17)(18)(19)(20)(21)(22) and merging, their use in late stage optimization has remained largely unexplored. Of course nothing prevented this, and indeed such an idea is implicit in the fragment approach and anticipated by computational design methods like LUDI, HOOK, GrowMol, and MCSS (36)(37)(38)(39).…”

“…Fragments can optimally explore receptor pockets (13)(14)(15)(16)(17)(18) and better cover chemical space (19)(20)(21)(22), and we wondered if they could guide a late-stage optimization that had thus far floundered. In a previous fragment-based docking screen against AmpC (22) and in a defragmentation study of a known AmpC inhibitor (23), crystal structures of three anionic fragments were determined: a thiophene carboxylic acid (F1), a benzoic acid (F2), and an aryl tetrazole (F3), all of which bind in the same distal region of the active site where the aryl carboxylate of compound 2 was placed ( Fig.…”

Structure-Based Approach for Binding Site Identification on AmpC β-Lactamase

“…Binding sites may be explored experimentally by using ligand molecules as probes. This can be done crystallographically, using solvent 14 or even larger molecules, 15 or by nuclear magmetic resonance (NMR), using small molecule fragments that can probe the ability of a particular site to bind to a particular functionality or group of functionalities. 16 Computational approaches are also used to identify hot spots for ligand binding.…”

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

“…Such an understanding, like that concerning the selectivity, requires both structural and mechanistic information. Recently, structures of several enzymes in neat organic solvents, namely those of subtilisin Carlsberg in acetonitrile (15,16) and dioxane (17), ␥-chymotrypsin in hexane (18,19), and elastase in acetonitrile (20), have been elucidated and found to be essentially the same as in water. Furthermore, the structures in hexane of a ␥-chymotrypsin-product complex has been determined (19) and that of a tetrahedral complex claimed (18).…”