Experimental and Computational Active Site Mapping as a Starting Point to Fragment‐Based Lead Discovery

Abstract

Small highly soluble probe molecules such as aniline, urea, N-methylurea, 2-bromoacetate, 1,2-propanediol, nitrous oxide, benzamidine, and phenol were soaked into crystals of various proteins to map their binding pockets and to detect hot spots of binding with respect to hydrophobic and hydrophilic properties. The selected probe molecules were first tested at the zinc protease thermolysin. They were then applied to a wider range of proteins such as protein kinase A, D-xylose isomerase, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, endothiapepsin, and secreted aspartic protease 2. The crystal structures obtained clearly show that the probe molecules populate the protein binding pockets in an ordered fashion. The thus characterized, experimentally observed hot spots of binding were subjected to computational active site mapping using HotspotsX. This approach uses knowledge-based pair potentials to detect favorable binding positions for various atom types. Good agreement between the in silico hot spot predictions and the experimentally observed positions of the polar hydrogen bond forming functional groups and hydrophobic portions was obtained. Finally, we compared the observed poses of the small-molecule probes with those of much larger structurally related ligands. They coincide remarkably well with the larger ligands, considering their spatial orientation and the experienced interaction patterns. This observation confirms the fundamental hypothesis of fragment-based lead discovery: that binding poses, even of very small molecular probes, do not significantly deviate or move once a ligand is grown further into the binding site. This underscores the fact that these probes populate given hot spots and can be regarded as relevant seeds for further design.

Insight into the structural similarity between HIV protease and secreted aspartic protease-2 and binding mode analysis of HIV-Candida albicans inhibitors

“…Nevertheless, the polar contact established with the carbonyl moiety of Gly27 in the interaction with HIV-1 protease, is preserved also in the complex with Sap2, where it interacts with the corresponding carbonyl oxygen of Gly220. Finally, our docking results are in agreement with the published crystal structure of C. albicans Sap2 in complex with ritonavir, which is a recent important entry to this topic 36 . In fact, as showed in the crystal structure, the arrangement of ritonavir hydrophobic residues into Sap2 subsites appears similar to that obtained through molecular docking in the present study, thus these data corroborate our docking calculations.…”

Extracellular proteinases of Candida species pathogenic yeasts

“…Three‐dimensional atomic resolution structures are available for C. albicans Sap1, Sap2, Sap3 and Sap5, for C. parapsilosis Sapp1 and one of the Sapp2 homologues, and for C. tropicalis Sapt1 . Candidapepsins are monomeric proteins with a highly conserved overall structure, consisting mainly of β‐sheets in which two (N‐ and C‐terminal) domains form a central binding cleft that binds a substrate (Figure ).…”

Advantages of crystallographic fragment screening: Functional and mechanistic insights from a powerful platform for efficient drug discovery

“…Superimposing crystal structures from soaking with different organic solvents can provide insight regarding the conformational landscape, chemical complementarity, plasticity, and surface hydration for a binding site of interest. In addition, the structural information, with the aid of molecular modeling, can be used to develop pharmacophoric models or improve binding affinity and specificity for lead optimization (Mattos et al, 2006; Behnen et al, 2012). However, difficulties in reliably predicting optimal binding modes and overcoming exquisitely demanding stereochemical requirements for linked molecules have limited the application of MSCS for drug discovery.…”