Experimental and computational active site mapping as a starting point to fragment-based lead discovery.

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.

DSX: a knowledge-based scoring function for the assessment of protein-ligand complexes.

We introduce the new knowledge-based scoring function DSX that consists of distance-dependent pair potentials, novel torsion angle potentials, and newly defined solvent accessible surface-dependent potentials. DSX pair potentials are based on the statistical formalism of DrugScore, extended by a much more specialized set of atom types. The original DrugScore-like reference state is rather unstable with respect to modifications in the used atom types. Therefore, an important method to overcome this problem and to allow for robust results when deriving pair potentials for arbitrary sets of atom types is presented. A validation based on a carefully prepared test set is shown, enabling direct comparison to the majority of other popular scoring functions. Here, DSX features superior performance with respect to docking- and ranking power and runtime requirements. Furthermore, the beneficial combination with torsion angle-dependent and desolvation-dependent potentials is demonstrated. DSX is robust, flexible, and capable of working together with special features of popular docking engines, e.g., flexible protein residues in AutoDock or GOLD. The program is freely available to the scientific community and can be downloaded from our Web site www.agklebe.de .

fconv: Format conversion, manipulation and feature computation of molecular data.

UNLABELLED: fconv is a program intended for parsing and manipulating multiple aspects and properties of molecular data. Up to now, it has been developed and extensively tested for 3 years. It has become a very robust and comprehensive tool involved in a broad range of computational workflows that are currently applied in our drug design environment. Typical tasks are as follows: conversion and error correction of formats such as PDB(QT), MOL2, SDF, DLG and CIF; extracting ligands from PDB as MOL2; automatic or ligand-based cavity detection; rmsd calculation and clustering; substructure searches; alignment and structural superposition; building of crystal packings; adding hydrogens; calculation of various properties like the number of rotatable bonds; molecular weights or vdW volumes. The atom type classification is based on a consistent assignment of internal atom types, which are by far more differentiated compared with e.g. Sybyl atom types. Apart from the predefined mapping of these types onto Sybyl types, the user is able to assign own mappings by providing modified template files, thus allowing for tailor-made atom type sets. AVAILABILITY: fconv is free software available under GNU General Public License. C++ sources and precompiled executables for LINUX/UNIX, Mac OS and Windows, as well as tutorials are available on http://www.agklebe.de.

How to replace the residual solvation shell of polar active site residues to achieve nanomolar inhibition of tRNA-guanine transglycosylase.

In a computational and structural study, we investigated a series of 4-substituted lin-benzoguanines that are potent inhibitors of tRNA-guanine transglycosylase (TGT), a putative target for the treatment of shigellosis. At first glance, it appears self-evident that the placement of a positively charged ligand functional group between the carboxylate groups of two adjacent aspartate residues in the glycosylase catalytic center leads to enhanced ligand binding. The concomitant displacement of water molecules that partially solvate the aspartates prior to ligand binding appears to result as a consequence of this. However, the case study presented herein shows that this premise is much too superficial. Placement of a likely positively charged amino group at such a pivotal position, interfering with the residual water solvation shell, is at best cost-neutral compared with the unsubstituted parent ligand not conflicting with the residual water shell. A ligand that orients a hydroxy group in this position shows even decreased binding. Based on the cost-neutral placement of the amino functionality, hydrophobic side chains can now be further attached to fill, with increasing potency, a small hydrophobic pocket remote to the aspartates. Any attempts to cross the pivotal position between both aspartates with nonpolar scaffolds reveals only decreased binding, even though the waters of the residual solvation shell are successfully repelled. This surprising observation fostered a detailed analysis of the role of water molecules involved in the residual solvation of polar active site residues. Their geometry and putative replacement in the binding pocket of TGT has been studied by a comparative database analysis, computational active site mapping, and a series of crystal structure analyses. Furthermore, conformational preferences of attached hydrophobic moieties explain their contribution to a gradual increase in binding affinity.

TransCent: computational enzyme design by transferring active sites and considering constraints relevant for catalysis.

BACKGROUND: Computational enzyme design is far from being applicable for the general case. Due to computational complexity and limited knowledge of the structure-function interplay, heuristic methods have to be used. RESULTS: We have developed TransCent, a computational enzyme design method supporting the transfer of active sites from one enzyme to an alternative scaffold. In an optimization process, it balances requirements originating from four constraints. These are 1) protein stability, 2) ligand binding, 3) pKa values of active site residues, and 4) structural features of the active site. Each constraint is handled by an individual software module. Modules processing the first three constraints are based on state-of-the-art concepts, i.e. RosettaDesign, DrugScore, and PROPKA. To account for the fourth constraint, knowledge-based potentials are utilized. The contribution of modules to the performance of TransCent was evaluated by means of a recapitulation test. The redesign of oxidoreductase cytochrome P450 was analyzed in detail. As a first application, we present and discuss models for the transfer of active sites in enzymes sharing the frequently encountered triosephosphate isomerase fold. CONCLUSION: A recapitulation test on native enzymes showed that TransCent proposes active sites that resemble the native enzyme more than those generated by RosettaDesign alone. Additional tests demonstrated that each module contributes to the overall performance in a statistically significant manner.