Discovering novel ligands for macromolecules using X-ray crystallographic screening

Abstract

The need to decrease the time scale for clinical compound discovery has led to innovations at several stages in the process, including genomics/proteomics for target identification, ultrahigh-throughput screening for lead identification, and structure-based drug design and combinatorial chemistry for lead optimization. A critical juncture in the process is the identification of a proper lead compound, because a poor choice may generate costly difficulties at later stages. Lead compounds are commonly identified from high-throughput screens of large compound libraries, derived from known substrates/inhibitors, or identified in computational prescreeusing X-ray crystal structures. Structural information is often consulted to efficiently optimize leads, but under the current paradigm, such data require preidentification and confirmation of compound binding. Here, we describe a new X-ray crystallography-driven screening technique that combines the steps of lead identification, structural assessment, and optimization. The method is rapid, efficient, and high-throughput, and it results in detailed crystallographic structure information. The utility of the method is demonstrated in the discovery and optimization of a new orally available class of urokinase inhibitors for the treatment of cancer.

Rational Design for Crystallization of β-Lactoglobulin and Vitamin D3 Complex: Revealing a Secondary Binding Site

“…In the case of a weak binding affinity, the concentrations of ligands (or ligand solubility) may have to be 10 mM or higher in order to observe crystallographic occupancy. 37,38 We suspect that the lower occupancy of vitamin D 2 might explain an early study in which no electron density was found beyond the calyx using the crystal structure of LG-vitamin D 2 complex. 10 To optimize the binding and occupancy of vitamin D 3 to LG, we prepared this complex at various pH values ranging from 4 to 8 with a vitamin D 3 /LG ratio ranging from 1 to 3.…”

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

“…This method has been used to sample large compound libraries and detects ligands by monitoring variations in the electron density map relative to the native form. Nevertheless, even using high concentrations of ligand there are several examples of failure of obtaining complex between a protein target and a high-affinity ligand (Nienaber et al 2000). The main reasons for this failure are the following: (1) low solubility of the ligand, (2) large conformational changes due to ligand binding, which may break crystals (in the case of soaking experiments), and (3) small solvent channels in the crystal or obstructed binding site.…”

“…Methods allowing high-throughput screening could be applied to speed up the process of determination of structural basis for specificity of ligand library for a selected protein target. The method is rapid, efficient, and high-throughput, and it results in detailed crystallographic structure information (Nienaber et al 2000).…”

“…The determination of the structural basis for specificity of ligands against protein targets is a scientific endeavor that makes use of computational (Kuntz et al 1994) and experimental approaches (Nienaber et al 2000). The interaction between protein and ligand is the key feature in the structure-based design of inhibitors.…”

“…In order to identify possible new lead compounds against diseases such as malaria and tuberculosis, we will use the crystallographic screening procedure (Nienaber et al 2000). The determination of the structural basis for specificity of ligands against protein targets is a scientific endeavor that makes use of computational (Kuntz et al 1994) and experimental approaches (Nienaber et al 2000).…”