Determining the structural basis for specificity of ligands using crystallographic screening.
ABSTRACT Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure- based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.
- [show abstract] [hide abstract]
ABSTRACT: The ubiquitous purine nucleoside phosphorylases (PNPs) play a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. This review updates the properties of the enzymes from eukaryotes and a wide range of prokaryotes, including a tentative classification of the enzymes from various sources, based on three-dimensional structures in the solid state, subunit composition, amino acid sequences, and substrate specificities. Attention is drawn to the compelling need of quantitative experimental data on subunit composition in solution, binding constants, and stoichiometry of binding; order of ligand binding and release; and its possible relevance to the complex kinetics exhibited with some substrates. Mutations responsible for PNP deficiency are described, as well as clinical methods, including gene therapy, for corrections of this usually fatal disease. Substrate discrimination between enzymes from different sources is also being profited from for development of tumour-directed gene therapy. Detailed accounts are presented of design of potent inhibitors, largely nucleosides and acyclonucleosides, their phosphates and phosphonates, particularly of the human erythrocyte enzyme, some with Ki values in nanomolar and picomolar range, intended for induction of the immunodeficient state for clinical applications, such as prevention of host-versus-graft response in organ transplantations. Methods of assay of PNP activity are reviewed. Also described are applications of PNP from various sources as tools for the enzymatic synthesis of otherwise inaccessible therapeutic nucleoside analogues, as coupling enzymes for assays of orthophosphate in biological systems in the micromolar and submicromolar ranges, and for coupled assays of other enzyme systems.Pharmacology [?] Therapeutics 01/2001; 88(3):349-425. · 7.79 Impact Factor
- Methods in Enzymology - METH ENZYMOLOGY. 01/1997; 277:505-524.
- [show abstract] [hide abstract]
ABSTRACT: The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for discovering small inhibitory molecules that can modify the degree of cell proliferation. The three-dimensional structure of CDK2 provides a structural foundation for understanding the mechanisms of activation and inhibition of CDK2 and for the discovery of inhibitors. In this article five structures of human CDK2 are summarised: apoprotein, ATP complex, olomoucine complex, isopentenyladenine complex, and des-chloro-flavopiridol complex.Progress in cell cycle research 01/1996; 2:137-45.