Biomedical subjects

Anne Cleasby

Publications and source records attributed to Anne Cleasby.

6 recordsLinked to original sources

PubMed2005

Structures for the potential drug target purine nucleoside phosphorylase from Schistosoma mansoni causal agent of schistosomiasis.

Despite the availability of effective chemotherapy, schistosomiasis continues to be one of the major parasitic infections to affect the human population worldwide. Currently, little is known of the structural biology of the parasites that are responsible for the disease and few attempts have been made to develop second generation drugs, which may become essential if resistance to those currently available becomes an issue. Here, we describe three crystal structures for the enzyme purine nucleoside phosphorylase (PNP) from Schistosoma mansoni, a component of the purine salvage pathway. PNP is known to be essential for the recovery of purine bases and nucleosides in schistosomes, due to an absence of the enzymes for de novo synthesis, making it a sensitive point in the parasite's metabolism. In all three structures reported here, acetate occupies part of the base-binding site and is directly bound to the conserved glutamic acid at position 203. One of the structures presents the crystallization additive sulfobetaine 195 (NDSB195) occupying simultaneously the ribose and phosphate binding sites, whilst a second presents only phosphate in the latter. The observation of sulfobetaine specifically bound to the protein active site was unexpected and is unique to this structure as far as we are aware. Considerable flexibility is observed in the active site, principally due to variable structural disorder in the regions centered on residues 64 and 260. This conformational plasticity extends to the way in which both NDSB195 and phosphate bind to the individual monomers of the trimeric structure reported here. Differences between the parasite and human enzymes are limited principally to the base-binding site, where the substitution of V245 in the mammalian enzymes by S247 introduces additional hydrogen bonding potential to the site. This is satisfied in the structures described here by a water molecule whose presence is normally observed only in complexes with 6-oxopurines. Residue Y202, which replaces F200 in human PNP, is able to reach over the ribose-binding site to interact with H259 and is predicted to form an additional hydrogen bond with the 5' hydroxyl of nucleoside substrates.

Amino Acid Sequence↗

PubMed2005

Identification of novel p38alpha MAP kinase inhibitors using fragment-based lead generation.

We describe the structure-guided optimization of the molecular fragments 2-amino-3-benzyloxypyridine 1 (IC(50) 1.3 mM) and 3-(2-(4-pyridyl)ethyl)indole 2 (IC(50) 35 microM) identified using X-ray crystallographic screening of p38alpha MAP kinase. Using two separate case studies, the article focuses on the key compounds synthesized, the structure-activity relationships and the binding mode observations made during this optimization process, resulting in two potent lead series that demonstrate significant increases in activity. We describe the process of compound elaboration either through the growing out from fragments into adjacent pockets or through the conjoining of overlapping fragments and demonstrate that we have exploited the mobile conserved activation loop, consisting in part of Asp168-Phe169-Gly170 (DFG), to generate significant improvements in potency and kinase selectivity.

Aminopyridines↗

PubMed2005

Fragment-based lead discovery using X-ray crystallography.

Fragment screening offers an alternative to traditional screening for discovering new leads in drug discovery programs. This paper describes a fragment screening methodology based on high throughput X-ray crystallography. The method is illustrated against five proteins (p38 MAP kinase, CDK2, thrombin, ribonuclease A, and PTP1B). The fragments identified have weak potency (>100 microM) but are efficient binders relative to their size and may therefore represent suitable starting points for evolution to good quality lead compounds. The examples illustrate that a range of molecular interactions (i.e., lipophilic, charge-charge, neutral hydrogen bonds) can drive fragment binding and also that fragments can induce protein movement. We believe that the method has great potential for the discovery of novel lead compounds against a range of targets, and the companion paper illustrates how lead compounds have been identified for p38 MAP kinase starting from fragments such as those described in this paper.

CDC2-CDC28 Kinases↗

PubMed2005

The discovery of novel protein kinase inhibitors by using fragment-based high-throughput x-ray crystallography.

This article describes the application of a high-throughput X-ray crystallographic fragment-based screening methodology to identify low-molecular-weight leads for structure-based optimisation into protein kinase inhibitors. The identification of two novel p38alpha MAP kinase inhibitors (with IC50=65 and 150 nM) starting from low-molecular-weight fragments is described.

Crystallography, X-Ray↗

PubMed2004

Apo and inhibitor complex structures of BACE (beta-secretase).

Human BACE, also known as beta-secretase, shows promise as a potential therapeutic target for Alzheimer's disease. We determined the apo structure of BACE to 1.75 A, and a structure of a hydroxyethylamine inhibitor complex derived by soaking. These show significant active-site movements compared to previously described BACE structures. Additionally, the structures reveal two pockets that could be targeted by structure-based drug design.

Amines↗

PubMed2003

Cloning, expression and preliminary crystallographic studies of the potential drug target purine nucleoside phosphorylase from Schistosoma mansoni.

The parasite Schistosoma mansoni, unlike its mammalian hosts, lacks the de novo pathway for purine biosynthesis and depends on salvage pathways for its purine requirements. The gene encoding one enzyme of this pathway, purine nucleoside phosphorylase from S. mansoni (SmPNP) was identified, fully sequenced and cloned into the bacterial expression vector pMAL c2G to produce a protein in fusion with maltose-binding protein. The recombinant fusion protein was expressed at high levels and was purified in a single step by amylose resin affinity chromatography. After factor Xa cleavage, SmPNP was purified using a cation-exchange column and crystallized by hanging-drop vapour diffusion using polyethylene glycol 1500 as precipitant in the presence of 20% glycerol in acetate buffer. The use of the non-detergent sulfobetaine 195 (NDSB 195) as an additive had a marked effect on the size of the resulting crystals. Two data sets were obtained, one from a crystal grown in the absence of NDSB 195 and one from a crystal grown in its presence. The crystals are isomorphous and belong to the space group P2(1)2(1)2(1). It is intended to use the structures in the discovery and development of specific inhibitors of SmPNP.

Amino Acid Sequence↗