Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei.

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.

Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening.

Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl-CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD-binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting a fragment-based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side-chain perturbations to key active-site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small-molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.

Inhibitor-bound complexes of dihydrofolate reductase-thymidylate synthase from Babesia bovis.

Babesiosis is a tick-borne disease caused by eukaryotic Babesia parasites which are morphologically similar to Plasmodium falciparum, the causative agent of malaria in humans. Like Plasmodium, different species of Babesia are tuned to infect different mammalian hosts, including rats, dogs, horses and cattle. Most species of Plasmodium and Babesia possess an essential bifunctional enzyme for nucleotide synthesis and folate metabolism: dihydrofolate reductase-thymidylate synthase. Although thymidylate synthase is highly conserved across organisms, the bifunctional form of this enzyme is relatively uncommon in nature. The structural characterization of dihydrofolate reductase-thymidylate synthase in Babesia bovis, the causative agent of babesiosis in livestock cattle, is reported here. The apo state is compared with structures that contain dUMP, NADP and two different antifolate inhibitors: pemetrexed and raltitrexed. The complexes reveal modes of binding similar to that seen in drug-resistant malaria strains and point to the utility of applying structural studies with proven cancer chemotherapies towards infectious disease research.

Leveraging structure determination with fragment screening for infectious disease drug targets: MECP synthase from Burkholderia pseudomallei.

As part of the Seattle Structural Genomics Center for Infectious Disease, we seek to enhance structural genomics with ligand-bound structure data which can serve as a blueprint for structure-based drug design. We have adapted fragment-based screening methods to our structural genomics pipeline to generate multiple ligand-bound structures of high priority drug targets from pathogenic organisms. In this study, we report fragment screening methods and structure determination results for 2C-methyl-D-erythritol-2,4-cyclo-diphosphate (MECP) synthase from Burkholderia pseudomallei, the gram-negative bacterium which causes melioidosis. Screening by nuclear magnetic resonance spectroscopy as well as crystal soaking followed by X-ray diffraction led to the identification of several small molecules which bind this enzyme in a critical metabolic pathway. A series of complex structures obtained with screening hits reveal distinct binding pockets and a range of small molecules which form complexes with the target. Additional soaks with these compounds further demonstrate a subset of fragments to only bind the protein when present in specific combinations. This ensemble of fragment-bound complexes illuminates several characteristics of MECP synthase, including a previously unknown binding surface external to the catalytic active site. These ligand-bound structures now serve to guide medicinal chemists and structural biologists in rational design of novel inhibitors for this enzyme.

Predicting the success of fragment screening by X-ray crystallography.

Fragment screening using X-ray crystallography is a method that can provide direct three-dimensional readouts of the structures of protein-small molecule complexes for lead development and fragment-based drug discovery. With current technology, an amenable crystal form can be screened crystallographically against a library of 1000-2000 fragments in 1-2 weeks. We have performed over a dozen crystallographic screening campaigns using our own compound collection called Fragments of Life™ (FOL). While the majority of our fragment screening campaigns have generated multiple hits, some unexpectedly turned out to be nonproductive, either yielding no bound ligands, or only those thought to be inadequate for lead development. In this chapter, we have attempted to identify one or more parameters which could be used to predict whether a crystallized protein target would be a good candidate for fragment hit discovery. Here, we describe the parameters of crystals from 18 fragment screening campaigns, including six unsuccessful targets. From this analysis, we have concluded that there are no parameters that are absolutely predictive of fragment screening success. However, we do describe a parameter we have termed pocket factor which provides a statistically significant variance between nonproductive targets and productive targets shown to bind fragments. The pocket factor is calculated using a novel method of consensus scoring from three distinct pocket-finding algorithms, and the results may be used to prioritize targets for fragment screening campaigns based on an initial crystal structure.

Fragment screening of infectious disease targets in a structural genomics environment.

Structural genomics efforts have traditionally focused on generating single protein structures of unique and diverse targets. However, a lone structure for a given target is often insufficient to firmly assign function or to drive drug discovery. As part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID), we seek to expand the focus of structural genomics by elucidating ensembles of structures that examine small molecule-protein interactions for selected infectious disease targets. In this chapter, we discuss two applications for small molecule libraries in structural genomics: unbiased fragment screening, to provide inspiration for lead development, and targeted, knowledge-based screening, to confirm or correct the functional annotation of a given gene product. This shift in emphasis results in a structural genomics effort that is more engaged with the infectious disease research community, and one that produces structures of greater utility to researchers interested in both protein function and inhibitor development. We also describe specific methods for conducting high-throughput fragment screening in a structural genomics context by X-ray crystallography.