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

Abstract

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.

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

“…Binding to only one active site per trimer is also observed crystallographically for certain fragments. 10 However, this result does not correlate with affinity, nor explain the 1:1 binding observed for CDP. We speculate that the apparent 1:1 binding observed for CDP and certain fragments may be due to differential affinity across the three active sites in the Bp IspF trimer.…”

“…Sequence analysis shows over 30% identity for IspF proteins across prokaryotic and eukaryotic species. 10 Structural studies show high conservation of residues essential in substrate-binding, catalysis, and other aspects of IspF enzymatic function. 8 Thus compounds which inhibit the IspF enzyme from any given pathogenic organism could possess some potential for cross-species activity.…”

“…Previously, we conducted fragment-based screens with IspF from B. pseudomallei ( Bp IspF) by nuclear magnetic resonance (NMR) spectroscopy, and by x-ray crystallography. 10 Screening generated a collection of hits (Figure 1), and led to 14 fragment-bound complexes with as many as three small molecules bound in unique sites of the protein.…”

“…In each case, an aromatic nitrogen occupies the available metal coordination site in the fragment-bound structure. 10 Structural characterization of these fragment hits provide chemical starting points as well as relative distance and orientation information, suitable to a linking strategy. We therefore set out to design and synthesize a series of molecules that connect the cytidine moiety to different zinc pocket fragment binders to form “fusion” ligands.…”

Synthesis and biological evaluation of pyrazolopyrimidines as potential antibacterial agents

“…22 In collaboration with the SSGCID, additional NMR-based screening campaigns were conducted against Mycobacterium paratuberculosis (Mp) IspD, Mycobacterium abscessus (Ma) IspE and Bp IspF with approximately 1000 compounds. From this effort, FOL7185 was identified as a clear fragment binder for both Mp IspD and Ma IspE, and a very weak binder below the STD-NMR hit threshold for Bp IspF (Figure 1).…”