Biochemistry and Pharmacology - Research Publications
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ItemFrom Knock-Out Phenotype to Three-Dimensional Structure of a Promising Antibiotic Target from Streptococcus pneumoniae(PUBLIC LIBRARY SCIENCE, 2013-12-13)Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k(cat) = 22 s(-1), K(M)(PYR) = 2.55 ± 0.05 mM and K(M)(ASA) = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T(M)(app)) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T(M)(app) = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K(D)(4→2) = 1.7 nM, which is considerably tighter than its E. coli ortholog (K(D)(4→2) = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å(2)) compared to its E. coli counterpart (500 Å(2)). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.
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ItemRepurposing the selective estrogen receptor modulator bazedoxifene to suppress gastrointestinal cancer growth.(EMBO Press, 2019)Excessive signaling through gp130, the shared receptor for the interleukin (IL)6 family of cytokines, is a common hallmark in solid malignancies and promotes their progression. Here, we established the in vivo utility of bazedoxifene, a steroid analog clinically approved for the treatment of osteoporosis, to suppress gp130-dependent tumor growth of the gastrointestinal epithelium. Bazedoxifene administration reduced gastric tumor burden in gp130Y757F mice, where tumors arise exclusively through excessive gp130/STAT3 signaling in response to the IL6 family cytokine IL11. Likewise, in mouse models of sporadic colon and intestinal cancers, which arise from oncogenic mutations in the tumor suppressor gene Apc and the associated β-catenin/canonical WNT pathway, bazedoxifene treatment reduces tumor burden. Consistent with the proposed orthogonal tumor-promoting activity of IL11-dependent gp130/STAT3 signaling, tumors of bazedoxifene-treated Apc-mutant mice retain excessive nuclear accumulation of β-catenin and aberrant WNT pathway activation. Likewise, bazedoxifene treatment of human colon cancer cells harboring mutant APC did not reduce aberrant canonical WNT signaling, but suppressed IL11-dependent STAT3 signaling. Our findings provide compelling proof of concept to support the repurposing of bazedoxifene for the treatment of gastrointestinal cancers in which IL11 plays a tumor-promoting role.
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ItemEPO does not promote interaction between the erythropoietin and beta-common receptors (vol 8, 12457, 2018)(NATURE PUBLISHING GROUP, 2019-05-21)A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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ItemEPO does not promote interaction between the erythropoietin and beta-common receptors(NATURE PORTFOLIO, 2018-08-20)A direct interaction between the erythropoietin (EPOR) and the beta-common (βc) receptors to form an Innate Repair Receptor (IRR) is controversial. On one hand, studies have shown a functional link between EPOR and βc receptor in tissue protection while others have shown no involvement of the βc receptor in tissue repair. To date there is no biophysical evidence to confirm a direct association of the two receptors either in vitro or in vivo. We investigated the existence of an interaction between the extracellular regions of EPOR and the βc receptor in silico and in vitro (either in the presence or absence of EPO or EPO-derived peptide ARA290). Although a possible interaction between EPOR and βc was suggested by our computational and genomic studies, our in vitro biophysical analysis demonstrates that the extracellular regions of the two receptors do not specifically associate. We also explored the involvement of the βc receptor gene (Csf2rb) under anaemic stress conditions and found no requirement for the βc receptor in mice. In light of these studies, we conclude that the extracellular regions of the EPOR and the βc receptor do not directly interact and that the IRR is not involved in anaemic stress.