7 research outputs found
Exploring The Binding Of Aminoglycosides And Effect Of Peptides On Functionally Important Regions Of The Ribosome
ABSTRACT
EXPLORING THE BINDING OF AMINOGLYCOSIDES AND EFFECT OF PEPTIDES
ON FUNCTIONALLY IMPORTANT REGIONS OF THE RIBOSOME
by
PRABUDDHA WADUGE
September 2019
Advisor: Prof. Christine S. Chow
Major: Chemistry
Degree: Doctor of Philosophy
Aminoglycosides (AGs) are potent antibiotics that target helix 44 (h44) of the functionally important aminoacyl-tRNA site (A site) of the bacterial ribosome. Despite potency in many cases, some limitations such as resistance and off-target binding restrict the use of AGs as antibiotics. Modification of currently available AGs is a major approach to overcome those limitations. Towards this end, it is of importance to examine the molecular interactions of modified AGs with their target ribosomal RNA (rRNA), h44. Therefore, we employed in vitro dimethyl sulfate (DMS) footprinting and a fluorescence assay to examine binding of AGs to bacterial ribosomes and minimal A-site RNA constructs, respectively. We utilized DMS’s ability to specifically methylate a h44 internal-loop residue, A1408, where binding of AGs has a negative effect on DMS reactivity. As observed in DMS footprinting patterns, chemical modifications on AGs altered h44 binding with bacterial ribosomes. The in vitro binding affinities determined from these probing experiments correlated well with the in vivo antibacterial activities of the tested compounds.
A fluorescence assay was employed to compare binding affinities of modified AGs to RNA constructs resembling the target bacterial A site and the human mitochondrial A site as an off target. Modified AGs showed higher specificity towards bacterial over mitochondrial A sites compared to the parent compound. The trends in binding affinities were consistent with previously reported in vitro translation inhibition of AGs, indicating the utility of the fluorescence assay with minimal RNA motifs in assessing the selectivity of modified AGs.
Helix 69 (H69) of 23S rRNA, which interacts with h44 during ribosome subunit assembly, was studied for its effects on AG antibacterial activity. H69 contains the modified nucleotide pseudouridine (Ψ), which is important for H69 structure and function. We determined minimum inhibitory concentration to evaluate the effects of Ψ modifications in H69 on modified AG activity. We observed that the loss of Ψ along with a defective ribosome release factor (RF2) rendered AG resistance. These results suggest the importance of H69 in AG activity through H69-RF2 interactions in translation termination.
In addition to A site and AGs, exploring novel antibiotic targets and antibiotic leads in the bacterial ribosome is important for overcoming antibiotic resistance. Towards this end, we employed an in vivo expression system to examine the in vivo effects and target binding of an in vitro selected peptide (TLWDLIP) targeting helix 31 (h31) of the bacterial ribosome. Our in vivo growth and in vivo DMS footprinting assays show that the expressed TLWDLIP moderately inhibits bacterial growth and induces conformational changes in the h31 loop region, respectively. Overall, the study of potential antibiotic leads binding to different functionally important regions of the ribosome using in vitro and in vivo systems yields a better understanding of rRNA interactions, which will be useful in developing both selective and potent antibiotics
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Comparative ligandomics implicates secretogranin III as a disease-restricted angiogenic factor in laser-induced choroidal neovascularization
Choroidal neovascularization (CNV) is a leading cause of vision loss in the elderly. All approved anti-angiogenic drug therapies for CNV target vascular endothelial growth factor (VEGF) but confer limited efficacy. Identification of other CNV-related angiogenic factors will facilitate the development of VEGF-independent alternative therapies. Here, we applied comparative ligandomics to live mice with or without laser-induced CNV for global mapping of CNV-selective endothelial ligands. Secretogranin III (Scg3) previously identified by the same approach as a diabetes-restricted angiogenic factor was mapped with a more than 935-fold increase in binding to CNV vessels compared to healthy choriocapillaris. A novel in vivo ligand binding assay independently confirmed a marked increase in Scg3 binding to CNV vessels, whereas VEGF showed no increase in CNV-selective binding. A new technique of functional immunohistochemistry allowed the visualization and confirmed the increase in in vivo Scg3 binding to CNV vasculatures, including CNV microcapillaries with detailed vascular structures, which was blocked by anti-Scg3 humanized antibody Fab fragment (hFab). The hFab effectively alleviated laser-induced CNV with an efficacy similar to the anti-VEGF drug aflibercept. Homozygous deletion of the Scg3 gene in mice significantly reduced the severity of CNV. Furthermore, the therapeutic activity of anti-Scg3 hFab, but not aflibercept, was abolished in Scg3
mice, suggesting the Scg3-dependent nature of the hFab-mediated therapy. These findings suggest that Scg3 plays an important role in CNV pathogenesis and is a promising disease-restricted angiogenic factor for ligand-guided disease-targeted anti-angiogenic therapy of CNV
Selectively targeting disease‐restricted secretogranin III to alleviate choroidal neovascularization
Choroidal neovascularization (CNV), a leading cause of blindness in the elderly, is routinely treated with vascular endothelial growth factor (VEGF) inhibitors that have limited efficacy and potentially adverse side effects. An unmet clinical need is to develop novel therapies against other angiogenic factors for alternative or combination treatment to improve efficacy and safety. We recently described secretogranin III (Scg3) as a disease‐selective angiogenic factor, causally linked to diabetic retinopathy and acting independently of the VEGF pathway. An important question is whether such a disease‐selective Scg3 pathway contributes to other states of pathological angiogenesis beyond diabetic retinopathy. By applying a novel in vivo endothelial ligand binding assay, we found that the binding of Scg3 to CNV vessels in live mice was markedly increased over background binding to healthy choriocapillaris and blocked by an Scg3‐neutralizing antibody, whereas VEGF showed no such differential binding. Intravitreal injection of anti‐Scg3 humanized antibody Fab (hFab) inhibited Matrigel‐induced CNV with similar efficacy to the anti‐VEGF drug aflibercept. Importantly, a combination of anti‐Scg3 hFab and aflibercept synergistically alleviated CNV. Homozygous deletion of the Scg3 gene markedly reduced CNV severity and abolished the therapeutic activity of anti‐Scg3 hFab, but not aflibercept, suggesting a role for Scg3 in VEGF‐independent CNV pathogenesis and therapy. Our work demonstrates the stringent disease selectivity of Scg3 binding and positions anti‐Scg3 hFab as a next‐generation disease‐targeted anti‐angiogenic therapy for CNV
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Secretogranin III stringently regulates pathological but not physiological angiogenesis in oxygen-induced retinopathy
Conventional angiogenic factors, such as vascular endothelial growth factor (VEGF), regulate both pathological and physiological angiogenesis indiscriminately, and their inhibitors may elicit adverse side effects. Secretogranin III (Scg3) was recently reported to be a diabetes-restricted VEGF-independent angiogenic factor, but the disease selectivity of Scg3 in retinopathy of prematurity (ROP), a retinal disease in preterm infants with concurrent pathological and physiological angiogenesis, was not defined. Here, using oxygen-induced retinopathy (OIR) mice, a surrogate model of ROP, we quantified an exclusive binding of Scg3 to diseased versus healthy developing neovessels that contrasted sharply with the ubiquitous binding of VEGF. Functional immunohistochemistry visualized Scg3 binding exclusively to disease-related disorganized retinal neovessels and neovascular tufts, whereas VEGF bound to both disorganized and well-organized neovessels. Homozygous deletion of the Scg3 gene showed undetectable effects on physiological retinal neovascularization but markedly reduced the severity of OIR-induced pathological angiogenesis. Furthermore, anti-Scg3 humanized antibody Fab (hFab) inhibited pathological angiogenesis with similar efficacy to anti-VEGF aflibercept. Aflibercept dose-dependently blocked physiological angiogenesis in neonatal retinas, whereas anti-Scg3 hFab was without adverse effects at any dose and supported a therapeutic window at least 10X wider than that of aflibercept. Therefore, Scg3 stringently regulates pathological but not physiological angiogenesis, and anti-Scg3 hFab satisfies essential criteria for development as a safe and effective disease-targeted anti-angiogenic therapy for ROP
An Advanced Apralog with Increased in vitro and in vivo Activity toward Gram-negative Pathogens and Reduced ex vivo Cochleotoxicity
We describe the convergent synthesis of a 5-O-β-D-ribofuranosyl-based apramycin derivative (apralog) that displays significantly improved antibacterial activity over the parent apramycin against wild-type ESKAPE pathogens. In addition, the new apralog retains excellent antibacterial activity in the presence of the only aminoglycoside modifying enzyme (AAC(3)-IV) acting on the parent, without incurring susceptibility to the APH(3') mechanism that disables other 5-O-β-D-ribofuranosyl 2-deoxystreptamine type aminoglycosides by phosphorylation at the ribose 5-position. Consistent with this antibacterial activity, the new apralog has excellent 30 nM activity (IC50 ) for the inhibition of protein synthesis by the bacterial ribosome in a cell-free translation assay, while retaining the excellent across-the-board selectivity of the parent for inhibition of bacterial over eukaryotic ribosomes. Overall, these characteristics translate into excellent in vivo efficacy against E. coli in a mouse thigh infection model and reduced ototoxicity vis à vis the parent in mouse cochlear explants
Design, Multigram Synthesis, and in Vitro and in Vivo Evaluation of Propylamycin: A Semisynthetic 4,5-Deoxystreptamine Class Aminoglycoside for the Treatment of Drug-Resistant Enterobacteriaceae and Other Gram-Negative Pathogens
Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant Enterobacteriaceae (CREs), present a major and growing threat to human health and society, providing an urgent need for the development of improved potent antibiotics for their treatment. We describe the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of propylamycin. Propylamycin is a 4'-deoxy-4'-alkyl paromomycin whose alkyl substituent conveys excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections, including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside modifying enzymes or rRNA methyl transferases. Importantly, propylamycin is demonstrated not to be susceptible to the action of the ArmA resistance determinant whose presence severely compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance when used in combination with carbapenems. Cell-free translation assays, quantitative ribosome footprinting, and X-ray crystallography support a model in which propylamycin functions by interference with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, which is better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of aminoglycoside therapy