Design et synthèse d'antibiotiques aminoglycosidiques

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition

The azide−alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide−alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na_2S_2O_4, 2% HOCH_2CH_2SH, 10% HCO_2H, 95% CF_3CO_2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO_2H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies

A BODIPY-Cyclooctyne for Protein Imaging in Live Cells

Cellular proteins that bear reactive azides can be imaged by fluorescence microscopy following strain-promoted ligation to cyclooctyne dyes. Here we describe BODIPY-cyclooctyne (BDPY), a membrane-permeant fluorophore that can be used to label intracellular proteins in live mammalian cells. Flow cytometry reveals fluorescence signals more than 25-fold above background after labeling of azide-tagged cells with BDPY

Live-Cell Imaging of Cellular Proteins by a Strain-Promoted Azide–Alkyne Cycloaddition

Live and let dye: Three coumarin-cyclooctyne conjugates have been used to label proteins tagged with azidohomoalanine in Rat-1 fibroblasts. All three fluorophores labeled intracellular proteins with fluorescence enhancements ranging from eight- to 20-fold. These conjugates are powerful tools for visualizing biomolecule dynamics in living cells

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Microbial pathogens use complex secretion systems to deliver virulence factors into host cells, where they disrupt host cell function. Understanding these systems is essential to the development of new treatments for infectious disease. A challenge in such studies arises from the abundance of host cell proteins, which interfere with detection of microbial effectors. Here we describe a metabolic labeling strategy that allows selective enrichment of microbial proteins from the host cell cytoplasm. The method enables efficient identification of microbial proteins that have been delivered to the host, identifies distinct secretion profiles for intracellular and extracellular bacteria, and allows for determination of the order of injection of microbial proteins into host cells

Antibacterial aminoglycosides with a modified mode of binding to the ribosomal-RNA decoding site.

Antibacterial aminoglycosides with a modified mode of binding to the ribosomal-RNA decoding site

Antibacterial Aminoglycosides with a Modified Mode of Binding to the Ribosomal-RNA Decoding Site

International audiencerésumé non disponibl

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Microbial pathogens use complex secretion systems to deliver virulence factors into host cells, where they disrupt host cell function. Understanding these systems is essential to the development of new treatments for infectious disease. A challenge in such studies arises from the abundance of host cell proteins, which interfere with detection of microbial effectors. Here we describe a metabolic labeling strategy that allows selective enrichment of microbial proteins from the host cell cytoplasm. The method enables efficient identification of microbial proteins that have been delivered to the host, identifies distinct secretion profiles for intracellular and extracellular bacteria, and allows for determination of the order of injection of microbial proteins into host cells

Structure-based design, synthesis, and A-site rRNA cocrystal complexes of functionally novel aminoglycoside antibiotics: C2" ether analogues of paromomycin.

International audienceA series of 2"-O-substituted ether analogues of paromomycin were prepared based on new site-selective functionalizations. X-ray cocrystal complexes of several such analogues revealed a new mode of binding in the A-site rRNA, whereby rings I and II adopted the familiar orientation and position previously observed with paromomycin, but rings III and IV were oriented differently. With few exceptions, all of the new analogues showed potent inhibitory activity equal or better than paromomycin against a sensitive strain of S. aureus. Single digit microM MIC values were obtained against E. coli, with some of the ether appendages containing polar or basic end groups. Two analogues showed excellent survival rate in a mouse septicemia protection assay. Preliminary histopathological analysis of the kidney showed no overt signs of toxicity, while controls with neomycin and kanamycin were toxic at lower doses

Discovery of an orally bioavailable and selective PKMYT1 inhibitor RP-6306

PKMYT1 is an important regulator of CDK1 phosphorylation and is a compelling therapeutic target for the treatment of certain types of DNA damage response cancers due to its established synthetic lethal relationship with CCNE1 amplification. To date, no selective inhibitors have been reported for this kinase that would allow for investigation of the pharmacological role of PKMYT1 in the treatment of cancer. To address this need we conducted a focused screening effort that identified compound 1 as a weak PKMYT1 inhibitor. Introduction of a dimethylphenol dramatically increased potency on PKMYT1. These dimethylphenol analogs were found to exist as Type III atropisomers that could be separated and profiled as single enantiomers. Structure-based drug design aided by co-crystal structures of several analogs enabled optimization of cell-based potency and kinase selectivity. Parallel optimization of ADME properties led to the identification of potent and selective inhibitors of PKMYT1 with favorable pharmacokinetics. RP-6306 inhibits the phosphorylation of CDK1 Thr14 in vivo in tumor tissue and inhibits CCNE1-amplified tumor cell growth in several preclinical xenograft models. The first-in-class clinical candidate RP-6306 is currently being evaluated in Phase 1 clinical trials (NCT04855656) for treatment of various solid tumors