Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Monday Speaker Abstracts

MEASUREMENT OF ACCUMULATION OF MOLECULES IN DIDERM BACTERIA, AND IN PHAGOCYTOSED S. AUREUS CELLS IN MACROPHAGES George M Ongwae 1 ; Zichen M Liu 1 ; Joey J Kelly 1 ; Marcos M Pires 1 ; 1 University of Virginia, Chemistry, Charlottesville, VA, USA 4 University of Massachusetts, Molecular and Cellular Biology, Amherst, MA, USA A significant bottleneck to drug discovery and development is that few methods exist for measuring the permeation of molecules across cell membranes. Traditional methods, such as Minimum Inhibitory Concentration (MIC), have limitations in estimating drug accumulation independently from drug potency. Although mass spectrometry methodologies offer certain advantages, they also possess inherent limitations, including restricted throughput capacity and an inability to definitively ascertain cytosolic accumulation. 1,2 The ChloroAlkane Penetration Assay (CAPA) pioneered by the Kritzer Lab has become widely adopted as a method for measuring apparent accumulation; it involves the application of chloroalkane-tagged test molecules (pulse step) to cytosolic HaloTag-expressing mammalian cells. 3 Subsequent detection of chloroalkane-fluorophore signals (chase step) reveals the penetration levels. Despite the wide adoption of CAPA 5, 6 , we recognized the potential confounding influence of the 15-atom long chloroalkane tag on penetration analysis in bacteria. In contrast, azides are known for their minimal size and relatively low disruptive impact as biorthogonal tags. 7 We have, therefore, introduced a robust assay, the CHloroakane Azide Membrane Permeability (CHAMP), for quantitative assessment of small molecule accumulation within Gram-negative bacteria that are engineered to express HaloTag protein. CHAMP employs biorthogonal epitopes anchored within HaloTag-expressing bacteria and measures permeation using azide-bearing test molecules through strain-promoted azide-alkyne cycloaddition(SPAAC). 8 In Mycobacterium tuberculosis (Mtb), the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We developed a novel assay that anchors a strained alkyne on the peptidoglycan, which sits directly beneath the mycomembrane, followed by Click chemistry with test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. 9 We have also developed an assay to measure the arrival of antibiotics within the phagosomes of infected macrophages by metabolically incorporating bioorthogonal reactive handles within the surface of S. aureus and adding Click chemistry complementary tags to antibiotics. 10 References(1) Sci Rep 2015, 5, 17968. (2) ACS Chem Biol 2014, 9 (11), 2535 2544. (3) J Am Chem Soc 2018, 140 (36), 11360-11369. (4) ACS Infect Dis 2023, 9 (1), 97-110. (5) Methods Mol Biol 2007, 356, 195-208. (6) J Am Chem Soc 2022, 144 (32), 14687-14697. (7) J Am Chem Soc 2004, 126 (46), 15046-15047. (8) ACS Chem Biol 2019, 14 (4), 725-734.(9) Angew Chem Int Ed Engl. 2023 62(20):e202217777. (10) Angew Chem Int Ed Engl. 2024 63(3): e202313870. 2 Lehigh University, Biology, Bethlehem, PA, USA 3 Shanghai Jiao Tong, Chemistry, Shanghai, China

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