Biophysical Society Thematic Meeting - November 16-20, 2015

Biophysics in the Understanding, Diagnosis, and Treatment of Infectious Diseases Speaker Abstracts

Post-Translational Modification of a Nucleoid Associated Protein Regulates Cell State in Mycobacteria Alex Sakatos, Michael Chase, Sarah Fortune . Harvard TH Chan School of Public Health, Boston, MA, USA. Although there are many known mechanisms by which eukaryotic cells achieve epigenetic inheritance, the mechanisms by which prokaryotic cells generate heritable, semi-stable differences in state remain poorly understood. Here we demonstrate that with a population of mycobacterial cells, there are privileged subpopulations of cells that are more tolerant of antibiotic killing than the majority of cells. We show that these differences in cell state are heritable and semi-stable. We further demonstrate that there is extensive post-translational modification of the nucleoid associated protein, HupB, at predicted DNA binding residues. Mutation of the sites of post-translational modification disrupts the formation of subpopulations of antibiotic tolerant cells. We propose that metabolically driven modification of HupB acts as an epigenetic regulator, controlling the formation of a specialized subpopulation of antibiotic tolerant cells. XChem: From Crystals to Potent Molecules with X-Rays and Poised Synthesis Frank von Delft 1,2 . 1 University of Oxford, Headington, United Kingdom, 2 Diamond Light Source Research Foundation, Oxfordshire, United Kingdom. Fragment-based lead discovery is now a well-established as a powerful approach to early drug or lead discovery: since small (<250Da) compounds (“fragments”) tend to bind relatively promiscuously, hits can be readily identified by screening against comparatively small compound libraries (100s-1000s). What remains challenging is that hits typically bind weakly: not only must the screening technique be sufficiently sensitive, but potency can only be achieved through considerable synthetic elaboration. Historically, the most sensitive primary screening technique of all, direct observation in crystal structures, has been too challenging to be achievable by but a few labs world-wide. Equally, no consensus has yet emerged on systematic strategies for synthetic follow-up. Now, beamline I04-1 at Diamond Light Source has established X-ray screening as a routine medium-throughput experiment with a capacity of up to 500 crystals/day (from soaking to dataset), a facility being offered to Diamond users since April 2015, with dedicated weekly beamtime. The highly streamlined process includes image recognition for crystal targeting, soaking by acoustic dispensing, robot-assisted harvesting, unattended X-ray data collection, automatic data integration, and pan-dataset electron density analysis for detecting hits. The technology was developed as a joint research project with the Protein Crystallography group of the SGC at Oxford University, and has been validated on a series of diverse targets, all of which have yielded hits. Moreover, a “poised” fragment library has been developed that provides clear and robust routes to first-shell follow-up: combined with new algorithms for prioritizing compounds, the ultimate ambition is to establish how potency can be achieved cheaply from very limited initial experiments. If achievable, this would have a major impact all aspects of biological research.

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