Structure-Based Drug Design Against a Biosecurity Pathogen

Coxiella burnetii, a gram-negative Gammaproteobacterium, causes the human disease Q-fever. It displays a biphasic lifestyle, with a metabolically constrained small cell variant form enabling survival in the environment. Infection through aerosolization triggers conversion to the metabolically active large cell variant. Disease begins in an acute phase, with flu-like symptoms and some cases develop into a chronic infection, the debilitating and potentially fatal form of Q-fever. C. burnetii is sensitive to the antibiotic doxycycline, which can clear acute disease. However, extended combinatorial treatment with hydroxychloroquine is required to manage chronic Q-fever. As a result, there is interest in developing novel anti-Coxiella inhibitors. Here, we approach this challenge from two directions. Firstly, 2-methylisocitrate lyase is identified as a potential target for a new C. burnetii inhibitor, including the first substrate bound structure. X-ray crystallography-based fragment screening is then applied, identifying a large number of binding fragments, bound not only to the active site but also an allosteric site. We show that binders inhibit enzyme activity, with promise for further development. Lastly, using cryo-EM we determined the structure of the C. burnetii ribosome (the target of doxycycline), revealing CLaSP, a previously unannotated gene encoding a large subunit peptide and a new prokaryotic hibernation factor, HPFcold. We compare doxycycline binding between the C. burnetii and E. coli ribosomes, revealing two new mechanisms of action for tetracyclines against the large subunit. These explain the potency of doxycycline against C. burnetii and, for E. coli, reveal a novel inactive conformation of the ribosome, potentially explaining observed bactericidal tetracycline behaviour. Both avenues of the project present significant advances in the pursuit of new antibiotics against C. burnetii and other pathogens.<p></p>

Author dc:creator

• William Stuart (21050243)

• pathogen
• coxiella burnetii
• structural biology
• drug development
• ribosome
• enzyme mechanism
• methylisocitrate cycle
• fragment based lead discovery
• X-ray crystallography
• cryo-electron microscopy
• doxycycline
• methylcitrate cycle
• bacteria
• escherichia coli

Statement dc:rights

• CC BY-NC-ND
• Open Access after 2028-03-30

Identifier

10779/exe.32050116.v1

OAI identifier oai:identifier

oai:figshare.com:article/32050116