Development of binding and activity inhibition assays for the antibiotic resistance-associated protein PhoQ

Addis, H.; Blankenship, D.; Carlson, E. E.

doi:10.64898/2026.06.15.732377

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bioRxiv · 10.64898/2026.06.15.732377

Development of binding and activity inhibition assays for the antibiotic resistance-associated protein PhoQ

Addis, H.·Blankenship, D.·Carlson, E. E.

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Abstract

Antimicrobial resistant infections present a growing threat to public health and were associated with or directly caused 6 million deaths globally in 2021. This huge death toll highlights the need for novel strategies to address AMR infections. Interfering with the regulation of resistance mechanisms could provide an alternative approach to treat drug-resistant infections. PhoQ, a sensor histidine kinase ubiquitous amongst gram-negative bacteria, regulates several virulence factors, as well as resistance to outer membrane-targeting antibiotics, making it an attractive target for adjuvant therapy development. However, the identification of potent small molecule inhibitors is limited by the assays available for in vitro assessment of binding and activity inhibition in PhoQ. Thus, we sought to investigate the use of a fluorescence-based assay to evaluate enzymatic activity, as well as a thermal shift assay to assess inhibitor-protein binding in PhoQ. Together, these newly implemented protocols are valuable contributors to the toolbox of methods available for the development of PhoQ-targeted inhibitors to block this major contributor to antimicrobial resistance.

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Addis, H., Blankenship, D., Carlson, E. E.. 2026-06-16. Development of binding and activity inhibition assays for the antibiotic resistance-associated protein PhoQ. https://doi.org/10.64898/2026.06.15.732377

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