The research and development of a new antimicrobial drug using a target‐based approach raises the question of whether any resulting hits will also show activity against the homologous target in other closely related organisms. While an assessment of the similarities of the predicted interactions between the identified inhibitor and the various targets is an obvious first step in answering this question, no clear and consistent framework has been proposed for how this should be done. Here we developed Multifaceted Target Specificity Analysis (MTSA) and applied it to type III pantothenate kinase (PanKIII) – an essential enzyme required for coenzyme A biosynthesis in a wide range of pathogenic bacteria – as a case study to establish if targeting a specific organism's PanKIII would lead to a narrow‐ or broad‐spectrum agent. We propose that MTSA is a useful tool and aid for directing new target‐based antimicrobial drug development initiatives.
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