Molecular Determinants Governing the Antitubercular Activity of Griselimycin

Tuberculosis, often considered the world's deadliest infectious disease, is associated with over one million deaths annually. The emergence of drug-resistant strains of <i>Mycobacterium tuberculosis</i> ( <i>Mtb</i> ) makes anti-tuberculosis drug development a critical priority. Griselimycin (GM) is a cyclic peptide that targets the essential DNA sliding clamp of <i>Mtb</i> . While GM is a promising <i>Mtb</i> antibiotic, its poorly understood structure-activity relationship has stalled derivatization. To investigate the contribution of each amino acid towards its activity, we assessed the antibiotic activity of an alanine scan library in <i>M. tuberculosis</i> and <i>M. smegmatis</i> . Residues essential for activity and tolerable to modification were identified, and the impact of backbone <i>N</i> -methylation at each position was determined. Edits to cyclization chemistry, unnatural amino acid incorporation, and replacing the acetylated <i>N</i> -terminus with a free amine were also investigated. Lastly, incorporation of an <i>N</i> -terminal fluorophore enabled visualization of GM accumulation inside of mycobacteria both in and outside of macrophage cells, where <i>Mtb</i> natively resides. These findings present the first comprehensive structure-activity investigation into GM and can be used to rationally design future analogues.