Mapping Griselimycin Functional Group Roles in Sliding Clamp Binding and Antimycobacterial Activity

Mycobacterial infections constitute a major public health burden, with antibiotic resistance contributing significantly to increasing mortality rates. "Anti-evolution" drugs that treat infections and limit new resistance represent promising next-generation antibiotics for these infections. The DNA replication machinery, or replisome, represents a prime anti-evolution drug target. Within the replisome, targeting the bacterial sliding clamp protein DnaN is particularly advantageous: it is essential for both normal and error-prone DNA replication and interacts with replication proteins through a short, conserved clamp binding motif (CBM) that is distinct from eukaryotic homologs. A promising starting point to target mycobacterial sliding clamp proteins is the cyclic depsipeptide natural product griselimycin (GM), which inhibits mycobacterial growth by interacting with the sliding clamp protein as a CBM mimic to inhibit DNA replication. Nonetheless, a detailed map of how GM's chemical structure influences its function is lacking. Here, we prepared a systematic series of GM variants to probe functional group roles in GM's interaction with bacterial sliding clamp proteins and in antibiotic activity against the model mycobacterial species Mycobacterium smegmatis. Overall, we determined that the N-terminal exocyclic region makes a limited contribution to both sliding clamp protein interaction and antibiotic activity. Modifications to the three native proline residues also contribute minimally to sliding clamp protein interaction but do improve antibiotic activity. Taken together, these studies reveal previously unknown functional group roles in GM and highlight the N-terminus and/or proline residues as favorable sites for GM optimization to improve antimycobacterial activity and/or reduce synthetic complexity.