The increasing prevalence of drug-resistant bacterial infections is widely recognized as an impending public health crisis. As a consequence, there is an urgent clinical need for the development of new antibacterial therapeutics, and a related need to characterize novel drug targets. The Clp proteases, a widely distributed class of ATP-fueled proteolytic machines, have emerged as promising targets in a cross section of important bacterial pathogens. These enzymes degrade cytosolic proteins through the cooperation of an ATP-dependent unfoldase (e.g., ClpX) and the barrel-shaped ClpP peptidase. Multiple classes of compounds that kill bacteria by specifically inhibiting or dysregulating ClpP have been identified. The most widely studied class of these are the acyldepsipeptides (ADEPs), which are derivatives of naturally occurring non-ribosomally encoded peptides produced by some <i>Streptomyces. </i>Structural and biochemical studies have shown that ADEPs bind to the surface of ClpP and dysregulate Clp protease assembly and function. ADEPs typically consist of a complex peptide macrocycle linked to an N-acylphenylalanine substituent. Here, we examine the activity of ADEP fragments lacking the full macrocycle. We find that fragments with partial macrocycles retain considerable ability to bind and dysregulate ClpP. Curiously, some fragments appear to inhibit proteolysis at high concentrations, suggesting an alternate binding site at the peptidase active sites. Further, we report crystal structures of ADEP fragments bound to ClpP, which provide additional evidence for secondary inhibitory effects. Our findings open new possibilities for future ADEP development and provide a new area for ADEP structure optimization targeting an alternate location within the Clp protease.

Identification of a secondary binding site for acyldepsipeptide fragments within the bacterial Clp protease

ACS Spring Meeting 2021

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