Bacterial pathogens have conserved complex molecular machines that are required to cause disease. Among these machines are those that make up a type IV pilus (T4P) biogenesis apparatus and the related type II secretion (T2S) machine. T4Ps are surface appendages essential for adherence and host colonization by many important bacterial pathogens; T2S systems export toxins that are critical for many diseases. Acinetobacter baumanii, Pseudomonas aeruginosa, and Neisseria gonorrhoeae all produce T4Ps. Among the components common to both machines is a pre-pilin peptidase enzyme that specifically removes the characteristic N-terminal signal sequence from pre-pilin proteins and N-methylates the nascent amino terminus. These enzymes are highly conserved, absolutely required for T4P and T2S function, and represent a novel class of neutral pH aspartic acid peptidases that do not resemble mammalian enzymes. As such, they are ideal targets for new drug discovery. We have devised an in vivo Fster resonance energy transfer (FRET) system comprised of three recombinant proteins expressed in E. coli: the pre-pilin peptidase, the pre-pilin protein fused to enhanced yellow fluorescent protein (eYFP), and an essential assembly protein to which the pre-pilin binds fused to dsRed. Cleavage of the pre-pilin fusion protein by the pre-pilin peptidase releases eYFP and prevents FRET with dsRed. If the pre-pilin peptidase is inactivated by mutation, then FRET occurs. We will adapt this extremely sensitive system and a related even simpler system that relies on fluorescence anisotropy to develop in vitro high-throughput small molecule screens to identify pre-pilin peptidase inhibitor candidates. Secondary assays will establish specificity and spectrum of activity. The initial effort will provie candidates that will subsequently subjected to further chemical refinement to enhance potency, specificity and activity against whole bacteria. Finally, (a) lead compound(s) will be advanced to preclinical testing using an established murine model of P. aeruginosa pneumonia in which T4Ps are required for disease. Ultimately, the goal is to discover an entirely novel therapeutic class of anti-virulence agents to improve human health.
Bacteria that cause human infections are increasingly resistant to antibiotics. Alarmingly, physicians are now powerless to treat some infections. Therefore, new medicines that attack different bacterial targets are urgently needed. We propose an innovative drug screen for molecules that block a specific protein that is required for many bacteria to cause disease. This drug target does not exist in people. This screen will take advantage of new advances and state-of-the-art equipment that can rapidly test thousands of compounds. Initial compounds will be modified for better properties and one lead compound will be tested in an animal model. These efforts may lead to promising new treatments for infections resistant to currently available antibiotics.
