This project has two main elements. The major effort involves collaboration with scientists at the NIH Chemical Genomics Center (NCGC) to conduct a high-throughput screen (HTS) of a large chemical library to search for compounds that activate ClpP peptidase and protease activity in a manner similar to the ADEP antibiotics. This project is partially funded through an R03 award (1 R03 MH095569) granted to me in 2012. The interactions between ADEP and ClpP, as shown by X-ray crystallography, suggest that there should be a high likelihood of finding organic molecules that display a rigid structure that mimics the aromatic/aliphatic part of ADEP, dock to ClpP, and exert allosteric effects on its activity. The primary contacts between ADEP and ClpP involve hydrophobic interactions between an aromatic ring in ADEP and a deep pocket on the apical surface of ClpP. In addition, there are hydrophobic interactions between an aliphatic chain in ADEP and a hydrophobic groove that extends from the hydrophobic pocket toward the axial channel of ClpP. Other minor interactions include hydrogen binding involving backbone atoms from a short peptide segment of ADEP. The depsipeptide portion of ADEP has very little interaction with ClpP and serves primarily to restrict the conformational flexibility of the aliphatic regions in ADEP, which are fixed in a configuration that locks into the docking site. The solution structure of ADEP alone confirms that there is little induced change in its upon binding to ClpP. Peptidase activity of ClpP will be measured using a FRET peptide that yields an easily quantifiable fluorescence signal when cleaved. The assay requires readily available chemicals, a modified peptide that has been synthesized in our laboratory, and purified ClpP protease, which is prepared in our laboratory. A preliminary screen of a small chemical library has allowed optimization of the assay and has identified a few leads for further study. The large scale screening of over 300,000 compounds is underway. Initial hits in the screen will be validated at NCGC by a second round of screening involving timed assays in order to eliminate false positives resulting from intrinsic fluorescence of the test chemicals. Validated hits will then be assayed further in my laboratory to obtain a more complete profile of binding affinity, activating effect on both peptide and protein substrates, and comparative specificity for human, E. coli, and B. subtilis ClpPs. Compounds will then be tested for antimicrobial activity against laboratory strains of E. coli and B. subtilis. Compounds will also be tested for their growth inhibitory activity against several human cancer cell lines. Once promising lead compounds have been identified and screened by the various secondary assays mentioned, the synthetic chemistry team at NCGC will begin designing synthetic strategies for making the compounds and variations of the compounds to develop new versions that are optimized for binding to ClpP and for effectiveness against cultures of bacteria. Our laboratory has begun to construct strains of E. coli to test candidate compounds identified in the HTS. ADEPs do not penetrate the outer membrane of Gram-negative bacteria. In addition, ADEPs are substrates for the multidrug transporter, AcrAB, which rapidly eliminates many drugs from the cell. We have constructed a strain that has been deleted for acrAB and have introduced a plasmid that expresses an outer membrane protein that alters the permeability of the outer membrane and allows compounds to enter E. coli cells. These strains will be useful for comparing the relative cell permeability and effectiveness as antibiotics of candidate compounds and ADEPs against E. coli. We have also arranged with Dr. Scott Stibitz from Center for Biologics Evaluation, FDA, to test the compounds against several Gram positive and Gram-negative pathogenic bacterial strains and to evaluate the rate at which resistance arises. Resistance could result from mutations in ClpP that cause impaired binding of the active compounds or severely compromised ClpP activity. Both types of mutants should be rare because they would be expected to lack important biological activities of ClpXP and ClpA/C-P and thus compromise the growth of the bacteria in natural environments. Development of resistance by acquisition of mutations in the targets can be expected to be rare, because of the multiple targets of dysregulated ClpP, which must include precursors many vital enzymatic or regulatory proteins. To complement the efforts to identify new compounds that mimic ADEPs in their binding to ClpP and activating its protease activity, we have initiated a genetic screen to obtain mutants of ClpP that have altered binding properties and possibly altered allosteric responses to binding of ligands. ADEPs bind to the docking site on the apical surface of ClpP used by ClpX and ClpA/C in forming the biologically functional ClpXP and ClpAP complexes. We have developed a sensitive selection procedure that will identify mutants of ClpP that bind ADEPs less well but continue to bind ClpX and ClpA/C and thus retain biological function. The selection is based on the ability of ClpXP to degrade proteins that have an 11-amino acid tag (called an SsrA tag) at the C-terminus. By engineering the tag at the C-terminus of a toxic protein, we have created a strain that can only grow when ClpXP is functional within the cell. We have used a similar strategy based on a different toxic protein in the past to successfully isolate mutants of ClpX with altered substrate recognition properties (Erica N. Jones and Michael R. Maurizi, manuscript in preparation). We have modified the selection system in order to first allow screening of a plasmid library expressing mutated ClpP for resistance to ADEP. This initial screen must be done under conditions in which the toxic protein is tightly repressed. Once the library has been enriched for plasmids expressing ClpP that is not activated by ADEP (thus allowing cell survival in the presence of ADEP) we will induce the SsrA-tagged toxic protein and look for survivors that retain ClpP activity as evidenced by their ability to degrade the SsrA-tagged toxic protein. Forms of ClpP that appear to display differential binding to ADEPs and ClpX will be characterized further by standard biochemical assays. The mutated ClpP will also be tested for their sensitivity to candidate compounds identified in the HTS. The goal of this work is to identify the critical residues in ClpP that are involved in both binding of ADEPs and ClpX and in the allosteric response that communicates to the axial channel and causes the channel to be expended and allow indiscriminate protein entry. Mutated forms of ClpP that respond differently to ADEP and ClpX could show different binding affinity or binding rates or could be affected in residues that make new interactions that stabilize the activated structure of ClpP.