Host Enzymes as Targets of Novel Host-Directed Antimicrobial Therapies

Carbapenem-resistant Gram-negative Enterobacteriaceae represent an escalating public health threat. According to The Centers for Disease Control and Prevention, resistant intracellular non-typhoidal Salmonella (NTS) is one of the most significant drug-resistant threats. Salmonellae account for the majority of cases of infectious diarrhea worldwide, leading to an estimated two million deaths annually. Multidrug-resistant (MDR) invasive NTS Salmonella strains are a significant cause of concern to military personnel. Warfighters are at high risk of contracting bloodstream infection, especially in Africa, or Southeast Asia. In Thailand, Warfighters are affected by NTS strains, among which 94.6% are resistant to at least 1-2 antimicrobials. New antimicrobials are urgently needed to tackle these MDR Gram-negative bacteria. Since conventional antibiotics fail to treat infections caused by MDR strains, innovative approaches are necessary to address this emerging threat. Drugs directly targeting bacterial metabolic processes have been extensively researched. The innovation of our idea is to use silencing and inhibition of host enzymes to develop entirely new therapies enhancing host-mediated clearance of bacteria. Since pathogens rapidly evolve new mechanisms to counteract antibiotics affecting their physiology, our host-directed therapy is expected to be superior as it does not affect pathogens directly and therefore can escape the microbial resistance mechanisms. We propose to target deubiquitinases, DUBs, which control the ubiquitin (Ub) system, which provides robust control of protein degradation, but it can also "fine-tune" regulation of the protein function. Ubiquitin system involves over 1,000 elements, including ~100 DUBs, which reverse the ubiquitylation. Expression and activities of host DUBs are rapidly altered during infection with pathogenic bacteria. DUBs regulate host inflammation and are some of the essential components of host protective responses against various infections. Being substrate-specific elective enzymes, DUBs represent attractive and druggable targets. Interference with the DUB function enables a highly targeted pharmacological intervention by small molecule agents. We have already identified two inhibitors, which are strong candidates for validation studies. Many DUB inhibitors are already known and evaluated for cancer therapies, and we will repurpose such inhibitors in this proposal to promote bacterial killing by macrophages. The overall objective of this proposal is to identify specific enzyme inhibitors supporting the bacteriocidal activity of macrophages and leading to bacterial clearance. Our application directly addresses novel approaches to antimicrobial drug developments, linking to the Fiscal Year 2019 Peer Reviewed Medical Research Program Topic Area of "Antimicrobial Resistance." We hypothesize that we can identify specific DUB targets and DUB inhibitors, which efficiently promote bacterial killing by macrophages, opening entirely novel targets for the development of host-directed therapeutics against S. typhimurium and other MDR Gram-negative infections. The premise is the burgeoning evidence of common mechanisms centered around Ub signaling, which is utilized by the host to clear bacterial infections. We propose the following specific aims to examine the hypothesis: Aim 1: Identify DUBs supporting bacterial killing by macrophages. We showed that distinct DUBs are regulated in macrophages infected with Gram-negative pathogens, thus controlling bacterial clearance. Using new generation active-site probes combined with chemoproteomics and siRNA screen, we will identify specific DUBs modulated by Salmonella infection, and contributing to bacterial killing by macrophages. Aim 2: Identify novel DUB inhibitors that enhance bacterial killing in macrophages. We observed that by inhibiting the activity of specific DUBs, we will increase bacterial killing. We will examine the cellular and in vivo effects of ~500 DUB inhibitors on induction of pro-inflammatory cytokines, cytotoxicity, and macrophage-mediated killing of S. typhimurium. A combination of methodologies will be used to identify host-directed therapies enhancing the antimicrobial functions of macrophage, including cell-based HTS combining siRNA libraries and molecularly diverse small molecule collections. The expected results are the identification of novel strategies targeting the ubiquitin system to combat bacterial infections, bypassing the innate bacterial MDR mechanisms and offering new druggable targets. Since some of these inhibitors are tested in clinical trials for other diseases, such as cancer, drug repurposing is expected. The expected impact is the identification of specific DUBs as novel drug targets for therapeutic strategies, which avoid bacterial resistance mechanisms, and identification of DUB inhibitors enhancing the bacterial killing by macrophages. Long term, we will develop synergistic antimicrobial therapies for MDR infections to reduce the required dose and increase the safety of each compound. The ubiquitin process sets the stage to an entirely novel and innovative approach in the antimicrobial therapeutic development, offering multiple and highly specific targets. Once specific ligands are successfully identified, it will revolutionize the development of antimicrobial drugs. Less