Development of synthetic methodologies for N- to C-terminal cyclization of peptidomimetic small molecules and in silico and in vitro studies of the role of cyclization on antibacterial and antiviral activities

Abstract

Among the different mechanisms of resistance in Gram-negative bacteria, the permeability barrier of the outer membrane and efflux pumps represent a significant challenge to address as they synergize to reduce accumulation of most antibiotics. As no predictive model of the structural and physicochemical properties governing efflux susceptibility and outer membrane permeability has been developed, there is a pressing need for strategies to enhance the accumulation of small molecules in Gram-negative bacteria. The Small-molecule Penetration & Efflux in Antibiotic-Resistant Gram-Negatives (SPEAR-GN) project takes a multidisciplinary approach to develop a class- and activity-independent model for antibiotic permeability by building small-molecule libraries. For my contribution to this project, I conducted the synthesis of a library centered around a piperazinone scaffold which is derived from the natural product acyldepsipeptide (ADEP), a potent activator of bacterial caseinolytic protease P (ClpP). The N- to C-terminal cyclization of the ADEP pharmacophore, N-heptenoyl 3,5-difluorophenylalanine, was hypothesized to constraint the conformation of the scaffold and improve its metabolic stability. From an optimized three-step synthetic route to produce the piperazinone core, I generated a library of 48 chemically diverse piperazinone analogs, for which I investigated and optimized different methodologies such as N-alkylation of secondary amides with inactivated alkyl halides using phase-transfer catalysis, photoinduced copper catalysis and synthetic handles. In parallel to the piperazinone library, Quentin Gibault and Katelyn Stevens generated a set of uncyclized or seco analogs to examine the role of the cyclization on antibacterial activity and accumulation in Gram-negatives. The Zgurskaya lab evaluated the antibacterial activity of the library by the measuring the minimum inhibitory concentration (MIC) in isogenic strain sets of wild-type, hyperporinated, efflux-deficient, and doubly compromised (i.e., hyperporinated and efflux-deficient) E. coli, P. aeruginosa, and A. baumannii. The promising biological results set the stage for future LC/MS accumulation studies and will lead to the identification of physicochemical properties or motifs governing efflux susceptibility and outer membrane permeability in the seco and piperazinone libraries. The persistent emergence of antimicrobial-resistant bacteria, paired with a dwindling pipeline of therapeutic treatments amplifies the urgency for novel antibacterials. However, antibiotics that exploit new mechanisms of action provide modern challenges to bacteria; and thus, require the development of a completely new resistance regime, potentially lengthening the duration of action. One promising target departing from traditional antibacterial discovery paradigm is caseinolytic protease P (ClpP). Essential in bacterial homeostasis and virulence, this protease can be chemo-activated by natural products such as acyldepsipeptide (ADEP), resulting in uncontrolled protein degradation and subsequent bacterial cell death. Although ADEP exhibit impressive potency against Gram-positive pathogens, its overall low stability and the synthetic challenge that represents the peptidolactone prevent further development as an antibacterial. To structurally simplify ADEP and maintain its potency associated with the peptidolactone, I investigated the introduction of structural constraint to the ADEP bioactive fragment, N-heptenoyl-3,5-difluorophenylalanine, via N- to C-terminal cyclization. To understand the conformational behavior of the cyclized ClpP activators ranging from six- to eight-membered ring, I conducted computational studies on the conformational space of each analog. The synthetic work I performed provided methodologies to access the six-membered rings (piperazinones and pyrazinones), the seven-membered ring (1,4-diazepan-2-one), and the eight-membered ring (1,4-diazecan-2-one), and the generation of the corresponding small-molecule ADEP analogs. To evaluate the capacity of this series to activate ClpP, I employed a fluorescence-based peptide degradation assay. Although all the cyclized analogs were inactive against Bacillus subtilis ClpP, the biological results demonstrated that, in conjunction with docking studies, a hydrogen bonding with ADEP and Tyr62 is essential for the chemo-activation of ClpP and conformational alteration of the scaffold cannot overcome the loss of this interaction. The COVID-19 pandemic culminated in more than 470 million cases and six million deaths worldwide since the outbreak of SARS-CoV-2 in December 2020. These numbers along with our individual experience during the last two years make the need for antiviral treatments for coronaviruses indisputable. As the pandemic was taking hold, Jessi Gardner, Katelyn Stevens and I investigated four structurally diverse scaffolds for potential non-covalent SARS-CoV-2 Main protease (Mpro) inhibitors. Among these four scaffolds I designed by leveraging existing literature on Mpro inhibitors and hits from a large-scale crystallographic fragment screen by Diamond Light Source, I conducted docking studies to validate a piperazine scaffold. Although the piperazine scaffold was not pursued because of its poor binding to Mpro, it informed the design of a substituted piperazinone scaffold. Docking studies indicated that this scaffold engaged in multiple protein-ligand interactions with Mpro resulting in good docking scores. Since the piperazinone ring is the result of a N- to C-terminal cyclization of a “peptidic” scaffold, I also conducted docking studies of an “uncyclized” peptidic series of analogs to compare the impact of the cyclization on the binding. Although the docking score of these analogs was weaker, I synthesized a preliminary library of piperazinone and peptidic potential Mpro inhibitors setting the stage for biochemical evaluation against SARS-CoV-2 Mpro.