Antibiotic resistance is one the largest health concerns of the modern era, threatening decades of progress in antibacterial research and development. β-lactams, the first line of defense against most Gram positive and negative pathogens, are increasingly ineffective in clinical settings, driven in large part by the proliferation of β-lactamases, enzymes which degrade β-lactams. While these proteins have evolved over millions of years with hundreds of known variants, clinical selection has led to the proliferation of extended spectrum β-lactamases and carbapenems like CTX-M and KPC-2, respectively. Previous generation β-lactamase inhibitors such as clavulanic acid and sulbactam are ineffective against these enzymes, creating a pressing need for new inhibitor development.

While the recent discovery and approval of covalent avibactam and vaborbactam have shown great promise in bridging this gap, further clinical selection will likely drive new enzymes and mutations resistant to these agents, such as the spread of metallo-lactamases, or mutations within the KPC-2 family. Given the paucity of information around non-covalent β-lactamase inhibition, we sought to further characterize the specific interactions of a potent, non-covalent CTX-M inhibitor, revealing the importance of amide-π stacking against the β3 backbone (Chapter 2). Building off of this work, we also discovered new non-covalent scaffolds for the inhibition of KPC-2 (Chapters 3 and 4). Our work revealed how the improved hydrophobicity and conformational flexibility of carbapenemases such as KPC-2 can be exploited for building non-covalent affinity, supporting future efforts towards combating emerging resistance.

Most cancer chemotherapeutics are administered at or near their maximum tolerated dose, yet poor efficacy and insufficient therapeutic index remain major causes of attrition in oncology drug development. New pharmacological approaches that selectively target tumor cells are therefore of significant interest. Emerging evidence suggests that an augmented pool of intracellular labile Fe(II) is a metabolic signature of many cancers and thus may represent a targetable chemical environment. We have leveraged Fe(II)-dependent, Fenton-type reactivity of the 1,2,4-trioxolane ring to develop highly selective, reactivity-based probes of the ferrous iron pool and a prototypical scaffold for Fe(II)-dependent drug delivery of chemotherapeutics. These probes reveal higher reactive Fe(II)-pools in cancer cells as compared to non-tumorigenic cells, changes that can be linked to alteration of iron metabolism. Our findings suggest that many cancers alter iron metabolism to increase intracellular Fe(II)-pools and these changes can be exploited for cell-selective drug delivery. By using this scaffold to mask a highly potent DNA-alkylator we illustrate that this prodrug system is capable of stabilizing and selectively delivering such potent cytotoxins in vivo. Overall our results establish that Fe(II)-dependent prodrug/delivery strategies can be used to enhance the therapeutic index of cancer chemotherapeutics. Encouraged by these results further efforts were made to apply trioxolane-based systems as novel, tumor-selective cleavable linkers for antibody-drug conjugate strategies. Preliminary results indicate trioxolane-based linkers are capable of efficiently releasing drugs from ADC’s in cancer cells but optimization will be required before further development.

Malaria, a disease caused by infection of protozoan parasites from genus Plasmodium, claims an estimated 600,000 lives annually, with the majority of these deaths in children. The utility of artemisinin and its derivates, which constitute the foundation of modern antimalarial care, is complicated by its suboptimal pharmacological parameters and by a concerning rise in genetically driven artemisinin partial resistance phenotypes in areas of high disease burden. Following limited clinical adoption of second generation synthetic trioxolane leads arterolane and artefenomel, small molecules which retain the endoperoxide-driven antiparasitic effect of artemisinin with improved properties and streamlined synthesis, there exists an urgent and undertargeted need for improved antimalarials in this class.Herein we report the design, synthesis, and in vitro and in vivo evaluation of a library of novel trans-3” substituted 1,2,4-trioxolanes. For carbamate substituted compounds in this class, we find promising antiparasitic efficacy, excellent metabolic stability, and a superior cure rate in a 2-day dosing regimen for lead compound 9d against the rodent P. berghei model of malaria, motivating future advancement of 9d into a humanized rodent malaria model (Chapter 2). We additionally report the synthesis and evaluation of trans-3”-aryl substituted congeners of artefenomel with a focus on carbamate-linked phenols bearing basic amine or other solubilizing functionality (Chapter 3). We find this class to broadly exhibit excellent antiparasitic efficacy and improved solubility and metabolic stability relevant to the 2nd generation trioxolanes. We advance the lead analog 17, which completely eliminates K13 C580Y parasites in vitro in the ring stage survival assay (RSA) and retains potent efficacy against high-RSA Ugandan clinical isolates. We furthermore report a stereoselective route to the (R,R)- and (S,S)- forms of 17, observing increased solubility of the separated enantiomeric forms versus the racemate. We extensively profile 17S as a clinical development candidate and found the compound to exhibit a protein binding and metabolic stability profile we predict should translate to extended drug exposures in human malaria patients. We additionally explore the P. falciparum stress response as driven by PK4-mediated eIF2α phosphorylation (Chapter 4). We identify dozens of drug-like kinase inhibitors from the GSK PERK kinase set with potent antiparasitic effect. We find these inhibitors phenocopy the antiparasitic effect of bona fide PfPK4 inhibitor GSK-2606414 and plausibly retain the target profile of this comparator. We leverage our medium-throughput in vitro P. falciparum library screening pipeline to screen the SelleckChem antikinase library, reporting dozens of potent hits against the parasite and suggesting additional points of entry for targeting the P. falciparum kinome.