A Trioxacarcin Prodrug with Accelerated Reversion and an Enterobacteriaceae-Selective Antibiotic with Low Induction of Resistance

Abstract

The trioxacarcins are highly oxygenated, complex bacterial fermentation products first isolated from Streptomyces bottropensis DO-45 in 1981 with remarkable broad-spectrum antiproliferative activity against both Gram-positive bacteria and eukaryotic cells. This antiproliferative activity motivated a phase I clinical trial for LL-D49194α1, a closely related natural product, which ended with one woman dying from myocarditis and the abandonment of further development. The more recent advent of antibody-drug conjugates (ADCs), a class of targeted chemotherapeutics consisting of a monoclonal antibody conjugated to a toxic payload through a linker system, has promise for attenuating the off-target toxicity of the trioxacarcins. However, to realize a stable trioxacarcin ADC, it has been found that a prodrug of the electrophilic spiro-epoxide functionality responsible for the activity of the class is necessary. In chapters one and two of this dissertation, I detail efforts to optimize a trioxacarcin prodrug for application in an ADC by developing a simplified diversifiable pharmacophore and probing structure-activity relationships (SAR) for both antiproliferative activity and reversion kinetics. In doing so, I identify an optimized pharmacophore possessing identical potency, a three-fold increase in prodrug reversion rate, and increased hydrophilicity, as compared to previous trioxacarcin prodrug ADC warheads. Antibiotic-resistant pathogens of the Gram-negative family Enterobacteriaceae are among the deadliest of the multidrug-resistant (MDR) bacteria emerging today, taking the top two spots in the 2024 edition of the Bacterial Priority Pathogens List published by the World Health Organization. In chapters three and four, I detail the discovery and gram-scale synthesis of ciprimelpin, a potent Enterobacteriaceae-selective antibiotic that acts by inhibiting the LolCDE component of the localization of lipoproteins pathway. Unlike previously reported LolCDE inhibitors, ciprimelpin has a low propensity for resistance development: it induces a very low frequency of spontaneous resistance (FoR), and isolated mutant strains contain at least two and as many as five mutations in the target protein complex yet remain highly susceptible. Ciprimelpin is well tolerated, orally bioavailable in mice, and highly efficacious in murine infection models of MDR Enterobacteriaceae infection, either by intravenous or oral administration. Ciprimelpin’s in vivo efficacy and tolerability, low propensity for resistance development, and novel mechanism of action make it a promising preclinical candidate for the treatment of infections caused by MDR-Enterobacteriaceae, including those for which there are currently no treatment options.