Developing Dithiocarbamate Derivatives as Copper-Dependent Antimicrobials Against S. pneumoniae and Other Pathogens

Abstract

From the ancient Egyptians in 2500 BC using copper pots to prevent contamination of drinking water to the Greek physician Hippocrates writing about the application of dry copper powder on leg wounds to improve wound healing, humans have long known about the antimicrobial properties of copper. Mirroring the natural use of copper outside the human body, copper plays a role in innate immunity and host defenses. In the context of infection, it has been shown that the concentration of free copper ions within the blood increases 4-fold during infection. As it has been established that copper itself is antimicrobial, there has been a renewed effort to combine the efforts of copper with antimicrobials for synergistic biocidal effect. There is a growing body of evidence to use increased copper concentration to directly intoxicate intracellular pathogens within macrophages and other phagocytes as an antibiotic mechanism. In 2014, Festa et al. developed a copper-dependent antimicrobial against the fungus Cryptococcus neoformans that increases the efficiency of macrophage fungicidal activity. Following this model, we sought to identify small molecules with copper-dependent toxicity (CDT) through a targeted screen of compounds for antibiotic efficacy. From this targeted screen, we identified N,N-dimethyldithiocarbamate (DMDC) as a potent bactericidal copper-dependent antibiotic against Streptococcus pneumoniae in vitro with efficacy in significantly decreasing bacterial burden in vivo. DMDC was also proven to be antifungal against Coccidioides immitis, antiparasitic against Schistosoma mansoni, and antibacterial against Staphylococcus aureus. To provide mechanistic insights at the host-pathogen interface, we investigated DMDC’s interaction with innate immune phagocytes and found robust macrophage clearing of bacteria incubated with a combination of copper and DMDC. Zinc intoxication, hydrogen peroxide, and nitric oxide contribute to this rapid in vitro killing. Extending from the drug screening identifying DMDC, compounds derived from DMDC were screened and two compounds within the dithiocarbamate class were identified as copper-dependent bactericidal antibiotics with efficacy in vitro. One compound from the screen, TLA4, was an effective antibiotic in vivo. These findings identify and investigate copper-dependent antibiotics in the dithiocarbamate class, serving as a model for future development of similar compounds. Despite the successful clinical implementation of pneumococcal vaccines, the rise in disease caused by non-vaccine strains and antimicrobial resistance within S. pneumoniae are growing trends that exacerbate the impetus to develop novel antimicrobial strategies. The work detailed in this dissertation serves to contribute to this need.