Synthesis and Derivatization of Compounds for the Treatment of Salmonella enterica Infections

Salmonella enterica serovars cause an estimated 93 million infections each year that result either in typhoid fever or salmonellosis. Among those subspecies that cause typhoid fever, Salmonella enterica serovar Typhi can form biofilms on gallstones in the gallbladders of acutely infected patients, leading to chronic carriage of the bacterium in 3-5% of patients. These biofilms are recalcitrant to antibiotic-mediated eradication, leading to chronic fecal shedding of the bacteria. As humans are the only known reservoir for this pathogen, this fecal shedding is responsible for the re-infection of poorly treated water supplies, and perpetuates the disease in endemic regions, specifically Sub-Saharan Africa and Southeast Asia. If gallstone-mediated biofilm formation can be prevented or disrupted, treatment and potential eradication of the disease becomes possible. Here, the synthesis and anti-biofilm activity of a library of small molecules is reported based on a previously identified hit that demonstrated the ability to both inhibit and disrupt S. Typhi and S. Typhimurium (a nontyphoidal model serovar for S. Typhi) biofilms. Lead compounds were identified that inhibit S. Typhimurium biofilm formation in vitro at sub-micromolar concentrations and are five-fold more potent at biofilm disruption than the parent compound. Three of the most promising compounds were tested in a murine model of chronic Salmonella carriage, and demonstrated synergy with ciprofloxacin. The most potent biofilm disruptor resulted in a 4.5-5 log-fold reduction in bacteria in the gallbladder when dosed with 1 mg/kg cipirofloxacin. Further in vivo testing with varying doses of ciprofloxacin and this novel anti-biofilm compound resulted in nearly undetectable levels of bacteria in the gallbladder and two distal organs, the liver and spleen, at the highest dose of ciprofloxacin (4 mg/kg). Further compound development focusing on different parts of the parent compound yielded improved activity in both inhibition and dispersion of S. Typhimurium biofilms. Additional work is proposed that continues modifying different areas of the parent scaffold to probe activity. Finally, a derivative of the parent compound was made with a biotin tag for use in pull-down assays to aid in the identification of the cellular target of the compounds and assist in elucidating the mechanism of action. This work furthers the development of effective anti-biofilm agents as a therapeutic against Salmonella chronic carriage.

Targeting a second mode of infection is imperative for treating non-chronic cases of S. enterica infection. S. Typhi and S. Typhimurium spread intracellularly through the infection of macrophages in the host defense system. A series of compounds were found that inhibit the spread of S. Typhimurium in mouse macrophages while reducing the incidence of host cell death and decreasing the bacterial load within the macrophages. This lead compound was found to work effectively both in vitro in human and mouse macrophages and in vivo in mouse models of S. Typhimurium infection. In the murine models, the mice survived lethal injections of the bacteria when treated with a sub-optimal dose of ciprofloxacin and the lead compound. Further exploration of the scaffold was carried out with a second library of compounds planned awaiting the results of the first library of derivatives. Additionally, initial efforts towards biotinylation of the lead compound were undertaken in order to identify the cellular targets of the lead compound. This work seeks to expand the number of treatment options for patients suffering from acute cases of S. Typhi and S. Typhimurium infection.