Dimensionally Enhanced Antibacterial Library Screening

Abstract

The emergence and spread of antimicrobial resistance is a major public health threat, and there is an urgent need to develop new strategies to address the issue. In this study, the possibility of enhancing a whole cell based antibacterial library screen by increasing the dimensionality of the screening effort is explored using methicillin-resistant Staphylococcus aureus (MRSA) as the target organism. One dimension involved generating and screening a human liver microsome metabolized FDA approved drug library. Comparative screening of the un-metabolized (UM) and pre-metabolized (PM) libraries allows identification of intrinsically active agents from the UM library screen and of agents with active metabolites from the PM library screen. To further enhance this screening effort, it was combined with a −/+ resistant to antibiotic screen (−/+ cefoxitin; Cef). This allows the identification of agents that can act synergistically with the resistant to antibiotic. This approach revealed five compounds with substantially improved activity after metabolism and four compounds with substantial synergistic activity with cefoxitin. Capecitabine in particular only had significant antibacterial activity after metabolism. Its metabolites were isolated, identified, and characterized for spectrum of activity along with several other anticancer drugs with anti-MRSA activity. Floxuridine, gemcitabine, novobiocin, and rifaximin were identified as having substantial synergy with cefoxitin from the −/+Cef screens. Checkerboard assays verified synergy for these agents. Floxuridine demonstrated a particularly high degree of synergy with cefoxitin (FIC = 0.14). This study demonstrates how a dimensionally enhanced comparative screening effort can identify new antibacterial agents and strategies for countering antibacterial agent resistance.

Library Screening for Synergistic Combinations of FDA-Approved Drugs and Metabolites with Vancomycin against VanA-Type Vancomycin-Resistant Enterococcus faecium

“…In a prior study, we demonstrated in MRSA how a two-dimensional screening strategy comparing an unmetabolized (UM) versus postmetabolized (PM) FDA library screen combined with a −/+ resistant-to antibiotic screen could enhance the ability to identify new agents and new synergistic combinations ( 23 , 32 ). This study identified gemcitabine as having generally good anti-MRSA activity, identified strong synergy between cefoxitin and floxuridine against MRSA, and also identified capecitabine as having several anti-MRSA metabolites.…”

“…Many drugs are also known to have active metabolites ( 21 , 22 ). Comparative screening of the unmetabolized (UM) and postmetabolized (PM) libraries allows agents with increased antibacterial activity to be identified for deconvolution and active metabolite identification, as recently demonstrated using a human liver microsome-metabolized FDA-approved drug library ( 23 ).…”

“…In a prior study we have demonstrated an approach in which FDA-approved drug library screening is performed against MRSA with both FDA-approved drugs and their metabolites and simultaneously in the absence and presence of a resistant-to antibiotic ( 23 ). This allows intrinsically active agents, agents with active metabolites, and agents synergistic with the resistant-to antibiotic to be identified in a single screen.…”

An Oral Fluorouracil Prodrug, Capecitabine, Mitigates a Gram-Positive Systemic Infection in Mice

“…The potential for S. aureus to evolve resistance to capecitabine is not practical to test in broth culture, because S. aureus is not growth inhibited by capecitabine at concentrations up to 200 μ M ( 39 ). However, S. aureus 5-FU-resistant isolates evolved in MHB harbor mutations in genes consistent with the roles of 5-FU in disrupting DNA and RNA metabolism.…”

“…Capecitabine was developed in the late 1990s and is used to treat colon, breast, and gastric cancers ( 21 , 37 , 38 ). It is not by itself considered antibacterial and requires three metabolic steps for conversion to 5-FU ( 29 , 39 ). To validate the identification of capecitabine as an inhibitor of S. Typhimurium growth, we compared growth in the presence of capecitabine in the minimal medium versus a rich medium.…”