High-throughput identification and rational design of synergistic small-molecule pairs for combating and bypassing antibiotic resistance

Abstract

Antibiotic-resistant infections kill approximately 23,000 people and cost $20,000,000,000 each year in the United States alone despite the widespread use of small-molecule antimicrobial combination therapy. Antibiotic combinations typically have an additive effect: the efficacy of the combination matches the sum of the efficacies of each antibiotic when used alone. Small molecules can also act synergistically when the efficacy of the combination is greater than the additive efficacy. However, synergistic combinations are rare and have been historically difficult to identify. High-throughput identification of synergistic pairs is limited by the scale of potential combinations: a modest collection of 1,000 small molecules involves 1 million pairwise combinations. Here, we describe a high-throughput method for rapid identification of synergistic small-molecule pairs, the overlap2 method (O2M). O2M extracts patterns from chemical-genetic datasets, which are created when a collection of mutants is grown in the presence of hundreds of different small molecules, producing a precise set of phenotypes induced by each small molecule across the mutant set. The identification of mutants that show the same phenotype when treated with known synergistic molecules allows us to pinpoint additional molecule combinations that also act synergistically. As a proof of concept, we focus on combinations with the antibiotics trimethoprim and sulfamethizole, which had been standard treatment against urinary tract infections until widespread resistance decreased efficacy. Using O2M, we screened a library of 2,000 small molecules and identified several that synergize with the antibiotic trimethoprim and/or sulfamethizole. The most potent of these synergistic interactions is with the antiviral drug azidothymidine (AZT). We then demonstrate that understanding the molecular mechanism underlying small-molecule synergistic interactions allows the rational design of additional combinations that bypass drug resistance. Trimethoprim and sulfamethizole are both folate biosynthesis inhibitors. We find that this activity disrupts nucleotide homeostasis, which blocks DNA replication in the presence of AZT. Building on these data, we show that other small molecules that disrupt nucleotide homeostasis through other mechanisms (hydroxyurea and floxuridine) also act synergistically with AZT. These novel combinations inhibit the growth and virulence of trimethoprim-resistant clinical Escherichia coli and Klebsiella pneumoniae isolates, suggesting that they may be able to be rapidly advanced into clinical use. In sum, we present a generalizable method to screen for novel synergistic combinations, to identify particular mechanisms resulting in synergy, and to use the mechanistic knowledge to rationally design new combinations that bypass drug resistance.

Thinking Outside the Box—Novel Antibacterials To Tackle the Resistance Crisis

“…[17,18] Target-based whole-cell screenings include comparative profiling of,f or example,g enetically modified versus wildtype bacterial strains or the rescue of an antibiotic effect upon external supplementation, for example,o fametabolite. [105][106][107][108][109] Numerous HTS campaigns have been performed in various genetically modified E. coli reporter strains.…”

“…Bioinformatic approaches aim to take advantage of these huge data sets to identify new antibacterial compounds and potential new targets or to help clarify the mode of action of new compounds. [105][106][107][108][109]…”

Synergistic and Antagonistic Drug Interactions in the Treatment of Systemic Fungal Infections

“…We showed that identifying these drug interactions can quickly lead to additional interacting pairs by examining structure (Fig. S3) or by investigating underlying mechanism (Wambaugh et al, 2017). Finally, our newly discovered interaction of dicyclomine and FLZ exhibited therapeutic potential in vivo, demonstrating the clinical potential of fluconazole-containing synergistic pairs in the clinic.…”

“…Synergistic combination therapies are increasingly important clinical options, especially for drug resistant microbes (Cowen and Lindquist, 2005;Kalan and Wright, 2011;Uppuluri et al, 2008;Zheng et al, 2018). Traditionally, synergistic drug pairs were discovered serendipitously, but new methods are improving our ability to uncover important interactions (Brown et al, 2014;Cokol et al, 2011;Cokol et al, 2018;Jansen et al, 2009;Robbins et al, 2015;Spitzer et al, 2011;Wambaugh et al, 2017;Wildenhain et al, 2015). In this study, we identified a wide variety of molecules that interact synergistically with the antifungal FLZ to inhibit fungal growth.…”

Über bisherige Denkweisen hinaus – neue Wirkstoffe zur Überwindung der Antibiotika‐Krise

“…Bioinformatische Methoden haben das Ziel, diese großen Datensätze zu nutzen, um neue antibakterielle Verbindungen und potentielle neue zelluläre Targets zu identifizieren oder den Wirkmechanismus von neuen Verbindungen aufzuklären. [105][106][107][108][109]…”