New Multidrug Efflux Inhibitors for Gram-Negative Bacteria

Abstract

Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors. IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.

Towards the sustainable discovery and development of new antibiotics

“…Examples of non-essential target inhibitors are represented by: Inhibitors of virulence-conferring factors or pathways (also known as anti-virulence compounds or pathoblockers 86 that target, for example, quorum sensing mechanisms 87 , biofilm formation 88 , bacterial secretion systems 89 , 90 , enzymes for tissue penetration 91 or intracellular survival 92 ). Efflux pump inhibitors 93 . Suicide substrates such as β-lactamase inhibitors 94 , 95 .…”

“…These factors can be addressed by suitable compound design, which generally remains rather empirical and challenging 254 – 257 . Other possibilities to address this key area would be to use these compounds in combination with outer membrane permeabilizing agents 258 , 259 or efflux inhibitors 93 , 260 . Alternative approaches targeting extracellular virulence factors, for example, extracellular lectins required for attachment and biofilm formation or secreted proteolytic enzymes, do not suffer from a possible lack of bacterial uptake 261 .…”

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“…Of the EIs identified, auranofin ( 8 , Figure 56 ), an antirheumatic agent, increased ethidium bromide accumulation and potentiated antibiotic activity in checkerboard assays. 898 While most drug screening efforts have focused on looking at inhibitors of RND inner membrane transporters, a recent study identified an inhibitor of the ABC type efflux pump MacAB in S. Typhimurium. Compound 9 ( Figure 56 ) was identified from screening a drug library for compounds that inhibited the growth of a macAB -overexpressing strain in the presence of erythromycin.…”

How researchers are revamping antimicrobial drugs

“…So when a substance activates ramA, the fluorescent protein is produced and the researchers can see the glow using high-throughput screening machinery. The team has now screened nearly 50,000 compounds, identifying 43 efflux-pump inhibitors, 11 of which have been shown to increase the efficacy of antibiotics in lab tests 2 .…”