Small molecules with antimicrobial activity against E. coli and P. aeruginosa identified by high-throughput screening

Abstract

Background and purpose: New antimicrobials are needed because of the emergence of organisms that are resistant to available antimicrobials. The purpose of this study was to evaluate a high-throughput screening approach to identify antibacterials against two common disease-causing bacteria, and to determine the frequency, novelty, and potency of compounds with antibacterial activity.

Experimental approach: A high-throughput, turbidometric assay of bacterial growth in a 96-well plate format was used to screen a diverse collection of 150,000 small molecules for antibacterial activity against E. coli and P. aeruginosa. The statistical Z'-factor for the assay was > or = 0.7.

Key results: Screening for inhibition of E. coli growth gave a 'hit' rate (> 60% inhibition at 12.5 microM) of 0.025%, which was more than 5-fold reduced for P. aeruginosa. The most potent antibacterials (EC50 < 0.5 microM) were of the nitrofuran class followed by naphthalimide, salicylanilide, bipyridinium and quinoazolinediamine chemical classes. Screening of > 250 analogs of the most potent antibacterial classes established structure-activity data sets.

Conclusions and implications: Our results validate and demonstrate the utility of a growth-based phenotype screen for rapid identification of small-molecule antibacterials. The favourable efficacy and structure-activity data for several of the antibacterial classes suggests their potential development for clinical use.

Figure 1

Antimicrobial screen against E. coli (top) and P. aeruginosa (bottom). (a) Bacterial growth kinetics. Growth of these bacteria is expressed as optical density at 600 nm (shown as a.u. (600 nm)) for cultures on 96-well plates over 48 h at 37°C without and with antibiotics (kanamycin for E. coli and carbenicillin for P. aeruginosa). Initial OD was 0.005 for E. coli and 0.1 for P. aeruginosa. (b) Histogram of ODs measured at 16 h for E. coli and 7 h for P. aeruginosa without and with antibiotic. (c) Histogram of bacterial growth inhibition from primary screening of compounds.

Figure 2

Concentration–response analysis of bacterial growth inhibition. Data shown for E. coli and P. aeruginosa for the most potent compound of each class: (a) benzoxazine; (b) bipyridinium; (c) nitrofurans; (d) salicylanilide; (e) guanidone; (f) quinazolindiamine; (g) naphthalimide. Compound structures shown with corresponding dose–response curves. (h) carbenicilin and (i) kanamycin are the antibiotic controls for P. aeruginosa and E. coli, respectively, shown for comparison.

Figure 3

SAR analysis of nitrofuranylamides, nitrofuranylethenyls, salicylanilides, N-benzylphenylethylamines, quinazolinediamines and naphthalimides.