Diaminoquinazoline MMV675968 from Pathogen Box inhibits Acinetobacter baumannii growth through targeting of dihydrofolate reductase

Warangkhana Songsungthong,Pimchai Chaiyen,Sunisa Prasopporn,Eric S Nicholson,Ubolsree Leartsakulpanich,Louise E Bohan,Suganya Yongkiettrakul

doi:10.1038/s41598-019-52176-8

Abstract

Antibiotic resistance in Acinetobacter baumannii is a major global health threat. New drugs with novel chemical structures are needed to overcome a myriad of resistance mechanisms in A. baumannii. In this study, we screened an open-source Pathogen Box library for anti-A. baumannii compounds. Compound MMV675968 (a diaminoquinazoline analog) was the only non-reference compound found to inhibit the growth of all four A. baumannii test strains with IC50 of 0.6–2.7 μM, IC90 of 0.7–3.9 μM, and MIC of 1.6–10 μM. We showed that MMV675968 targeted A. baumannii dihydrofolate reductase (AbDHFR) as determined by an E. coli surrogate whose growth was dependent on AbDHFR function and by an in vitro DHFR activity assay. Additionally, chemical scaffolds of DHFR inhibitors that are effective as antibiotics against A. baumannii were identified using an in vitro DHFR activity assay and A. baumannii growth inhibition. MMV675968 was the most potent among DHFR inhibitors tested in inhibiting A. baumannii growth. This study shows for the first time that MMV675968 inhibits A. baumannii growth via selective inhibition of AbDHFR and is therefore a promising scaffold for further antibiotic development against A. baumannii.

Highlights

IC50, IC90, and Minimum inhibitory concentration (MIC) values were 0.6–2.7 μM (0.22–0.97 mg/L), 0.7–3.9 μM (0.25–1.40 mg/L), and 1.6–10 μM (0.58–3.60 mg/L), respectively (Fig. 1), which are comparable to corresponding values of effective antibiotics against A. baumannii[9,10,11]
Using equal amount of total protein, dihydrofolate reductase (DHFR) activity present in the lysate of E. coli BL21(DE3) containing pET15b accounted for only 3% and 6% of that detected in the lysate of E. coli BL21(DE3) overexpressing A. baumannii dihydrofolate reductase (AbDHFR) and hDHFR, respectively (Fig. 3A,B). These results suggest that most of DHFR activity measured in E. coli BL21(DE3) expressing AbDHFR and hDHFR comes from overexpressed enzymes not endogenous EcDHFR
An open-source Pathogen Box was screened for bioactive compounds against A. baumannii

Summary

Introduction

The growth of the E. coli surrogates were tested in the presence of MMV675968, trimethoprim (TMP), a bacterial DHFR inhibitor, or methotrexate (MTX), a known hDHFR inhibitor[16,17]. To determine which chemical scaffolds are the most effective antibiotic candidates against A. baumannii, MMV675968 and seven other antimicrobial DHFR inhibitors (Fig. 4A) were tested for their efficacy in inhibiting AbDHFR in vitro and in vivo and in inhibiting A. baumannii growth. MMV675968 was the only compound that completely inhibited the growth of all four A. baumannii strains (Fig. 4D).

Results

Conclusion