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

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.

A novel bicyclic 2,4-diaminopyrimidine inhibitor of Streptococcus suis dihydrofolate reductase

“…DHFRs from various bacteria species do not necessarily cluster to the same clade. Specifically, Ss DHFR does not belong in the same clade as DHFRs of Gram-negative bacteria previously shown to be inhibited by MMV675968 ( Nelson & Rosowsky, 2001 ; Songsungthong et al, 2019 ). Consequently, it cannot be assumed a priori that MMV675968 will effectively inhibit DHFR from S. suis .…”

“…The mechanisms of action of most non-reference hits are unknown. MMV675968, one of the hits, has been shown to inhibit the growth of protozoan parasites and Gram-negative bacteria ( Lau et al, 2001 ; Nelson & Rosowsky, 2001 ; Popov et al, 2006 ; Songsungthong et al, 2019 ) via inhibition of dihydrofolate reductase (DHFR), an enzyme in the thymidylate cycle.…”

“…MMV675968 inhibits DHFR of protozoan parasites and of Gram-negative bacteria ( Lau et al, 2001 ; Nelson & Rosowsky, 2001 ; Popov et al, 2006 ; Songsungthong et al, 2019 ). We therefore hypothesized that MMV675968 inhibited S. suis growth through inhibition of S. suis DHFR ( Ss DHFR).…”

“…Other bicyclic 2,4-diaminopyrimidines were able to inhibit Streptococcus mutans growth ( Zhang et al, 2015 ), confirming this hypothesis. Since methotrexate is a known inhibitor of human DHFR, whereas MMV675968 shows 59-83 fold selectivity for bacterial DHFRs over human DHFR ( Nelson & Rosowsky, 2001 ; Songsungthong et al, 2019 ), and is not cytotoxic against human HL-60 and HepG2 cell lines ( https://www.mmv.org/mmv-open/pathogen-box/about-pathogen-box ), MMV675968 might be a better starting compound for antibiotic discovery.…”

“…Since the amount of NADPH consumed is directly proportional to the amount of THF product, monitoring NADPH consumed using absorbance at 340 nm (A 340 ; ε 340 of 12,300 M −1 cm −1 ) is indicative of DHFR enzymatic activity. The DHFR activity assay was performed as previously described ( Tirakarn et al, 2012 ; Songsungthong et al, 2019 ). Briefly, using kinetics mode of a UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA), A 340 value of the reaction mixture (50 mM TES, pH 7.0, 75 mM β -mercaptoethanol, 1 mM EDTA, 1 mg/mL BSA, 0.1 mM NADPH, 0.1 mM dihydrofolate) was set to blank (A 340 = 0).…”