Fragment-Based Design of Symmetrical Bis-benzimidazoles as Selective Inhibitors of the Trimethoprim-Resistant, Type II R67 Dihydrofolate Reductase

Bastien, Dominic; Ebert, Maximilian; Forge, Delphine; J, Toulouse; Kadnikova, Natalia; Perron, Florent; Mayence, Annie; Huang, Tien L.; Eynde, Jean Jacques Vanden; Pelletier, Joelle N.

doi:10.1021/jm201645r

Cited by 27 publications

(43 citation statements)

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“…Among these, the DfrB1 protein (also known as R67 DHFR) is the best-studied type II DHFR (13)(14)(15)(16)(17). It is proposed to be recently evolved, and it confers a significant survival advantage under TMP exposure to microbes that harbor it (18).…”

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confidence: 99%

Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance

Edens²,

Alejaldre

et al. 2017

Antimicrob Agents Chemother

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Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a Ͼ2,000-fold increased trimethoprim resistance on overexpression in E. coli. Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.KEYWORDS type II dihydrofolate reductase, trimethoprim resistance, E. coli clinical isolates, dfrB4, antibiotic-resistant genes, class I integron, urinary tract infection P ublic health agencies worldwide rank trimethoprim (TMP) a broad-spectrum antibiotic of importance in human medicine (1). Widely used as a result of its low cost and effectiveness, TMP inhibits the activity of many microbial chromosomal dihydrofolate reductases (DHFRs); thus, DHFRs have long served as prioritized targets of antiproliferative drugs (2). Although the majority of living cells harbor a chromosomal member of the type I DHFR family, encoded by a dfrA homolog, the dfrB genes encode a family of plasmid-borne type II DHFRs that are evolutionarily unrelated to type I DHFRs. The dfrB genes have been found in pathogenic bacteria recovered from many food sources, including fish (3), pigs (4, 5), and cows (6), where they confer TMP resistance. Bacteria carrying dfrB genes have also been identified in wastewater samples (7). Over the past decade, dfrB genes have been tracked indirectly in antibiotic resistance studies through identification of integron-related elements (8-10). Therefore, the importance of dfrB genes in TMP resistance in human pathogens may be underappreciated (11,12).To date, only seven members of the dfrB gene family are known, and they are highly homologous (77% to 94% genetic identity, 77% to 99% amino acid identity) ( Table 1). Among these, the DfrB1 protein (also known as R67 DHFR) is the best-studied type II DHFR (13)(14)(15)(16)(17). It is proposed to be recently evolved, and it confers a significant survival advantage under TMP exposure to microbes that harbor it (18). To date, the family of dfrB genes has consistently been reported to be contained within the following mobile

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Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance

Edens²,

Alejaldre

et al. 2017

Antimicrob Agents Chemother

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“…Our results have direct implications on the threat that R67 DHFR holds with respect to antibiotic resistance. Already natively resistant to trimethoprim as a result of its fold being unrelated to that of DfrA DHFRs, R67 DHFR may continue to evolve even as we direct inhibitors to it, and will need to be closely monitored.…”

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Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active‐site substitutions

et al. 2014

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Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar K M values but k cat values 5-to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (T m~1 2 C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no activesite substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation.

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“…20 Among the weakly inhibiting, low molecular weight fragments that were identified, 1 H -benzimidazole-5-carboxylic acid was selected to build symmetrical compounds that reflect the symmetry of the DfrB1 active site. 20 The resulting symmetrical bisbenzimidazoles offered competitive inhibition that was 100- to 1000-fold more efficient than the initial fragment, with K i in the low micromolar range (Scheme 1). 20 Preliminary crystallographic results and kinetic analyses demonstrated that two molecules of 1 bind simultaneously inside the DfrB1 tunnel.…”

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confidence: 99%

Structure-Based Design of Dimeric Bisbenzimidazole Inhibitors to an Emergent Trimethoprim-Resistant Type II Dihydrofolate Reductase Guides the Design of Monomeric Analogues

Yachnin

Ruediger

et al. 2019

ACS Omega

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The worldwide use of the broad-spectrum antimicrobial trimethoprim (TMP) has induced the rise of TMP-resistant microorganisms. In addition to resistance-causing mutations of the microbial chromosomal dihydrofolate reductase (Dfr), the evolutionarily and structurally unrelated type II Dfrs (DfrBs) have been identified in TMP-resistant microorganisms. DfrBs are intrinsically TMP-resistant and allow bacterial proliferation when the microbial chromosomal Dfr is TMP-inhibited, making these enzymes important targets for inhibitor development. Furthermore, DfrBs occur in multiresistance plasmids, potentially accelerating their dissemination. We previously reported symmetrical bisbenzimidazoles that are the first selective inhibitors of the only well-characterized DfrB, DfrB1. Here, their diversification provides a new series of inhibitors ( K i = 1.7–12.0 μM). Our results reveal two prominent features: terminal carboxylates and inhibitor length allow the establishment of essential interactions with DfrB1. Two crystal structures demonstrate the simultaneous binding of two inhibitor molecules in the symmetrical active site. Observations of those dimeric inhibitors inspired the design of monomeric analogues, binding in a single copy yet offering similar inhibition potency ( K i = 1.1 and 7.4 μM). Inhibition of a second member of the DfrB family, DfrB4, suggests the generality of these inhibitors. These results provide key insights into inhibition of the highly TMP-resistant DfrBs, opening avenues to downstream development of antibiotics for combatting this emergent source of resistance.

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Fragment-Based Design of Symmetrical Bis-benzimidazoles as Selective Inhibitors of the Trimethoprim-Resistant, Type II R67 Dihydrofolate Reductase

Cited by 27 publications

References 46 publications

Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance

Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance

Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active‐site substitutions

Structure-Based Design of Dimeric Bisbenzimidazole Inhibitors to an Emergent Trimethoprim-Resistant Type II Dihydrofolate Reductase Guides the Design of Monomeric Analogues

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