Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistance. We investigate their inhibition with known and novel bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). Importantly, all are inhibited by the HPPK inhibitors, making these molecules dual-target inhibitors of two folate pathway enzymes that are strictly microbial.

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

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.

Transglutaminase-Catalyzed Bioconjugation Using One-Pot Metal-Free Bioorthogonal Chemistry.

General approaches for controlled protein modification are increasingly sought-after in the arena of chemical biology. Here, using bioorthogonal reactions, we present combinatorial chemoenzymatic strategies to effectuate protein labeling. A total of three metal-free conjugations were simultaneously or sequentially incorporated in a one-pot format with microbial transglutaminase (MTG) to effectuate protein labeling. MTG offers the particularity of conjugating residues within a protein sequence rather than at its extremities, providing a route to labeling the native protein. The reactions are rapid and circumvent the incompatibility posed by metal catalysts. We identify the tetrazine ligation as most-reactive for this purpose, as demonstrated by the fluorescent labeling of two proteins. The Staudinger ligation and strain-promoted azide-alkyne cycloaddition are alternatives. Owing to the breadth of labels that MTG can use as a substrate, our results demonstrate the versatility of this system, with the researcher being able to combine specific protein substrates with a variety of labels.

Investigation of Classical Organic and Ionic Liquid Cosolvents for Early-Stage Screening in Fragment-Based Inhibitor Design with Unrelated Bacterial and Human Dihydrofolate Reductases.

Drug design by methods such as fragment screening requires effective solubilization of millimolar concentrations of small organic compounds while maintaining the properties of the biological target. We investigate four organic solvents and three 1-butyl-3-methylimidazolium (BMIm)-based ionic liquids (ILs) as cosolvents to establish conditions for screening two structurally unrelated dihydrofolate reductases (DHFRs) that are prime drug targets. Moderate concentrations (10%-15%) of cosolvents had little effect on inhibition of the microbial type II R67 DHFR and of human DHFR (hDHFR), while higher concentrations of organic cosolvents generally decreased activity of both DHFRs. In contrast, a specific IL conserved the activity of one DHFR, while severely reducing the activity of the other, and vice versa, illustrating the differing effect of ILs on distinct protein folds. Most of the cosolvents investigated preserved the fold of R67 DHFR and had little effect on binding of the cofactor NADPH, but reduced the productive affinity for its substrate. In contrast, cosolvents resulted in modest structural destabilization of hDHFR with little effect on productive affinity. We conclude that the organic cosolvents, methanol, dimethylformamide, and dimethylsulfoxide, offer the most balanced conditions for early-stage compound screening as they maintain sufficient biological activity of both DHFRs while allowing for compound dissolution in the millimolar range. However, IL cosolvents showed poor capacity to solubilize organic compounds at millimolar concentrations, mitigating their utility in early-stage screening. Nonetheless, ILs could provide an alternative to classical organic cosolvents when low concentrations of inhibitors are used, as when characterizing higher affinity inhibitors.

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

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 >2,000-fold increased trimethoprim resistance on overexpression in E. coli Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.

Miniature multi-channel SPR instrument for methotrexate monitoring in clinical samples.

A multi-channel fully integrated SPR biosensor was applied for the analysis of an anti-cancer drug, methotrexate (MTX) as a potential analytical tool used in clinical chemistry laboratories for therapeutic drug monitoring (TDM). MTX concentrations in a patient's serum undergoing chemotherapy treatments can be determined by surface plasmon resonance (SPR) sensing using folic acid-functionalized gold nanoparticles (FA-AuNP) in competition with MTX for the bioreceptor, human dihydrofolate reductase (hDHFR) immobilized on the SPR sensor chip. To validate this biosensor, 13 nm FA-AuNP were shown to interact with immobilized hDHFR in the absence of MTX and this interaction was inhibited in the presence of MTX. The sensor was calibrated for MTX in phosphate buffer at different dynamic range by varying nanoparticle sizes (5, 13, 23 nm) and by modifying the Kd of the bioreceptor using wild-type and mutant hDHFR. Furthermore, initial binding rate data analyzes demonstrated quantitative and fast sensor response under 60s. This MTX assay was subsequently adapted to a fully integrated multi-channel SPR system built in-house and calibrated in human serum with a dynamic range of 28-500 nM. The SPR system was applied to analyzes of actual clinical samples and the results are in good agreement with fluorescence polarization immunoassay (FPIA) and LC-MS/MS. Finally, the prototype system was tested by potential clinical users in a hospital setting at the biochemistry laboratory of a Montreal hospital (Hôpital Maisonneuve-Rosemont).