Kinetic Barrier to Enzyme Inhibition Is Manipulated by Dynamical Local Interactions in E. coli DHFR.

Dihydrofolate reductase (DHFR) is an important drug target and a highly studied model protein for understanding enzyme dynamics. DHFR's crucial role in folate synthesis renders it an ideal candidate to understand protein function and protein evolution mechanisms. In this study, to understand how a newly proposed DHFR inhibitor, 4'-deoxy methyl trimethoprim (4'-DTMP), alters evolutionary trajectories, we studied interactions that lead to its superior performance over that of trimethoprim (TMP). To elucidate the inhibition mechanism of 4'-DTMP, we first confirmed, both computationally and experimentally, that the relative binding free energy cost for the mutation of TMP and 4'-DTMP is the same, pointing the origin of the characteristic differences to be kinetic rather than thermodynamic. We then employed an interaction-based analysis by focusing first on the active site and then on the whole enzyme. We confirmed that the polar modification in 4'-DTMP induces additional local interactions with the enzyme, particularly, the M20 loop. These changes are propagated to the whole enzyme as shifts in the hydrogen bond networks. To shed light on the allosteric interactions, we support our analysis with network-based community analysis and show that segmentation of the loop domain of inhibitor-bound DHFR must be avoided by a successful inhibitor.

Discovery of Homogentisic Acid as a Precursor in Trimethoprim Metabolism and Natural Product Biosynthesis.

Opportunistic infections by Burkholderia cenocepacia are life threatening for patients suffering from cystic fibrosis and chronic granulomatous disease. These infections are often associated with variable clinical outcomes, prompting an interest in molecular investigations of phenotypes associated with disease severity. The production of the pyomelanin pigment is one such phenotype, which was recently linked to the ability of clinical strains to carry out biotransformation of the antibiotic trimethoprim. However, this biotransformation product was not identified, and differences in metabolite production associated with pyomelanin pigmentation are poorly understood. Here, we identify several key metabolites produced exclusively by the pyomelanin-producing strains. To provide insight into the structures and biosynthetic origin of these metabolites, we developed a mass spectrometry-based strategy coupling unsupervised in silico substructure prediction with stable isotope labeling referred to as MAS-SILAC (Metabolite Annotation assisted by Substructure discovery and Stable Isotope Labeling by Amino acids in Cell culture). This approach led to discovery of homogentisic acid as a precursor for biosynthesis of several natural products and for biotransformation of trimethoprim, representing a previously unknown mechanism of antibiotic tolerance. This work presents application of computational methods for analysis of untargeted metabolomic data to link the chemotype of pathogenic microorganisms with a specific phenotype. The observations made in this study provide insights into the clinical significance of the melanated phenotype.

Chiral evasion and stereospecific antifolate resistance in Staphylococcus aureus.

Antimicrobial resistance presents a significant health care crisis. The mutation F98Y in Staphylococcus aureus dihydrofolate reductase (SaDHFR) confers resistance to the clinically important antifolate trimethoprim (TMP). Propargyl-linked antifolates (PLAs), next generation DHFR inhibitors, are much more resilient than TMP against this F98Y variant, yet this F98Y substitution still reduces efficacy of these agents. Surprisingly, differences in the enantiomeric configuration at the stereogenic center of PLAs influence the isomeric state of the NADPH cofactor. To understand the molecular basis of F98Y-mediated resistance and how PLAs' inhibition drives NADPH isomeric states, we used protein design algorithms in the osprey protein design software suite to analyze a comprehensive suite of structural, biophysical, biochemical, and computational data. Here, we present a model showing how F98Y SaDHFR exploits a different anomeric configuration of NADPH to evade certain PLAs' inhibition, while other PLAs remain unaffected by this resistance mechanism.

MANORAA: A machine learning platform to guide protein-ligand design by anchors and influential distances.

The MANORAA platform uses structure-based approaches to provide information on drug design originally derived from mapping tens of thousands of amino acids on a grid. In-depth analyses of the pockets, frequently occurring atoms, influential distances, and active-site boundaries are used for the analysis of active sites. The algorithms derived provide model equations that can predict whether changes in distances, such as contraction or expansion, will result in improved binding affinity. The algorithm is confirmed using kinetic studies of dihydrofolate reductase (DHFR), together with two DHFR-TS crystal structures. Empirical analyses of 881 crystal structures involving 180 ligands are used to interpret protein-ligand binding affinities. MANORAA links to major biological databases for web-based analysis of drug design. The frequency of atoms inside the main protease structures, including those from SARS-CoV-2, shows how the rigid part of the ligand can be used as a probe for molecular design (http://manoraa.org).

Dihydrofolate Reductase Inhibitors: The Pharmacophore as a Guide for Co-Crystal Screening.

In this work, co-crystal screening was carried out for two important dihydrofolate reductase (DHFR) inhibitors, trimethoprim (TMP) and pyrimethamine (PMA), and for 2,4-diaminopyrimidine (DAP), which is the pharmacophore of these active pharmaceutical ingredients (API). The isomeric pyridinecarboxamides and two xanthines, theophylline (THEO) and caffeine (CAF), were used as co-formers in the same experimental conditions, in order to evaluate the potential for the pharmacophore to be used as a guide in the screening process. In silico co-crystal screening was carried out using BIOVIA COSMOquick and experimental screening was performed by mechanochemistry and supported by (solid + liquid) binary phase diagrams, infrared spectroscopy (FTIR) and X-ray powder diffraction (XRPD). The in silico prediction of low propensities for DAP, TMP and PMA to co-crystallize with pyridinecarboxamides was confirmed: a successful outcome was only observed for DAP + nicotinamide. Successful synthesis of multicomponent solid forms was achieved for all three target molecules with theophylline, with DAP co-crystals revealing a greater variety of stoichiometries. The crystalline structures of a (1:2) TMP:THEO co-crystal and of a (1:2:1) DAP:THEO:ethyl acetate solvate were solved. This work demonstrated the possible use of the pharmacophore of DHFR inhibitors as a guide for co-crystal screening, recognizing some similar trends in the outcome of association in the solid state and in the molecular aggregation in the co-crystals, characterized by the same supramolecular synthons.

A genetically targeted reporter for PET imaging of deep neuronal circuits in mammalian brains.

Positron emission tomography (PET) allows biomolecular tracking but PET monitoring of brain networks has been hampered by a lack of suitable reporters. Here, we take advantage of bacterial dihydrofolate reductase, ecDHFR, and its unique antagonist, TMP, to facilitate in vivo imaging in the brain. Peripheral administration of radiofluorinated and fluorescent TMP analogs enabled PET and intravital microscopy, respectively, of neuronal ecDHFR expression in mice. This technique can be used to the visualize neuronal circuit activity elicited by chemogenetic manipulation in the mouse hippocampus. Notably, ecDHFR-PET allows mapping of neuronal projections in non-human primate brains, demonstrating the applicability of ecDHFR-based tracking technologies for network monitoring. Finally, we demonstrate the utility of TMP analogs for PET studies of turnover and self-assembly of proteins tagged with ecDHFR mutants. These results establish opportunities for a broad spectrum of previously unattainable PET analyses of mammalian brain circuits at the molecular level.

Rapid determination of sulfamethoxazole and trimethoprim illegally added to health products using excitation-emission matrix fluorescence coupled with the second-order calibration method.

In this work, a simple and fast analytical method based on a self-weighted alternating trilinear decomposition (SWATLD) algorithm coupled with excitation-emission matrix (EEM) fluorescence was developed for the simultaneous determination of sulfamethoxazole (SMZ) and trimethoprim (TMP) illegally added to health products. With the second-order advantage, the proposed method obtained satisfactory results in the presence of peak overlap and unknown interferences. The analysis time for a single sample is only 0.8 minutes. The average spiked recoveries of SMZ and TMP in three health product spiked samples were in the range of 91.0-106.2% and 86.8-107.8%, respectively. The relative standard deviations (RSDs) were lower than 8.6%. In addition, verification parameters including sensitivity (SEN), selectivity (SEL), the limit of detection (LOD), the limit of quantification (LOQ), intra-day precision, and inter-day precision were calculated, and the results show that the proposed method is feasible. The quantitative results of the proposed method were further confirmed by the LC-MS/MS method, which proved that the proposed method was efficient and green for drug-abuse monitoring of SMZ and TMP in health products.

Improving the accuracy and convergence of drug permeation simulations via machine-learned collective variables.

Understanding the permeation of biomolecules through cellular membranes is critical for many biotechnological applications, including targeted drug delivery, pathogen detection, and the development of new antibiotics. To this end, computer simulations are routinely used to probe the underlying mechanisms of membrane permeation. Despite great progress and continued development, permeation simulations of realistic systems (e.g., more complex drug molecules or biologics through heterogeneous membranes) remain extremely challenging if not intractable. In this work, we combine molecular dynamics simulations with transition-tempered metadynamics and techniques from the variational approach to conformational dynamics to study the permeation mechanism of a drug molecule, trimethoprim, through a multicomponent membrane. We show that collective variables (CVs) obtained from an unsupervised machine learning algorithm called time-structure based Independent Component Analysis (tICA) improve performance and substantially accelerate convergence of permeation potential of mean force (PMF) calculations. The addition of cholesterol to the lipid bilayer is shown to increase both the width and height of the free energy barrier due to a condensing effect (lower area per lipid) and increase bilayer thickness. Additionally, the tICA CVs reveal a subtle effect of cholesterol increasing the resistance to permeation in the lipid head group region, which is not observed when canonical CVs are used. We conclude that the use of tICA CVs can enable more efficient PMF calculations with additional insight into the permeation mechanism.

Photodegradation of Ciprofloxacin, Clarithromycin and Trimethoprim: Influence of pH and Humic Acids.

In view of the rising relevance of emerging pollutants in the environment, this work studies the photodegradation of three antibiotics, evaluating the effects of the pH of the medium and the concentration of dissolved organic matter. Simulated light (with a spectrum similar to that of natural sunlight) was applied to the antibiotics Ciprofloxacin (Cip), Clarithromycin (Cla) and Trimethoprim (Tri), at three different pH, and in the presence of different concentrations of humic acids. The sensitivity to light followed the sequence: Cip > Cla > Tri, which was inverse for the half-life (Tri > Cla > Cip). As the pH increased, the half-life generally decreased, except for Cla. Regarding the kinetic constant k, in the case of Cip and Tri it increased with the rise of pH, while decreased for Cla. The results corresponding to total organic carbon (TOC) indicate that the complete mineralization of the antibiotics was not achieved. The effect of humic acids was not marked, slightly increasing the degradation of Cip, and slightly decreasing it for Tri, while no effect was detected for Cla. These results may be relevant in terms of understanding the evolution of these antibiotics, especially when they reach different environmental compartments and receive sunlight radiation.

Mussel-Inspired Immobilization of Photocatalysts with Synergistic Photocatalytic-Photothermal Performance for Water Remediation.

The serious problem of pharmaceutical and personal care product pollution places great pressure on aquatic environments and human health. Herein, a novel coating photocatalyst was synthesized by adhering Ag-AgCl/WO3/g-C3N4 (AWC) nanoparticles on a polydopamine (PDA)-modified melamine sponge (MS) through a facile layer-by-layer assembly method to degrade trimethoprim (TMP). The formed PDA coating was used for the anchoring of nanoparticles, photothermal conversion, and hydrophilic modification. TMP (99.9%; 4 mg/L) was removed in 90 min by the photocatalyst coating (AWC/PDA/MS) under visible light via a synergistic photocatalytic-photothermal performance route. The stability and reusability of the AWC/PDA/MS have been proved by cyclic experiments, in which the removal efficiency of TMP was still more than 90% after five consecutive cycles with a very little mass loss. Quantitative structure-activity relationship analysis revealed that the ecotoxicities of the generated intermediates were lower than those of TMP. Furthermore, the solution matrix effects on the photocatalytic removal efficiency were investigated, and the results revealed that the AWC/PDA/MS still maintained excellent photocatalytic degradation efficiency in several actual water and simulated water matrices. This work develops recyclable photocatalysts for the potential application in the field of water remediation.

A trimethoprim derivative impedes antibiotic resistance evolution.

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.

Chemical Dimerization-Induced Protein Condensates on Telomeres.

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.

A Chemical Approach for Programmable Protein Outputs Based on Engineered Cell Interactions.

Cell-cell interactions and communication are crucial to the proper function of complex mammalian physiology including neurocognitive and immune system functions. While many tools are available for observing and perturbing intracellular processes, relatively few exist to probe intercellular processes. Current techniques for studying interactions often rely on direct protein contact, and few can manipulate diverse, functional outputs with tunable protein expression. To address these limitations, we have developed a small-molecule approach based on a trimethoprim prodrug-enzyme pair capable of reporting the presence of two different engineered cell populations with programmable protein outputs. The approach relies on bacterial nitroreductase enzyme catalysis, which is orthogonal to normal mammalian biology, and diffusion of trimethoprim from "activator" cells to "receiver" cells. We test this strategy, which can theoretically regulate many different types of proteins, using biochemical and in vitro culture assays with optical and cytokine protein readouts. This describes the first small-molecule approach capable of detecting and controlling engineered cell-cell outputs, and we anticipate future applications that are especially relevant to the field of immuno-oncology.

Effects of the antibiotics trimethoprim (TMP) and sulfamethoxazole (SMX) on granulation, microbiology, and performance of aerobic granular sludge systems.

This work assessed the effect of the antibiotics trimethoprim (TMP) and sulfamethoxazole (SMX) on the granulation process, microbiology, and organic matter and nutrient removal of an aerobic granular sludge (AGS) system. In addition, after the maturation stage, the impact of the redox mediator anthraquinone-2,6-disulfonate (AQDS) (25 µM) on the biotransformation of the antibiotics was evaluated. The reactor R1 was maintained as a control, and the reactor R2 was supplemented with TMP and SMX (200 µg L-1). The ability to remove C, N, and P was similar between the reactors. However, the structural integrity of the AGS was impaired by the antibiotics. Low TMP (∼30%) and SMX (∼60%) removals were achieved when compared to anaerobic or floccular biomass aerobic systems. However, when the system was supplemented with AQDS, an increase in the removal of TMP (∼75%) and SMX (∼95%) was observed, possibly due to the catalytic action of the redox mediator on cometabolic processes. Regarding the microbial groups, whereas Proteobacteria and Bacterioidetes increased, Planctomycetes decreased in both reactors. However, TMP and SMX presence seemed to inhibit or favor some genera during the formation of the granules, possibly due to their bactericidal action.

Drug-Drug Binary Solids of Nitrofurantoin and Trimethoprim: Crystal Engineering and Pharmaceutical Properties.

With the aim of developing multidrug solids through a tuned crystal engineering approach, we have selected two antiurinary infective drugs, namely, nitrofurantoin (NF) and trimethoprim (TMP) and isolated eight binary drug-drug solid solvates along with a nonsolvated cocrystal. Crystal structure analyses were performed for eight of these solids and rationalized in terms of known supramolecular synthons formed by pyrimidine, imide, and amine functionalities. Notably, the TMP-NF anhydrous cocrystal and its ionic cocrystal hydrate exhibit enhanced equilibrium solubilities compared to pure NF or the simple NF hydrate. Furthermore, the ionic cocrystal hydrate exhibits greater antibacterial activity against the Gram-negative bacteria, E. coli, compared to the parent TMP and NF at the lowest concentration of 3.9 µg/mL. This study indicates initial pathways using the cocrystal methodology that would help to eventually arrive at an antiurinary cocrystal with optimal properties.

Golgi recruitment assay for visualizing small-molecule ligand-target engagement in cells.

The development of methods that allow detection of ligand-target engagement in cells is an important challenge in chemical biology and drug discovery. Here, we present a Golgi recruitment (G-REC) assay in which the ligand binding to the target protein can be visualized as Golgi-localized fluorescence signals. We show that the G-REC assay is applicable to the detection of various ligand-target interactions, ligand affinity comparison among distinct protein isoforms, and the monitoring of unmodified drug-target engagement in cells.

Inhibitor design to target a unique feature in the folate pocket of Staphylococcus aureus dihydrofolate reductase.

Staphylococcus aureus (Sa) is a serious concern due to increasing resistance to antibiotics. The bacterial dihydrofolate reductase enzyme is effectively inhibited by trimethoprim, a compound with antibacterial activity. Previously, we reported a trimethoprim derivative containing an acryloyl linker and a dihydophthalazine moiety demonstrating increased potency against S. aureus. We have expanded this series and assessed in vitro enzyme inhibition (Ki) and whole cell growth inhibition properties (MIC). Modifications were focused at a chiral carbon within the phthalazine heterocycle, as well as simultaneous modification at positions on the dihydrophthalazine. MIC values increased from 0.0626-0.5 µg/mL into the 0.5-1 µg/mL range when the edge positions were modified with either methyl or methoxy groups. Changes at the chiral carbon affected Ki measurements but with little impact on MIC values. Our structural data revealed accommodation of predominantly the S-enantiomer of the inhibitors within the folate-binding pocket. Longer modifications at the chiral carbon, such as p-methylbenzyl, protrude from the pocket into solvent and result in poorer Ki values, as do modifications with greater torsional freedom, such as 1-ethylpropyl. The most efficacious Ki was 0.7 ± 0.3 nM, obtained with a cyclopropyl derivative containing dimethoxy modifications at the dihydrophthalazine edge. The co-crystal structure revealed an alternative placement of the phthalazine moiety into a shallow surface at the edge of the site that can accommodate either enantiomer of the inhibitor. The current design, therefore, highlights how to engineer specific placement of the inhibitor within this alternative pocket, which in turn maximizes the enzyme inhibitory properties of racemic mixtures.

Sonochemical degradation of trimethoprim in water matrices: Effect of operating conditions, identification of transformation products and toxicity assessment.

The sonochemical degradation of trimethoprim (TMP), a widely used antibiotic, in various water matrices was investigated. The effect of several parameters, such as initial TMP concentration (0.5-3 mg/L), actual power density (20-60 W/L), initial solution pH (3-10), inorganic ions, humic acid and water matrix on degradation kinetics was examined. The pseudo-first order degradation rate of TMP was found to increase with increasing power density and decreasing pH, water complexity (ultrapure water > bottled water > secondary wastewater) and initial TMP concentration. TMP degradation is accompanied by the formation of several transformation products (TPs) as evidenced by LC-QToF-MS analysis. Nine such TPs were successfully identified and their time-trend profiles during degradation were followed. An in silico toxicity evaluation was performed showing that several TPs could potentially be more toxic than the parent compound towards Daphnia magna, Pimephales promelas and Pseudokirchneriella subcapitata.

Micro vs. nano: PLGA particles loaded with trimethoprim for instillative treatment of urinary tract infections.

Recurring infections and increasing resistances continue to complicate treatment of urinary tract infections. To investigate alternative treatment options, trimethoprim loaded micro- (D[4;3] of 1-9 µm) and nanoparticles (Z-Avg of 200-400 nm) were prepared from two types of poly(d,l-lactic-co-glycolic acid) (PLGA) for instillative therapy. While PLGA 503H microparticles could not be loaded with more than 2.6% trimethoprim, PLGA 2300 entrapped 22%. When preparing nanoparticles, both types displayed an even higher drug load of up to 29% using PLGA 2300, while PLGA 503H drug load stagnated at 10%. After eight hours, drug release from microparticles amounted to 55% (503H) and 35% (2300) whereas total drug release occurred after 8 (503H) and 9 days (2300). In case of nanoparticles, trimethoprim was liberated much faster with 60% after 2 h and a complete release after 24 h from both polymers. PLGA 2300 seems to be the better choice for entrapment of trimethoprim in microparticles considering the drug load. Both polymers, however, seem to be viable options for nanoparticles. Due to the higher overall drug load, nanoparticles seem to be advantageous over microparticles for instillative therapy, especially when prepared with PLGA 2300.

Synthesis, crystal structure and leishmanicidal activity of new trimethoprim Ru(III), Cu(II) and Pt(II) metal complexes.

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania, which has very limited treatment options and affects poor and underdeveloped populations. The current treatment is plagued by many complications, such as high toxicity, high cost and resistance to parasites; therefore, novel therapeutic agents are urgently needed. Herein, the synthesis, characterization and in vitro leishmanicidal potential of new complexes with the general formula [RuCl3(TMP)(dppb)] (1), [PtCl(TMP)(PPh3)2]PF6 (2) and [Cu(CH3COO)2(TMP)2]·DMF (3) (dppb = 1,4-bis(diphenylphosphino)butane, PPH3 = triphenylphosphine and TMP = trimethoprim) were evaluated. The complexes were characterized by infrared, UV-vis, cyclic voltammetry, molar conductance measurements, elemental analysis and NMR experiments. Also, the geometry of (2) and (3) were determined by single crystal X-ray diffraction. Despite being less potent against promastigote L. amazonensis proliferation than amphotericin B reference drug (IC50 = 0.09 ± 0.02 µM), complex (2) (IC50 = 3.6 ± 1.5 µM) was several times less cytotoxic (CC50 = 17.8 µM, SI = 4.9) in comparison with amphotericin B (CC50 = 3.3 µM, SI = 36.6) and gentian violet control (CC50 = 0.8 µM). Additionally, complex (2) inhibited J774 macrophage infection and amastigote number by macrophages (IC50 = 6.6 and SI = 2.7). Outstandingly, complex (2) was shown to be a promising candidate for a new leishmanicidal therapeutic agent, considering its biological power combined with low toxicity.