Trimethoprim

Molecular Basis of Dihydrofolate Reductase (DHFR) Inhibition

The primary molecular target of trimethoprim is dihydrofolate reductase (DHFR), an enzyme vital for bacterial survival. drugbank.compatsnap.com

Downstream Biochemical Consequences of DHFR Inhibition

The inhibition of DHFR by this compound sets off a cascade of biochemical events that are detrimental to the bacterial cell.

Investigating Alternative or Ancillary Mechanisms of Action

While the primary mechanism of this compound is the inhibition of DHFR, research has explored other potential ways the drug and its derivatives exert their effects.

Emergence and Spread of Resistance

The emergence and dissemination of trimethoprim resistance are complex processes driven by various genetic and environmental factors.

Horizontal gene transfer (HGT) is a predominant mechanism facilitating the rapid spread of antibiotic resistance genes, including those conferring this compound resistance, among bacterial populations. bioguardlabs.commdpi.comnih.govfrontiersin.org This process involves the transfer of genetic material between bacteria through three main mechanisms:

Conjugation: Considered the most prevalent method for the dissemination of antibiotic resistance genes, conjugation involves direct cell-to-cell contact and the transfer of DNA, typically via plasmids. bioguardlabs.commdpi.comnih.govfrontiersin.org

Transduction: Involves bacteriophages (viruses that infect bacteria) transferring bacterial DNA from one bacterium to another. bioguardlabs.commdpi.comnih.govfrontiersin.org

Transformation: Refers to the uptake of naked extracellular DNA fragments by a recipient bacterium. mdpi.comnih.govfrontiersin.org

Mobile genetic elements (MGEs) such as plasmids, integrons, and transposons play a crucial role in mobilizing and disseminating this compound resistance genes. nih.govfrontiersin.orgmicrobiologyresearch.orgoup.comnih.govuit.nomdpi.com Specifically, dihydrofolate reductase (dfr) genes, which confer resistance to this compound by encoding resistant DHFR enzymes, are frequently found on these MGEs. frontiersin.orgoup.comnih.govuit.no Class 1 and 2 integrons are particularly associated with dfrA genes, often alongside sul genes (conferring sulfamethoxazole resistance), forming "clinical integrons" that contribute to multidrug resistance. frontiersin.orgoup.com The selective pressure exerted by the widespread use of this compound and its combination with sulfamethoxazole (cotrimoxazole) promotes the evolution of increasingly compact gene arrangements carried by these mobile genetic elements, facilitating their movement within and between bacterial genomes. frontiersin.orgnih.gov For instance, studies on uropathogenic Escherichia coli have identified dfrA14 or dfrA1 gene cassettes integrated into conserved sul2-strA-strB clusters on transferable plasmids, ensuring co-resistance. frontiersin.orgnih.gov

Global and regional surveillance systems are critical for monitoring the prevalence and trends of antimicrobial resistance (AMR), including resistance to this compound. The World Health Organization's (WHO) Global Antimicrobial Resistance and Use Surveillance System (GLASS) is a key initiative that collects and reports national data on AMR and antimicrobial use in humans. who.intwho.int

Data from surveillance efforts highlight the significant burden of bacterial AMR, particularly in urinary tract infections (UTIs), where Escherichia coli and Klebsiella pneumoniae are major pathogens. nih.govnih.gov In 2019, this compound/sulfamethoxazole-resistant Escherichia coli alone was attributed to 3,453 deaths globally. nih.gov

Regional surveillance data reveal varying patterns of this compound resistance. For example, a systematic review of antimicrobial resistance among uropathogens in the Asia-Pacific region (APAC) from 2008 to 2020 indicated a very high prevalence of resistance to this compound/sulfamethoxazole, ranging between 33% and 90%. nih.gov The highest prevalence rates were reported from countries such as Bangladesh, India, Sri Lanka, and Indonesia. nih.gov

The following table illustrates the point prevalence estimates of resistance to co-trimoxazole (this compound/sulfamethoxazole) in E. coli and K. pneumoniae in selected Asia-Pacific countries:

Overcoming Resistance: Research Strategies

To combat the escalating problem of this compound resistance, research efforts are focused on two primary strategies: identifying novel DHFR inhibitors and developing new this compound analogs.

Research is actively pursuing the discovery of new compounds that can effectively inhibit both wild-type bacterial DHFR and its this compound-resistant mutant variants. elifesciences.orgnih.govasm.orgresearchgate.netrsc.orgnih.gov This involves screening large compound databases and employing structure-based design approaches. elifesciences.orgnih.govresearchgate.net

One promising approach involves identifying inhibitors with novel chemical scaffolds. For instance, a compound designated CD15-3 was identified through an integrated computational and experimental approach. elifesciences.org CD15-3 demonstrated inhibition of wild-type E. coli DHFR and its this compound-resistant variants (P21L, A26T, and L28R) with comparable IC50 values (50–75 µM). elifesciences.org Notably, the emergence of resistance to CD15-3 was significantly delayed compared to this compound in in vitro evolution experiments, and whole-genome sequencing of resistant strains showed no mutations in the target folA locus. elifesciences.org

Another class of compounds under development is propargyl-linked antifolates (PLAs). These inhibitors are designed to be potent against a range of DHFR-containing bacteria, including wild-type and this compound-resistant strains of Methicillin-Resistant Staphylococcus aureus (MRSA). asm.orgresearchgate.netrsc.orgnih.gov Studies have shown that PLAs can retain potency against resistant strains, with some compounds exhibiting low mutational frequencies and maintaining effective MIC values. asm.org

Furthermore, ionized non-classical antifolates (INCAs) represent a novel class of DHFR inhibitors developed using a structure-based approach. nih.gov These compounds are engineered to broadly inhibit resistance-conferring DHFR isoforms, including those found in clinical Staphylococcus aureus isolates like DfrG and DfrK, which render this compound and iclaprim ineffective. nih.gov INCAs have demonstrated low nanomolar enzymatic activity and potent cellular activity with human selectivity against clinically relevant this compound-resistant MRSA isolates. nih.gov

Computational methods, such as density-functional calculations, molecular docking, and molecular dynamics simulations, are increasingly utilized to design lead compounds that target atypical plasmid-encoded DHFRs, such as R67 DHFR (Type II DHFR), which bears no structural resemblance to classical DHFRs and confers this compound resistance. mdpi.com

The development of new this compound analogs involves modifying the existing molecular structure to enhance its activity against resistant strains and improve its pharmacodynamic properties without compromising its affinity for bacterial DHFR. mdpi.com

One innovative strategy explores the development of photoresponsive this compound analogs, where the antibacterial activity can be modulated by light. mdpi.com Researchers have reported red-shifted responsive azobenzene photoswitches containing this compound as a core, demonstrating the potential for controlled antibacterial action. mdpi.com

This compound derivatives are also being developed to enhance the drug's ability to suppress DHFR and overcome bacterial resistance. These modifications aim to increase the probability of binding to target proteins and improve treatment efficacy. Examples include:

Halogenated this compound Derivatives: Iodinated this compound (TMP-I) has shown promise as an effective derivative, exhibiting resistance-overcoming properties and synergistic effects with sulfamethoxazole, similar to this compound. Its ease of synthesis from inexpensive starting materials makes it an attractive candidate for further development against MRSA infections.

C7-substituted Analogs: Research is also exploring C7-substituted analogs of this compound, aiming to identify compounds with enhanced affinity and selectivity for bacterial DHFR, thereby circumventing the impact of resistance mutations.

The goal of these analog development efforts is to create robust drugs that are less susceptible to the rapid emergence of resistance, ensuring continued therapeutic options for bacterial infections.

Pharmacodynamic Indices and Their Correlation with Efficacy

Key pharmacodynamic parameters have been identified to correlate with the therapeutic efficacy of antibiotics in both in vitro and in vivo animal infection models tandfonline.com. For trimethoprim, especially in combination with sulfamethoxazole (TMP/SMX), the relationship between drug exposure and bacterial killing can exhibit characteristics of both time-dependent and concentration-dependent activity, depending on the pathogen and specific conditions asm.orgumich.edu.

The free area under the concentration-time curve to minimum inhibitory concentration ratio (fAUC/MIC) is a pharmacodynamic index that combines both the duration and extent of drug exposure researchgate.net. It is considered a predictive pharmacokinetic/pharmacodynamic (PK/PD) index for potentiated sulfonamides like TMP/SMX researchgate.net.

Studies have shown that for Escherichia coli, fAUC/MIC ratios of 40.7, 59.5, and 86.3 were required for stasis, 1-log₁₀, and 2-log₁₀ CFU reductions, respectively, when treated with TMP/SMX nih.govresearchgate.net. In contrast, for Stenotrophomonas maltophilia, no quantifiable fAUC/MIC thresholds for stasis or CFU reductions were observed, indicating a lack of consistent activity against this pathogen even at varying MICs nih.govresearchgate.netresearchgate.net.

For this compound as a component of TMP/SMX, stasis against Stenotrophomonas maltophilia in an in vitro chemostat model was achieved at an fAUC/MIC of 67.4 researchgate.netnih.govoup.com. However, clinically meaningful exposures required to achieve 1-log₁₀ CFU reductions were not quantifiable against this pathogen researchgate.netnih.gov.

In a neutropenic murine thigh infection model using TMP/SMX against Staphylococcus aureus, the free AUC/MIC of sulfamethoxazole (SMX) was found to be a better predictor of in vivo antimicrobial activity than Cmax/MIC, with an R² value of 0.69 nih.gov. For this compound, the 24-hour fAUC was 11.3 μg·h/ml (range 7.2–15.2), and the fAUC/MIC ratio for E. coli was 23 (range 16.4–29.2) researchgate.net.

Table 1: fAUC/MIC Ratios for TMP/SMX Against E. coli and S. maltophilia

The free maximum concentration to minimum inhibitory concentration ratio (fCmax/MIC) is another pharmacodynamic index used to predict antimicrobial efficacy redemc.net. For TMP/SMX, fCmax/MIC has been identified as an equivalent pharmacodynamic driver to fAUC/MIC in some studies researchgate.netnih.govoup.com. In an in vitro chemostat model, both fAUC/MIC and fCmax/MIC were identified as pharmacodynamic drivers for TMP/SMX against Stenotrophomonas maltophilia, with stasis achieved at an fAUC/MIC of 67.4 and an fCmax/MIC that correlated similarly researchgate.netnih.govoup.com.

In a study examining TMP/SMX against glycopeptide-intermediate Staphylococcus aureus (GISA), at simulated peak serum concentrations (Cmax), TMP/SMX was bactericidal against two of three strains, suggesting a concentration-dependent antibacterial activity umich.edu. High drug concentration:MIC ratios (9.8–39.2 x MIC) resulted in complete eradication of the organism in two of three strains and a greater than 2-log CFU/ml reduction in the third umich.edu.

The time that the free drug concentration remains above the minimum inhibitory concentration (%T > MIC) is a pharmacodynamic parameter often correlated with the efficacy of time-dependent antibiotics mdpi.comtandfonline.com. While TMP/SMX can exhibit concentration-dependent killing, some data suggest that for antibiotics with a time-dependent action, such as TMP/SMX, improvements in clinical outcomes plateau when the time period in which the concentration exceeds the MIC₉₀ is more than 60% asm.org.

However, in a study using a neutropenic murine thigh infection model for TMP/SMX against Staphylococcus aureus, TMP/SMX did not show antimicrobial activity when the free %T > MIC was less than 100% nih.gov. This suggests that for optimal activity, a high percentage of time above MIC might be necessary for TMP/SMX against certain pathogens in vivo nih.gov.

In Vitro Pharmacodynamic Models

In vitro pharmacodynamic models are valuable tools for simulating in vivo human pharmacokinetic profiles and assessing the efficacy of antibiotics, including the potential for drug resistance development plos.org. These models allow for controlled study of the drug-pathogen interaction.

Chemostat models are a type of in vitro pharmacodynamic model used to study bacterial growth and kill curves under controlled conditions, simulating drug concentrations over time nih.govresearchgate.netoup.complos.org.

In studies using an in vitro chemostat model, TMP/SMX demonstrated significantly greater killing for Escherichia coli than for Stenotrophomonas maltophilia at various MICs nih.govresearchgate.netresearchgate.net. For E. coli, mean changes in 24-hour CFU ranged from -4.49 to +1.83 log₁₀ CFU depending on the MIC nih.govresearchgate.net. However, against S. maltophilia, TMP/SMX showed no stasis or CFU reductions regardless of the MIC, and no pharmacodynamic thresholds were quantifiable nih.govresearchgate.netresearchgate.net. This suggests limited activity of TMP/SMX monotherapy against susceptible S. maltophilia even at higher doses researchgate.netnih.govoup.com.

Chemostat models have also been used to investigate the efficacy of TMP/SMX in combination with other antibiotics, such as rifampicin, against methicillin-resistant Staphylococcus aureus (MRSA) plos.org. These models can demonstrate the usefulness of combination therapy, particularly against resistant strains, where regrowth observed with monotherapy might be inhibited by the combination plos.org.

In Vivo Pharmacodynamic Models

In vivo pharmacodynamic models, often utilizing animal infection models, are crucial for evaluating the efficacy of antibiotics in a living system, providing insights that complement in vitro findings.

A neutropenic murine thigh infection model has been used to examine the pharmacokinetics/pharmacodynamics of TMP/SMX against Staphylococcus aureus researchgate.netnih.gov. In this model, the antimicrobial efficacy was calculated as the change in bacterial density after 24 hours of treatment nih.gov. The free AUC/MIC and free %T > MIC of sulfamethoxazole were found to correlate better with in vivo antimicrobial activity than Cmax/MIC nih.gov. Specifically, the free AUC/MIC had an R² of 0.69, and free %T > MIC had an R² of 0.71, compared to Cmax/MIC with an R² of 0.53 nih.gov. The study also indicated that distributed doses of TMP/SMX showed higher antimicrobial activity than a single dosage nih.gov. However, antimicrobial activity was not observed when the free %T > MIC was below 100% nih.gov.

For this compound, typical values for apparent volume of distribution, clearance, and terminal half-life were 1.99 L/kg, 0.33 L/h·kg, and 4.2 hours, respectively, in a non-linear mixed effects model fitted to experimental data researchgate.net. The 24-hour fAUC for this compound was 11.3 μg·h/ml researchgate.net.

Table 2: Correlation of PK/PD Indices with in vivo Antimicrobial Activity of TMP/SMX Against S. aureus (Murine Thigh Infection Model) nih.gov

Structure-Activity Relationship (SAR) Studies

Trimethoprim's core structure, 2,4-diamino-5-(3,4,5-trimethoxybenzyl) pyrimidine, is fundamental to its inhibitory activity. mdpi.comnih.govmdpi.com The 2,4-diaminopyrimidine moiety is crucial for forming hydrogen bonds with key residues in the DHFR active site. basicmedicalkey.com For instance, in bacterial DHFR, this moiety interacts with residues such as Asp27, Ile5, and Phe92. In human DHFR, important residues for ligand binding include Ile-7, Glu-30, Phe-31, Phe-34, Leu-67, Arg-70, and Val-115. mdpi.com

The trimethoxybenzyl group also plays a significant role, contributing to the selective binding of this compound to various DHFR enzymes. basicmedicalkey.com The main moieties involved in the interaction between TMP and the receptor are the methoxy groups, which act as hydrogen bond acceptors, and the amine and peptide groups, which serve as hydrogen bond donors. mdpi.com Additionally, the aromatic rings of this compound are often involved in π-π stacking interactions within the enzyme's active site. mdpi.com

Structural modifications to the this compound scaffold can significantly impact its potency and selectivity. The introduction of an amide bond into this compound analogs, for example, has been shown to increase their affinity for human DHFR compared to the unmodified compound. mdpi.comnih.govmdpi.com This modification can also lead to stronger inhibitory properties against human DHFR. mdpi.com

Replacing the 4-methoxy group with benzyloxy or phenylethanone groups can improve interactions within the DHFR binding site, thereby increasing activity. However, the effect of increasing the size of the aliphatic chain within TMP analogs is not always straightforward and requires further investigation. mdpi.comnih.gov

For bacterial DHFR, 2,4-diamino-5-(3',4',5'-trisubstituted benzyl)pyrimidines, particularly 3',5'-dialkoxy-4'-trisubstituted analogs, have demonstrated high antibacterial activity and selectivity. sci-hub.se While some 3',5'-dialkyl derivatives show less selectivity than this compound, they can still be at least 100-fold less active against mammalian DHFR than against microbial enzymes. sci-hub.se The selectivity for bacterial DHFRs can be dependent on the nature of the 4'-substituent, with amino analogs showing high selectivity. sci-hub.se Propargyl-linked inhibitors, for instance, have shown significantly increased potency, with some compounds exhibiting 3500-fold greater potency than this compound against human DHFR. mdpi.com

Modifying the pyrimidine ring, such as replacing it with a benzene ring, can slightly reduce affinity but derivatives may still interact with key residues like Glu-30 and Phe-34. mdpi.comnih.gov Inserting an amide bond in place of the methylene bridge can also allow for binding to the minor groove of DNA, indicating a dual activity. mdpi.comnih.gov

Rational drug design approaches are employed to enhance the biological activity and safety profile of this compound derivatives. this compound serves as a valuable model compound for the development of new DHFR inhibitors. mdpi.comnih.gov Strategies include incorporating structural elements like amide bonds, which are typical of DNA-binding ligands such as netropsin, into TMP analogs. mdpi.comnih.gov This can lead to compounds with dual activity, acting as both DHFR inhibitors and DNA-binding agents. mdpi.comnih.gov

The design process often involves optimizing hydrophobic and hydrophilic interactions within the DHFR active site. mdpi.com For example, molecular models of E. coli DHFR have been used to design analogs incorporating 3'-carboxyalkoxy moieties to generate ionic interactions with positively charged active-site residues like Arg-57, leading to exceptionally high affinities. sci-hub.se The ultimate goal of rational design is to develop compounds that can broadly inhibit both wild-type and resistant DHFR isoforms, addressing the challenge of antibiotic resistance. nih.gov

Computational Modeling and Simulation

Computational methods, including molecular docking and molecular dynamics simulations, are indispensable tools for elucidating the molecular interactions of this compound and its analogs with DHFR. mdpi.comnih.gov

Molecular docking studies are used to explore the potential binding modes of ligands within the DHFR active site and to confirm their affinity for the enzyme. nih.govmdpi.com These studies provide insights into the binding energies and specific interactions formed between this compound or its analogs and DHFR. mdpi.comnih.govmdpi.com

For instance, molecular docking results show that the introduction of an amide bond into TMP analogs generally increases their affinity for human DHFR compared to unmodified TMP. mdpi.comnih.govmdpi.comnih.gov Methotrexate (MTX), a potent DHFR inhibitor, typically exhibits a lower binding energy (e.g., -9.5 kcal/mol) compared to this compound and its analogs, reflecting its high inhibitory activity. mdpi.comnih.govmdpi.com

Key amino acid residues frequently observed to interact with this compound and its derivatives in DHFR include Glu-30, Phe-34, Ile-7, Ala-9, Ser-59, Thr-56, Tyr-121, Asp-145, Thr-146, Arg-70, and Val-115. mdpi.comnih.govmdpi.commdpi.comnih.gov These interactions often involve hydrogen bonds and π-π stacking interactions. mdpi.comnih.govmdpi.comnih.gov For example, compound 2, a TMP analog with an amide bond, showed a binding energy of -8.3 kcal/mol and formed five hydrogen bonds with Ala-9, Thr-56, Tyr-121, Asp-145, and Thr-146. nih.gov Molecular docking studies have also revealed that increasing the number of methylene groups in the chain linking two aromatic rings in TMP analogs can cause an inversion in the binding mode. nih.gov

Table 1: Binding Energies of Selected DHFR Inhibitors and Analogs

Molecular dynamics (MD) simulations are employed to provide a dynamic view of protein-ligand interactions, revealing structural changes, binding stability, and conformational dynamics over time. mdpi.comoup.comrsc.orgmorressier.comresearchgate.netresearchgate.net These simulations analyze parameters such as Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Solvent Accessible Surface Area (SASA), and Radius of Gyration (Rg). mdpi.comresearchgate.net

MD simulations have shown that DHFR inhibitors can stabilize the enzyme, with some analogs leading to lower RMSD values compared to the apo-protein, indicating a stabilizing effect. mdpi.commdpi.com For instance, a derivative (compound 13) was found to have a significant impact on protein stabilization, with RMSD oscillating around 1.1 Å over a 20 ns simulation. mdpi.com

The binding of drugs like this compound and methotrexate induces conformational changes in DHFR, particularly affecting flexible regions such as the Met20 loop. rsc.orgnih.gov These changes can decouple global motion and perturb fast motions at distal regions of DHFR, establishing a dynamic link between the substrate binding site and catalytic residues. nih.gov MD simulations are also crucial for understanding how mutations in DHFR confer resistance to this compound by inducing distinct structural changes, which can be exploited for rational drug design. oup.comrsc.org For example, the L28R mutation in E. coli DHFR, which confers this compound resistance, leads to increased substrate affinity due to newly formed hydrogen bonds with the aminobenzoyl glutamate tail of DHF. rsc.org

MD studies can further elucidate the role of water molecules at the binding interface, showing that negative entropy associated with this compound binding in wild-type DHFR can be due to water organization. rsc.org Simulations have also been used to study the dynamics of atypical DHFRs, such as the R67 DHFR, which confers resistance to this compound and is structurally unrelated to canonical DHFRs. tennessee.edunih.gov

Table 2: Molecular Dynamics Simulation Parameters for this compound and Analogs

Interactions with Biological Macromolecules (Excluding Target Enzyme)

Beyond its primary target enzyme, dihydrofolate reductase, this compound also interacts with other biological macromolecules, notably human serum albumin (HSA). These interactions are crucial for understanding the drug's transport and distribution within the body.

Mechanisms of Adverse Reactions Related to Reactive Metabolites

Trimethoprim's bioactivation to reactive metabolites is considered a potential contributing factor to idiosyncratic adverse drug reactions (IADRs) acs.orgnih.govannualreviews.org. These reactive metabolites can covalently bind to proteins, a process hypothesized to initiate immune responses or direct cellular toxicity nih.gov.

One significant mechanism of this compound bioactivation involves its oxidation to a reactive pyrimidine iminoquinone methide intermediate acs.orgku.eduoup.comnih.govscholaris.caacs.org. This intermediate has a protonated molecular ion of m/z 289 and is highly electrophilic nih.govscholaris.ca. The formation of this iminoquinone methide can occur through direct oxidation of this compound or via sequential secondary oxidation of primary, demethylated this compound metabolites ku.edunih.gov.

Glutathione (GSH) plays a crucial role in detoxifying reactive metabolites of this compound acs.orgnih.govnih.govacs.orgnih.gov. In the presence of GSH, human liver microsomes (HLM) produce GSH conjugates, indicating the trapping of these reactive intermediates acs.orgnih.govacs.orgresearchgate.net. Two major GSH adducts, TMPG-1 and TMPG-2, are believed to originate from the iminoquinone methide intermediate acs.org. Additionally, minor GSH adducts (TMPG-3, -4, and -5) have been identified, suggesting the formation of other reactive intermediates like ortho- or para-quinone methides, potentially following multiple O-demethylations acs.orgnih.govacs.org. The scavenging of reactive this compound metabolites by glutathione is considered a protective mechanism in vivo, leading to the formation of urinary mercapturic acids nih.gov.

Cytochrome P450 (CYP) enzymes are extensively involved in the metabolism and bioactivation of this compound ku.edunih.govnih.govdrugbank.commdpi.comnih.gov. Studies using heterologously expressed human P450s have provided insights into the specific roles of various CYP isoforms:

CYP1A2 and CYP3A4: These enzymes are capable of forming the iminoquinone methide intermediate ku.edunih.gov. They also contribute to the production of major GSH adducts (TMPG-1 and TMPG-2) acs.orgnih.govacs.org. CYP3A4 is particularly significant, catalyzing multiple O-demethylation reactions that can lead to quinone methide intermediates ku.edunih.gov.

CYP2D6: This enzyme is primarily responsible for catalyzing both demethylation and the subsequent secondary oxidation step to form quinone-methide species ku.edunih.gov. CYP2D6 also contributes to the formation of minor GSH adducts, such as TMPG-3 acs.orgnih.govacs.org.

Here is a summary of the involvement of key CYP enzymes in this compound metabolism and bioactivation:

The formation of reactive metabolites, such as the iminoquinone methide, suggests a mechanism by which this compound could contribute to idiosyncratic toxic responses acs.orgnih.govannualreviews.org. Idiosyncratic adverse drug reactions (IADRs) are unpredictable and can be severe nih.govchildrensmercy.org. The involvement of CYP2D6 in the formation of reactive metabolites is particularly noteworthy because this enzyme displays wide interindividual variability due to genetic polymorphism acs.orgmdpi.com. This genetic variability in CYP2D6 activity may be a factor in the rare adverse drug reactions observed with this compound acs.orgtandfonline.com. Furthermore, the covalent binding of chemically reactive metabolites to cellular proteins can lead to direct organ toxicity or the formation of neoantigens, potentially stimulating an immune response nih.gov.

Metabolic Pathways and Metabolite Identification

This compound undergoes oxidative metabolism, primarily in the liver, yielding a variety of stable metabolites acs.orgku.edunih.govdrugbank.comresearchgate.netjst.go.jp.

The major metabolic pathways for this compound include O-demethylation and N-oxidation acs.orgku.edunih.govdrugbank.comresearchgate.netjst.go.jpnih.govtandfonline.com.

O-Demethylation: This pathway involves the oxidative removal of methyl groups from the trimethoxybenzyl moiety of this compound ku.edunih.govdrugbank.comresearchgate.netjst.go.jpnih.govtandfonline.com. The most abundant O-demethylated metabolites identified in humans are 3'-desmethyl-trimethoprim and 4'-desmethyl-trimethoprim ku.edunih.govdrugbank.com. These two metabolites account for approximately 65% and 25% of the total metabolite formation, respectively nih.govdrugbank.com. Further O-demethylation reactions can occur, potentially leading to up to three free hydroxyl groups on the benzylic ring ku.edunih.gov.

N-Oxidation: This pathway involves the oxidation of nitrogen atoms in the pyrimidine ring ku.edunih.govdrugbank.comresearchgate.netjst.go.jpnih.govtandfonline.com. Stable N-oxide metabolites identified include this compound 1-N-oxide (1-NO-TMP) and this compound 3-N-oxide (3-NO-TMP) ku.edunih.govtandfonline.com. N-oxide metabolites generally constitute a minor proportion of the total metabolites, typically less than 5% drugbank.com.

Other identified metabolites include benzylic alcohol (Cα-OH-TMP), formed via oxidation of the methylene bridge, and its N-acetyl cysteine (NAC) adduct (Cα-NAC-TMP), which serves as evidence of this compound bioactivation ku.edunih.gov.

Here is a summary of stable this compound metabolites and their formation pathways:

Mechanism of Folate-Related Adverse Effects (e.g., Megaloblastic Anemia, Thrombocytopenia)

This compound's therapeutic action is rooted in its ability to inhibit bacterial dihydrofolate reductase (DHFR) wikipedia.orgelsevier.esmhmedical.compatsnap.commdpi.com. This enzyme is crucial for the conversion of dihydrofolic acid (DHF) to tetrahydrofolic acid (THF), a vital coenzyme essential for the synthesis of purine nucleotides, thymidine (a precursor for DNA replication), and certain amino acids wikipedia.orgpatsnap.commdpi.com. By blocking this critical step, this compound disrupts bacterial DNA synthesis, leading to impaired bacterial cell growth and proliferation wikipedia.orgpatsnap.commdpi.com.

While this compound exhibits a significantly higher inhibitory activity for bacterial DHFR compared to human DHFR (approximately sixty thousand times greater), it can still bind to the human enzyme, albeit with lower affinity wikipedia.orgmhmedical.comenghusen.dk. This interaction with human DHFR can lead to a reduction in serum folate levels and, in some patients, result in folate deficiency enghusen.dk.

Megaloblastic Anemia: Megaloblastic anemia is a type of macrocytic anemia characterized by impaired DNA synthesis during red blood cell production elsevier.eswikipedia.org. When DNA synthesis is compromised, the cell cycle cannot progress effectively, leading to continued cell growth without division, which manifests as macrocytosis and ineffective erythropoiesis elsevier.eswikipedia.org. This compound, by inhibiting human DHFR and consequently depleting tetrahydrofolate, can induce a functional folate deficiency that impairs DNA synthesis in rapidly dividing cells, including hematopoietic precursor cells in the bone marrow elsevier.esacpjournals.orgresearchgate.net. This can result in megaloblastic changes in red blood cells, particularly in individuals with pre-existing low folate stores enghusen.dkacpjournals.org.

Thrombocytopenia: Thrombocytopenia, characterized by a decreased number of circulating platelets, has been reported as a potential adverse effect associated with this compound, particularly when used in combination with sulfamethoxazole (TMP/SMX) wikipedia.orgada.orgada.orgdroracle.ainih.gov. The mechanism underlying this compound-induced thrombocytopenia is largely considered to be an immune-mediated disorder ada.orgdroracle.ainih.gov. It is hypothesized that the drug can induce the formation of autoantibodies that bind to platelet membrane proteins, leading to increased platelet destruction ada.orgnih.gov. This reaction is thought to fall within the "quinine type" immune pathogenesis category, where the presence of the drug enhances the structural affinity between autoantibodies and platelet glycoproteins, thereby facilitating binding and subsequent platelet destruction ada.org. This adverse effect can be severe and potentially life-threatening, and its onset is considered to be dose- and duration-independent in some cases droracle.ainih.gov.

Mechanisms of Other Systemic Effects (e.g., Hyperkalemia, Creatinine Competition)

Beyond its direct effects on folate metabolism, this compound can exert other systemic effects through distinct mechanisms, notably impacting potassium homeostasis and creatinine excretion.

Hyperkalemia: this compound is a recognized cause of hyperkalemia, an elevation in serum potassium levels wikipedia.orggpnotebook.comnih.govunmc.edunih.govtg.org.aukarger.comacpjournals.orgpathway.mdbmj.com. The primary mechanism involves this compound's action on the renal tubules, where it reduces potassium excretion wikipedia.orggpnotebook.comnih.govtg.org.auacpjournals.org. This occurs through the competitive inhibition of epithelial sodium channels (ENaC) located in the distal nephron wikipedia.orgnih.govunmc.edunih.govtg.org.aukarger.comacpjournals.org. This compound's structural similarity to the potassium-sparing diuretic amiloride allows it to mimic amiloride's effect wikipedia.orgnih.govunmc.edunih.govtg.org.aukarger.comacpjournals.org. By blocking ENaC, this compound reduces the reabsorption of sodium, which in turn diminishes the lumen-negative transepithelial voltage, thereby inhibiting potassium secretion into the urine nih.govacpjournals.org. The risk of developing hyperkalemia is heightened in patients receiving higher dosages of this compound, those with underlying renal impairment, or individuals concurrently taking other medications that can impede renal potassium excretion, such as renin-angiotensin system inhibitors or spironolactone gpnotebook.comunmc.edunih.govtg.org.aukarger.compathway.mdbmj.com.

Creatinine Competition: this compound can lead to an apparent increase in serum creatinine levels, which does not necessarily reflect a true decrease in glomerular filtration rate (GFR) wikipedia.orggpnotebook.comtg.org.aubmj.comnih.govcuriousclinicians.comresearchgate.netdroracle.ai. This phenomenon is due to this compound's competitive inhibition of creatinine secretion in the renal tubules wikipedia.orggpnotebook.comtg.org.aubmj.comnih.govcuriousclinicians.comresearchgate.net. Creatinine is not only filtered by the glomeruli but also actively secreted by the proximal tubules via organic cation transporters, specifically OCT2 and MATE1 curiousclinicians.comresearchgate.net. As a weak organic base, this compound competes with creatinine for these same transporters curiousclinicians.com. This competition reduces the tubular secretion of creatinine, causing it to accumulate in the bloodstream and leading to an elevated serum creatinine concentration nih.govcuriousclinicians.comresearchgate.net. This rise in serum creatinine is typically reversible, often appearing within 1-3 days of initiating this compound therapy and resolving within approximately a week after discontinuation nih.govdroracle.ai.

Table 1: Key Metabolic and Toxicological Mechanisms of this compound

Synergistic Interactions with Sulfonamides (e.g., Sulfamethoxazole)

The combination of trimethoprim with sulfonamides, such as sulfamethoxazole, is a well-established example of antimicrobial synergism, often referred to as co-trimoxazole. This combination is widely used to treat various bacterial and some fungal infections. nih.govjove.com

The synergistic effect between this compound and sulfamethoxazole stems from their ability to inhibit successive steps in the bacterial tetrahydrofolate (THF) synthesis pathway, which is crucial for bacterial growth and replication. nih.govwikipedia.orgdrugbank.comaap.org

Sulfamethoxazole's Action : Sulfamethoxazole, a sulfonamide, acts as a competitive inhibitor of dihydropteroate synthase. This enzyme is responsible for converting para-aminobenzoic acid (PABA) to dihydrofolic acid (DHF), an early precursor in the folate synthesis pathway. By competing with PABA, sulfamethoxazole prevents the initial synthesis of DHF. wikipedia.orgdrugbank.comaap.orgwikipedia.orgsketchy.com

This compound's Action : this compound then targets a subsequent step by competitively inhibiting dihydrofolate reductase (DHFR). This enzyme is essential for reducing DHF to tetrahydrofolic acid (THF), the biologically active form of folate. wikipedia.orgaap.orgsketchy.comwikipedia.orgmims.comnih.govdrugbank.com

This sequential blockade effectively halts the production of THF, which is vital for the synthesis of purines, thymidine, and methionine, all necessary for bacterial DNA and protein production. jove.comwikipedia.orgdrugbank.comsketchy.com While this compound alone is generally bacteriostatic, its combination with sulfamethoxazole can result in bactericidal activity. drugbank.com

The combined use of this compound and sulfamethoxazole has been shown to slow the development of bacterial resistance compared to using either drug alone. drugbank.commsdvetmanual.com This is attributed to the dual-target mechanism, making it more challenging for bacteria to develop resistance to both drugs simultaneously. However, despite this advantage, increased bacterial resistance to this compound-sulfamethoxazole has been observed, particularly with long-term use, such as in prophylaxis for Pneumocystis jirovecii pneumonia in HIV-infected patients. oup.comoup.comresearchgate.netnih.gov Resistance mechanisms can include alterations to the bacterial cell wall, overproduction of DHFR, or the production of resistant DHFR enzymes. drugbank.comtandfonline.com The prevalence of resistance genes, such as sul genes (for sulfonamide resistance) and dfr genes (for this compound resistance), has been documented in various bacterial isolates. nih.gov

Interactions with Other Antimicrobial Agents

Beyond sulfonamides, this compound can exhibit synergistic interactions with other antimicrobial agents, influencing treatment outcomes and interkingdom dynamics in complex infections.

Studies have demonstrated in vitro synergism between this compound and aminoglycosides like amikacin against various Gram-negative bacilli, including Klebsiella pneumoniae, Serratia marcescens, and Escherichia coli. nih.govasm.org This synergy can lead to a greater reduction in bacterial growth and killing compared to either drug used individually. For instance, serum bactericidal titers were found to be significantly higher with the combination of this compound and amikacin than with either drug alone. nih.gov The mechanism for this synergy is not fully elucidated but may involve the ability of beta-lactams to increase the uptake of aminoglycosides, although this specific interaction with this compound is still under investigation. mdpi.com

The following table illustrates the synergistic inhibitory and bactericidal effects of this compound and amikacin against certain Gram-negative bacteria:

Note: Synergy was defined as an FIC or FBC index equal to or less than 0.5 in some studies nih.gov, while others used a threshold of ≤ 0.25 for strong synergy asm.org.

This compound can influence interkingdom interactions, particularly in dual infections involving bacteria and fungi. Research has shown that this compound exposure can alter the production of small-molecule natural products by both bacteria and fungi in co-cultivation. For example, in studies involving Burkholderia cenocepacia (bacterium) and Aspergillus fumigatus (fungus), this compound affected the biochemical pathway for DHN-melanin biosynthesis in A. fumigatus, altering fungal conidia pigmentation. researcher.lifeacs.orgacs.org Additionally, this compound was observed to inactivate fragin, a bacterially produced antifungal compound, by A. fumigatus. researcher.lifeacs.org These findings highlight how therapeutic compounds like this compound can shape microbial and fungal metabolomes, influencing interkingdom interactions and the expression of virulence factors in complex infection environments. researcher.lifeacs.orgacs.org

Environmental Fate and Degradation Mechanisms

Trimethoprim (TMP) is generally considered recalcitrant to biodegradation in standard ready and inherent biodegradability tests nih.gov. However, it can undergo degradation through various aerobic and anaerobic biological mechanisms, as well as physico-chemical transformations in surface waters nih.gov.

Occurrence and Distribution in Environmental Samples

This compound is frequently detected in various environmental samples globally, often in the nanogram per liter (ng/L) to microgram per liter (µg/L) range nih.gov. Its ubiquitous presence is largely attributed to incomplete removal in conventional wastewater treatment processes mdpi.commdpi.com.

Reported concentrations of this compound in different environmental matrices include:

Untreated Municipal Wastewaters: Concentrations typically range from 0.17 µg/L to 8.8 µg/L diva-portal.org.

Hospital Effluents: Can contain higher concentrations, up to 11.3 µg/L diva-portal.org.

Surface Waters: Concentrations can reach up to 0.48 mg/L (480 µg/L) in general surface waters mdpi.com, and up to 28 µg/L in surface waters contaminated by industrial discharges diva-portal.org. In the Thames catchment, elevated concentrations reaching 2000 ng/L (2 µg/L) have been observed, with one specific site recording 3000 ng/L (3 µg/L) biorxiv.org.

Wastewater Treatment Plant Effluents: The highest mean concentration found in Finnish WWTP effluent was 532 ng/L oup.com. In South India, TMP was present at concentrations up to 1 µg/L in STP outlets researchgate.netnih.gov.

Urban Lakes: In Hanoi, Vietnam, the average concentration of this compound in surface water was 8.93 ng/L, with a detection frequency of 100% and a range of 0.65 to 67.9 ng/L semanticscholar.org.

Nationwide Studies (China): A review of antibiotic distribution in China's aquatic environment reported a highest concentration of 924.79 ng/L for TMP in 2009 mdpi.com.

Other Samples: this compound has also been detected in Swedish treated wastewater, surface water, and fish janusinfo.se.

Table 2: Reported Concentrations of this compound in Environmental Samples

Contribution to Antibiotic Resistance in the Environment

This compound, like other antibiotics, is a significant contributor to the selection, maintenance, and increase of antibiotic resistance in environmental bacteria nih.govjanusinfo.seexeter.ac.uk. The presence of even low concentrations of antibiotics in the environment can trigger the development of antibiotic resistance janusinfo.se.

The primary mechanism of this compound resistance in bacteria is the acquisition of mobile dfrA genes frontiersin.orgasm.orgresearchgate.netnih.govsciforum.net. These genes encode variants of the dihydrofolate reductase (DHFR) enzyme that are insensitive to this compound, bypassing the drug's inhibitory effect frontiersin.orgasm.org. dfrA genes are frequently found as gene cassettes within mobile genetic elements such as class 1 and class 2 integrons, as well as transposons and plasmids frontiersin.orgsciforum.net. This association with mobile genetic elements facilitates the rapid horizontal transfer and spread of this compound resistance among diverse bacterial species in the environment frontiersin.orgsciforum.net.

Wastewater treatment plants (WWTPs) are considered crucial hotspots for the dissemination of antibiotic resistance into the environment researchgate.net. They provide favorable conditions for the proliferation of antibiotic-resistant bacteria and for the horizontal transfer of antibiotic resistance genes (ARGs) among different microorganisms researchgate.net. Studies have indicated that concentrations of this compound found in Swedish wastewater treatment plants are likely to select for bacterial resistance janusinfo.se. Consequently, this compound has been added to the Water Framework Directive's Hazardous Compounds Watch List, signifying its potential threat to environmental and human health due to its role in driving resistance exeter.ac.uk. Research has shown a correlative relationship between the residue of TMP and the prevalence of sulfonamide antibiotic resistance genes, such as sul1, in river environments downstream of WWTPs biorxiv.orgresearchgate.net. To mitigate the selection for resistance in aquatic environments, a protective exposure limit of 1 µg/L for this compound has been proposed diva-portal.org.

Repurposing Trimethoprim for Non-Antimicrobial Applications

Drug repurposing, the identification of new uses for existing drugs, offers a promising strategy for developing potent and less toxic therapeutic agents. This compound is being investigated for applications beyond its conventional antimicrobial role. researchgate.net

Recent in silico studies have explored this compound's potential as a colchicine-binding site (CBS) inhibitor, suggesting its repurposing as an anticancer agent. This compound shares structural similarities and pharmacophoric features with colchicine and combretastatins, which are potent antimitotic agents targeting the CBS. researchgate.netresearchgate.net

Molecular docking studies have shown that this compound exhibits a good binding affinity to the CBS, with an average CDOCKER_ENERGY of -33.75 kcal/mol. researchgate.netresearchgate.net Molecular dynamics simulations, conducted over 100 nanoseconds, further confirmed the stability of the this compound-tubulin complex, with a root mean square deviation (RMSD) of less than 2.5 Å for the protein backbone. researchgate.netresearchgate.net The binding energy, calculated using the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) approach, was -27.3 kcal/mol, corroborating the docking results. researchgate.netresearchgate.net These findings suggest that this compound has the potential to inhibit tubulin polymerization, a key process in cell division, thereby exhibiting anticancer properties. researchgate.net

Table 1: Computational Binding Data of this compound to Colchicine-Binding Site

Synthesis and Characterization of Metal Complexes and Nanoparticles with this compound

This compound's molecular structure, particularly its pyrimidine ring and amino groups, allows it to act as a chelating agent for metal ions. researchgate.net This property has led to the synthesis and characterization of various metal complexes and nanoparticles incorporating this compound, with investigations into their enhanced biological activities.

Complexes of this compound with transition metals such as Cu(II), Zn(II), Pt(II), Ru(III), Fe(III), Cr(III), Mn(II), Co(II), Cd(II), and Hg(II) have been synthesized. researchgate.netjscimedcentral.comijcsi.projscimedcentral.com Characterization techniques, including UV-Vis, IR, mass spectrometry, and 1H NMR spectroscopy, elemental analysis, electrochemical studies, and thermal analysis, have been employed to elucidate their structures and properties. researchgate.netjscimedcentral.comijcsi.projscimedcentral.comnih.govnsps.org.ngresearchgate.net

These studies have revealed that this compound can act as a monodentate or bidentate ligand, coordinating with metal ions through its amino groups and pyrimidine nitrogen atoms. researchgate.netjscimedcentral.comnih.gov For instance, spectral studies indicate that this compound can link with metal ions as a bidentate ligand through the nitrogen atom N(7) of the amino group and the nitrogen atom N(2) of the pyrimidine ring. jscimedcentral.com Proposed geometries for these complexes include square planar, tetrahedral, and octahedral. researchgate.netjscimedcentral.comijcsi.pronih.govmdpi.com

Furthermore, this compound derivatives and their metal complexes have been functionalized with nanoparticles, such as zinc oxide (ZnO) and gold (Au) nanoparticles. researchgate.netmdpi.com The synthesis of nano-sized complexes of this compound with Fe(II) and Cu(II) using sonication methods has also been reported, with resulting nanocomplexes having sizes of 57.56 nm and 69.88 nm, respectively. nsps.org.ng These nanoparticle functionalizations aim to enhance properties for drug delivery, therapy, and imaging. mdpi.com

Table 2: Examples of this compound Metal Complexes and Nanoparticle Functionalizations

Targeting Human DHFR with this compound Derivatives

While this compound primarily targets bacterial DHFR with high selectivity, research is ongoing to develop this compound derivatives that can effectively inhibit human dihydrofolate reductase (hDHFR). mdpi.commdpi.comresearchgate.netnih.gov Inhibiting hDHFR is a promising strategy for cancer therapy, as this enzyme is essential for cellular processes, including DNA synthesis in rapidly proliferating cells like cancer cells. mdpi.comresearchgate.netnih.gov

Studies have shown that structural modifications to this compound, such as incorporating amide bonds or methylene bridges, can improve binding to the active site of hDHFR and maintain interactions with key residues like Glu-30 and Phe-34. mdpi.com Several synthesized this compound analogs have demonstrated greater hDHFR inhibitory activity than this compound itself. mdpi.commdpi.comnih.gov For example, a series of benzamide this compound derivatives showed hDHFR IC50 values ranging from 4.72 to 20.17 µM, which is significantly more active than this compound's IC50 of 55.26 µM. mdpi.comnih.gov Derivatives like JW2 and JW8 were identified as particularly promising, with JW2 interacting strongly with Gly-117 and JW8 with Asn-64 and Arg-70. mdpi.comnih.gov Other analogs, such as compounds 2 and 3, exhibited hDHFR IC50 values of 0.99 µM and 0.72 µM, respectively, highlighting their enhanced inhibitory properties compared to standard this compound. mdpi.comnih.gov

Table 3: Inhibitory Activity of this compound Derivatives against Human DHFR

Investigation of this compound's Influence on Host-Pathogen Interactions

Beyond its direct antimicrobial action, this compound has been investigated for its influence on host-pathogen interactions, including potential immunomodulatory effects. tandfonline.comcm-uj.krakow.pl While primarily known for inhibiting bacterial dihydrofolate reductase (DHFR) and thus bacterial nucleic acid synthesis, there are reports suggesting that this compound, particularly in combination with sulfamethoxazole (TMP-SMX), can exert anti-inflammatory and immunomodulatory effects. tandfonline.comcm-uj.krakow.pl

For instance, TMP-SMX has been reported to have a beneficial effect on phyto-hemagglutinin-induced lymphocyte transformation. tandfonline.com Studies have indicated that this compound, at therapeutically relevant dosages, can suppress anti-inflammatory responses. tandfonline.com The combination of this compound and sulfamethoxazole has shown more potent suppressant effects than either drug alone. tandfonline.com

Furthermore, research has explored how this compound exposure can influence chemical interactions between pathogens. In the context of cystic fibrosis, this compound has been shown to induce secondary metabolite production in Burkholderia and affect interkingdom interactions between B. cenocepacia and Aspergillus fumigatus. acs.org This suggests that this compound can modulate the metabolic profiles of pathogens, potentially influencing the resolution of infections. acs.org

In an immunodeficient mouse model of melanoma skin cancer, TMP-SMX significantly improved survival rates, reduced tumor weight and growth, and decreased vascular endothelial growth factor levels. nih.gov This anti-cancer effect was linked to the triggering of an allergic reaction and the promotion of immunity, with TMP-SMX increasing the infiltration of mast cells and the release of allergy-related and immune-enhancing mediators. nih.gov

Q. What experimental models are most effective for studying trimethoprim’s antimicrobial efficacy, and how can reproducibility be ensured?

Methodological Answer:

• Use standardized in vitro susceptibility testing (e.g., broth microdilution) with quality-controlled bacterial strains (e.g., E. coli ATCC 25922) to ensure consistency .
• For in vivo models, consider sea urchin embryos to assess developmental toxicity, but restrict concentration ranges (e.g., ≤450 µM) to avoid confounding mortality effects. Document environmental variables (pH, temperature) rigorously .
• Validate reproducibility via inter-laboratory calibration, especially for endpoints like radialization rates or MIC values .

Q. How can researchers optimize LC-MS protocols for quantifying this compound and its metabolites in biological samples?

Methodological Answer:

• Construct calibration curves using ≥20 gradient concentrations of this compound, validated with internal standards. Include quality controls (QCs) to assess intra-day and inter-day precision .
• For metabolite identification, combine MassQL queries (to screen MS2 fragmentation patterns for this compound substructures) with manual validation to reduce false positives. For example, MassQL identified 33/47 candidate features as true this compound derivatives in a recent study .

Q. What statistical approaches are recommended for analyzing dose-response relationships in this compound toxicity studies?

Methodological Answer:

• Use nonlinear regression models (e.g., sigmoidal curves) to fit dose-response data, accounting for inhomogeneous variances. Tools like GraphPad Prism or R’s drc package are suitable .
• Apply principal component analysis (PCA) to FT-IR spectral data to differentiate metabolic responses under varying pH conditions .

Advanced Research Questions

Q. How can network analysis and chemical-genomic datasets elucidate this compound’s synergistic mechanisms with other antibiotics?

Methodological Answer:

• Leverage synthetic lethality concepts: Identify gene pairs whose co-inhibition amplifies this compound’s effect. For example, this compound + sulfamethizole disrupts folate biosynthesis via metabolite accumulation, while this compound + AZT targets distinct pathways .
• Use E. coli K12 chemical-genomic data to predict "two-hit" synergies. Validate hypotheses with growth inhibition assays under combinatorial drug exposure .

Q. What experimental designs mitigate confounding factors when studying this compound resistance reversibility in bacterial populations?

Methodological Answer:

• Conduct longitudinal studies with control cohorts (e.g., a Swedish intervention reduced this compound use by 85% but saw no resistance decline in E. coli). Include phenotypic (e.g., clonal analysis) and genotypic (e.g., dfr gene profiling) metrics .
• Account for co-resistance patterns (e.g., this compound resistance linked to fluoroquinolone resistance) by stratifying isolates using whole-genome sequencing .

Q. How can advanced metabolomics techniques improve the detection of this compound biotransformation products in complex matrices?

Methodological Answer:

• Combine MS2LDA (to extract Mass2Motifs) with MassQL for substructure-specific queries. For instance, MassQL identified a this compound derivative (m/z 549.129) missed by MS2LDA, demonstrating enhanced sensitivity .
• Integrate stable isotope labeling (e.g., ¹³C-trimethoprim) to track metabolic fate in microbial communities or host tissues .

Q. What biochemical assays are critical for characterizing novel this compound-resistant dihydrofolate reductase (DHFR) variants?

Methodological Answer:

• Perform enzyme kinetics (Km, Vmax) using purified DHFR variants under physiological pH (5–7). For example, DfrB7 showed altered substrate affinity compared to wild-type DHFR .
• Use X-ray crystallography or cryo-EM to resolve structural changes in mutant DHFRs that confer this compound resistance .

Contradictory Data Resolution

Q. Why do some studies report pH-dependent this compound efficacy while others show no significant variation?

Methodological Answer:

• Conflicting results may arise from differences in bacterial species (e.g., E. coli vs. Streptococcus pyogenes) or experimental conditions. Standardize pH buffers (e.g., MES for pH 5, HEPES for pH 7) and pre-acclimate cultures to target pH levels before exposure .
• Re-evaluate historical data using multivariate tools like PC-DFA to isolate pH-specific metabolic responses from noise .

Q. How should researchers address discrepancies in this compound resistance gene prevalence across geographical regions?

Methodological Answer:

• Conduct meta-analyses of resistance gene databases (e.g., CARD, ResFinder), stratifying by region and host species. For example, dfrA1 predominates in Europe, while dfrB7 is emerging in Asia .
• Use machine learning (e.g., random forests) to model resistance gene spread, incorporating variables like antibiotic usage patterns and horizontal gene transfer rates .

Methodological Best Practices

Q. What guidelines ensure rigor in reporting this compound-related research?

Methodological Answer:

• Follow the FINER criteria (Feasible, Interesting, Novel, Ethical, Relevant) when formulating research questions .
• Adhere to journal-specific formatting (e.g., ≤3 heading levels, metric units) and statistical reporting standards (e.g., define "significant" only with p-values) .
• Deposit raw data (e.g., LC-MS spectra, resistance gene sequences) in public repositories like MetaboLights or NCBI .