Antibiotic Trimethoprim: Definition, Mechanism and Research

What is trimethoprim?

Trimethoprim (TMP), a lipophilic weakly alkaline pyrimethamine bacteriostatic agent, also known as sulfonamide booster, is white or white crystalline powder at room temperature. It is effective against a variety of gram-positive and negative bacteria such as Escherichia coli, Proteus mirabilis, pneumococcus, etc., but ineffective against pseudomonas aeruginosa infection.

Trimethoprim uses

The lowest antibacterial concentration of trimethoprim is often lower than 10mg/L, and it is easy to cause bacterial resistance when used alone, so it is generally not used alone, mainly composed of compound preparations with sulfanilamide drugs. It is used in the treatment of urinary tract infection (UTI), intestinal infection, respiratory infection, dysentery, enteritis, typhoid fever, meningitis, otitis media, meningitis, sepsis and soft tissue infection. The therapeutic effect on typhoid fever and paratyphi is no less than that of ampicillin, and it can also be combined with long-acting sulfonamides for the prevention and treatment of drug-resistant falciparum malaria.

Mechanism of action of trimethoprim

5,6,7, 8-tetrahydrofolate (THF) and its derivatives are essential precursors for the biosynthesis of DNA bases and amino acids, and dihydrofolate reductase (DHFR) catalyzed 5, 6-dihydrofolate (DHF) to reduce it to THF via NADPH cofactors. By inhibiting the dihydrofolate reductase of bacteria, Trimethoprim blocks the synthesis of tetrahydrofolate, thus interfering with the folate metabolism of bacteria, and ultimately inhibiting the growth and reproduction of bacteria. Compared to mammals, trimethoprim has a stronger affinity with bacterial dihydrofolate reductase, which allows trimethoprim to selectively interfere with bacterial biosynthesis processes.

(a) DHFR is an essential enzyme that plays a central role in nucleotide and amino acid biosynthesis; (b) Trimethoprim (TMP) is a bacteriostatic antibiotic molecule that competitively inhibits DHFR activity. (Manna, M. S., 2021)

Trimethoprim-sulfamethoxazole

The antibacterial spectrum of trimethoprim is similar to that of sulfanilamide agents but with stronger potency, and it is effective against a variety of gram-positive and negative bacteria.. Because the bacteria are easily resistant to trimethoprim, it is not suitable for use as an antimicrobial drug alone. The combination of trimethoprim and sulfanilamide can enhance the antibacterial effect several times to tens of times.

When trimethoprim is used in combination with sulfamethoxazole (SMX), trimethoprim prevents the conversion of DHF to THF by inhibiting DHFR in bacteria, while sulfamethoxazole inhibits dihydrofolate synthetase in bacteria, preventing the production of DHF. This double blocking effect prevents the bacteria from synthesizing folic acid effectively, which inhibits its growth and reproduction. The combination of trimethoprim and sulfamethoxazole can reduce the minimum inhibitory concentration (MIC), increase the inhibitory zone, and enhance the bactericidal activity.

TMP inhibits Na+/K+-ATPase in the basement membrane of distal renal tubular epithelial cells. However, patients taking TMP-SMX may experience hyponatremia and hyperkalemia. In addition, because TMP inhibits drug transporters such as organic cationic transporter 2 and multidrug and toxin extruders 2-K in the proximal tubules, the concentration of serum creatinine (SCr), the substrate of these transporters, may be reversible. Researchers studied variability in SCr, serum sodium (Na+), and serum potassium (K+) concentrations after TMP-SMX treatment began, and assessed the risk of hyponatremia and hyperkalemia in patients with increased SCr but no change in glomerular filtration rate (GFR). The results suggest that TMP may increase the risk of hyponatremia and hyperkalemia in a dose-dependent manner without altering GFR.

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Resistance to trimethoprim

Gene mutation and gene expression regulation: Bacteria develop low levels of resistance to TMP through mutations in the folA gene or non-allelic variants on their chromosomes. This mutation causes the dihydrofolate reductase (DHFR) in bacteria to be less sensitive to TMP. In addition, overexpression of the dfr gene promoter and amino acid mutations at key sites can also restrict the binding of TMP molecules, resulting in acquired resistance.

Plasmid-mediated drug resistance: Clinical drug resistance often results from plasmid-mediated DHFR, which can be integrated into chromosomes via transposons. This mechanism allows bacteria to acquire resistance through horizontal gene transfer.

Altered cell permeability: Bacteria reduce the entry of TMP by altering the permeability of the cell membrane, thereby reducing its antibacterial effectiveness.

Exogenous thymine dependent mutations: Some bacterial mutants depend on exogenous thymine and thymine for growth, and such mutants exhibit intrinsic resistance to TMP.

Emergence of novel resistant enzymes: For example, the dfrB7 enzyme encoded by the DfrB7 gene has a low affinity that confers the host bacteria with acquired resistance to TMP.

Research progress of trimethoprim

Trimethoprim derivatives impede antibiotic resistance evolution

Antibiotic resistance has become an emerging public health crisis due to the significant increase in mortality and morbidity of bacterial infections associated with antibiotic resistance in recent years. By comprehensively understanding the molecular evolution of TMP resistance in E.coli, Madhu et al. identified and targeted L28R mutations in dihydrofolate reductase, thereby effectively slowing the evolution of antibiotic resistance. The researchers used a derivative of TMP, 4' -demethyltrimethoprim (4'-DTMP), to eliminate the genetic trail that accumulated the L28R mutation, the most favorable drug-resistant DHFR mutation for TMP. L28R mutations have positive epistatic interactions with other drug-resistant DHFR mutations because L28R compensates for the catalytic defects caused by these mutations. Thus, blocking the L28R mutation shifts the evolutionary trajectory toward genotypes with different DHFR mutations. The results show how information from laboratory evolutionary experiments and structural analysis of resistant mutations can guide the development of new antibiotic molecules and the improvement of existing antibiotics. In addition, for drug targets (DHFR) known to be evolutionarily adaptable, closely monitoring bacterial evolution and developing mutation-specific antibiotic molecules could not only eliminate resistant bacteria, but also improve the long-term efficacy of antibiotic therapy by blocking the evolutionary trajectories that lead to resistant genotypes.

Research of trimethoprim and cyclodextrin

Trimethoprim is a broad spectrum, high efficiency and low toxicity antibiotic, but its strong hydrophobicity makes the dissolution rate of trimethoprim low and the bioavailability low, which limits the scope of clinical application. Cyclodextrin is a new pharmaceutical excipient and can be used as a carrier. A large number of studies have confirmed that the tendency of water molecules to form hydrogen bonds in the lipophile cavity of cyclodextrin in aqueous solution can not be fully satisfied when the preparation methods such as saturated aqueous solution method and stirring method are used for inclusion. Therefore, the whole molecule or the liposome part of the drug can replace the water molecule in the cyclodextrin, thereby reducing the energy of the system to form the inclusion compound, which can significantly increase the solubility or dissolution rate of the drug, improve the bioavailability and efficacy of the drug, and has a broad development prospect

Regulation of eDHFR-tagged proteins using TMP-PROTAC

Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, the control of engineered proteins can be used for therapeutic applications. Etersque et al. covalently linked the antibiotic trimethopridine to pomadomide, a ligand of E3 ligase cereblon, thereby developing a molecule called TMP-PROTAC that induces the degradation of proteins of interest (POI) that fuse with the small protein domain (E.coli dihydrofolate reductase eDHFR) gene. Thus, a robust method for selective and reversible degradation of EDHFR-labeled proteins was constructed.

References

1. Manna, M. S., et al. A trimethoprim derivative impedes antibiotic resistance evolution. Nature Communications. 2021, 12(1): 2949.
2. Etersque, J. M., et al. Regulation of eDHFR-tagged proteins with trimethoprim PROTACs. Nature Communications. 2023, 14(1): 7071.