Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

Abstract

Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa Cryptosporidium and Toxoplasma as well as the fungus Candida glabrata. A comparison of the structures of DHFR from the fungal species Candida glabrata and Pneumocystis suggests that the compounds may also potently inhibit Pneumocystis DHFR.Reduced folate cofactors such as tetrahydrofolate are required for critical cellular functions, such as the production of dTMP, several amino acids, and purines. Dihydrofolate reductase (DHFR), the sole source of tetrahydrofolate, is one of several enzymes in the folate biosynthetic pathway. DHFR has been a recognized and validated drug target since the 1960s, with the discovery of methotrexate (5). Fortunately, since pathogenic and human forms of DHFR exhibit several critical sequence differences, it has also been possible to develop speciesselective antifolates for several infectious diseases, including malaria, toxoplasmosis, and urinary tract infections. Trimethoprim (TMP) (Fig. 1) is a commonly administered antifolate, primarily in combination with sulfamethoxazole (TMP-SMZ), which inhibits dihydropteroate synthase (DHPS), another enzyme in the folate pathway (14). TMP-SMZ is most effective against prokaryotic pathogens. However, it is also recognized as first-line therapy in treating and preventing the common eukaryotic opportunistic pathogen Pneumocystis jirovecii, which causes life-threatening pneumonia in immunocompromised patients (20).Interestingly, while TMP inhibits bacterial species of DHFR at concentrations in the low nanomolar range, it inhibits many eukaryotic species of the enzyme at concentrations in the micromolar range, resulting in 3 orders of magnitude lower potency. Even the use of TMP-SMZ as a prophylactic agent against pneumocystis relies heavily on the sulfa component (31) and only on relatively weak binding between TMP and P. jirovecii DHFR (6,18). In fact, studies have reported that mutations conferring resistance to TMP-SMZ arise in DHPS, not in DHFR (17, 28). In contrast, when the DHFR inhibitor pyrimethamine, which is four times more potent than TMP (18), is used in combination with sulfadiazine against pneumocystis, mutations arise in DHFR as well as DHPS (20). A logical conclusion of these studies is that the low potency of TMP against P. jirovecii DHFR is preventing it from reaching its full potential as an effective therapy for this eukaryotic opportunistic pathogen. In contrast, high-affinity DHFR inhib...

Genetic modification of the diarrhoeal pathogen Cryptosporidium parvum

“…Cryptosporidium is remarkably resistant to antifolates, a mainstay of treatment against other apicomplexans, and this resistance has been attributed to differences in the target enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) 24 . However, Cryptosporidium is unique among apicomplexans in that it acquired a thymidine kinase (TK) by horizontal gene transfer from bacteria 25 .…”

Dihydrofolate Reductase Is a Valid Target for Antifungal Development in the Human Pathogen Candida albicans

“…However, given that Dfr1p is present in humans and fairly well conserved between mammals and fungi, selectivity is likely to be an issue. Interestingly, Anderson and colleagues (18,53) described trimethoprim derivatives with an extended central linker with dramatically enhanced potency against C. glabrata DHFR. However, despite exhibiting selective inhibition of the fungal enzyme over the human enzyme in cell-free biochemical assays, toxicity to mammalian cells remained an issue (54).…”

“…In its reduced form, tetrahydrofolate (THF) is an essential coenzyme for a number of cellular enzymes, serving as a carrier for the transfer of one-carbon (1C) units as well as their interconversion between various oxidation states (18). THF is required for the synthesis of dTMP, purines, and methionine, as well as a multitude of other important metabolites.…”

Targeted Mutations of Bacillus anthracis Dihydrofolate Reductase Condense Complex Structure−Activity Relationships

“…For example, mutations in the BaDHFR active site revealed that the polar character of Y102 negatively impacts the enzyme’s affinity with TMP and the propargyl-linked compounds, thus aiding in elucidating one factor of the enzyme’s innate resistance to TMP. While polar propargylic substitutions may appear appealing, such substitutions have been shown in other species to have a negative impact on cell permeability 17 . Mutational studies also helped further interpret information gathered from structure-based drug design studies.…”

“…The studies of mutant BaDHFR enzymes also helped further analyze the complex SAR data to understand the role of substituents on the propargyl linker of the lead compounds, as well as the effect of substitution patterns around the aryl ring. Currently, inhibitor development has been directed towards a methyl substituent on the propargyl bridge due to the efficacy seen in other DHFR species such as SaDHFR, C. hominus DHFR, and C. glabrata DHFR 6a, 7b, 17–18 . These mutational studies indicate that a propargylic hydrogen would be a more favorable substituent for BaDHFR.…”