Dihydrofolate Reductase In Cells Research Articles

Investigating the anti-cancer potential of pyrimethamine analogues through a modern chemical biology lens

Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents.

Cycloguanil and Analogues Potently Target DHFR in Cancer Cells to Elicit Anti-Cancer Activity

Dihydrofolate reductase (DHFR) is an established anti-cancer drug target whose inhibition disrupts folate metabolism and STAT3-dependent gene expression. Cycloguanil was proposed as a DHFR inhibitor in the 1950s and is the active metabolite of clinically approved plasmodium DHFR inhibitor Proguanil. The Cycloguanil scaffold was explored to generate potential cancer therapies in the 1970s. Herein, current computational and chemical biology techniques were employed to re-investigate the anti-cancer activity of Cycloguanil and related compounds. In silico modeling was employed to identify promising Cycloguanil analogues from NCI databases, which were cross-referenced with NCI-60 Human Tumor Cell Line Screening data. Using target engagement assays, it was found that these compounds engage DHFR in cells at sub-nanomolar concentrations; however, growth impairments were not observed until higher concentrations. Folinic acid treatment rescues the viability impairments induced by some, but not all, Cycloguanil analogues, suggesting these compounds may have additional targets. Cycloguanil and its most promising analogue, NSC127159, induced similar metabolite profiles compared to established DHFR inhibitors Methotrexate and Pyrimethamine while also blocking downstream signaling, including STAT3 transcriptional activity. These data confirm that Cycloguanil and its analogues are potent inhibitors of human DHFR, and their anti-cancer activity may be worth further investigation.

Soluble oligomerization provides a beneficial fitness effect on destabilizing mutations

Mutations create the genetic diversity on which selective pressures can act, yet also create structural instability in proteins. How, then, is it possible for organisms to ameliorate mutation-induced perturbations of protein stability while maintaining biological fitness and gaining a selective advantage? Here we used site-specific chromosomal mutagenesis to introduce a selected set of mostly destabilizing mutations into folA--an essential chromosomal gene of Escherichia coli encoding dihydrofolate reductase (DHFR)--to determine how changes in protein stability, activity, and abundance affect fitness. In total, 27 E. coli strains carrying mutant DHFR were created. We found no significant correlation between protein stability and its catalytic activity nor between catalytic activity and fitness in a limited range of variation of catalytic activity observed in mutants. The stability of these mutants is strongly correlated with their intracellular abundance, suggesting that protein homeostatic machinery plays an active role in maintaining intracellular concentrations of proteins. Fitness also shows a significant correlation with intracellular abundance of soluble DHFR in cells growing at 30 °C. At 42 °C, the picture was mixed, yet remarkable: A few strains carrying mutant DHFR proteins aggregated, rendering them nonviable, but, intriguingly, the majority exhibited fitness higher than wild type. We found that mutational destabilization of DHFR proteins in E. coli is counterbalanced at 42 °C by their soluble oligomerization, thereby restoring structural stability and protecting against aggregation.

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-angstroms resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.