Structural analysis of dihydrofolate reductases enables rationalization of antifolate binding affinities and suggests repurposing possibilities

Abstract

Antifolates are competitive inhibitors of dihydrofolate reductase (DHFR), a conserved enzyme that is central to metabolism and widely targeted in pathogenic diseases, cancer and autoimmune disorders. Although most clinically used antifolates are known to be target specific, some display a fair degree of cross-reactivity with DHFRs from other species. A method that enables identification of determinants of affinity and specificity in target DHFRs from different species and provides guidelines for the design of antifolates is currently lacking. To address this, we first captured the potential druggable space of a DHFR in a substructure called the 'supersite' and classified supersites of DHFRs from 56 species into 16 'site-types' based on pairwise structural similarity. Analysis of supersites across these site-types revealed that DHFRs exhibit varying extents of dissimilarity at structurally equivalent positions in and around the binding site. We were able to explain the pattern of affinities towards chemically diverse antifolates exhibited by DHFRs of different site-types based on these structural differences. We then generated an antifolate-DHFR network by mapping known high-affinity antifolates to their respective supersites and used this to identify antifolates that can be repurposed based on similarity between supersites or antifolates. Thus, we identified 177 human-specific and 458 pathogen-specific antifolates, a large number of which are supported by available experimental data. Thus, in the light of the clinical importance of DHFR, we present a novel approach to identifying differences in the druggable space of DHFRs that can be utilized for rational design of antifolates.

In Silico Study Identified Methotrexate Analog as Potential Inhibitor of Drug Resistant Human Dihydrofolate Reductase for Cancer Therapeutics

“…The conserved hydrogen bond with OE1 atom of catalytic residue Glu30 was formed with the pterin moiety of Hit molecule [ 42 , 55 ]. The additional oxygen atom in the structure of Hit compound formed a hydrogen bond with Ser59 in both wild type and mutant hDHFR, while Ser59 belonged to the coenzyme NADPH binding site [ 56 ]. It was speculated that the hydrogen bond between Ser59 and the modified p -ABA moiety of Hit compound contributed to the strong binding of the Hit compound with the mutant structure of hDHFR.…”

Efficacy and Mechanism of Traditional Medicinal Plants and Bioactive Compounds against Clinically Important Pathogens

“…Helicases consititute another significant replicative enzyme responsible for separating and/or rearranging DNA double-strands [153]. Furthermore, myricetin (43) and baicalein (44) have been proposed as potent inhibitors of numerous DNA and RNA polymerases, as well as viral reverse transcriptase, resulting in the inhibition of bacterial growth [154]. EGCG (20), myricetin (43), and robinetin (53) have been demonstrated as inhibitors of dihydrofolate reductase in Streptomonas maltophilia, P.vulgaris, S. aureus, M. tuberculosis, and E. coli [43,155,156].…”

Trimethoprim: An Old Antibacterial Drug as a Template to Search for New Targets. Synthesis, Biological Activity and Molecular Modeling Study of Novel Trimethoprim Analogs

“…Inhibition of folate-metabolizing enzymes leads to an imbalance in the pathways involved in active synthesizing thymidylate, disrupts DNA replication, and eventually causes cell death [10]. Therefore, DHFR is a very good example of a well-established molecular target of new active compounds, which could be approved as antibacterial drugs and therapeutic agents against a variety of fatal disorders e.g., cancer [11,12]. Several classes of compounds have been explored for their potential antifolate activity; among the most outstanding are diaminopyrimidine [13,14], diaminoquinazolin [15], diaminopteridine [16], and also diaminotriazines [17].…”