Current Enzyme Inhibition (v.8, #2)

Dihydrofolate reductase (DHFR) enzyme catalyzes the reduction of dihydrofolate to tetrahydrofolate using NADPH as a cofactor to restore the reduced folate pools for reactions requiring one carbon transfer. Folates are required for de novo synthesis of purines and thymidylate, as well as glycine, methionine and serine. For this reason, DHFR has been an important target for chemotherapy for many diseases including cancer. Cell death ensues after the inhibition of DHFR due to the inhibition of nucleotide synthesis and DNA replication. Methotrexate (MTX), an analog of dihydrofolate is a tight binding inhibitor of DHFR. Since its analog aminopterin’s success in the clinic in the treatment of acute lymphocytic leukemia almost 65 years ago, MTX has also been used to treat non-Hodgkin’s lymphoma, osteosarcoma, choriocarcinoma, head and neck, and breast cancer. However, the development of side effects and both intrinsic and acquired drug resistance to MTX and other antifolates are the main clinical limitations. Myelosuppression and stomatitis are due to cell death of not only cancer cells but also rapidly dividing normal cells such as bone marrow cells and epithelial cells of the gut, respectively. Due to these limitations, a detailed understanding of DHFR at every level has been undertaken such as structure-functional analysis, mechanisms of action, transcriptional and translation regulation of DHFR to develop more effective antifolates. In this paper, we review novel therapeutic approaches to regulate DHFR activity and its expression to overcome resistance or toxicity.

Chemoresistance is one of the major reasons for the failure of anticancer chemotherapy in treating advanced stage cancer. The mechanism of chemoresistance to fluoropyrimidines and antifolates has been extensively investigated in the past 40 years. It has been well established that thymidylate synthase (TYMS, TS) and dihydrofolate reductase (DHFR) are two major targets for fluoropyrimidines and antifolates, respectively. The regulatory mechanism of TS and DHFR expression is rather complex involving transcriptional, post-transcriptional and translational regulations. Our recent understanding of the chemoresistance mechanism has been extended beyond the simple one target/drug view. In this review, we will focus on the recent advancement of non-coding microRNAs (miRNAs) in contributing to the regulations of TS and DHFR expression, and to the chemoresistance mechanism of fluoropyrimidines and antifolates.

The ubiquitous enzyme dihydrofolate reductase (DHFR) is responsible for the reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate in an NADPH-dependent manner. The enzymes DHFR and thymidylate synthase (TS), which converts deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), are coupled in the folate pathway as the product of TS (dihydrofolate) is the substrate for DHFR. Because of their crucial roles in the production of the precursors of RNA and DNA for protein synthesis in every organism, DHFR and TS are key pharmacological targets for the treatment of cancer, as well as bacterial and opportunistic infections. The effectiveness of antifolates lies in their ability to selectively disrupt folate pathways that ultimately lead to cell death. However, in many instances the efficacy of clinically available antifolates is limited by their ineffectiveness against many pathogenic organisms or by the increase in drug-resistance, mainly due to the rise of mutations observed in clinical isolates. This review surveys more than 300 DHFR structures and over 200 TS structures representing 28 species of enzyme. Novel antifolates continue to be synthesized in an effort to enhance species selectivity and to increase potency without added toxicity. The focus of this review will be on DHFR and TS enzymes from mammalian and pathogenic organisms that have become the target of bioterrorism or have become a major medical concern as drug-resistance has overtaken the efficacy of many current treatments.

This brief review will cover recent advances and applications of molecular dynamics simulations of dihydrofolate reductase over the course of the past few years. The application of the technique to the study of kinetic isotope effects, binding free energy of drugs, impact of ligand binding on protein conformation and study of coupled motions in hydrogen tunneling reactions will be discussed.

Human malaria parasites, in particular the most dangerous species, Plasmodium falciparum, are responsible for more than a million deaths per year, mainly of young African children, as well as causing enormous social and economic problems in endemic countries. In the absence of efficacious vaccines, the main weapon against malaria infections is intervention with drugs. Within the antimalarial drugs currently deployed clinically, the antifolates are amongst the oldest synthetic compounds, having been in use for over six decades and still of major importance today, despite widespread parasite resistance to this class of inhibitor. To date, only two enzymes of folate metabolism have ever been targeted in malaria chemotherapy and prophylaxis, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). The products of this pathway, reduced folate cofactors, are essential for DNA synthesis and the metabolism of certain amino acids. However, the de novo synthesis and interconversions of reduced folate derivatives involve a number of other enzymes that have not as yet been exploited as drug targets, despite the well established position of folate metabolism as a clinically validated point of intervention. Our current understanding of this area of metabolism in the parasite and its potential for providing novel targets for badly needed new antimalarial drugs are reviewed here.

Cyclic AMP is an ubiquitous molecule that serves as an important second messenger for multiple signaling pathways. At least nine membrane-bound mammalian isoforms of adenylyl cyclase have been identified, and each isoform has a distinct pattern of tissue distribution as well as interaction with regulatory proteins within local cytosolic environment. Adenylyl cyclase is coupled to G-protein receptors and serves to convert ATP to cAMP. Although many of the upstream G-protein receptors and cAMP regulators such as phosphodiesterase have been utilized as targets of pharmacotherapy, the attempt of pharmacologic regulation of adenylyl cyclase itself has not been successful. Recent studies have characterized the distinct physiologic effect of each adenylyl cyclase isoform within different organ systems, suggesting the potential therapeutic utility of isoform-specific regulators of adenylyl cyclase. In the current review, we aim to discuss the effects of genetic regulation for each adenylyl cyclase isoform as well as the development of isoformspecific adenylyl cyclase regulators including their therapeutic potential.