Synthesis and Characterization of Naproxen-Salicylate Derivatives as Potential Dual-Targeted Inhibitors of Dihydrofolate Reductase

Dihydrofolate reductase (DHFR) is an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF). Chemotherapy drugs such as methotrexate help to slow the progression of cancer by limiting the ability of dividing cells to make nucleotides by competitively inhibiting DHFR. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been previously reported to exhibit competitive inhibition of DHFR, in addition to their primary action on cyclooxygenase enzymes. This interaction interferes with the enzymatic reduction of dihydrofolate to tetrahydrofolate, thereby impeding the folate metabolism pathway essential for nucleotide synthesis and cell proliferation. This activity stems from their structural resemblance to the p-aminobenzoyl-l-glutamate (pABG) moiety of folate, a substrate of DHFR. It has been established that NSAIDs containing a salicylate group (which has structural similarities to pABG), such as diflunisal, exhibit stronger DHFR-binding activity. In this study, we synthesized salicylate derivatives of naproxen with the aim of exploring their potential as inhibitors of DHFR. The interactions between these derivatives and human DHFR were characterized using a combination of biochemical, biophysical, and structural methods. Through polyacrylamide gel electrophoresis (PAGE) analysis, enzymatic assays, and quantitative ELISA, we investigated the binding affinity and inhibitory potency of the synthesized salicylate derivatives towards DHFR. The findings of this study suggest the potential of salicylate derivatives of naproxen as promising candidates for the inhibition of DHFR, thereby offering novel therapeutic opportunities for modulating the inflammatory process through multiple pathways. Further optimization of these derivatives could lead to the development of more efficacious dual-targeted analogs with enhanced therapeutic benefits.

Dihydrofolate reductase-like protein inactivates hemiaminal pharmacophore for self-resistance in safracin biosynthesis

Dihydrofolate reductase(DHFR),a housekeeping enzyme in primary metabolism,has been extensively studied as a model of acid-base catalysis and a clinic drug target.Herein,we investigated the enzymology of a DHFR-like protein SacH in safracin(SAC)biosynthesis,which reductively inactivates hemiaminal pharmacophore-containing biosynthetic intermediates and antibiotics for self-resistance.Furthermore,based on the crystal structure of SacH−NADPH−SAC-A ternary complexes and mutagenesis,we proposed a catalytic mechanism that is distinct from the previously characterized short-chain dehydrogenases/reductases-mediated inactivation of hemiaminal pharmacophore.These findings expand the functions of DHFR family proteins,reveal that the common reaction can be catalyzed by distinct family of enzymes,and imply the possibility for the discovery of novel antibiotics with hemiaminal pharmacophore.

Synthesis and evaluation of 8-deaza-5,6,7,8-tetrahydromethotrexate derivatives as dihydrofolate reductase inhibitors

A series of N5-substituted 8-deaza-5,6,7,8-tetrahydromethotrexate derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase（DHFR）.The results indicated that modification of the pteridine ring of methotrexate（MTX） rendered poor activity against human DHFR.

Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at the active site first, until to a critical concentration of the denaturant the overall conformation change of the molecule may occur cooperatively. Activation of DHFR in low GuHCl concentration is due to the subtle conformational change at the active site of the enzyme. Although the conformational change at the active site decreases the affinity of the enzyme for the substrates, it accelerates the velocity of the limiting step in the catalytic reaction, i.e. the velocity of the disso-ciation of products from the ternary complex.

Calorimetric study on thermally-induced conformational change of dihydrofolate reductase

Dihydrofolate reductase(DHFR)(EC 1.5.1.3)exists in all biobodies.It is an importantcomponent of the chemistry of DNA synthesis in cells.Methotrexate,a potent inhibitor ofDHFR,is a well-known anticancer agent.It has been employed extensively in clinicaltreatment.Therefore,the study on the conformational change of DHFR gives occasion topeople’s close attention.The thermally-induced conformational change of DHFR

3D-QSAR Study of 7,8-Dialkyl-l,3-diaminopyrrolo-[3,2-f] Quinazolines with Anticancer Activity as DHFR Inhibitors

A three-dimensional quantitative structure-activity relationship （3D-QSAR） study of a series of 7,8-dialkyl-l,3-diaminopyrrolo-[3,2-f] quinazolines with anticancer activity as dihydrofo- late reductase （DHFR） inhibitors was carried out by using the comparative molecular field analysis （CoMFA）, on the basis of our reported 2D-QSAR of these compounds. The es- tablished 3D-QSAR model has good quality of statistics and good prediction ability; the non cross-validation correlation coefficient and the cross-validation value of this model are 0.993 and 0.619, respectively, the F value is 193.4, and the standard deviation SD is 0.208. This model indicates that the steric field factor plays a much more important role than the electrostatic one, in satisfying agreement with the published 2D-QSAR model. However, the 3D-QSAR model offers visual images of the steric field and the electrostatic field. The 3D-QSAR study further suggests the following： to improve the activity, the substituent R^1 should be selected to be a group with an adaptive bulk like Et or i-Pr, and the substituent R should be selected to be a larger alkyl. In particular, based on our present 3D-QSAR as well as the published 2D-QSAR, the experimentMly-proposed hydrophobic binding mechanism on the receptor-binding site of the DHFR can be further explained in theory. Therefore, the QSAR studies help to further understand the ＂hydrophobic binding＂ action mechanism of this kind of compounds, and to direct the molecular design of new drugs with higher activity.

Synthesis and Structure-Antitumor Activity of 4,6-Diamino-1,2-Dihydro-2 ,2-Dimethyl-1-(Substituted-Naphthyl- 2)-1,3,5-Triazines

Introduction 4,6-Diamino-1,2-dihydro-2,2-dimethyl-1-substituted-1,3,5-triazines (Ⅰ) are dihydrofolate reductase(DHFR) inhibitors, hence they possess the inhibition to the growth of bacteria and cancer cells. Baker’s antifolate (Ⅱ) has shown promise as an antitumor agertt in clinical trials. The study of the structure-activity relationships of I shows that the inhibition to vertebrate DHFR is significantly influenced by the hydrophobicity of 1-substituent, i.e., the stronger the hydrophobicity of the 1-substituent, the more potent the inhibition of the compound to vertebrate DHFR.

Computational Study of the Interactions between Antimalarial Chemotherapies with Folate Pathway Receptors and Telomerase Reverse Transcriptase

Malaria is a life-threatening disease responsible for half a million death annually, and with nearly half of the world’s population at risk. The rapid drop in observed cases of malaria in the last two decades has been due to a combination of preventive and therapeutic remedies. However, the absence of a vaccine, new antimalarial chemotherapies and increased parasitic resistance have led to a plateau of infections and renewed research interest in target human and Plasmodium (the malaria parasite) receptors and new drugs. In this study, the impact of mutation on the affinity on antimalarial drugs with the bifunctional enzyme complex, dihydrofolate reductase (DHFR) is explored. In addition, homology modeling is used to build the three-dimensional models of the enzymes Plasmodium telomerase reverse-transcriptase (pf-TERT) and Plasmodium dihydropteroate synthetase (pf-DHPS) to determine their affinity with antimalarial drugs. The interaction energies and stable complexes formed between these enzymes and antimalarial drugs (chloroquine, artemisinin, primaquine, pyrimethamine, sulfadoxine and pentamidine) were modelled using AutoDock vina. Our data indicate that pf-TERT and pf-DHPS form stable complexes with the antimalarial ligands with affinity ranging from −4.0 to −6.9 kcal/mol. The affinity with crystal structures of DHFR receptors was higher ranging from −6.0 to −10.0 kcal/mol. The affinity to DHFR also decreases with the mutation a confirmation of the source of resistance. The highest affinity interaction for all the receptors modeled is observed with Artemisinin a benchmark antimalarial drug. This can be attributed to the size, shape and dipolar surface of the ligand. The observed complexes are stabilized by strategic active site polar and non-polar contacts.

Syntheses and Antiproliferative Evaluation of 6-Thienyl, 6-Polyphenyl Aryl and 6-Naphthyl Derivatives of 2,4-Diaminopyrido [3,2-d] pyramidine as Non-classical Antifolate Targeting DHFR

A series of 6-thienylethenyl, 6-polyphenyl arylethenyl, 6-thienylethyl and 6-polyphenyl arylethyl deriva- tives of 2,4-diaminopyrido[3,2-d]pyrimidine for targeting dihydrofolate reductase（DHFR） was designed and synthe- sized as non-classical antifolates in order to overcome drug resistance. The compounds were evaluated for in vitro antitumor activities, rhDHFR and antimicrobial activities. All the compounds exhibited antitumor activities, with ICs0 values in the range of 0.13--17.8 gmol/L against HL-60, HeLa and A549. Both the types of aryl groups and the orientation of polyphenyl aryl made an impact on the biological activities. 6-Naphthylethyl derivatives 5c and 5d were proved to be the most active Dt-IFR inhibitors, which were more potent than 6-phenylethyl, 6-thienylethyl and 6-biphenylethyl derivatives. Docking studies reveal that flexible saturated carbon-carbon bond of C9--C10 is essential for biological activities in molecular backbone. Antimicrobial test shows that most of the compounds exhibit antibacterial activities.

High multiple mutations of Plasmodium falciparum-resistant genotypes to sulphadoxine-pyrimethamine in Lagos, Nigeria

Background Plasmodium falciparum-resistance to sulphadoxine-pyrimethamine(SP)has been largely reported among pregnant women.However,the profile of resistance markers to SP dihydrofolate reductase(dhfr)and dihydropteroate synthase(dhps)in the general population are varied and not frequently monitored.Currently,SP is used as partner drug for artemisinin combination therapy(SP-artesunate)in some sub-Saharan African countries or as a prophylactic drug in intermittent preventive treatment of malaria during pregnancy and infants and in seasonal malaria chemoprevention(SMC).Profiling of P.falciparum-resistant genotypes to SP is dynamic and critical in providing data that would be useful for malaria control programmes.This study assessed the profile of dhfr and dhps genes genotypes among individuals with malaria in Lagos,Nigeria.Methods Molecular markers of SP resistance were identified by nested PCR and sequenced among malaria positive dried blood spots(DBS)that were collected from individuals attending health facilities from January 2013 to February 2014 and during community surveys from October 2010 to September 2011 across different Local Government Areas of Lagos State,Nigeria.Results A total of 242 and 167 samples were sequenced for dhfr and dhps,respectively.Sequence analysis of dhfr showed that 95.5%(231/242),96.3%(233/242)and 96.7%(234/242)of the samples had N51I,C59R and S108N mutant alleles,respectively.The prevalence of dhps mutation at codons A437G,A613S,S436A,A581G,I431V and K540E were 95.8%(160/167),41.9%(70/167),41.3%(69/167),31.1%(52/167),25.1%(42/167),and 1.2%(2/167)respectively.The prevalence of triple mutations(CIRNI)in dhfr was 93.8%and 44.3%for the single dhps haplotype mutation(SGKAA).Partial SP-resistance due to quadruple dhfr-dhps haplotype mutations(CIRNI-SGKAA)and octuple haplotype mutations(CIRNI-VAGKGS)with rate of 42.6%and 22.0%,respectively has been reported.Conclusions There was increased prevalence in dhfr triple haplotype mutations when compared with previous reports in the same environment but aligned with high prevalence in other locations in Nigeria and other countries in Africa.Also,high prevalence of dhfr and dhps mutant alleles occurred in the study areas in Lagos,Nigeria five to eight years after the introduction of artemisinin combination therapy underscores the need for continuous monitoring.

High expression of EPO gene using un-dhfr negative cell

Erythropoietin （EPO） genomic gene was cloned and its expression vector pOP13/EPO was constructed. CHOK12 cell was transfected by this vector using lipofectin method. A stable expression cell strain C10 cell with the EPO production at 160IU/d in 10\+6 cells were obtained at 400 μg/mL G418. Based on the C10 cell, another vector pHY/dhfr （dihydrofolate reductase） that carries a dhfr gene and a selecting marker of hygromycin B resistant gene was transferred to this cell. Several cell clones were obtained at 200 μg/mL hygromycin B. These cell clones that can express both EPO gene and exogenous dhfr gene were selected under the progressively increased concentration to 1 μmol methotrexate（MTX）. Some high EPO expression cell clones were obtained, the highest expression was 2 400 IU/d in 10\+6 cells, 15 times higher than that without MTX pressure. Then, a method of EPO high expression by using undhfr negative cell was primarily established. EPO bioactivity was found by using TF1 cell.