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...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.展开更多
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 synt...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.展开更多
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 pr...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.展开更多
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...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.展开更多
文摘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.
基金Supported by the National Natural Science Foundation of China（Nos.21172014, 21302007）.
文摘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.
基金supported by the National Natural Science Foundation of China(Nos.31930002,21621002,21877002,81991525,82273829 and 22077007)the key project at central government level:the ability establishment of sustainable use for valuable Chinese medicine resources(2060302-2201-17).
文摘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.
文摘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.
