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.

Targeting folate metabolism for therapeutic option: A bioinformatics approach.

Lymphatic filariasis, commonly called elephantiasis, poses a burden of estimated level of 5.09 million disability adjusted life year. Limitations of its sole drug, diethylcarbamazine (DEC) drive exploration of effective filarial target. A few plant extracts having polyphenolic ingredients and some synthetic compounds possess potential dihydrofolate reductase (DHFR) inhibitory effect. Here, we postulated a plausible link between folates and polyphenolics based on their common precursor in shikimate metabolism. Considering its implication in structural resemblance based antagonism, we have attempted to validate parasitic DHFR protein as a target. The bioinformatics approach, in the absence of crystal structure of the proposed target, used to authenticate and for virtual docking with suitable tested compounds, showed remarkably lower thermodynamic parameters as opposed to the positive control. A comparative docking analysis between human and Brugia malayi DHFR also showed effective binding parameters with lower inhibition constants of these ligands with parasitic target, but not with human counterpart highlighting safety and efficacy. This study suggests that DHFR could be a valid drug target for lymphatic filariasis, and further reveal that bioinformatics may be an effective tool in reverse pharmacological approach for drug design.

The 19-bp deletion polymorphism of dihydrofolate reductase (DHFR) and nonsyndromic cleft lip with or without cleft palate: evidence for a protective role

Objective Nonsyndromic cleft lip with or without cleft palate (NS-CL/P) are among the most common congenital birth defects worldwide. Several lines of evidence point to the involvement of folate, as well as folate metabolizing enzymes in risk reduction of orofacial clefts. Dihydrofolate reductase (DHFR) enzyme participates in the metabolic cycle of folate and has a crucial role in DNA synthesis, a fundamental feature of gestation and development. A functional polymorphic 19-bp deletion within intron-1 of DHFR has been associated with the risk of common congenital malformations. The present study aimed to evaluate the possible association between DHFR 19-bp deletion polymorphism and susceptibility to NS-CL/P in an Iranian population. Material and Methods The current study recruited 100 NS-CL/P patients and 100 healthy controls. DHFR 19-bp deletion was determined using an allele specific-PCR method. Results We observed the DHFR 19-bp homozygous deletion genotype (D/D) vs. homozygous wild genotype (WW) was more frequent in controls than in NS-CL/P patients (25% vs. 13%), being associated with a reduced risk of NS-CL/P in both codominant (OR=0.33, P=0.027) and recessive (OR=0.45, P=0.046) tested inheritance models. We also stratified the cleft patients and reanalyzed the data. The association trend for CL+CL/P group compared to the controls revealed that the DD genotype in both codominant (OR=0.30, P=0.032) and recessive models (OR=0.35, P=0.031) was associated with a reduced risk of CL+CL/P. Conclusions Our results for the first time suggested the DHFR 19-bp D/D genotype may confer a reduced risk of NS-CL/P and might act as a protective factor against NS-CL/P in the Iranian subjects. .

Detection of RFC,DHFR and GST-? expression in human osteosarcoma cell line with MTX-resistence by FQ-PCR / 中国矫形外科杂志

[Objective]To study the difference in expression of RFC, GST-?, DHFRmRNA between human osteosarcoma U2-OS cell line and the MTX-resisitant variants U2-OS/R1-R3, and to investigate the significance in MTX resistance for human osteosarcoma. [Methods]Three resistant MTX human osteosarcoma cell lines were established by pulse exposure parental cell line(U2-OS) in gradually increased dose of MTX . The expression of RFC,GST-?,DHFRmRNA were assayed by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR).[Results]Three MTX-resistant variants(U2-OS/R1-R3) were successfully established , the results of the FQ-PCR revealed that the MTX resistance was associated with the decreased expression of the RFC mRNA and increased expression of DHFR mRNA and GST-? mRNA.[Conclusion]The author investigated the MTX resistant mechanism of human osteosarcoma cell line at a gene level. The decreased expression of RFC mRNA and the increased expression of DHFR mRNA and GST-? mRNA participate in the MTX resistance in human osteosarcoma cell lines U-2 OS. This provides the evidence for exploring the MTX resistance mechanism in clinical osteosarcoma patients ,and helps to screen the patients who are insensitive to MTX chemotherapy.