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.

In silico study to predict promiscuous T cell and B cell-epitopes derived from the vaccine candidate antigens of Plasmodium vivax binding to MHC class-II alleles

Malaria is one of the major causes of health and disability globally, even after tremendous efforts to eradicate it. Till date no highly effective vaccine is available for its control. The primary reason for the low efficacy of vaccines is extensive polymorphism in potential vaccine candidate antigen genes and HLA polymorphisms in the human population. This problem can be resolved by developing a vaccine using promiscuous peptides to combine the number of HLA alleles. This study predicted T and B cell epitopes (promiscuous peptides) by targeting PPPK-DHPS and DHFR-TS proteins of Plasmodium vivax, using different in silico tools. Selected peptides were characterized as promiscuous peptides on the basis of their immunogenicity, antigenicity and hydrophobicity. Furthermore, to confirm their immunogenicity, these peptides were utilized for molecular modelling and docking analysis. For determining the requisite affinity with distinct HLA Class-I, and HLA Class-II alleles, only five peptides for DHFR-TS and 3 peptides for PPPK-DHPS were chosen as promiscuous peptides. The D1 peptide has the maximum binding energy with HLA alleles, according to HLA-peptide complex modelling and binding interaction analyses. These findings could lead to the development of epitope-based vaccinations with improved safety and efficacy. These epitopes could be major vaccine targets in P. vivax as they possess a higher number of promiscuous peptides. Also, the B cell epitopes possess maximum affinity towards different alleles as analyzed by docking scores. However, further investigation is warranted in vitro and in vivo.

Elevated folic acid results in contrasting cancer cell line growth with implications for mandatory folic acid fortification / 한국영양학회지

PURPOSE: The initiation of mandatory folic acid fortification using pteroylmonoglutamic acid (PteGlu) has reduced the rate of congenital malformations. However, it also appears to be responsible for several adverse effects, including increased cancer incidence. This may be related to physicho-chemical characteristics of PteGlu. This study examines the potential effect of high concentrations of PteGlu on a population subjected to mandatory folic acid fortification using an in vitro model. METHODS: Caco-2 (colorectal cancer) and MCF7 (breast cancer) cell lines were cultured at 6 different PteGlu concentrations (0, 0.1, 1, 50, 250, and 500µg/ml) for 6 days. Cell growth was determined using thiazolyl blue tetrazolium bromide assay. The genotype of dihydrofolate reductase 19bp deletion/insertion (DHFR 19-del) was also scored in cell lines using a restriction fragment length polymorphism technique to examine whether genetic variations may factor in cell proliferation. RESULTS: PteGlu exhibited differential growth promoting properties between cell lines. Caco-2 cells did not show a significant growth difference at low concentrations compared to control, however, at higher concentrations, the growth showed a contrasting trend in the early experimental period, while MCF7 showed enhanced cell growth at all concentrations. The DHFR 19-del genotype differed in the two cell lines. CONCLUSION: Altered response to PteGlu by Caco-2 and MCF7 may reflect a tissue specific disease aetiology or genotype specific differential enzyme activity, for example by DHFR, to critical levels of PteGlu. As folic acid fortification is a blanket intervention, and DHFR and other enzyme activities vary between individuals, PteGlu intake may have an as yet undefined effect on health. These findings may be relevant when considering mandatory folic acid fortification for disease prevention.

Association of rs1 1 742688 polymorphism of DHFR gene with non-syndromie cleft lip with or without cleft palate in the northeast of China / 实用口腔医学杂志

Objective:To investigate the association between dihyrofolate reductase(DHFR)gene rs1 1 742688 polymorphism and non-syndrom cleft lip with or without cleft palate (NSCL/P)in northest Chinese population.Methods:PCR-restriction fragment length polymorphism(PCR-RFLP)was used to identify the rs1 1 742688 polymorphism of DHFR gene of 220 NSCL/P patients(inclu-ding 1 38 core families)and 1 80 healthy controls.Hardy-Weinberg test and SPSS statistical software were used to calculate the data, OR and 95% confidence intervalarents.Results:In case-contral analysis,there was no significant difference in TT genotype of rs1 1 742688 between NSCL/P subjects and the controls(χ2 =0.439,P >0.05)in.Conclusion:The polymorphism of rs1 1 742688 in DHFR gene is not associated with NSCL/P in northest Chinese population.

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. .

High-throughput virtual screening for novel dhfr inhibitors of P. Jiroveci from the zinc database.

Many of the opportunistic infections that occur at this late stage can be fatal and since that Pneumocystis carinii pneumonia (PCP) is a leading opportunistic infection found among immunocompromised (CD4 cell < 200) patients worldwide. DHFR is responsible for the growth and maturation of sporozoites stage (life cycle) in Pneumocystis as reported. Currently, 13 million chemical compounds are available for virtual screening in ZINC database. The biological information of four known drug molecules like TMP/SMX, Dapsone, Atovaquone and Pentamidine were collected from the PubChem compound database. Q-Site Finder online tool was used to determine the active site of DHFR in P. jiroveci. LogP values of chemical compounds were identified with the Atom-additive method. Since, existing drugs are synthetic chemicals that give more side effects in Pneumocystis affected patients. Polar surface area value of oxamide (86.18) was predicted to be in the ranges of existing drug values. Pentamidine was proved to be a more efficient ligand based on the dock score of -26.3398 still could not be considered as the natural compound oxamide also was highly comparable with the value of -20.3173. The binding affinity of the selected molecule was analyzed through Pose View and LigPlot.

Status of dhps and dhfr genes of Plasmodium falciparum in Colombia before artemisinin based treatment policy / Estado de los genes dhps y dhfr de Plasmodium falciparum en Colombia antes de la recomendación de tratamiento basado en artemisinina

Introduction: Surveillance of the genetic characteristics of dhps and dhfr can be useful to outline guidelines for application of intermittent preventive therapy in Northwest Colombia and to define the future use of antifolates in artemisinin-based combination therapy schemes. Objective: To evaluate the frequency of mutations in dhps and dhfr and to characterize parasite populations using msp-1, msp-2 and glurp in historic samples before artemisinin-based therapy was implemented in the country. Methods: A controlled clinical study was carried out on randomly selected Plasmodium falciparum infected volunteers of Northwest Colombia (Turbo and Zaragoza). A sample size of 25 subjects per region was calculated. Treatment efficacy to antifolates was assessed. Molecular analyses included P. falciparum genotypes by msp-1, msp-2 and glurp and evaluation of the status of codons 16, 51, 59, 108 and 164 of dhfr and 436, 437, 540, 581 and 613 of dhps. Results: In total 78 subjects were recruited. A maximum number of 4 genotypes were detected by msp-1, msp-2 and glurp. Codons 16, 59 and 164 of the dhfr gene exhibited the wild-type form, while codons 51 and 108 were mutant. In the dhps gene, the mutant 437 glycine was detected in 85% on day 0, while codons 436, 540, 581 and 613 were wild-type. Conclusions: Plasmodium falciparum populations were very homogeneous in this region of Colombia, and the triple mutants of dhfr and dhps Asn108, Ile51 and Gly437 were predominant in clinical isolates.

Introducción. La vigilancia de las características genéticas de dhps y dhfr puede utilizarse para delinear guías de aplicación de terapia preventiva intermitente en el nordeste de Colombia y para definir el uso futuro de los antifolatos en esquemas terapéuticos basados en artemisinina. Objetivo. Evaluar la frecuencia de mutaciones en dhps y dhfr, y caracterizar las poblaciones parasitarias usando msp-1, msp-2 y glurp, en muestras históricas obtenidas antes de la implementación en el país de la terapia basada en artemisinina. Métodos. Se llevó a cabo un estudio clínico controlado en voluntarios infectados con Plasmodium falciparum seleccionados aleatoriamente y provenientes del nordeste de Colombia (Turbo y Zaragoza). Se calculó una muestra de 25 sujetos por región. Se evaluó la eficacia al tratamiento con antifolatos. Los análisis moleculares incluyeron la obtención de genotipos de msp-1, msp-2 y glurp y el estado de los codones 16, 51, 59, 108 y 164 de dhfr, y 436, 437, 5540, 581 y 613 de dhps. Resultados. Se estudiaron 78 sujetos. Se detectó un número máximo de 4 genotipos con msp-1, msp-2 y glurp. Los codones 16, 59 y 164 del gen dhfr se encontraron en su forma silvestre, mientras que los codones 51 y 108 estaban mutados. En el gen dhps, la forma mutante (glicina) en el codón 437, se detectó en 85% el día 0, mientras que los codones 436, 540, 581 y 613 se encontraron silvestres. Conclusiones. Las poblaciones de P. falciparum son muy homogéneas en esta región de Colombia y las triple mutantes de dhfr y dhps Asn108, Ile51 and Gly437, predominaron en los aislamientos clínicos.

Expression of truncated fragment of human OPG in CHO-DHFR- cells and its bioactivity characterization / 细胞与分子免疫学杂志

AIM: To obtain high level expression of recombinant human truncated osteoprotegerin (TOPG) with higher bioactivity in CHO-DHFR~-cells. METHODS: The recombinant vector pcDNA3.1/DHFR-TOPG was constructed and transfected into CHO-DHFR~- Cells by the directions of LipofectAMINE~(TM)2000 for stable expression. The stable expression cell strains were screened by selective medium IMDM with 50 mL/L FCS, then serially passed in methotraxate (MTX) for gene amplification. The expression were analyzed by ELISA and RT-PCR. At last, the bioactivity analysis was performed in vitro. RESULTS: The expression level of recombinant truncated human OPG was up to 6 mg/L·72 h, and it had significant suppression effect on the formation of OLC(P<0.05). CONCLUSION: Recombinant truncated human OPG has high expression and bioactivity. The results make it possible for further studying and clinical implying of OPG.

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.

Construction and Eukaryotic Expressing of GPI-Anchored Met-RANTES / 中国生物工程杂志

Objective: To express glycosyl-phosphatidylinositol (GPI) modified Met- RANTES fusion protein on Chinese hamster ovary (CHO) cells and to develop a novel immunosuppressant GPI anchored form of Met-RANTES. Methods: The eukaryotic expression vector PEF/GPI-Met-RANTES were constructed and transfected into CHO cells by electroporation. The transfectants were selected with methotrexate (MTX). Expression of the recombinant protein was assessed by flow cytometric analysis, cell immunofluorescence staining and immunogold electron microscopy. Results: The chimeric molecules of GPI anchored form of Met-RANTES including the whole reading frame were constructed, and the sequence was identical to the designed sequence. GPI anchored form of Met-RANTES was stably expressed on CHO- DHFR- cells. Conclusion: A large amount of GPI modified Met-RANTES fusion protein was expressed on CHO cells. GPI anchored form of Met-RANTES may be used as novel immunosuppressant for suppressing reaction in graft rejection.

Preliminary study of hypothermia cell living system in CHO(dhfr-) cells culture / 中国康复理论与实践

@#ObjectiveTo validate the feasibility of the outer space carrying system in culturing the CHO(dhfr-)cells in hypothermia, and to observe the effects of this carrying system on the growth characteristics of the CHO(dhfr-) cells.MethodsThe growth characteristics of the CHO(dhfr-) cells were observed and analyzed with cell morphological observation, MTT assay, FCM,3H incorporation and chromosome after the cells were cultured for 25 days in the carrying system. ResultsComparing with the control group, the CHO(dhfr-) cells appeared multiple cell morphological changes, the difference of cell cycle was not significant, the broken chromosome was not seen, the cell growth speed decreased markedly and big molecular biosynthesis increased obviously. ConclusionThe outer space carrying system has no outstanding effects on the survival and heredity of the CHO(dhfr-) cells, so that it can be used in cell carry.

Characteristic of growth decreased CHO(dhfr-) cell line mutated by outer space / 中国康复理论与实践

@#ObjectiveTo investigate the changes of the morphology, growth and cycle of CHO(dhfr-) cells after space flight.MethodsCHO(dhfr-) cells were carried in the No.18 recoverable satellite and monocloned harvesting cells before multiplying. 4 cell lines were selected randomly,and the growth characteristics of the most slowly growing one at the fifth passage was observed by methods of MTT and FCM as well as the cells' shape.Results159 cell strains were obtained after monocloning and multiplying. The cells' morphology changes, growth speed decrease and the number of G1 phase increased markedly.ConclusionSpace flight induced morphological changes of cells and it is impossible to screen out finer bioengineering cells.

Expression on dhfr-CHO cells and activity study of CD137 (human 4-1BB) / 中国免疫学杂志

Objective:To set up a eukaryotic system for high expressing human CD137 and to investigate the role of CD137 and CD137L on signals transduction of cells.Methods:The pCDNA3 plasmid containing full length of human CD137 cDNA sequence(CMV ILA SEN,CIS) and pSV2 dhfr plasmid were cotransfected into dhfr CHO cells by lipoid mediating method. The positive clone was selected with G418. Expression of CD137 on dhfr CHO cells were induced by MTX and detected by RT PCR, immunocytochemistry and flow cytometry.It's activity study was done by method of incorporating 3H TdR.Results:CD137 expressed on the surface of dhfr CHO, expression rate was 96.07%, it's activity study indicated that CD137 increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.Conclusion:dhfr CHO cells that highly express CD137 were established. CD137 can increase the proliferation of PBMC stimulated by anti CD3 monoclonal antibody.

Gene Point Mutation in the Dihydrofolate Reductase-thymidylate Synthase Gene of Plasmodium falciparum / 中国寄生虫学与寄生虫病杂志

Objective To investigate the gene point mutation in the dihydrofolate reductase thymidylate synthase (dhfr) gene of Plasmodium falciparum isolate from Yunnan Province strongly associated with pyrimethamine and cycloguanil resistance. Methods Nested PCR and restriction endonuclease digestion were applied to detect the gene mutation using dried blood filter paper collected from the fields in Yunnan Province. Results Different mutations were found in 4 amino acids at positions 16, 51, 108 and 164 of dhfr gene, particularly, Asn 108 and Ile 51, the mutaiton frequency being 94.1% and 90.1%, respectively. The frequency of the wild type genotype (3D7 type) Ser 108 appeared lower ( 9.1%) , while the frequency of the Ala 16 was high( 61.8%); the mutation type was very high, the ratio of HB3 type, 7G8 type/FCR3 type and Cambodian type was 1∶21∶7.5. Conclusion The investigation first demonstrated that Plasmodium falciparum Yunnan isolate dihydrofolate reductase thymidylate synthase gene(dhfr) at positions 16, 51 ,108 and 164 exhibited different degrees of point mutation. The frequency of mutation of the 7D8 type involved in pyrimethamine resistance was higher, while that of the FCR3 type involved in cycloquanil resistance was lower.