75 results about "Dihydrofolic acid" patented technology

Methods for producing modified polypeptides containing amino acid analogues are disclosed. The invention further provides purified dihydrofolate reductase polypeptides, produced by the methods of the invention, in which the methionine residues have been replaced with homoallyglycine, homoproparglycine, norvaline, norleucine, cis-crotylglycine, trans-crotylglycine, 2-aminoheptanoic acid, 2-butynylglycine and allylglycine.

The invention discloses a Chinese hamster ovary (CHO) genetic engineering cell line for performing high-level secretory expression on AcAPc2 and relates to a CHO cell line for performing high-level secretory expression on the AcAPc2. The invention aims to solve the problems that the expression level of an exogenous gene product expressed by using CHO is low at present and the industrialized production cannot be realized. The CHO genetic engineering cell line for performing high-level secretory expression on the AcAPc2 is obtained by the following step of: screening dihydrofolate reductase-deficient CHO cells which are taken as host cells, and amplifying to obtain the cell line for performing stable and high-level expression on the AcAPc2. The CHO cell line can perform high-level expression on the AcAPc2; the expression level can be up to 10mg / L.72h; and the CHO cell line can be industrially produced. The cell line is used for resisting blood coagulation and treating tumor, septicaemiaand the like.

Sensitivity of a patient's cancer to treatment with 10-propargyl-10-deazaaminopterin is assessed and patients are selected for treatment of cancer with 10-propargyl-10-deazaaminopterin, by determining the amount of a selected polypeptide expressed by the cancer and comparing the amount with the amount of the selected polypeptide expressed by a reference cancer. The polypeptide includes a member of a folate pathway polypeptide within a cell and may include at least one of reduced folate carrier-1 enzyme (RFC-1), dihydrofolate reductase (DHFR), folylpoly-gamma-glutamate synthetase (FPGS), thymidylate synthase (TS), γ-glutamyl hydrolase (GGH), and glycinamide ribonucleotide formyltransferase (GARFT).

Compounds of formula (I) or (II) are dihydrofolate reductase inhibitors, useful for the treatment of, for example, cell proliferative diseases:wherein A and D are independently —CHR7— or —NR7—; E and G are independently ═CR7— or ═N—; each R6 independently represents hydrogen or —OR7; R7 is hydrogen or C1-C6 alkyl; R1 is a carboxylic acid group (—COOH), or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group; R2 is the side chain of a natural or non-natural alpha amino acid which does not contain a carboxyl, or carboxyl ester group; Y is a bond, —C(═O)—, —S(═O)2—, —C(═O)NR3—, —C(═S)—NR3, —C(═NH)NR3 or —S(═O)2NR3— wherein R3 is hydrogen or optionally substituted C1-C6 alkyl; L1 is a divalent radical of formula -(Alk1)m(Q)n(Alk2)p- wherein m, n and p are independently 0 or 1, and Q, Alk1 and Alk2 are as defined in the claims; X1 represents a bond; —C(═O); or —S(═O)2—; —NR4C(═O)—, —C(═O)NR4—, —NR4C(═O)NR5—, —NR4S(═O)2—, or —S(═O)2NR4— wherein R4 and R5 are independently hydrogen or optionally substituted C1-C6 alkyl; and z is 0 or 1.

A Chinese hamster ovary genetic engineering cell line capable of efficiently expressing Ancylostoma caninumanticoagulant peptide 5 (AcAP5) in secretion mode relates to a Chinese hamster ovary (CHO) cell line capable of efficiently expressing the AcAP5 in secretion mode. The cell line resolves the problem that at present, expression amount is low by using CHO to express exogenous gene products, and requirements of industrial production cannot be met. The Chinese hamster ovary genetic engineering cell line for the efficient secretion expression AcAP5 utilizes dihydrofolic acid reductase defect type Chinese hamster ovary cells as host cells, and the cell line capable of stably and efficiently expressing AcAP5 is obtained after the host cells are screened and amplified. The CHO cell line can efficiently express the AcAP5, can be produced in industrialization mode and is used for resisting blood coagulation and thrombus. Furthermore, expression amount can reach to 12 mg / L / 72h.

The invention provides a biosynthesis method for increasing accumulation of L-5-methyltetrahydrofolate, and an L-5-methyltetrahydrofolate synthetase system co-expressed recombinant plasmid and a construction method and application thereof. The L-5-methyltetrahydrofolate synthetase system co-expressed recombinant plasmid comprises DHFR (dihydrofolate reductase) gene folA and a MTHFR (methylenetetrahydrofolate reductase) gene metF sequence. The biosynthesis method for increasing accumulation of L-5-methyltetrahydrofolate includes converting the L-5-methyltetrahydrofolate synthetase system co-expressed recombinant plasmid to accumulate an original strain of the L-5-methyltetrahydrofolate so as to obtain a recombinant strain, and fermenting the recombinant strain. The accumulation of the L-5-methyltetrahydrofolate in final fermentation product is evidently higher than that of the L-5-methyltetrahydrofolate in the original strain. Utilization rate of raw materials is increased, production cost and energy consumption are reduced, and a foundation for industrial biosynthesis of the L-5-methyltetrahydrofolate is laid.

The present inventors conducted dedicated studies and successfully constructed expression vectors that enable high-level production of foreign gene-derived proteins in mammalian host cells, which comprise a translation-impaired dihydrofolate reductase gene cistron whose expression has been attenuated by altering the codons to the least frequently used codons in mammals; and a gene cassette which has a cloning site for incorporation of a foreign gene between a highly transcriptionally active promoter and a highly stable polyadenylation signal.

A group of 12 carriers with efficient expression for preparing the humanized antibody of mammalian is prepared by directional combination of different elements, that is, the eukaryotic cell expression elements including strong promoter EF-1 alpha, the gene in light-or heavy-chain constent region of the antibody molecular for human genom DNA or message RNA, intrinsic ribosomal recognition site and dihydrofolate reductase gene. It can be used to clone antibody virable region or Fab gene and introduce it into CHO cell to obtain permanently efficiency expression to different antibody genes.

The compositions and methods described herein discloses the design, synthesis and testing of compounds that act as inhibitors of DHFR. The basic scaffold of these inhibitors includes a 2,4-diaminopyrimidine ring with a propargyl linker to another substituted aryl, bicyclo or heteroaryl ring. These DHFR inhibitors are potent and selective for many different pathogenic organisms, including the DHFR enzyme from bacteria such as Bacillus anthracis and methicillin-resistant Staphylococcus aureus, fungi such as Candida glabrata, Candida albicans and Cryptococcus neoformans and protozoa such as Cryptosporidium hominis and Toxoplasma gondii. These compounds and other similar compounds are also potent against the mammalian enzyme and may be useful as anti-cancer therapeutics.

The invention discloses a GS-DHFRmut double-gene screening expression vector, and a preparation method and an application thereof, and belongs to the biotech field. The expression vector contains a promoter controlling the expression of a glutamine synthetase gene, the glutamine synthetase gene, a promoter controlling the expression of a mutant dihydrofolate reductase gene, and the mutant dihydrofolate reductase gene, and the downstream of each of two screening genes has a PolyA signal sequence. The invention also discloses the preparation method of the expression vector and the application of the expression vector. The GS-DHFRmut double-gene screening expression vector improves the expression level of an exogenous protein, enlarges the use resource of host cell strains in the genetic engineering, and has an important application value in the industrialized development of the genetic engineering.

Compounds of the formula I: wherein R1, R2, R1' and R2' are independently hydrogen or a group releasing the free amine in vivo, R6 is a substituted phenyl or an optionally substituted, bicyclic or tricyclic aryl ring system or an optionally substituted mono, bi- or tricyclic heteroaryl ring system having utility as DHFR inhibitors with favourable pharmacokinetic properties.

The invention provides a combined medicine having efficacy of antineoplastic drugs. The combined medicine consists of chlorogenic acid and a dihydrofolate reductase inhibitor of unit preparations in same or different specifications, and a pharmaceutically acceptable carrier, wherein the chlorogenic acid and the dihydrofolate reductase inhibitor are applied in a simultaneous or separated mode. According to the combined medicine, a synergistic effect can be developed when the chlorogenic acid and the DHFR (dihydrofolate reductase) inhibitor are used in a combined mode, and hematopoietic function injury and weight loss caused by the DHFR inhibitor can be relieved; toxic and side effects of the DHFR inhibitor can be effectively reduced; and the medicine, which combines the chlorogenic acid and the DHFR inhibitor is excellent in curative effect, low in toxicity and good in prospect of clinical application.

The present disclosure provides compounds of Formula I:or a pharmaceutically acceptable salt thereof, wherein R5, R6 and Z are as described herein. The disclosure also provides pharmaceutical compositions thereof; and methods for inhibiting DHFR activity; and methods for treating cell proliferative diseases, autoimmune disease, inflammatory disease or bacterial, fungal or parasitic infection by administering a compound of Formula I.

Compounds of formula (I) or (II) are dihydrofolate reductase inhibitors, useful for the treatment of, for example, cell proliferative diseases:wherein A and D are independently —CHR7— or —NR7—; E and G are independently ═CR7— or ═N—; each R6 independently represents hydrogen or —OR7; R7 is hydrogen or C1-C6 alkyl; R1 is a carboxylic acid group (—COOH), or an ester group which is hydrolysable by one or more intracellular carboxylesterase enzymes to a carboxylic acid group; R2 is the side chain of a natural or non-natural alpha amino acid which does not contain a carboxyl, or carboxyl ester group; Y is a bond, —C(═O)—, —S(═O)2—, —C(═O)NR3—, —C(═S)—NR3, —C(═NH)NR3 or —S(═O)2NR3— wherein R3 is hydrogen or optionally substituted C1-C6 alkyl; L1 is a divalent radical of formula -(Alk1)m(Q)n(Alk2)p- wherein m, n and p are independently 0 or 1, and Q, Alk1 and Alk2 are as defined in the claims; X1 represents a bond; —C(═O); or —S(═O)2—; —NR4C(═O)—, —C(═O)NR4—, —NR4C(═O)NR5—, —NR4S(═O)2—, or —S(═O)2NR4— wherein R4 and R5 are independently hydrogen or optionally substituted C1-C6 alkyl; and z is 0 or 1.

A process for the large-scale chemoenzymatic production of (6S)-5-methyl-5,6,7,8-tetrahydrofolic acid, also known as (6S)-5-methylTHFA, the process comprising the steps of: (1) reducing folic acid (FA) so as to yield dihydrofolic acid (DHFA); (2) stereoselectively reducing DHFA with dihydrofolate reductase (DHFR) in the presence of NADP / NADPH, glucose and GluDH so as to yield (6S)-THFA; (3) converting (6S)-THFA to (6S)-5-methylTHFA; and (4) isolating (6S)-5-methylTHFA.

The invention discloses derivatives and salts of damino dihydrotriazine, and a preparation method, a composition and application thereof. According to the invention, the preparation method of the damino dihydrotriazine derivative and the damino dihydrotriazine salt can be realized by adopting a method I or a method II, wherein the method I includes the step of obtaining a general formula I compound prepared through the reaction between a general formula IV compound and a general formula V compound, while the method II includes the step of mixing a general formula VIII compound with a general formula II compound under an acidic condition, and obtaining the compound shown in the general formula I through a cyclization reaction of the mixture. The invention also provides application of derivatives and salts of the damino dihydrotriazine in preparation of human dihydrofolate reductase inhibitors, preventing and curing drugs for tumor or bacterial infection diseases. The invention further provides a drug composition, which comprises an effective amount of the derivatives and / or salts of the damino dihydrotriazine, as well as pharmaceutically acceptable carriers. According to the invention, spiro heterocyclic ring derivatives of the damino dihydrotriazine have an excellent inhibitory activity on human dihydrofolate reductase, tumor cells and bacteria.

Cells and cell lines are disclosed that are able to produce therapeutic proteins, antibodies, vectors, and viral vectors such as lentiviral vectors and adeno-associated viral (AAV) vectors. The cells and / or cell lines can have mutations or deletions in either one or both of the endogenous di-hydrofolate reductase (DHFR− / −) or glutamine synthetase (GS− / −) genes such that DHFR and / or GS expression or function is substantially reduced or eliminated.

A novel screening assay for identifying therapeutic agents and their cellular targets is described. The assay is useful in developing new antibacterial, antifungal, antiparasitic and anti cancer therapeutics. New inhibitors of dihydrofolate reductase (DHFR) have been identified and their cellular target confirmed using the assay of the present invention. Methods of treating diseases that benefit from an inhibition of DHFR are also described.

The invention discloses a recombinant CHO cell strain capable of stably expressing GPC3 and an application thereof. The preservation number of the CHO cell strain is CGMCC No.18881, the GPC3 is encoded by a cDNA sequence shown in the formula of SEQ ID No. 1, an antigen produced by the recombinant CHO cell strain is used for preparing a GPC3 monoclonal antibody, and the recombinant CHO cell strainis used for preparing a GPC3 antibody. The folate reductase defect type CHO cells are lack of endogenous dihydrofolate reductase, when an expression vector containing a dihydrofolate reductase gene and a target gene is co-transfected, the dihydrofolate reductase gene and the target gene are generally integrated at the same site of a host chromosome, and then in the presence of a competitive inhibitor-methotrexate of dihydrofolate reductase, the increasing of the copy number of the dhfr gene is driven to achieve the purpose of increasing the expression quantity of the target gene, so that a stable cell line for stably and efficiently expressing GPC3 is constructed, and the technical blank is filled.

The invention relates to a reagent (kit) for measuring formaldehyde by using an enzyme-multiplied method, an enzyme colorimetric method and an enzyme coupling method as well as a method for measuring the concentration of the formaldehyde, and composition and ingredients of the reagent, belonging to the technical field of food / environmental test. The reagent (kit) comprises the main ingredients of buffer solution, coenzyme, adenosine triphosphoric acid, tetrahydrofolic acid, glyceric aldehyde-3-phosphoric acid, formaldehyde dehydrogenase, formic acid-dihydrofolic acid ligase, glyceric aldehyde-3-glycerol phosphate dehydrogenase and stabilizing agent. The concentration of the formaldehyde is measured by mixing a sample and the reagent according to a certain volume ratio to carry out a series of enzymatic reaction, placing the reactant under an ultraviolet / visible light analyzer and detecting the ascending degree of absorbance at a dominant wavelength of 340nm.

The invention belongs to the technical field of enzymatic synthesis, and relates to a method for preparing L-5-calcium methyl tetrahydrofolate through an enzymic method. The method comprises the following steps that 1, folic acid serves as the raw material, and zinc powder is used for making zinc powder reduced to be dihydrofolic acid; 2, dihydrofolic acid reductase and glucose dehydrogenase are added in dihydrofolic acid, and a reaction is conducted to obtain L-tetrahydrofolic acid; 3, formaldehyde and hydroboron are added in L-tetrahydrofolic acid, and a reaction is conducted for obtaining L-5-methyltetrahydrofolate; 4, L-5-methyltetrahydrofolate is salified, and L-5-calcium methyl tetrahydrofolate is obtained. Accordingly, the effective method for preparing L-5-calcium methyl tetrahydrofolate through the enzymic method is provided, the preparation method is free of splitting, mild in reaction, high in yield, high in product purity and friendly to environment, and has a very good industrial prospect.

The invention relates to a homocysteine diagnosis / determination reagent (kit) using enzyme-colorimetry and enzyme-linked method technology. Meanwhile, the invention also relates to a homocysteine concentration determination method, reagent composition and component, which belongs to the technical filed of medical test and determination. The reagent (box) of the invention mainly includes: buffer solution, coenzyme, 5-methyltetrahydrofolate, methionine synzyme, dihydrofolate reductase and stabilizing agent; samples and reagent are mixed according to a certain volume ratio to generate a series of enzymatic reactions; then the reactants are arranged under an ultraviolet / visible light analyzer to detect the increasing degree of absorbance at the 340nm position of a dominant wave so as to measure and calculate the concentration of the homocysteine.

A method for identifying a protein as being able to bind a ligand comprising, providing a molecule composed of a methotrexate moiety that is covalently bonded to the ligand; introducing the molecule into a cell which a) expresses a first fusion protein comprising a dihydrofolate reductase capable of binding methotrexate, expresses b) a second fusion protein comprising the protein, wherein one of the first and second fusion proteins also comprises a transcription activator domain and the other comprises a DNA-binding domain, and c) has a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first fusion protein to the second fusion protein; permitting the molecule to bind to the first fusion protein and to the second fusion protein so as to activate the expression of the reporter gene; and selecting the cell if it expresses the reporter gene.

The invention aims at providing an application of dihydrofolate synthetase in detection of sulphonamide residue and a rapid sulphonamide detection method constructed by utilizing the dihydrofolate synthetase. The dihydrofolate synthetase capable of detecting the sulphonamide residue is an amino acid sequence shown in a sequence SEQ ID NO.1. Compared with the traditional detection method, the dihydrofolate synthetase has the advantages that firstly the dihydrofolate synthetase is wide in source, protein fragments just containing a dihydrofolate synthetase core sequence can be applied, and the dihydrofolate synthetase can be rapidly and stably obtained by adopting a genetic engineering method; and secondly, a rapid detection method for all the sulphonamides containing a p-aminobenzene sulphonamide parent core structure can be constructed by utilizing the new application of the dihydrofolate synthetase.

Novel medicinal compositions containing anti-Fas antibody which are useful as preventives or remedies for autoimmune diseases or rheumatoid arthritis. More particularly, medicinal compositions containing as the active ingredients antihuman Fas antibody having an activity of inducing apoptosis and a compound having an antagonism to folic acid or an effect of inhibiting dihydrofolate reductase. By using these medicinal compositions, the content of anti-Fas antibody can be reduced in preventives or remedies for autoimmune diseases or rheumatoid arthritis containing anti-Fas antibody. Thus, the possibility of the appearance of the tolerance in the patient's body due to the expression of an antibody against the anti-Fas antibody, etc., which makes it possible to provide excellent preventives or remedies usable in long-term administration.

The invention provides a DHFR-based multi-residue fluorescence polarization immunoassay method for sulfonamide synergist drugs, which is characterized in that dihydrofolate reductase DHFR is derived from sulfanilamide synergist drug sensitive strains, including staphylococcus aureus, streptococcus pneumoniae and mycobacterium tuberculosis. According to the invention, a sulfanilamide synergist drug multi-residue fluorescence polarization immunoassay method based on the sulfanilamide synergist drug receptor protein dihydrofolate reductase is established for the first time, the established method is applied to multi-residue detection of sulfanilamide synergist drugs in milk, and a method technical support is provided for screening of sulfanilamide synergist drugs in food. According to the invention, DHFR from sulfanilamide synergist drug sensitive strains is creatively adopted, and the method is initiated in the field. The method breaks through the limitation of the conventional method, expands the application range, and has the advantages of rapidness, simplicity, accuracy and the like.

The invention discloses a method for preparing a receptor-ligand binding sulfonamide antibiotic protein. The method comprises the following steps: firstly preparing a prokaryotic expression vector, and then isolating and purifying Escherichia coli strains to obtain dihydrofolate synthase; with a genome of Escherichia coli as a template, taking the obtained dihydrofolate synthetase gene fragment and cloning into the prokaryotic expression vector to obtain a cloning vector, and extracting recombinant plasmids from the cloning vector and the prokaryotic expression vector; culturing the recombinant plasmids and Escherichia coli in a culture medium to obtain a positive clone strain, and then continuously culturing the positive clone strain in the culture medium and centrifuging to obtain a thallus; concentrating the thallus and polyethylene glycol octylphenyl ether, and centrifuging to obtain a protein; and finally purifying the protein. According to the method for preparing the receptor-ligand binding sulfonamide antibiotic protein provided by the invention, a rapid detection product produced under the action of high binding force has sensitivity higher than that of the same product produced by an antibody binding principle.

The invention provides a sgRNA sequence for specifically knocking-out a DHFR (Dihydrofolate Reductase) gene. The sequence is as shown in SEQ ID NO:3. The invention also provides the application of the sgRNA sequence and a kit for knocking-out the DHFR gene. The sgRNA which is provided by the invention can be used for knocking-out the DHFR gene in a CHO (Chinese Hamster Ovary) cell quickly, efficiency and specifically, and obtaining a DHFR gene-deleted cell line; the yield of foreign protein can be greatly improved; the application prospect is good.