Folate Complex Research Articles

Двусторонняя посттромботическая ретинопатия как проявление ретиноваскулита на фоне генетически обусловленной тромбофилии

Purpose. To analyze current literature data on the topic of thrombotic and vasoocclusive lesions of retinal vessels in conditions of thrombophilia and endotheliopathy to determine the tactics of management of patients with this pathology. Material and methods. A search and analysis of current research published in the databases PubMed, eLibrary, Springer, ScienceDirect and Google Scholar has been performed. A clinical case of bilateral postthrombotic retinopathy with the phenomena of vasculitis and fibrovascular proliferation, which developed against the background of thrombophilia in a 34-year-old woman, is also described. Results. Retinal vasoocclusion occurred in conditions of thrombophilia due to hyperproduction of factor VIII and von Willebrand factor (vWF), in the presence of genetic markers of thrombogenic risk and a history of pulmonary embolism. Mutations F13, PAI-1, FBG, ITGA2 and ITGB3 demonstrate a moderate association with vasoocclusive retinal damage, while the genes of the folate complex enzymes MTRR and MTHFR are associated with thrombophilia due to hyperhomocysteinemia, endotheliopathy and the development of heart defects that increase the risk of occlusion of the CRV and its branches. Dynamic assessment of the condition of retinal vessels according to optical coherence tomography in angiography mode allowed us to confirm the effectiveness of antiVEGF therapy in combination with panretinal laser coagulation, which was accompanied by the restoration of lost visual functions. Conclusion. In order to prevent retinal vascular catastrophes for patients with a similar comorbid background, it is preferable to use multi-stage transpuppillary retinal laser coagulation and anti-VEGF therapy, thereby avoiding vitreoretinal surgical interventions to limit intraoperative and postoperative complications. Key words: postthrombotic retinopathy, neoangiogenesis, thrombophy

Supramolecular complexation between cucurbit[7]uril and folate and analytical applications

Folate (FA) plays a key role in the biosynthesis of amino acids, purines, and pyrimidines in the human body, and intracellular folate metabolism has become an attractive target of tumor chemotherapy. In this work, an inclusion interaction was found between FA and cucurbit[7]uril (CB[7]), and the formation of a CB[7]-FA 2:1 supramolecular inclusion complex was confirmed by fluorescence spectra, UV-Vis absorption spectroscopy, 1H NMR, and molecular modeling calculations. In addition, FA is generally determined through the indirect fluorescent method because it shows weak fluorescence in aqueous solution. Therefore, a simple, direct fluorescence probe method for rapidly measuring FA was investigated, and the linear equation of FA was ΔF = 14.691C + 37.366 within the concentration ranges of 0.82 ~ 18.31 µg mL–1. The proposed direct fluorescence method was applied to the determination of spiked plasma. We demonstrated that this method could provide an experimental basis for the targeted administration of the CB[7]-FA complex, and it could be extended as a promising fluorescence detection method for drugs in vivo.

Evaluation of Silver Nanoparticles Caffeic folate (NSFC) Complex Compound as New Potential Therapeutic Agent against Cancer Xenograft in Mice: Thesis Abstract

Thesis Abstract In the last few decades the properties of silver compounds have been of interest as potential cancer treatments. The present work was designed to study the effect of new conjugated caffeic and folic acid with silver nanoparticle with definite molecular size applied with and without gamma radiation exposure, as an antitumor agent against experimentally induced Ehrlich tumor and attempted to identify their potential molecular mechanisms of action throughout determination of anti-tumor activities using MTT cytotoxic assay against two human carcinoma cell lines in vitro, Apoptosis analysis by flow cytometry through caspase-8, caspase-3 and TNF determine in vivo . Adult female albino mice were used and divided into five groups, Animals were scarified and the following parameters were estimated, glutathione, glutathione peroxidase, superoxide dismutase in blood and caspase8, caspase 3 and TNF from scarified tumor tissue. The tumor specimens were processed for histopathological examination. Nano-silver folate caffeic (NSFC) complex compound treatment resulted in a growth inhibition in Hep-G2 and MCF-7 cells (IC50 7.70 µM and 14.50 µM, respectively). Flow cytometric analysis revealed that (NSFC) with radiation exposed has apoptotic effect at caspases 8, 3 and TNF more than any compound of them alone. That disturbance was found to be associated with a kinetic induction of apoptosis and showed modulation of antioxidant system beside its high percentage of necrotic cells by histopathological studies. the novel synthetic Nano-silver folate Caffeic complex compound may potentially present a new hope for the development of breast cancer therapeutics, which should attract further scientific and pharmaceutical interest.

<p>Synthesis of a vanadyl (IV) folate complex for the treatment of diabetes: spectroscopic, structural, and biological characterization</p>

BackgroundThis study aimed to design a compound with folic acid (FAH2) and vanadyl (IV) for use in the treatment of diabetes.Materials and methodsA novel vanadyl (IV) FAH2 complex was synthesized and characterized [(FA2−)(VO2+)]⋅3H2O. The speculated structure of this folate complex was determined using physicochemical techniques including microanalytical analysis, conductivity studies, spectroscopic examination, magnetic measurements, thermogravimetric analyses, and morphological X-ray powder diffraction, and scanning and transmission electron microscopies. The anti-diabetic therapeutic potential of the complexes was tested in a 30-day streptozotocin-induced diabetes rat model.ResultsThe conductivity test of the complex implied electrolyte behavior. The spectroscopic assessments of the isolated dark yellow solid complex revealed that FAH2 acts as a bidentate ligand. The coordination process with two vanadyl (IV) ions occurred through the deprotonation of both carboxyl groups of FAH2 in a regular square pyramid arrangement at a 2(FA)2−: 2(VO)2+ molar ratio. XRD, SEM, and TEM analyses revealed the complex crystalline nature of the complex. Treating diabetic rats with vanadyl (IV) FAH2 complex significantly improved many biological parameters relevant to diabetes pathology with minimal toxicity.ConclusionThe data generated in this study indicate that the synthesized vanadyl (IV) folate complex acts as a model of anti-diabetic agent.

Folic acid-conjugated chitosan oligosaccharide-magnetic halloysite nanotubes as a delivery system for camptothecin

In this research, to achieve enhanced intracellular uptake of anticancer drug carriers for efficient chemotherapy, folic acid conjugated chitosan oligosaccharides assembled magnetic halloysite nanotubes (FA-COS/MHNTs) have been tailored as multitask drug delivery system towards camptothecin (CPT). Besides magnetic targeting, the nanocomposites have been reacted with folate complex in order to selectively target cancer cells over expressing the folic acid receptor. HNTs showed to have a high storage capacity of CPT. In vitro, the release results indicated that CPT outflow from the nanocarriers at pH 5 was much greater than that at both pH 6.8 and 7.4. MTT assays showed that the CPT-loaded nanocarriers exhibited stronger cell growth inhibitory against colon cancer cell. Furthermore, nanocarriers gained specificity to target cancer cells because of the enhanced cell uptake mediated by FA moiety and presence of COS. Therefore, the rational designed HNTs nanocarrier for chemotherapy drug showed great potential as tumor-targeted drug delivery carrier.

Synthesis, spectroscopic characterizations and biological activities of vanadyl(II) folate compound as a new anti-DNA damage and antioxidant agent

New oxovanadium(IV) folate [(VO)2(FO)(NH4)2(SO4)2] complex was synthesized by the reaction between vanadyl(II) sulfate and folic acid vitamin B9 drug in an alkaline media. Elemental analysis shows 1:2 ligand to metal ion stoichiometry and the conductance data deduced that oxovanadium(IV) folate complex has non-electrolytic nature. IR spectrum of oxovanadium(IV) complex reveal that folic acid acts as a binuclear chelate via deprotonation of both carboxylic groups. The biological section aimed to test the toxicity of carbofuran orally for male rat and oxidative stress of the sub-lethal (2.4mg/kg b.w, 1/25 LD50) dose on the lipid peroxidation level (LPO), reduced glutathione content (GSH), antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities of testicular tissue. The protective efficiency of vanadylfolate (50mg/kg b.w) only or combined together carbofuran was tested. The taken of carbofuran orally led to elevation in LPO level by 2.10 fold in comparable with control. The efficient of antioxidant enzymes using of vanadylfolate in combined with carbofuran or its only were decreased by (25.48%, 18.60%, 22.46% and 16.62%) but the GSH level increased by 37.18% in testicular tissue in comparison with controller sample. The DNA damaging of carbofuran and vanadylfolate were assessed using single cell gel electrophoresis (SCGE) data.

Sequential backbone resonance assignments of the E. coli dihydrofolate reductase Gly67Val mutant: folate complex.

Occasionally, a mutation in an exposed loop region causes a significant change in protein function and/or stability. A single mutation Gly67Val of E. coli dihydrofolate reductase (DHFR) in the exposed CD loop is such an example. We have carried out the chemical shift assignments for H(N), N(H), C(α) and C(β) atoms of the Gly67Val mutant of E. coli DHFR complexed with folate at pH 7.0, 35°C, and then evaluated the H(N), N(H), C(α) and C(β) chemical shift changes caused by the mutation. The result indicates that, while the overall secondary structure remains the same, the single mutation Gly67Val causes site-specific conformational changes of the polypeptide backbone restricted around the adenosine-binding subdomain (residues 38-88) and not in the distant catalytic domain.

Structural and dynamic investigation of bovine folate receptor alpha (FOLR1), and role of ultra-high temperature processing on conformational and thermodynamic characteristics of FOLR1–folate complex

The folate receptor alpha (FOLR1) present in milk has widely been studied to investigate the effects of pasteurization, ultra-high temperature (UHT) processing and fermentation on net folate concentration. However, the folate binding mechanism with FOLR1, and effect of temperature on FOLR1–folate complex is poorly explored till now in bovine milk which is a chief resource of folate. Despite of enormous importance of folic acid and the routine intake of bovine milk, folic acid deficiency diseases are common in human race. To understand the folate deficiency in milk after processing, in absence of experimental structure, 3D model of bovine FOLR1 (bvFOLR1) was built followed by 40ns molecular dynamics (MD) simulation. The folate and its derivatives binding sites in bvFOLR1 were anticipated by molecular docking using AutoDock 4.2. Essential MD studies suggested the presence of a longer signal peptide (22 residues) and a short propeptide (7 residues) at the C-terminus that may cleaved during post-translational modification. MD analysis of bvFOLR1–folate complex at 298, 323, 353, 373 and 408K followed by binding energy (BE) calculation showed maximum binding affinity at ∼353K. However, at 373K and UHT (408K), the folate BE is significantly decreased with substantial conformational alteration. Heating at UHT followed by cooling within 298–408K range demoed no structural reformation with temperature reduction, and the folate was displaced from the active site. This study presented the disintegration of folate from bvFOLR1 during high temperature processing and revealed a lower folate concentration in UHT milk and dairy products.

Flavin-Dependent Thymidylate Synthase as a Drug Target for Deadly Microbes: Mutational Study and a Strategy for Inhibitor Design

The identification of flavin-dependent thymidylate synthase (FDTS) as an essential enzyme and its occurrence in several pathogenic microbes opens opportunities for using FDTS enzyme as an excellent target for new antimicrobial drug discovery. In contrast to the human thymidylate synthase enzyme that utilizes methylene-tetrahydrofolate (CH2H4 folate) for the conversion of dUMP to dTMP, the microbial enzymes utilize an additional non-covalently bound FAD molecule for the hydride transfer from NAD(P)H. The structural and mechanistic differences between the human and microbial enzymes present an attractive opportunity for the design of antimicrobial compounds specific for the pathogens. We have determined the crystal structure of FDTS enzyme in complex with the methyl donor, CH2H4 folate. We describe here the structure of a FDTS mutant and compare it with other FDTS complex structures, including a FDTS-CH2H4 folate complex. We identified a conformational change essential for substrate binding and propose a strategy for the design of FDTS specific inhibitors.

Comparing anti-hyperglycemic activity and acute oral toxicity of three different trivalent chromium complexes in mice

Three different ligands (rutin, folate and stachyose) of chromium(III) complexes were compared to examine whether they have similar effect on anti-hyperglycemic activity as well as the acute toxicity status. Anti-hyperglycemic activities of chromium rutin complex (CrRC), chromium folate complex (CrFC) and chromium stachyose complex (CrSC) were examined in alloxan-induced diabetic mice with daily oral gavage for a period of 2weeks at the dose of 0.5–3.0mg Cr/kg. Acute toxicities of CrRC and CrFC were tested using ICR mice at the dose of 1.0–5.0g/kg with a single oral gavage and observed for a period of 2weeks. Biological activities results indicated that only CrRC and CrFC could decrease blood glucose level, reduce the activities of aspartate transaminase, alanine transaminase, alkaline phosphatase, and increase liver glycogen level. In acute toxicity study, LD50 values for both CrRC and CrFC were above 5.0g/kg. The minimum lethal dose for CrFC was above 5.0g/kg, while that for CrRC was 1.0g/kg. Anti-diabetic activity of those chromium complexes was not similar and their acute toxicities were also different. CrFC represent an optimal chromium supplement among those chromium complexes with potential therapeutic value to control blood glucose in diabetes and non-toxicity in acute toxicity.

Targeting Tumor Cells through Chitosan-Folate Modified Microcapsules Loaded with Camptothecin

Poly(vinyl alcohol) microcapsules have been tailored as carriers to deliver camptothecin, an anticancer drug poorly soluble in water. The capsules have been reacted with a chitosan--folate complex in order to selectively target cancer cells overexpressing the folic acid receptor. Microcapsules decorated with the chitosan--folate complex have been characterized in their uptake and release of camptothecin, following the absorption band at λ = 370 nm diagnostic of the drug molecule. The selectivity of chitosan-folate microcapsules in targeting cancer cells has been demonstrated by fluorescence microscopy using HeLa cells, overexpressing the folate receptor and NIH3t3 fibroblasts as a negative control. The chitosan--folate microcapsules loaded with camptothecin significantly reduce the proliferation of HeLa tumor cells, while they have a negligible effect on fibroblasts. This work demonstrates that the chitosan--folate microcapsules represent a promising system to selectively target hydrophobic drugs, such as camptothecin, to tumor cells.

Functional Role for the Conformationally Mobile Phenylalanine 223 in the Reaction of Methylenetetrahydrofolate Reductase from Escherichia coli

The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) by NADH via a ping-pong reaction mechanism. Structures of the reduced enzyme in complex with NADH and of the oxidized Glu28Gln enzyme in complex with CH(3)-H(4)folate [Pejchal, R., Sargeant, R., and Ludwig, M. L. (2005) Biochemistry 44, 11447-11457] have revealed Phe223 as a conformationally mobile active site residue. In the NADH complex, the NADH adopts an unusual hairpin conformation and is wedged between the isoalloxazine ring of the FAD and the side chain of Phe223. In the folate complex, Phe223 swings out from its position in the NADH complex to stack against the p-aminobenzoate ring of the folate. Although Phe223 contacts each substrate in E. coli MTHFR, this residue is not invariant; for example, a leucine occurs at this site in the human enzyme. To examine the role of Phe223 in substrate binding and catalysis, we have constructed mutants Phe223Ala and Phe223Leu. As predicted, our results indicate that Phe223 participates in the binding of both substrates. The Phe223Ala mutation impairs NADH and CH(2)-H(4)folate binding each 40-fold yet slows catalysis of both half-reactions less than 2-fold. Affinity for CH(2)-H(4)folate is unaffected by the Phe223Leu mutation, and the variant catalyzes the oxidative half-reaction 3-fold faster than the wild-type enzyme. Structures of ligand-free Phe223Leu and Phe223Leu/Glu28Gln MTHFR in complex with CH(3)-H(4)folate have been determined at 1.65 and 1.70 A resolution, respectively. The structures show that the folate is bound in a catalytically competent conformation, and Leu223 undergoes a conformational change similar to that observed for Phe223 in the Glu28Gln-CH(3)-H(4)folate structure. Taken together, our results suggest that Leu may be a suitable replacement for Phe223 in the oxidative half-reaction of E. coli MTHFR.

NMR assignments of the binary hvDHFR1:folate complex

A better understanding of how salt affects enzyme activity can be gained via NMR studies of binary hvDHFR1:folate complex. Chemical shift assignments of the 17.9 kDa enzyme with bound substrate prepare the way for ongoing research of the effects of salt on enzyme flexibility through relaxation studies.

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-angstroms resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.

Structures of NADH and CH3-H4Folate Complexes of Escherichia coli Methylenetetrahydrofolate Reductase Reveal a Spartan Strategy for a Ping-Pong Reaction,

Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a (betaalpha)(8) barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH(3)-H(4)folate have now been determined at resolutions of 1.95 and 1.85 A, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH(3)-H(4)folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops beta2-alpha2 (L2), beta3-alpha3 (L3), and beta4-alpha4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open" conformation to allow NADH to bind.

Aspartate 120 of Escherichia coli Methylenetetrahydrofolate Reductase: Evidence for Major Roles in Folate Binding and Catalysis and a Minor Role in Flavin Reactivity,

Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the NADH-linked reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as cofactor. MTHFR is unusual among flavin oxidoreductases because it contains a conserved, negatively rather than positively charged amino acid (aspartate 120) near the N1-C2=O position of the flavin. At this location, Asp 120 is expected to influence the redox properties of the enzyme-bound FAD. Modeling of the CH(3)-H(4)folate product into the enzyme active site suggests that Asp 120 may also play crucial roles in folate binding and catalysis. We have replaced Asp 120 with Asn, Ser, Ala, Val, and Lys and have characterized the mutant enzymes. Consistent with a loss of negative charge near the flavin, the midpoint potentials of the mutants increased from 17 to 30 mV. A small kinetic effect on the NADH reductive half-reaction was also observed as the mutants exhibited a 1.2-1.5-fold faster reduction rate than the wild-type enzyme. Catalytic efficiency (k(cat)/K(m)) in the CH(2)-H(4)folate oxidative half-reaction was decreased significantly (up to 70000-fold) and in a manner generally consistent with the negative charge density of position 120, supporting a major role for Asp 120 in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. Asp 120 is also intimately involved in folate binding as increases in the apparent K(d) of up to 15-fold were obtained for the mutants. Examining the E(red) + CH(2)-H(4)folate reaction at 4 degrees C, we obtained, for the first time, evidence for the rapid formation of a reduced enzyme-folate complex with wild-type MTHFR. The more active Asp120Ala mutant, but not the severely impaired Asp120Lys mutant, demonstrated the species, suggesting a connection between the extent of complex formation and catalytic efficiency.

Protonation state of methyltetrahydrofolate in a binary complex with cobalamin-dependent methionine synthase.

N5-Methyltetrahydrofolate (CH(3)-H(4)folate) donates a methyl group to the cob(I)alamin cofactor in the reaction catalyzed by cobalamin-dependent methionine synthase (MetH, EC 2.1.1.3). Nucleophilic displacement of a methyl group attached to a tertiary amine is a reaction without an obvious precedent in bioorganic chemistry. Activation of CH(3)-H(4)folate by protonation prior to transfer of the methyl group has been the favored mechanism. Protonation at N5 would lead to formation of an aminium cation, and quaternary amines such as 5,5-dimethyltetrahydropterin have been shown to transfer methyl groups to cob(I)alamin. Because CH(3)-H(4)folate is an enamine, protonation could occur either at N5 to form an aminium cation or on a conjugated carbon with formation of an iminium cation. We used (13)C distortionless enhancement by polarization transfer (DEPT) NMR spectroscopy to infer that CH(3)-H(4)folate in aqueous solution protonates at N5, not on carbon. CH(3)-H(4)folate must eventually protonate at N5 to form the product H(4)folate; however, this protonation could occur either upon formation of the binary enzyme-CH(3)-H(4)folate complex or later in the reaction mechanism. Protonation at N5 is accompanied by substantial changes in the visible absorbance spectrum of CH(3)-H(4)folate. We have measured the spectral changes associated with binding of CH(3)-H(4)folate to a catalytically competent fragment of MetH over the pH range from 5.5 to 8.5. These studies indicate that CH(3)-H(4)folate is bound in the unprotonated form throughout this pH range and that protonated CH(3)-H(4)folate does not bind to the enzyme. Our observations are rationalized by sequence homologies between the folate-binding region of MetH and dihydropteroate synthase, which suggest that the pterin ring is bound in the hydrophobic core of an alpha(8)beta(8) barrel in both enzymes. The results from these studies are difficult to reconcile with an S(N)2 mechanism for methyl transfer and suggest that the presence of the cobalamin cofactor is important for CH(3)-H(4)folate activation. We propose that protonation of N5 occurs after carbon-nitrogen bond cleavage, and we invoke a mechanism involving oxidative addition of Co(1+) to the N5-methyl bond to rationalize our results.

Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+.

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.

Effects of fluorine substitution on the structure and dynamics of complexes of dihydrofolate reductase (Escherichia coli)

Fluorine NMR experiments with a protein containing fluorinated amino acid analogs can often be used to probe structure and dynamics of the protein as well as conformational changes produced by binding of small molecules. The relevance of NMR experiments with fluorine-containing materials to characteristics of the corresponding native (nonfluorinated) proteins depends upon the extent to which these characteristics are altered by the presence of fluorine. The present work uses molecular dynamics simulations to explore the effects of replacement of tryptophan by 6-fluorotryptophan in folate and methotrexate complexes of the enzyme dihydrofolate reductase (DHFR) (Escherichia coli). Simulations of the folate-native enzyme complex produce local correlation times and order parameters that are generally in good agreement with experimental values. Simulations of the corresponding fluorotryptophan-containing system indicate that the structure and dynamics of this complex are scarcely changed by the presence of fluorinated amino acids. Calculations with the pharmacologically important methotrexate-enzyme complex predict dynamical behavior of the protein similar to that of the folate complex for both the fluorinated and native enzyme. It thus appears that, on the time scale sampled by these computer simulations, substitution of 6-fluorotryptophan for tryptophan has little effect on either the structures or dynamics of DHFR in these complexes.

Synthesis, purification, and tumor cell uptake of 67Ga-deferoxamine--folate, a potential radiopharmaceutical for tumor imaging.

The vitamin folic acid was covalently linked to the chelating agent deferoxamine (DF) via an amide bond using a simple carbodiimide coupling reaction. A mixture of two isomers, DF--folate(alpha) and DF--folate(gamma), was produced involving the alpha- and gamma-carboxyl group of folic acid, respectively. These two isomers were separated by anion-exchange chromatography using a NH4HCO3 gradient. Competitive binding studies revealed that only the DF-folate(gamma) is recognized by the folate receptor on KB cells, interacting with an affinity comparable to unconjugated folic acid. The DF--folate conjugates were radiolabeled with the gamma-emitting radionuclide 67Ga3+ and tested for uptake by cultured KB cells overexpressing the folate receptor. The cellular accumulation of 67Ga-DF-folate(gamma) tracer exhibited rapid uptake kinetics in cell culture with a t1/2 of approximately 3 min. The KB cell association of 67Ga-DF--folate(gamma) was competitively blocked by free folic acid, indicating that uptake of the 67Ga-DF--folate(gamma) was specifically mediated by the folate receptor. Since the folate receptor is overexpressed on the surfaces of many neoplastic cells, these results suggest that 67Ga-DF--folate(gamma) complex might be useful as a diagnostic agent for noninvasive imaging of folate receptor-positing tumors.