Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division1,2. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu3. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell1,3, as well as chemotherapeutic agents such as methotrexate4-6. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance4-8. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.

Drug targeting is a progressive area of research with folate receptor alpha (FRα) receiving significant attention as a biological marker in cancer drug delivery. The binding affinity of folic acid (FA) to the FRα active site provides a basis for recognition of FRα. In this study, FA was conjugated to beta-cyclodextrin (βCD) and subjected to in silico analysis (molecular docking and molecular dynamics (MD) simulation (100 ns)) to investigate the affinity and stability for the conjugated system compared to unconjugated and apo systems (ligand free). Docking studies revealed that the conjugated FA bound into the active site of FRα with a docking score (free binding energy < -15 kcal/mol), with a similar binding pose to that of unconjugated FA. Subsequent analyses from molecular dynamics (MD) simulations, root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg) demonstrated that FA and FA-βCDs created more dynamically stable systems with FRα than the apo-FRα system. All systems reached equilibrium with stable RMSD values ranging from 1.9-2.4 Å and the average residual fluctuation values of the FRα backbone atoms for all residues (except for terminal residues ARG8, THR9, THR214, and LEU215) were less than 2.1 Å with a consistent Rg value of around 16.8 Å throughout the MD simulation time (0-100 ns). The conjugation with βCD improved the stability and decreased the mobility of all the residues (except residues 149-151) compared to FA-FRα and apo-FRα systems. Further analysis of H-bonds, binding free energy (MM-PBSA), and per residue decomposition energy revealed that besides APS81, residues HIS20, TRP102, HIS135, TRP138, TRP140, and TRP171 were shown to have more favourable energy contributions in the holo systems than in the apo-FRα system, and these residues might have a direct role in increasing the stability of holo systems.

Mice are arguably the most important tool in the preclinical evaluation of liposomes; however, the effects of inter-strain physiological variabilities on in vivo performance of liposomes have been seriously overlooked. The present study validated that plasma proteins (PPs) and the capability of mononuclear phagocyte system (MPS) (typically expressed by phagocytosis rate, K) were mice strain-dependent. Physiological variabilities in PPs and the phagocytosis rate jointly contributed to the inter-strain inconsistency of pharmacokinetic (PK) profiles of liposomes. For the PPs sensitive liposomes (such as plain PEGylated liposomes and folic acid functionalized PEGylated liposomes), inter-strain variabilities in PK profiles could be calibrated using the corrected phagocytic rate (KC = K×(c × Ig)/(alb×apo)), where c, Ig, alb and apo were respective the total content of complement proteins, immunoglobulins, albumin and apolipoproteins. While for the PPs insensitive liposomes (e.g., cRGD functionalized liposomes), phagocytic rate could be directly used to calibrate inter-strain difference of liposome PK profiles. Our data also warn that the reciprocal interaction between payloads and organisms would be much more complicated than that between liposomes and organisms, thus independent investigation should be conducted for each individual therapeutic agent.

Serine hydroxymethyltransferase (SHMT) is the major source of 1-carbon units required for nucleotide synthesis. Humans have cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms, which are upregulated in numerous cancers, making the enzyme an attractive drug target. Here, we show that the antifolates lometrexol and pemetrexed are inhibitors of SHMT2 and solve the first SHMT2-antifolate structures. The antifolates display large differences in their hydrogen bond networks despite their similarity. Lometrexol was found to be the best hSHMT1/2 inhibitor from a panel antifolates. Comparison of apo hSHMT1 with antifolate bound hSHMT2 indicates a highly conserved active site architecture. This structural information offers insights as to how these compounds could be improved to produce more potent and specific inhibitors of this emerging anti-cancer drug target.

A class of gold(I) phosphane complexes have been identified as inhibitors of dihydrofolate reductase (DHFR) from E. coli, an enzyme that catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), using NADPH as a coenzyme. In this work, to comprehend the nature of the interaction at the basis of these inhibitory effects, the binding properties of bis- and tris-phosphane gold(I) chloride compounds in regards to DHFR have been studied by emission spectroscopy and spectrophotometric assays. The lack of cysteine and seleno-cysteine residues in the enzyme active site, the most favorable sites of attack of Au(I) moieties, makes this work noteworthy. The interaction with the gold compounds results into the quenching of the DHFR tryptophan's emissions and in an enhancement of their intrinsic emission intensities. Moreover, a modulating action of NADPH is highlighted by means of an increase of the gold compound affinity toward the enzyme; in fact, the dissociation constants calculated for the interactions between DHFR and each gold compound in the presence of saturating NADPH were lower than the ones observed for the apo-enzyme. The fluorimetric data afforded to Kd values ranged from 2.22 ± 0.25 µM for (PPh₃)₂AuCl in the presence of NADPH to 21.4 ± 3.85 µM for ⁴L₃AuTf in the absence of NADPH. By elucidating the energetic aspects of the binding events, we have attempted to dissect the role played by the gold phosphane/protein interactions in the inhibitory activity, resulting in an exothermic enthalpy change and a positive entropic contribution (ΔH° = -5.04 ± 0.08 kcal/mol and ΔS° = 7.34 ± 0.005 cal/mol·K).

Hybrid supramolecular spherical nanoassembly of hen egg white lysozyme and bovine apo α lactalbumin (SNLYZ-BLA) was prepared with a mean size of ˜55.2 nm using an optimized desolvation method via chemical crosslinking. The nanoassembly, SNLYZ-BLA demonstrated dose-dependent reactive oxygen species (ROS) mediated cytotoxicity in multiple cancer cells such as MCF-7, MDA-MB231, HeLa and MG 63. It also demonstrated high loading capacity of a phytochemical based anticancer agent, curcumin (248.8 mg/g) and target-based pH-responsive in vitro drug release with around 85.8% curcumin release observed under acidic condition. Moreover, curcumin loaded SNLYZ-BLA (SNLYZ-BLA-CUR) induced cell viability reduction in all cancer cells including mouse melanoma (B16F10) by more than 90% within 24 h. Further, SNLYZ-BLA and SNLYZ-BLA-CUR when conjugated with folic acid enhanced the cytotoxicity via folate receptor-based targeting. Both drug loading and release induced conformational change and folding reconstitution of the protein nano-assembly, respectively, which made the whole system an efficient therapeutic agent that works via a dual mode of action. We demonstrated that SNLYZ-BLA and SNLYZ-BLA-CUR were highly biocompatible in vitro. Therefore, our supramolecular protein nanoassembly loaded with curcumin could emerge as a comprehensive cancer therapeutics that acts via a strategic mode of dual therapeutic mechanisms.

Aims: Acute increases in left ventricular end diastolic pressure (LVEDP) can induce pulmonary edema (PE). The mechanism(s) for this rapid onset edema may involve more than just increased fluid filtration. Lung endothelial cell permeability is regulated by pressure-dependent activation of nitric oxide synthase (NOS). Herein, we demonstrate that pressure-dependent NOS activation contributes to vascular failure and PE in a model of acute heart failure (AHF) caused by hypertension.Methods and results: Male Sprague-Dawley rats were anesthetized and mechanically ventilated. Acute hypertension was induced by norepinephrine (NE) infusion and resulted in an increase in LVEDP and pulmonary artery pressure (Ppa) that were associated with a rapid fall in PaO2, and increases in lung wet/dry ratio and injury scores. Heart failure (HF) lungs showed increased nitrotyrosine content and ROS levels. L-NAME pretreatment mitigated the development of PE and reduced lung ROS concentrations to sham levels. Apocynin (Apo) pretreatment inhibited PE. Addition of tetrahydrobiopterin (BH4) to AHF rats lung lysates and pretreatment of AHF rats with folic acid (FA) prevented ROS production indicating endothelial NOS (eNOS) uncoupling.Conclusion: Pressure-dependent NOS activation leads to acute endothelial hyperpermeability and rapid PE by an increase in NO and ROS in a model of AHF. Acute increases in pulmonary vascular pressure, without NOS activation, was insufficient to cause significant PE. These results suggest a clinically relevant role of endothelial mechanotransduction in the pathogenesis of AHF and further highlights the concept of active barrier failure in AHF. Therapies targetting the prevention or reversal of endothelial hyperpermeability may be a novel therapeutic strategy in AHF.

Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis and role as co-factor are well understood, knowledge about uptake of cobalamin by prokaryotic auxotrophs is scarce. Here, we characterize a cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, and demonstrate that it mediates the specific, ATP-dependent uptake of cobalamin. We solved the crystal structure of ECF-CbrT in an apo conformation to 3.4 Å resolution. Comparison with the ECF transporter for folate (ECF-FolT2) from the same organism, reveals how the identical ECF module adjusts to interact with the different substrate binding proteins FolT2 and CbrT. ECF-CbrT is unrelated to the well-characterized B12 transporter BtuCDF, but their biochemical features indicate functional convergence.

This study aimed to assess whether adolescents have an increased risk of adverse pregnancy outcomes (APO) compared to adult women. We used data on 43,327 births from the population-based Arkhangelsk County Birth Registry, Northwest Russia, for 2012-2014. The perinatal outcomes included stillbirth, preterm birth (<37 and <32 weeks), low and very low birthweight, 5 min Apgar score <7 and <4, perinatal infections, and the need for neonatal transfer to a higher-level hospital. Multivariable logistic regression was applied to assess the associations between age and APO. Altogether, 4.7% of deliveries occurred in adolescents. Both folic acid intake and multivitamin intake during pregnancy were more prevalent in adults. Adolescents were more likely to be underweight, to smoke, and to have infections of the kidney and the genital tract compared to adult women. Compared to adults, adolescents were at lower risk of low birthweight, a 5 min Apgar score <7, and need for neonatal transfer. Adolescents had no increased risk of other APO studied in the adjusted analysis, suggesting that a constellation of other factors, but not young age per se, is associated with APO in the study setting.

The objective of the study was to analyse leptin, IGF-1, Apo A, lipoproteins, haem oxygenase-1 (HO-1) in maternal sera and venous umbilical cord sera of newborn babies of 25 preeclamptics (group II), and 25 normotensive pregnant women (group I) as markers of obesity and growth in preeclamptic and normotensive pregnant women. Apo A I and II levels were estimated by competitive immunoassay using direct chemiluminiscence technology. Haem oxygenase-1 (HO-1), leptin and IGF-1 were analysed by ELISA. Maternal and cord blood levels of homocysteine, folic acid, lipid profile (namely, total cholesterol, triglycerides, LDL-C, VLDL-C and HDL-C), Haem oxygenase 1 were higher in preeclamptic women as compared to normotensive pregnant women. Serum and cord blood Apo A-I and Apo B, leptin levels, IGF-I were lower in preeclamptic women as compared to normotensive pregnant. The findings of high serum HO-1 levels in maternal and cord blood in preeclampsia supports the role of oxidative stress and excessive inflammatory response in the pathogenesis of preeclampsia. It seems likely that IGF-1 and leptin play a central role in controlling foetal growth. There is increasing evidence that the foundations of life-long health are, in part, laid in the uterus. Findings of present study suggest that alterations in biochemical markers of growth and obesity occur in mothers and foetuses and modifications of uterine environment can be of help to prevent future cardiovascular risk. Impact statement Preeclampsia has been reported to be associated with an increased risk of later life cardiovascular disease. However, information regarding how obesity increases the risk of preeclampsia is limited. Atherogenic milieu occurring during pregnancy persists into adulthood and foetal growth retardation is strongly associated with adult atherosclerosis. There is conflicting evidence regarding alterations of IGFs in preeclamptic pregnancies and deficit in circulating and cord blood IGF-1 levels in intrauterine growth restricted newborns and a correlation between IGF-1 levels and birth weight have been reported. Leptin is a predictor of cardiovascular risk independent of insulin resistance. Emerging evidence supports an important role for the haem oxygenase system (HO-1) in the maintenance of a healthy pregnancy, especially during pathological challenge. Conflicting data are available regarding HO-1, leptin and IGF -1 in preeclamptic mothers. The extent to which they mediate foetal growth and developmental abnormalities remains to be clarified. Serum IGF-1 levels were significantly decreased in preeclamptics and maternal IGF-1 showed a strong inverse correlation with leptin levels. High serum HO-1 levels in maternal and cord blood in preeclampsia were observed in the present study. Findings of the present study suggest that alterations in biochemical markers of growth and obesity occur in mothers and foetuses and modifications of the uterine environment can be of help to prevent future cardiovascular risk.

Recently, myoinositol (myo-ins) and folic acid combination has gained an important role for treating Polycystic Ovary Syndrome (PCOS), in addition to combined oral contraceptives (COC). We aimed to examine myo-ins effects on anti-Mullerian hormone (AMH) levels and compare them with those ones obtained administering COC. In this prospective study, 137 PCOS patients, diagnosed according to Rotterdam criteria and admitted to the Reproductive Endocrinology and Infertility Outpatient Clinic at Dokuz Eylul University (Izmir, Turkey), were included. After randomization to COC (n = 60) and myo-ins (n = 77) arms, anthropometric measurements, blood pressure, Modified Ferriman Gallwey scores were calculated. Biochemical and hormonal analysis were performed, and LH/FSH and Apo B/A1 ratios were calculated. Data analysis was carried out in demographically and clinically matched 106 patients (COC = 54; myo-ins = 52). After 3-month treatment, increase in HDL and decreases in LH and LH/FSH ratio were statistically more significant only in COC group when compared with baseline (in both cases p > 0.05). In myo-ins group, fasting glucose, LDL, DHEAS, total cholesterol, and prolactin levels decreased significantly (for all p < 0.05). Progesterone and AMH levels, ovarian volume, ovarian antral follicle, and total antral follicle counts lessened significantly in both groups (for all p < 0.05). In PCOS treatment, MYO is observed more effective in reductions of total ovarian volume and AMH levels.

Energy-coupling factor (ECF) transporters mediate uptake of micronutrients in prokaryotes. The transporters consist of an S-component that binds the transported substrate and an ECF module (EcfAA'T) that binds and hydrolyses ATP. The mechanism of transport is poorly understood but presumably involves an unusual step in which the membrane-embedded S-component topples over to carry the substrate across the membrane. In many ECF transporters, the S-component dissociates from the ECF module after transport. Subsequently, substrate-bound S-components out-compete the empty proteins for re-binding to the ECF module in a new round of transport. Here we present crystal structures of the folate-specific transporter ECF-FolT from Lactobacillus delbrueckii. Interaction of the ECF module with FolT stabilizes the toppled state, and simultaneously destroys the high-affinity folate-binding site, allowing substrate release into the cytosol. We hypothesize that differences in the kinetics of toppling can explain how substrate-loaded FolT out-competes apo-FolT for association with the ECF module.

The stepwise reduction of dihydrofolate to tetrahydrofolate entails significant conformational changes of dihydrofolate reductase (DHFR). Binary and ternary complexes of DHFR containing cofactor NADPH, inhibitor methotrexate (MTX), or both NADPH and MTX were characterized by 193 nm ultraviolet photodissociation (UVPD) mass spectrometry. UVPD yielded over 80% sequence coverage of DHFR and resulted in production of fragment ions that revealed the interactions between DHFR and each ligand. UVPD of the binary DHFR·NADPH and DHFR·MTX complexes led to an unprecedented number of fragment ions containing either an N- or C-terminal protein fragment still bound to the ligand via retention of noncovalent interactions. In addition, holo-fragments retaining both ligands were observed upon UVPD of the ternary DHFR·NADPH·MTX complex. The combination of extensive holo and apo fragment ions allowed the locations of the NADPH and MTX ligands to be mapped, with NADPH associated with the adenosine binding domain of DHFR and MTX interacting with the loop domain. These findings are consistent with previous crystallographic evidence. Comparison of the backbone cleavage propensities for apo DHFR and its holo counterparts revealed significant variations in UVPD fragmentation in the regions expected to experience conformational changes upon binding NADPH, MTX, or both ligands. In particular, the subdomain rotation and loop movements, which are believed to occur upon formation of the transition state of the ternary complex, are reflected in the UVPD mass spectra. The UVPD spectra indicate enhanced backbone cleavages in regions that become more flexible or show suppressed backbone cleavages for those regions either shielded by the ligand or involved in new intramolecular interactions. This study corroborates the versatility of 193 nm UVPD mass spectrometry as a sensitive technique to track enzymatic cycles that involve conformational rearrangements.

This review analyzes how interplay between folate binding and changes in folate binding protein (FBP) conformation/self-association affects the biological function of FBP. Concentration-dependent, reversible self-association of hydrophobic apo-FBP at pI=7.4 is associated with decreased affinity for folate, probably due to shielding of binding sites between interacting hydrophobic patches. Titration with folate removes apo-monomers, favoring dissociation of self-associated apo-FBP into apo-monomers. Folate anchors to FBP through a network of hydrogen bonds and hydrophobic interactions, and the binding induces a conformational change with formation of hydrophilic and stable holo-FBP. Holo-FBP exhibits a ligand-mediated concentration-dependent self-association into multimers of great thermal and chemical stability due to strong intermolecular forces. Both ligand and FBP are thus protected against biological/physicochemical decomposition. In biological fluids with low FBP concentrations, e.g., saliva, semen and plasma, hydrophobic apo-monomers and hydrophilic holo-monomers associate into stable asymmetrical complexes with aberrant binding kinetics unless detergents, e.g., cholesterol or phospholipids are present.

10-Formyltetrahydrofolate dehydrogenase (FDH), which is composed of a small N-terminal domain (Nt-FDH) and a large C-terminal domain, is an abundant folate enzyme in the liver and converts 10-formyltetrahydrofolate (10-FTHF) to tetrahydrofolate (THF) and CO2. Nt-FDH alone possesses a hydrolase activity, which converts 10-FTHF to THF and formate in the presence of β-mercaptoethanol. To elucidate the catalytic mechanism of Nt-FDH, crystal structures of apo-form zNt-FDH from zebrafish and its complexes with the substrate analogue 10-formyl-5,8-dideazafolate (10-FDDF) and with the products THF and formate have been determined. The structures reveal that the conformations of three loops (residues 86-90, 135-143 and 200-203) are altered upon ligand (10-FDDF or THF) binding in the active site. The orientations and geometries of key residues, including Phe89, His106, Arg114, Asp142 and Tyr200, are adjusted for substrate binding and product release during catalysis. Among them, Tyr200 is especially crucial for product release. An additional potential THF binding site is identified in the cavity between two zNt-FDH molecules, which might contribute to the properties of product inhibition and THF storage reported for FDH. Together with mutagenesis studies and activity assays, the structures of zNt-FDH and its complexes provide a coherent picture of the active site and a potential THF binding site of zNt-FDH along with the substrate and product specificity, lending new insights into the molecular mechanism underlying the enzymatic properties of Nt-FDH.

A dietary supplement (AP, Armolipid Plus) that combines red yeast rice extract, policosanol, berberine, folic acid, coenzyme Q10 and asthaxantine can have beneficial effects on cardiovascular disease (CVD) biomarkers. The aim of this study was to assess whether the intake of AP, in combination with dietary recommendations, reduces serum low density lipoprotein cholesterol (LDL-c) concentrations and other CVD biomarkers in patients with hypercholesterolemia. Eligible patients were recruited from the outpatient clinics of six Spanish hospitals Hospital Virgen del Rocío (Sevilla); Hospital San Jorge (Huesca); Hospital San Pedro (Logroño); Hospital Gregorio Marañón (Madrid), Hospital la Fe (Valencia) and Hospital Universitari Sant Joan (Reus) as recruiting and coordinating center. 102 participants (mean age ± SD; 50.91 ± 11.61; 32 men) with low CVD, with mild-to-moderately elevated LDL-c (between 3.35 mmol/L and 4.88 mmol/L) without hypolipemic therapy were randomized in a double-blind, parallel, controlled, multicenter trial commencing January 2012 and ending December 2012. Among the exclusion criteria were any concomitant chronic disease, triglycerides (TG) >3.97 mmol/L, pregnant or lactating, and history of CVD. At 12 weeks, compared to placebo, AP reduced LDL-c by -6.9%, apolipoprotein (Apo) B-100 by -6.6% and total cholesterol/HDL-c ratio by -5.5%, the ApoB/ApoA1 ratio by -8.6%, while increasing ApoA1 by +2.5% (p<0.05). AP consumption was associated with modest mean weight loss of -0.93 kg (95%CI: -1.74 to -0.12; P = 0.02) compared with control group while dietary composition remained unchanged in the AP group. The AP product was well tolerated. In conclusion, AP, combined with dietary recommendations, reduced LDL-c levels as well as total cholesterol/HDL-c and ApoB/ApoA1 ratios, while increasing Apo A1, all of which are improvements in CVD risk indicators. AP is a product which could benefit patients having moderate hyperlipidemia and excess body weight.

Though acute exposure to hypobaric hypoxia is reported to impair cognitive performance, the effects of prolonged exposure on different cognitive domains have been less studied. The present study aimed at investigating the time dependent changes in cognitive performance on prolonged stay at high altitude and its correlation with electroencephalogram (EEG) and plasma homocysteine. The study was conducted on 761 male volunteers of 25-35 years age who had never been to high altitude and baseline data pertaining to domain specific cognitive performance, EEG and homocysteine was acquired at altitude ≤240 m mean sea level (MSL). The volunteers were inducted to an altitude of 4200-4600 m MSL and longitudinal follow-ups were conducted at durations of 03, 12 and 18 months. Neuropsychological assessment was performed for mild cognitive impairment (MCI), attention, information processing rate, visuo-spatial cognition and executive functioning. Total homocysteine (tHcy), vitamin B12 and folic acid were estimated. Mini Mental State Examination (MMSE) showed temporal increase in the percentage prevalence of MCI from 8.17% on 03 months of stay at high altitude to 18.54% on 18 months of stay. Impairment in visuo-spatial executive, attention, delayed recall and procedural memory related cognitive domains were detected following prolonged stay in high altitude. Increase in alpha wave amplitude in the T3, T4 and C3 regions was observed during the follow-ups which was inversely correlated (r = -0.68) to MMSE scores. The tHcy increased proportionately with duration of stay at high altitude and was correlated with MCI. No change in vitamin B12 and folic acid was observed. Our findings suggest that cognitive impairment is progressively associated with duration of stay at high altitude and is correlated with elevated tHcy in the plasma. Moreover, progressive MCI at high altitude occurs despite acclimatization and is independent of vitamin B12 and folic acid.

We are addressing bacterial resistance to antibiotics by repurposing a well-established classic antimicrobial target, the dihydrofolate reductase (DHFR) enzyme. In this work, we have focused on Enterococcus faecalis, a nosocomial pathogen that frequently harbors antibiotic resistance determinants leading to complicated and difficult-to-treat infections. An inhibitor series with a hydrophobic dihydrophthalazine heterocycle was designed from the anti-folate trimethoprim. We have examined the potency of this inhibitor series based on inhibition of DHFR enzyme activity and bacterial growth, including in the presence of the exogenous product analogue folinic acid. The resulting preferences were rationalized using a cocrystal structure of the DHFR from this organism with a propyl-bearing series member (RAB-propyl). In a companion apo structure, we identify four buried waters that act as placeholders for a conserved hydrogen-bonding network to the substrate and indicate an important role in protein stability during catalytic cycling. In these structures, the nicotinamide of the nicotinamide adenine dinucleotide phosphate cofactor is visualized outside of its binding pocket, which is exacerbated by RAB-propyl binding. Finally, homology models of the TMP(R) sequences dfrK and dfrF were constructed. While the dfrK-encoded protein shows clear sequence changes that would be detrimental to inhibitor binding, the dfrF-encoded protein model suggests the protein would be relatively unstable. These data suggest a utility for anti-DHFR compounds for treating infections arising from E. faecalis. They also highlight a role for water in stabilizing the DHFR substrate pocket and for competitive substrate inhibitors that may gain advantages in potency by the perturbation of cofactor dynamics.

The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH7.4 (pI=7-8) ligand binding increased concentration-dependent self-association of FBP into stable multimers of holo-FBP. DSC of 3.3μM holo-FBP showed Tm (76°C) and molar enthalpy (146kcalM(-1)) values increasing to 78°C and 163kcalM(-1) at 10μM holo-FBP, while those of apo-FBP were 55°C and 105kcalM(-1). Besides ligand binding, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10μM) in DLS after a step-wise rise in temperature to 78°C≈Tm. Stable holo-FBP multimers may protect naturally occurring labile folates against decomposition or bacterial utilization. DSC established an interrelationship between diminished folate binding at pH5, especially in NaCl-free buffers, and low thermostability. Positively charged apo-FBP was almost completely unfolded and aggregated at pH5 (Tm 38°C) and holo-FBP, albeit more thermostable, was labile with aggregation tendency. Addition of 0.15M NaCl increased thermostability of apo-FBP drastically, and even more so that of holo-FBP. Electrostatic forces thus seem to contribute to a diminished thermostability at low pH. Fluorescence spectroscopy after irreversible thermal unfolding of FBP revealed a weak-affinity folate binding.

A combination of high folate with low vitamin B12 plasma status has been associated with cognitive impairment in a population exposed to mandatory folic acid fortification. The objective of the present study was to examine the interactions between plasma concentrations of folate and vitamin B12 markers in relation to cognitive performance in Norwegian elderly who were unexposed to mandatory or voluntary folic acid fortification. Cognitive performance was assessed by six cognitive tests in 2203 individuals aged 72-74 years. A combined score was calculated using principal component analysis. The associations of folate concentrations, vitamin B12 markers (total vitamin B12, holotranscobalamin (holoTC) and methylmalonic acid (MMA)) and their interactions in relation to cognitive performance were evaluated by quantile regression and least-squares regression, adjusted for sex, education, apo-ɛ4 genotype, history of CVD/hypertension and creatinine. Cross-sectional analyses revealed an interaction (P= 0·009) between plasma concentrations of folate and vitamin B12 in relation to cognitive performance. Plasma vitamin B12 concentrations in the lowest quartile (< 274 pmol/l) combined with plasma folate concentrations in the highest quartile (>18·5 nmol/l) were associated with a reduced risk of cognitive impairment compared with plasma concentrations in the middle quartiles of both vitamins (OR 0·22, 95 % CI 0·05, 0·92). The interaction between folate and holoTC or MMA in relation to cognitive performance was not significant. In conclusion, this large study population unexposed to mandatory folic acid fortification showed that plasma folate, but not plasma vitamin B12, was associated with cognitive performance. Among the elderly participants with vitamin B12 concentrations in the lower range, the association between plasma folate and cognitive performance was strongest.

As the second essential enzyme of the folate biosynthetic pathway, the potential antimicrobial target, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase), catalyzes the Mg(2+-)dependant transfer of pyrophosphate from the cofactor (ATP) to the substrate, 6-hydroxymethyl-7,8-dihydropterin. Recently, we showed that 8-mercaptoguanine (8-MG) bound at the substrate site (KD ∼13 µM), inhibited the S. aureus enzyme (SaHPPK) (IC50 ∼ 41 µM), and determined the structure of the SaHPPK/8-MG complex. Here we present the synthesis of a series of guanine derivatives, together with their HPPK binding affinities, as determined by SPR and ITC analysis. The binding mode of the most potent was investigated using 2D NMR spectroscopy and X-ray crystallography. The results indicate, firstly, that the SH group of 8-MG makes a significant contribution to the free energy of binding. Secondly, direct N(9) substitution, or tautomerization arising from N(7) substitution in some cases, leads to a dramatic reduction in affinity due to loss of a critical N(9)-H···Val46 hydrogen bond, combined with the limited space available around the N(9) position. The water-filled pocket under the N(7) position is significantly more tolerant of substitution, with a hydroxyl ethyl 8-MG derivative attached to N(7) (compound 21a) exhibiting an affinity for the apo enzyme comparable to the parent compound (KD ∼ 12 µM). In contrast to 8-MG, however, 21a displays competitive binding with the ATP cofactor, as judged by NMR and SPR analysis. The 1.85 Å X-ray structure of the SaHPPK/21a complex confirms that extension from the N(7) position towards the Mg(2+)-binding site, which affords the only tractable route out from the pterin-binding pocket. Promising strategies for the creation of more potent binders might therefore include the introduction of groups capable of interacting with the Mg(2+) centres or Mg(2+)-binding residues, as well as the development of bitopic inhibitors featuring 8-MG linked to a moiety targeting the ATP cofactor binding site.

High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 nm to 460-470 nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements.