ABSTRACT
A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4-ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, (R)-IPO and (S)-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated (R)- and (S)-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. (R)-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of (S)-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO's bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies.
Subject(s)
Glucuronides/metabolism , Microsomes/metabolism , Terpenes/chemistry , Terpenes/metabolism , Toxins, Biological/chemistry , Toxins, Biological/metabolism , Dose-Response Relationship, Drug , Hep G2 Cells , Humans , Microsomes/drug effects , Stereoisomerism , Terpenes/toxicity , Toxins, Biological/toxicityABSTRACT
Cytochrome P450 4B1 (CYP4B1) has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new "pro-toxicant" substrates for CYP4B1. In the present study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1-C8). Compounds with C3-C6 carbon chain lengths showed similar potency to IPO (LD50 ≈ 5 µM). Short chain analogs (<3 carbons) and long chain analogs (>6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative enedial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased ω-hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here.
Subject(s)
Amides/toxicity , Aryl Hydrocarbon Hydroxylases/metabolism , Furans/toxicity , Activation, Metabolic , Amides/chemical synthesis , Amides/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/antagonists & inhibitors , Aryl Hydrocarbon Hydroxylases/chemistry , Catalytic Domain , Cytochrome P-450 Enzyme Inhibitors/chemical synthesis , Cytochrome P-450 Enzyme Inhibitors/metabolism , Cytochrome P-450 Enzyme Inhibitors/toxicity , Furans/chemical synthesis , Furans/metabolism , Hep G2 Cells , Humans , Hydroxylation , Kinetics , Molecular Docking Simulation , Molecular Structure , Protein Binding , Rabbits , Structure-Activity Relationship , Terpenes/chemistry , Terpenes/toxicityABSTRACT
Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1â4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.
Subject(s)
Biofilms , DNA, Bacterial/metabolism , Polysaccharides, Bacterial/metabolism , Pseudomonas aeruginosa/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cations/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Extracellular Matrix/metabolism , Extracellular Space/genetics , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Immunoblotting , Microscopy, Confocal , Mutation , Plant Lectins/chemistry , Plant Lectins/metabolism , Polysaccharides, Bacterial/chemistry , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Receptors, N-Acetylglucosamine/chemistry , Receptors, N-Acetylglucosamine/metabolism , Staining and Labeling/methodsABSTRACT
The structural diversity of carbohydrates presents a major challenge for glycobiology and the analysis of glycoconjugates. Mass spectrometry has become a primary tool for glycan analysis thanks to its speed and sensitivity, but the information content regarding the glycan structure of protonated glycoconjugates is hindered by the inability to differentiate linkage and stereoisomers. Here, we examine a variety of protonated carbohydrate structures by gas-phase hydrogen/deuterium exchange (HDX) to discover that the exchange rates are distinct for isomeric carbohydrates with even subtle structural differences. By incorporating an internal exchange standard, HDX could effectively distinguish all linkage and stereoisomers that were examined and presents a mass spectrometry-based approach for glycan structural analysis with immense potential.
ABSTRACT
The myeloablative agent busulfan (1,4-butanediol dimethanesulfonate) is an old drug that is used routinely to eliminate cancerous bone marrow prior to hematopoietic stem cell transplant. The myeloablative activity and systemic toxicity of busulfan have been ascribed to its ability to cross-link DNA. In contrast, here we demonstrate that incubation of busulfan with the thiol redox proteins glutaredoxin or thioredoxin at pH 7.4 and 37 °C results in the formation of putative S-tetrahydrothiophenium adducts at their catalytic Cys residues, followed by ß-elimination to yield dehydroalanine. Both proteins contain a second Cys, in their catalytic C-X-X-C motif, which reacts with the dehydroalanine, the initial Cys adduct with busulfan, or the S-tetrahydrothiophenium, to form novel intramolecular cross-links. The reactivity of the dehydroalanine (DHA) formed is further demonstrated by adduction with glutathione to yield a lanthionine and by a novel reaction with the reducing agent tris(2-carboxyethyl)phosphine (TCEP), which yields a phosphine adduct via Michael addition to the DHA. Formation of a second quaternary organophosphonium salt via nucleophilic substitution with TCEP on the initial busulfan-protein adduct or on the THT(+)-Redoxin species is also observed. These results reveal a rich potential for reactions of busulfan with proteins in vitro, and likely in vivo. It is striking that several of the chemically altered protein products retain none of the atoms of busulfan, in contrast to typical drug-protein adducts or traditional protein modification reagents. In particular, the ability of a clinically used drug to convert Cys to dehydrolanine in intact proteins, and its subsequent reaction with biological thiols, is unprecedented.
Subject(s)
Alanine/analogs & derivatives , Busulfan/chemistry , Cysteine/chemistry , Myeloablative Agonists/chemistry , Sulfides/chemistry , Alanine/chemistry , Humans , Tandem Mass SpectrometryABSTRACT
Cytochrome P4503A4 (CYP3A4) is a peripheral membrane protein that plays a major role in enzymatic detoxification of many drugs and toxins. CYP3A4 has an integral membrane N-terminal helix and a localized patch comprised of the G' and F' helix regions that are embedded in the membrane, but the effects of membrane composition on CYP3A4 function are unknown. Here, circular dichroism and differential scanning calorimetry were used to compare the stability of CYP3A4 in lipid bilayer nanodiscs with varying ratios of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These lipids differ in the acyl-chain length and their degree of unsaturation. The thermal denaturation of CYP3A4 in nanodiscs occurs in a temperature range distinct from that of the nanodisc denaturation so it can be monitored calorimetrically. Melting temperatures (Tm), heat capacities (ΔCp), and calorimetric enthalpies (ΔHcal) for denaturation of CYP3A4 each increased with an increasing fraction of DMPC, with a maximum at 50% DMPC, before decreasing at 75% DMPC. Addition of the inhibitor ketoconazole results in increased thermal stability, and larger ΔCp and ΔHcal values, with different sensitivities to lipid composition. Effects of lipid composition on ligand binding dynamics were also studied. Equilibrium binding affinities of both ketoconazole (KTZ) and testosterone (TST) were minimally affected by lipid composition. However, stopped-flow analyses indicate that the rates of KTZ binding reach a maximum in membranes containing 50% DMPC, whereas the rate of TST binding decreases continuously with an increasing DMPC concentration. These results indicate that CYP3A4 is highly sensitive to the acyl-chain composition of the lipids and fluidity of the membrane in which it is embedded.
Subject(s)
Cytochrome P-450 CYP3A/chemistry , Lipids/chemistry , Membrane Fluidity , Nanostructures/chemistry , Temperature , Calorimetry, Differential Scanning , Circular Dichroism , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 CYP3A Inhibitors/metabolism , Cytochrome P-450 CYP3A Inhibitors/pharmacology , Dimyristoylphosphatidylcholine/chemistry , Enzyme Stability/drug effects , Humans , Ketoconazole/metabolism , Ketoconazole/pharmacology , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Models, Molecular , Phosphatidylcholines/chemistry , Protein Binding , Protein Denaturation , Protein Domains , Testosterone/metabolism , ThermodynamicsABSTRACT
Cytosolic glutathione transferases (GSTs) comprise a large family of enzymes with canonical structures that diverge functionally and structurally among mammals, invertebrates and plants. Whereas mammalian GSTs have been characterized extensively with regard to their structure and function, invertebrate GSTs remain relatively unstudied. The invertebrate GSTs do, however, represent potentially important drug targets for infectious diseases and agricultural applications. In addition, it is essential to fully understand the structure and function of invertebrate GSTs, which play important roles in basic biological processes. Invertebrates harbor delta- and epsilon-class GSTs, which are not found in other organisms. Drosophila melanogaster GSTs (DmGSTs) are likely to contribute to detoxication or antioxidative stress during development, but they have not been fully characterized. Here, the structures of two epsilon-class GSTs from Drosophila, DmGSTE6 and DmGSTE7, are reported at 2.1 and 1.5â Å resolution, respectively, and are compared with other GSTs to identify structural features that might correlate with their biological functions. The structures of DmGSTE6 and DmGSTE7 are remarkably similar; the structures do not reveal obvious sources of the minor functional differences that have been observed. The main structural difference between the epsilon- and delta-class GSTs is the longer helix (A8) at the C-termini of the epsilon-class enzymes.
Subject(s)
Drosophila Proteins/chemistry , Drosophila melanogaster/chemistry , Glutathione Transferase/chemistry , Amino Acid Sequence , Animals , Binding Sites , Drosophila Proteins/metabolism , Glutathione/metabolism , Glutathione Transferase/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Stability , Sequence Alignment , TemperatureABSTRACT
The DNA alkylating agent busulfan is used to 'precondition' patients with leukemia, lymphomas and other hematological disorders prior to hematopoietic stem cell transplants. Busulfan is metabolized via conjugation with glutathione (GSH) followed by intramolecular rearrangement to the GSH analog γ-glutamyl-dehydroalanyl -glycine (EdAG). EdAG contains the electrophilic dehydroalanine, which is expected to react with protein nucleophiles, particularly proteins with GSH binding sites such as glutaredoxins (Grx's). Incubation of EdAG with human Grx-1 or Grx-2 results in facile adduction of cys-23 and cys-77, respectively, as determined by ESI-MS/MS. The resulting modified proteins are catalytically inactive. In contrast, the glutathione transferase A1-1 includes a GSH binding site with a potentially reactive tyrosinate (Tyr-9) but it does not react with EdAG. Similarly, Cys-112 of GSTA1-1, which lies outside the active site and is known to form disulfides with GSH, does not react with EdAG. The results provide the first demonstration of the reactivity of any busulfan metabolites with intact proteins, and they suggest that GSH-binding sites containing thiolates are most susceptible. The adduction of Grx's by EdAG suggests the possible alteration of proteins that are normally regulated via Grx-dependent reversible glutathionylation or deglutathionylation. Dysregulation of Grx-dependent processes could contribute to cellular toxicity of busulfan.
Subject(s)
Busulfan/metabolism , Glutaredoxins/metabolism , Glutathione/analogs & derivatives , Glutathione/metabolism , Amino Acid Sequence , Catalysis , Glutaredoxins/chemistry , Glutathione/pharmacology , Glutathione Transferase/drug effects , Proton Magnetic Resonance Spectroscopy , Tandem Mass SpectrometryABSTRACT
To provide high-resolution X-ray crystallographic structures of a peptide with the Trp-cage fold, we prepared a cyclized version of this motif. Cyclized Trp-cage is remarkably stable and afforded two crystal forms suitable for X-ray diffraction. The resulting higher resolution crystal structures validate the prior NMR models and provide explanations for experimental observations that could not be rationalized by NMR structural data, including the structural basis for the increase in fold stability associated with motif cyclization and the manner in which a polar serine side chain is accommodated in the hydrophobic interior. A hexameric oligomer of the cyclic peptide is found in both crystal forms and indicates that under appropriate conditions, this minimized system may also serve as a model for protein-protein interactions.
Subject(s)
Peptides/chemistry , Protein Folding , Protein Multimerization , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, QuaternaryABSTRACT
P-glycoprotein (P-gp) is a member of the ABC transporter family that confers drug resistance to many tumors by catalyzing their efflux, and it is a major component of drug-drug interactions. P-gp couples drug efflux with ATP hydrolysis by coordinating conformational changes in the drug binding sites with the hydrolysis of ATP and release of ADP. To understand the relative rates of the chemical step for hydrolysis and the conformational changes that follow it, we exploited isotope exchange methods to determine the extent to which the ATP hydrolysis step is reversible. With γ(18)O4-labeled ATP, no positional isotope exchange is detectable at the bridging ß-phosphorus-O-γ-phosphorus bond. Furthermore, the phosphate derived from hydrolysis includes a constant ratio of three (18)O/two (18)O/one (18)O that reflects the isotopic composition of the starting ATP in multiple experiments. Thus, H2O-exchange with HPO4(2-) (Pi) was negligible, suggesting that a [P-gp·ADP·Pi] is not long-lived. This further demonstrates that the hydrolysis is essentially irreversible in the active site. These mechanistic details of ATP hydrolysis are consistent with a very fast conformational change immediately following, or concomitant with, hydrolysis of the γ-phosphate linkage that ensures a high commitment to catalysis in both drug-free and drug-bound states.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/chemistry , Binding Sites , Biocatalysis , Humans , Hydrolysis , Oxygen Isotopes , Protein ConformationABSTRACT
The fold stabilities and folding dynamics of a series of mutants of a model hairpin, KTW-NPATGK-WTE (HP7), are reported. The parent system and the corresponding DPATGK loop species display submicrosecond folding time constants. The mutational studies revealed that ultrafast folding requires both some prestructuring of the loop and a favorable interaction between the chain termini in the transition state. In the case of YY-DPETGT-WY, another submicrosecond folding species [Davis, C. M., Xiao, S., Raleigh, D. P., and Dyer, R. B. (2012) J. Am. Chem. Soc. 134, 14476-14482], a hydrophobic cluster provides the latter. In the case of HP7, the Coulombic interaction between the terminal NH3(+) and CO2(-) units provides this; a C-terminal Glu to amidated Ala mutation results in a 5-fold retardation of the folding rate. The effects of mutations within the reversing loop indicate the balance between loop flexibility (favoring fast conformational searching) and turn formation in the unfolded state is a major factor in determining the folding dynamics. The -NAAAKX- loops examined display no detectable turn formation propensity in other hairpin constructs but do result in stable analogues of HP7. Peptide KTW-NAAAKK-WTE displays the same fold stability as HP7, but both the folding and unfolding time constants are greater by a factor of 20.
Subject(s)
Mutation , Peptide Fragments/chemistry , Protein Folding , Amino Acid Sequence , Hydrophobic and Hydrophilic Interactions , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/genetics , Protein ConformationABSTRACT
All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the ß-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Molecular Docking Simulation , Tretinoin/analogs & derivatives , Cytochrome P-450 Enzyme System/chemistry , Humans , Hydroxylation/physiology , Molecular Structure , Stereoisomerism , Tretinoin/pharmacokineticsABSTRACT
The present studies have shown that (13)C=O, (13)C(α) and (13)C(ß) of H-bonded strand residues in ß-hairpins provide additional probes for quantitating the extent of folding in ß-hairpins and other ß-sheet models. Large differences in the structuring shifts (CSDs) of these (13)C sites in H-bonded versus non-H-bonded sites are observed: the differences between H-bonded and non-H-bonded sites are greater than 1.2 ppm for all three (13)C probes. This prompts us to suggest that efforts to determine the extent of hairpin folding from (13)C shifts should be based exclusively on the observation at the cross-strand H-bonded sites. Furthermore, the statistics suggest the (13)C' and (13)C(ß) CSDs will provide the best differentiation with 100%-folded CSD values approaching -2.6 and +3 ppm, respectively, for the H-bonded sites. These conclusions can be extended to edge-strands of protein ß-sheets. Our survey of reported (13)C shifts in ß-proteins indicates that some of the currently employed random coil values need to be adjusted, particularly for ionization-induced effects.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Proteins/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Carbon Isotopes , Databases, Protein , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Protein Structure, SecondaryABSTRACT
High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for 'forward' Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict 'one way' detoxication enzyme.
Subject(s)
Aldehydes , Glutathione Transferase , Catalysis , Aldehydes/chemistry , Glutathione Transferase/metabolism , Antioxidants , Glutathione/metabolism , LipidsABSTRACT
Vitamin D(3) is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D(3) (25OHD(3)), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD(3) was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD(3) hydroxylation by human liver microsomes, with the formation of 4ß,25-dihydroxyvitamin D(3) [4ß,25(OH)(2)D(3)] dominating (V(max)/K(m) = 0.85 ml · min(-1) · nmol enzyme(-1)). 4ß,25(OH)(2)D(3) was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D(3), and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4ß,25(OH)(2)D(3) in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4ß,25(OH)(2)D(3), but not CYP24A1-dependent 24R,25-dihydroxyvitamin D(3) formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD(3) metabolism may play an important role in the regulation of vitamin D(3) in vivo and in the etiology of drug-induced osteomalacia.
Subject(s)
Cytochrome P-450 CYP3A/metabolism , Vitamin D/metabolism , Chromatography, High Pressure Liquid , Humans , Microsomes, Liver/enzymology , Tandem Mass SpectrometryABSTRACT
The (13)C chemical shifts measured for designed ß-hairpins indicate that the structuring shifts (upfield for Cα and C', downfield for Cß) previously reported as diagnostic for ß-structuring in proteins appear only at the H-bonded strand residues. The resulting periodicity of structuring shift magnitudes is not, however, a consequence of H-bonding status; rather, it reflects a previously unrecognized alternation in the backbone torsion angles of ß-strands. This feature of hairpins is also likely to be present in proteins. The study provides reference values for the expectation shifts for (13)C sites in ß-structures that should prove useful in the characterization of the folding equilibria of ß-sheet models.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Proteins/chemistry , Hydrogen Bonding , Protein Structure, SecondaryABSTRACT
Monoclonal antibody (mAb) 10E8 recognizes a highly conserved epitope on HIV and is capable of neutralizing > 95% of circulating viral isolates making it one of the most promising Abs against HIV. Solution instability and biochemical heterogeneity of 10E8 has hampered its development for clinical use. We identify the source of 10E8 heterogeneity being linked to cis/trans isomerization at two prolines within the YPP motif in the CRD3 loop that exists as two predominant conformers that interconvert on a slow timescale. The YtransP conformation conformer can bind the HIV gp41 epitope, while the YcisP is not binding competent and shows a higher aggregation propensity. The high barrier of isomerization and propensity to adopt non-binding competent proline conformers provides novel insight into the slow binding kinetics, low potency, and poor solubility of 10E8. This study highlights how proline isomerization should be considered a critical quality attribute for biotherapeutics with paratopes containing potential cis proline amide bonds.
Subject(s)
Antibodies, Monoclonal/chemistry , Isomerism , Proline/chemistryABSTRACT
This article describes data related to a research article titled "The Busulfan Metabolite EdAG Irreversibly Glutathionylates Glutaredoxins" [1]. EdAG is an electrophilic GSH analog formed in vivo from busulfan, which is used in hematopoietic stem cell transplants. EdAG glutathionylates Glutaredoxins (Grx's) but not glutathione transferase A1-1 (GSTA1-1) in vitro. This article includes a complete NMR characterization of synthetic EdAG including homonuclear and heteronuclear correlation spectra. Also included are mass spectra of peptides from Grx's or GSTA1-1 that have cys residues that do not react with EdAG.
ABSTRACT
Vitamin K sequentially undergoes ω-oxidation followed by successive rounds of ß-oxidation to ultimately produce two chain-shortened carboxylic acid metabolites, vitamin K acid 1 and vitamin K acid 2. Two facile syntheses of these acid metabolites are described, each starting from commercially available menadione-cyclopentadiene adduct 3. Vitamin K acid 1 was synthesized in five steps via alkylation with a geranyl halide followed by subsequent oxidation reactions, while fully retaining the trans configuration of the side chain 2',3'-double bond. Vitamin K acid 2 was synthesized in 5 steps from 3via alkylation with dimethylallyl chloride and subsequent oxidation reactions.
ABSTRACT
Sirtuins are a family of NAD(+)-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 µM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition.