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1.
Biophys J ; 123(1): 68-79, 2024 01 02.
Article in English | MEDLINE | ID: mdl-37978799

ABSTRACT

Measuring protein thermostability provides valuable information on the biophysical rules that govern the structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently developed nonfluorescent membrane scaffold protein to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the "dark" nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs loaded with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol (DMPG) lipids show a complex biphasic thermal unfolding pattern with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5°C and 77.5°C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a DMPC and DMPG nanodisc. To extend the utility of this method, we evaluate and compare the thermostability of DsbB in different lipid environments. We introduce this method as a new tool that can be used to understand how compositionally and biophysically complex lipid environments drive membrane protein stability.


Subject(s)
Dimyristoylphosphatidylcholine , Membrane Proteins , Dimyristoylphosphatidylcholine/chemistry , Temperature , Fluorometry , Disulfides , Lipid Bilayers/chemistry
2.
bioRxiv ; 2023 May 09.
Article in English | MEDLINE | ID: mdl-37214798

ABSTRACT

Measuring protein thermostability provides valuable information on the biophysical rules that govern structure-energy relationships of proteins. However, such measurements remain a challenge for membrane proteins. Here, we introduce a new experimental system to evaluate membrane protein thermostability. This system leverages a recently-developed non-fluorescent membrane scaffold protein (MSP) to reconstitute proteins into nanodiscs and is coupled with a nano-format of differential scanning fluorimetry (nanoDSF). This approach offers a label-free and direct measurement of the intrinsic tryptophan fluorescence of the membrane protein as it unfolds in solution without signal interference from the "dark" nanodisc. In this work, we demonstrate the application of this method using the disulfide bond formation protein B (DsbB) as a test membrane protein. NanoDSF measurements of DsbB reconstituted in dark nanodiscs show a complex biphasic thermal unfolding pattern in the presence of lipids with a minor unfolding transition followed by a major transition. The inflection points of the thermal denaturation curve reveal two distinct unfolding midpoint melting temperatures (Tm) of 70.5 °C and 77.5 °C, consistent with a three-state unfolding model. Further, we show that the catalytically conserved disulfide bond between residues C41 and C130 drives the intermediate state of the unfolding pathway for DsbB in a nanodisc. We introduce this method as a new tool that can be used to understand how compositionally, and biophysically complex lipid environments drive membrane protein stability.

3.
Structure ; 31(6): 713-723.e3, 2023 06 01.
Article in English | MEDLINE | ID: mdl-37119820

ABSTRACT

In-frame deletion mutations can result in disease. The impact of these mutations on protein structure and subsequent functional changes remain understudied, partially due to the lack of comprehensive datasets including a structural readout. In addition, the recent breakthrough in structure prediction through deep learning demands an update of computational deletion mutation prediction. In this study, we deleted individually every residue of a small α-helical sterile alpha motif domain and investigated the structural and thermodynamic changes using 2D NMR spectroscopy and differential scanning fluorimetry. Then, we tested computational protocols to model and classify observed deletion mutants. We show a method using AlphaFold2 followed by RosettaRelax performs the best overall. In addition, a metric containing pLDDT values and Rosetta ΔΔG is most reliable in classifying tolerated deletion mutations. We further test this method on other datasets and show they hold for proteins known to harbor disease-causing deletion mutations.


Subject(s)
Computational Biology , Proteins , Proteins/chemistry , Mutation , Computer Simulation , Sequence Deletion , Magnetic Resonance Spectroscopy
4.
J Biomol NMR ; 77(3): 69-82, 2023 Jun.
Article in English | MEDLINE | ID: mdl-37016190

ABSTRACT

A single experimental method alone often fails to provide the resolution, accuracy, and coverage needed to model integral membrane proteins (IMPs). Integrating computation with experimental data is a powerful approach to supplement missing structural information with atomic detail. We combine RosettaNMR with experimentally-derived paramagnetic NMR restraints to guide membrane protein structure prediction. We demonstrate this approach using the disulfide bond formation protein B (DsbB), an α-helical IMP. Here, we attached a cyclen-based paramagnetic lanthanide tag to an engineered non-canonical amino acid (ncAA) using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry reaction. Using this tagging strategy, we collected 203 backbone HN pseudocontact shifts (PCSs) for three different labeling sites and used these as input to guide de novo membrane protein structure prediction protocols in Rosetta. We find that this sparse PCS dataset combined with 44 long-range NOEs as restraints in our calculations improves structure prediction of DsbB by enhancements in model accuracy, sampling, and scoring. The inclusion of this PCS dataset improved the Cα-RMSD transmembrane segment values of the best-scoring and best-RMSD models from 9.57 Å and 3.06 Å (no NMR data) to 5.73 Å and 2.18 Å, respectively.


Subject(s)
Lanthanoid Series Elements , Membrane Proteins , Membrane Proteins/chemistry , Amino Acids , Lanthanoid Series Elements/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Magnetic Resonance Spectroscopy , Protein Conformation
5.
Biochemistry ; 62(3): 700-709, 2023 02 07.
Article in English | MEDLINE | ID: mdl-36626571

ABSTRACT

Fragment-based drug discovery begins with the identification of small molecules with a molecular weight of usually less than 250 Da which weakly bind to the protein of interest. This technique is challenging for computational docking methods as binding is determined by only a few specific interactions. Inaccuracies in the energy function or slight deviations in the docking pose can lead to the prediction of incorrect binding or difficulties in ranking fragments in in silico screening. Here, we test RosettaLigand by docking a series of fragments to a cysteine-depleted variant of the TIM-barrel protein, HisF (UniProtKB Q9X0C6). We compare the computational results with experimental NMR spectroscopy screens. NMR spectroscopy gives details on binding affinities of individual ligands, which allows assessment of the ligand-ranking ability using RosettaLigand and also provides feedback on the location of the binding pocket, which serves as a reliable test of RosettaLigand's ability to identify plausible binding poses. From a library screen of 3456 fragments, we identified a set of 31 ligands with intrinsic affinities to HisF with dissociation constants as low as 400 µM. The same library of fragments was blindly screened in silico. RosettaLigand was able to rank binders before non-binders with an area under the curve of the receiver operating characteristics of 0.74. The docking poses observed for binders agreed with the binding pocket identified by NMR chemical shift perturbations for all fragments. Taken together, these results provide a baseline performance of RosettaLigand in a fragment-based drug discovery setting.


Subject(s)
Drug Discovery , Proteins , Ligands , Drug Discovery/methods , Proteins/chemistry , Protein Binding , Binding Sites
6.
PLoS Pathog ; 18(5): e1010518, 2022 05.
Article in English | MEDLINE | ID: mdl-35584193

ABSTRACT

The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 -membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope.


Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Animals , Antibodies, Neutralizing , Antibodies, Viral , Epitopes , Glycoproteins , Rabbits
7.
Ophthalmic Genet ; 43(4): 530-533, 2022 08.
Article in English | MEDLINE | ID: mdl-35315300

ABSTRACT

PURPOSE: To provide the initial confirmation of the c.1772C>T (p.Ser591Phe) mutation in the transforming growth factor-ß-induced (TGFBI) gene as being associated with variant lattice corneal dystrophy (LCD). METHODS: Ophthalmologic examination of the proband was performed with slit lamp biomicroscopy. Saliva was collected as a source of DNA for screening all 17 exons of TGFBI, after which three family members were selectively screened for variants in exon 13. Rosetta-based structure prediction was used to calculate changes in TGFBI protein (TGFBIp) stability secondary to the c.1772C>T (p.Ser591Phe) missense mutation. RESULTS: Slit lamp examination of the 38-year-old proband revealed a clear cornea right eye and unilateral, discrete, and branching lattice lines in the anterior and mid-stroma of the central cornea left eye. Screening of TGFBI in the proband revealed a heterozygous missense mutation in exon 13 (c.1772C>T (p.Ser591Phe)) that was also identified in her affected mother but not in her brother or maternal grandmother. Calculated energy change in Rosetta (ΔΔG) for the TGFBIp variant p.Ser591Phe was 23.5, indicating a thermodynamic destabilization resulting from energetic frustration. CONCLUSIONS: The p.Ser591Phe mutation in TGFBI is associated with an unilateral variant of LCD. Rosetta-predicted stability changes indicate that the p.Ser591Phe variant is destabilizing, which is consistent with other observations for LCD-causing mutations.


Subject(s)
Amyloid Neuropathies, Familial , Corneal Dystrophies, Hereditary , Extracellular Matrix Proteins , Transforming Growth Factors , Adult , Corneal Dystrophies, Hereditary/diagnosis , Corneal Dystrophies, Hereditary/genetics , DNA Mutational Analysis , Extracellular Matrix Proteins/genetics , Female , Humans , Male , Mutation , Mutation, Missense , Pedigree , Transforming Growth Factor beta , Transforming Growth Factors/genetics
8.
Elife ; 102021 05 18.
Article in English | MEDLINE | ID: mdl-34002696

ABSTRACT

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.


Subject(s)
Dopamine Plasma Membrane Transport Proteins/genetics , Drosophila melanogaster , Dystonic Disorders/genetics , Parkinson Disease/genetics , Psychomotor Disorders/genetics , Animals , Chloroquine/pharmacology , Disease Models, Animal , Dopamine/metabolism , Dopamine Plasma Membrane Transport Proteins/deficiency , Dopamine Plasma Membrane Transport Proteins/drug effects , Dystonic Disorders/drug therapy , Flight, Animal/drug effects , HEK293 Cells , Humans , Molecular Structure , Mutation, Missense , Parkinson Disease/drug therapy , Psychomotor Disorders/drug therapy
9.
J Biomol NMR ; 73(6-7): 347-364, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31243635

ABSTRACT

The translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), is a membrane protein located on the outer mitochondrial membrane. Experimentally-derived structures of mouse TSPO (mTSPO) and its homologs from bacterial species have been determined by NMR spectroscopy and X-ray crystallography, respectively. These structures and ligand interactions within the TSPO binding pocket display distinct differences. Here, we leverage experimental and computational studies to derive a unified structural model of mTSPO in the presence and absence of the TSPO ligand, PK11195, and study the effects of DPC detergent micelles on the TSPO structure and ligand binding. From this work, we conclude that that the lipid-mimetic system used to solubilize mTSPO for NMR studies thermodynamically destabilizes the protein, introduces structural perturbations, and alters the characteristics of ligand binding. Furthermore, we used Rosetta to construct a unified mTSPO model that reconciles deviating features of the mammalian and bacterial TSPO. These deviating features are likely a consequence of the detergent system used for structure determination of mTSPO by NMR. The unified mTSPO model agrees with available experimental NMR data, appears to be physically realistic (i.e. thermodynamically not frustrated as judged by the Rosetta energy function), and simultaneously shares the structural features observed in sequence-conserved regions of the bacterial proteins. Finally, we identified the binding site for an imaging ligand VUIIS8310 that is currently positioned for clinical translation using NMR spectroscopy and propose a computational model of the VUIIS8310-mTSPO complex.


Subject(s)
Models, Molecular , Protein Conformation , Receptors, GABA/chemistry , Animals , Bacterial Proteins/chemistry , Ligands , Mammals , Mice , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Permeability Transition Pore , Molecular Imaging , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Receptors, GABA/metabolism
10.
J Pharm Sci ; 107(7): 1937-1947, 2018 07.
Article in English | MEDLINE | ID: mdl-29499278

ABSTRACT

P-glycoprotein (Pgp) is a multidrug resistance transporter that limits the penetration of a wide range of neurotherapeutics into the brain including opioids. The diphenylpropylamine opioids methadone and loperamide are structurally similar, but loperamide has about a 4-fold higher Pgp-mediated transport rate. In addition to these differences, they showed significant differences in their effects on Pgp-mediated adenosine triphosphate (ATP) hydrolysis. The activation of Pgp-mediated ATP hydrolysis by methadone was monophasic, whereas loperamide activation of ATP hydrolysis was biphasic implying methadone has a single binding site and loperamide has 2 binding sites on Pgp. Quenching of tryptophan fluorescence with these drugs and digoxin showed competition between the opioids and that loperamide does not compete for the digoxin-binding site. Acrylamide quenching of tryptophan fluorescence to probe Pgp conformational changes revealed that methadone- and loperamide-induced conformational changes were distinct. These results were used to develop a model for Pgp-mediated transport of methadone and loperamide where opioid binding and conformational changes are used to explain the differences in the opioid transport rates between methadone and loperamide.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Analgesics, Opioid/metabolism , Loperamide/metabolism , Methadone/metabolism , ATP Binding Cassette Transporter, Subfamily B/chemistry , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Analgesics, Opioid/chemistry , Animals , Binding Sites , Biological Transport , Hydrolysis , Loperamide/chemistry , Methadone/chemistry , Mice , Protein Conformation
11.
FASEB J ; 32(2): 669-680, 2018 02.
Article in English | MEDLINE | ID: mdl-28939593

ABSTRACT

ATP binding cassette type-C (ABCC) transporters move molecules across cell membranes upon hydrolysis of ATP; however, their coupling of ATP hydrolysis to substrate transport remains elusive. Drosophila multidrug resistance-associated protein (DMRP) is the functional ortholog of human long ABCC transporters, with similar substrate and inhibitor specificity, but higher activity. Exploiting its high activity, we kinetically dissected the catalytic mechanism of DMRP by using E2-d-glucuronide (E2G), the physiologic substrate of human ABCC. We examined the DMRP-mediated interdependence of ATP and E2G in biochemical assays. We observed E2G-dependent ATPase activity to be biphasic at subsaturating ATP concentrations, which implies at least 2 E2G binding sites on DMRP. Furthermore, transport measurements indicated strong nonreciprocal cooperativity between ATP and E2G. In addition to confirming these findings, our kinetic modeling with the Complex Pathway Simulator indicated a 10-fold decrease in the E2G-mediated activation of ATP hydrolysis upon saturation of the second E2G binding site. Surprisingly, the binding of the second E2G allowed for substrate transport with a constant rate, which tightly coupled ATP hydrolysis to transport. In summary, we show that the second E2G binding-similar to human ABCC2-allosterically stimulates transport activity of DMRP. Our data suggest that this is achieved by a significant increase in the coupling of ATP hydrolysis to transport.-Karasik, A., Ledwitch, K. V., Arányi, T., Váradi, A., Roberts, A., Szeri, F. Boosted coupling of ATP hydrolysis to substrate transport upon cooperative estradiol-17-ß-D-glucuronide binding in a Drosophila ATP binding cassette type-C transporter.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Adenosine Triphosphate/chemistry , Drosophila Proteins/chemistry , Estradiol/analogs & derivatives , ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster , Estradiol/chemistry , Estradiol/metabolism , Humans , Hydrolysis , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/metabolism
12.
AAPS J ; 19(2): 409-420, 2017 03.
Article in English | MEDLINE | ID: mdl-28028729

ABSTRACT

P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that plays a major role in cardiovascular drug disposition by effluxing a chemically and structurally diverse range of cardiovascular therapeutics. Unfortunately, drug-drug interactions (DDIs) with the transporter have become a major roadblock to effective cardiovascular drug administration because they can cause adverse drug reactions (ADRs) or reduce the efficacy of drugs. Cardiovascular ion channel inhibitors are particularly susceptible to DDIs and ADRs with Pgp because they often have low therapeutic indexes and are commonly coadministered with other drugs that are also Pgp substrates. DDIs from cardiovascular ion channel inhibitors with the transporter occur because of inhibition or induction of the transporter and the transporter's tissue and cellular localization. Inhibiting Pgp can increase absorption and reduce excretion of drugs, leading to elevated drug plasma concentrations and drug toxicity. In contrast, inducing Pgp can have the opposite effect by reducing the drug plasma concentration and its efficacy. A number of in vitro and in vivo studies have already demonstrated DDIs from several cardiovascular ion channel inhibitors with human Pgp and its animal analogs, including verapamil, digoxin, and amiodarone. In this review, Pgp-mediated DDIs and their effects on pharmacokinetics for different categories of cardiovascular ion channel inhibitors are discussed. This information is essential for improving pharmacokinetic predictions of cardiovascular therapeutics, for safer cardiovascular drug administration and for mitigating ADRs emanating from Pgp.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cardiovascular Agents/administration & dosage , ATP Binding Cassette Transporter, Subfamily B, Member 1/drug effects , Animals , Biological Transport , Cardiovascular Agents/adverse effects , Cardiovascular Agents/pharmacokinetics , Drug Interactions , Humans , Ion Channels/antagonists & inhibitors
13.
Biochem Pharmacol ; 118: 96-108, 2016 Oct 15.
Article in English | MEDLINE | ID: mdl-27531061

ABSTRACT

The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/agonists , Adenosine Triphosphate/metabolism , Anti-Arrhythmia Agents/metabolism , Calcium Channel Blockers/metabolism , Models, Molecular , Verapamil/metabolism , ATP Binding Cassette Transporter, Subfamily B/chemistry , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/chemistry , Adenylyl Imidodiphosphate/chemistry , Adenylyl Imidodiphosphate/metabolism , Algorithms , Animals , Anti-Arrhythmia Agents/chemistry , Anti-Arrhythmia Agents/pharmacology , Binding Sites , Biocatalysis/drug effects , Calcium Channel Blockers/chemistry , Calcium Channel Blockers/pharmacology , Computer Simulation , Hydrolysis/drug effects , Ligands , Liposomes , Mice , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation/drug effects , Protein Folding/drug effects , Protein Stability/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Verapamil/chemistry , Verapamil/pharmacology
14.
Biosci Rep ; 36(2)2016 Jan 28.
Article in English | MEDLINE | ID: mdl-26823559

ABSTRACT

Drug-drug interactions (DDIs) and associated toxicity from cardiovascular drugs represents a major problem for effective co-administration of cardiovascular therapeutics. A significant amount of drug toxicity from DDIs occurs because of drug interactions and multiple cardiovascular drug binding to the efflux transporter P-glycoprotein (Pgp), which is particularly problematic for cardiovascular drugs because of their relatively low therapeutic indexes. The calcium channel antagonist, verapamil and the cardiac glycoside, digoxin, exhibit DDIs with Pgp through non-competitive inhibition of digoxin transport, which leads to elevated digoxin plasma concentrations and digoxin toxicity. In the present study, verapamil-induced ATPase activation kinetics were biphasic implying at least two verapamil-binding sites on Pgp, whereas monophasic digoxin activation of Pgp-coupled ATPase kinetics suggested a single digoxin-binding site. Using intrinsic protein fluorescence and the saturation transfer double difference (STDD) NMR techniques to probe drug-Pgp interactions, verapamil was found to have little effect on digoxin-Pgp interactions at low concentrations of verapamil, which is consistent with simultaneous binding of the drugs and non-competitive inhibition. Higher concentrations of verapamil caused significant disruption of digoxin-Pgp interactions that suggested overlapping and competing drug-binding sites. These interactions correlated to drug-induced conformational changes deduced from acrylamide quenching of Pgp tryptophan fluorescence. Also, Pgp-coupled ATPase activity kinetics measured with a range of verapamil and digoxin concentrations fit well to a DDI model encompassing non-competitive and competitive inhibition of digoxin by verapamil. The results and previous transport studies were combined into a comprehensive model of verapamil-digoxin DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/chemistry , Digoxin/chemistry , Models, Chemical , Verapamil/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Biological Transport, Active , Digoxin/pharmacokinetics , Drug Interactions , Mice , Verapamil/pharmacokinetics
15.
Biochim Biophys Acta ; 1860(4): 669-77, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26723172

ABSTRACT

BACKGROUND: Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure. METHODS: The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures. RESULTS: Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F-G loop regions and active site cavity volumes for the F87A mutated structures. CONCLUSIONS: Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site. GENERAL SIGNIFICANCE: The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.


Subject(s)
Bacillus megaterium/enzymology , Bacterial Proteins/chemistry , Cytochrome P-450 Enzyme System/chemistry , NADPH-Ferrihemoprotein Reductase/chemistry , Quinones/chemistry , Bacillus megaterium/genetics , Bacterial Proteins/genetics , Binding Sites , Cytochrome P-450 Enzyme System/genetics , NADPH-Ferrihemoprotein Reductase/genetics , Phenylalanine/chemistry , Phenylalanine/genetics , Protein Binding , Structure-Activity Relationship , Threonine/chemistry , Threonine/genetics
16.
Bioorg Med Chem Lett ; 24(1): 371-7, 2014 Jan 01.
Article in English | MEDLINE | ID: mdl-24269164

ABSTRACT

A number of delivery agents, such as proteins, liposomes, micelles, and nanoparticles, are utilized for transporting pharmaceutical agents in a physiological environment. This Letter focuses on the use of the copper(II) ion and its potential role as a delivery agent for the taxanes and taxol couple to a malaria drug. Nuclear magnetic resonance (NMR, (1)H, (13)C, (15)N), Mass Spectrometry (LC-MS, MALDI-TOF, FT-ICR) and computational methods are used to examine the structure of the complex. The National Cancer Institute's benchmark 60 cell line panel is used to compare the efficacy of the copper-taxol and copper-taxol-hydroxychloroquin complexes to that of iron-taxol and pure taxol.


Subject(s)
Antimalarials/chemistry , Antineoplastic Agents/chemistry , Copper/chemistry , Drug Carriers/chemistry , Organometallic Compounds/chemistry , Taxoids/chemistry , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Delivery Systems , Drug Screening Assays, Antitumor , Humans , Hydroxychloroquine/chemistry , Ions/chemistry , Molecular Structure , Organometallic Compounds/chemical synthesis , Paclitaxel/chemistry , Structure-Activity Relationship
17.
Mini Rev Med Chem ; 13(4): 509-21, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23373651

ABSTRACT

In medicinal chemistry, one of the most studied molecules in recent history is taxol. Taxol is a versatile natural product that is used in various cancer treatment regimens. It is administered to patients with breast, lung, and ovarian cancers, and is currently being studied for the treatment of squamous cell carcinoma of the oral cavity and tongue. Taxol has been tested in a number of research and clinical phase trials to determine feasibility, toxicity, and cytotoxicity against oral squamous cell carcinoma as a single drug regimen and as a contributing drug component in treatment plans. This paper reviews over forty articles that examine cell lines, murine models, and human results for the response of taxol against squamous cell carcinoma (SCC) of the oral cavity and the tongue.


Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Mouth Neoplasms/drug therapy , Paclitaxel/chemistry , Tongue Neoplasms/drug therapy , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Squamous Cell/mortality , Cell Survival/drug effects , Clinical Trials as Topic , Disease Models, Animal , Drug Therapy, Combination , Humans , Mouth Neoplasms/mortality , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Platinum Compounds/chemistry , Platinum Compounds/pharmacology , Platinum Compounds/therapeutic use , Survival Rate , Tongue Neoplasms/mortality
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