Spencer S Ericksen | University of Wisconsin-Madison (original) (raw)
Papers by Spencer S Ericksen
The Dopamine Receptors, 2010
G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins representing the larges... more G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins representing the largest and most universally expressed cell surface receptors and are present in almost all species and in a wide variety of cells. Here we will focus our attention on the catecholamine-binding GPCRs and in particular on the dopamine receptors. The catecholamine-binding GPCRs form a group of rhodopsin-like GPCRs composed of adrenoceptors, which are endogenously activated by epinephrine and norepinephrine, and dopamine receptors. We review the different "molecular switches" involved in GPCR activation and we emphasize the importance of extracellular loop 2 (ECL2) in ligand binding. A better understanding of the functional role of ECL2 can be achieved after the release of the crystal structures of B2AR and rhodopsin, which are consistent with dopamine D2 receptor substituted cysteine accessibility method (SCAM) experimental data. Even though reconstituted GPCR monomers appear sufficient to activate a G protein, in the native setting their dimerization/oligomerization may modulate activation through changes at the dimerization interface or a larger-scale reorientation of the protomers. Therefore, the structural aspects of oligomerization and their importance for receptor activation and signaling are also addressed.
Journal of Pharmacology and Experimental Therapeutics, 2009
We have uncovered a significant allosteric response of the D 2 dopamine receptor to physiological... more We have uncovered a significant allosteric response of the D 2 dopamine receptor to physiologically relevant concentrations of sodium (140 mM), characterized by a sodium-enhanced binding affinity for a D 4 -selective class of agonists and antagonists. This enhancement is significantly more pronounced in a D 2 -V2.61(91)F mutant and cannot be mimicked by an equivalent concentration of the sodium replacement cation N-methyl-D-glucamine. This phenomenon was explored computationally at the molecular level by analyzing the effect of sodium binding on the dynamic properties of D 2 receptor model constructs. Normal mode analysis identified one mode (M 19 ), which is involved in the open/closed motions of the binding cleft as being particularly sensitive to the sodium effect. To examine the consequences for D 2 receptor ligand recognition, one of the ligands, L-745,870 [3-{[4-(4-chlorophenyl) piperazin-1-yl]-methyl}-1H-pyrrolo[2,3-b]pyridine or CPPMA, chlorophenyl-piperazinyl methylazaindole], was docked into conformers along the M 19 trajectory. Structurally and pharmacologically well established ligand-receptor interactions, including the ionic interaction with D3.32(114) and interactions between the ligand aryl moieties and V2.61(91)F, were achieved only in "open" phase conformers.
Molecular Neurobiology, 2007
Receptor subunits in the Cys-loop superfamily assemble to form channels as hom opentamers or hete... more Receptor subunits in the Cys-loop superfamily assemble to form channels as hom opentamers or heteropentamers, expanding functional diversity through modularity. Expression of two or more compatible subunit types can lead to various receptor assemblies or subtypes. However, what may be good for diversity in vivo may be undesirable for the bench scientist, because we often wish to reduce our analyses to a single receptor subtype. By linking two or more subunits, creating tandems or concatamers, we can control stoichiometry and limit expression to exactly one receptor subtype. In this fashion, receptors with mixed subunit subtypes and heterozygous mutations can be separated from a mixture and can be described in detail. However, several recent studies have shown that this may be more easily conceived than accomplished, because several unforeseen problems have arisen. Concatamers can degrade, linkers can sometimes be clipped after or during translation, and one subunit may “loop out” or even become part of a second (now linked) pentamer with different characteristics. Some strategies have been developed to overcome these drawbacks, and the resultant new information that has begun to emerge has revitalized the study of these receptors in heterologous expression systems.
Journal of Neurochemistry, 2009
The D2 dopamine receptor is an important therapeutic target for the treatment of psychotic, agita... more The D2 dopamine receptor is an important therapeutic target for the treatment of psychotic, agitated, and abnormal behavioral states. To better understand the specific interactions of subtype-selective ligands with dopamine receptor subtypes, seven ligands with high selectivity (>120-fold) for the D4 subtype of dopamine receptor were tested on wild-type and mutant D2 receptors. Five of the selective ligands were observed to have 21-fold to 293-fold increases in D2 receptor affinity when three non-conserved amino acids in TM2 and TM3 were mutated to the corresponding D4 amino acids. The two ligands with the greatest improvement in affinity for the D2 mutant receptor [i.e., 3-{[4-(4-iodophenyl) piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine (L-750,667) and 1-[4-iodobenzyl]-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine (RBI-257)] were investigated in functional assays. Consistent with their higher affinity for the mutant than for the wild-type receptor, concentrations of L-750,667 or RBI-257 that produced large reductions in the potency of quinpirole’s functional response in the mutant did not significantly reduce quinpirole’s functional response in the wild-type D2 receptor. In contrast to RBI-257 which is an antagonist at all receptors, L-750,667 is a partial agonist at the wild-type D2 but an antagonist at both the mutant D2 and wild-type D4 receptors. Our study demonstrates for the first time that the TM2/3 microdomain of the D2 dopamine receptor not only regulates the selective affinity of ligands, but in selected cases can also regulate their function. Utilizing a new docking technique that incorporates receptor backbone flexibility, the three non-conserved amino acids that encompass the TM2/3 microdomain were found to account in large part for the differences in intermolecular steric contacts between the ligands and receptors. Consistent with the experimental data, this model illustrates the interactions between a variety of subtype-selective ligands and the wild-type D2, mutant D2, or wild-type D4 receptors.
Drug Metabolism and Disposition, 2003
This article is available online at http://dmd.aspetjournals.org
Journal of Pharmacology and Experimental Therapeutics, 2009
We have uncovered a significant allosteric response of the D 2 dopamine receptor to physiological... more We have uncovered a significant allosteric response of the D 2 dopamine receptor to physiologically relevant concentrations of sodium (140 mM), characterized by a sodium-enhanced binding affinity for a D 4 -selective class of agonists and antagonists. This enhancement is significantly more pronounced in a D 2 -V2.61(91)F mutant and cannot be mimicked by an equivalent concentration of the sodium replacement cation N-methyl-D-glucamine. This phenomenon was explored computationally at the molecular level by analyzing the effect of sodium binding on the dynamic properties of D 2 receptor model constructs. Normal mode analysis identified one mode (M 19 ), which is involved in the open/closed motions of the binding cleft as being particularly sensitive to the sodium effect. To examine the consequences for D 2 receptor ligand recognition, one of the ligands, L-745,870 [3-{[4-(4-chlorophenyl) piperazin-1-yl]-methyl}-1H-pyrrolo[2,3-b]pyridine or CPPMA, chlorophenyl-piperazinyl methylazaindole], was docked into conformers along the M 19 trajectory. Structurally and pharmacologically well established ligand-receptor interactions, including the ionic interaction with D3.32(114) and interactions between the ligand aryl moieties and V2.61(91)F, were achieved only in "open" phase conformers.
G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins representing the larges... more G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins representing the largest and most universally expressed cell surface receptors and are present in almost all species and in a wide variety of cells. Here we will focus our attention on the catecholamine-binding GPCRs and in particular on the dopamine receptors. The catecholamine-binding GPCRs form a group of rhodopsin-like GPCRs composed of adrenoceptors, which are endogenously activated by epinephrine and norepinephrine, and dopamine receptors. We review the different “molecular switches” involved in GPCR activation and we emphasize the importance of extracellular loop 2 (ECL2) in ligand binding. A better understanding of the functional role of ECL2 can be achieved after the release of the crystal structures of B2AR and rhodopsin, which are consistent with dopamine D2 receptor substituted cysteine accessibility method (SCAM) experimental data. Even though reconstituted GPCR monomers appear sufficient to activate a G protein, in the native setting their dimerization/oligomerization may modulate activation through changes at the dimerization interface or a larger-scale reorientation of the protomers. Therefore, the structural aspects of oligomerization and their importance for receptor activation and signaling are also addressed.
Journal of Biomolecular Structure & Dynamics, 2005
Cytochrome P450 1A1 oxidizes a diverse range of substrates, including the procarcinogenic xenobio... more Cytochrome P450 1A1 oxidizes a diverse range of substrates, including the procarcinogenic xenobiotic benzo[a]pyrene (B[a]P) and endogenous fatty acid precursors of prostaglandins, such as arachidonic acid (AA) and eicosapentaenoic acid (EA). We have investigated the extent to which enzyme-substrate interactions govern regio- and stereoselectivity of oxidation of these compounds by using docking and molecular dynamics (MD) simulations to examine the likelihood of substrate oxidation at various sites. Due to structural differences between the substrates analyzed, B[a]P and its diols (planar, rigid), and the fatty acids AA and EA (long, flexible), different docking strategies were required. B[a]P, B[a]P-7,8-diols, (+) 7S,85- and (-) 7R,8R-diols, were docked into the active site of a homology model of P450 1A1 using an automated routine. Affinity (Accelrys, San Diego, CA). AA and EA, on the other hand, required a series of restrained MD simulations to obtain a variety of productive binding modes. All complexes were evaluated by MD-based in silico site scoring to predict product profiles based on certain geometric criteria, such as angle and distance of a given substrate atom from the ferryl oxygen. For all substrates studied, the in vitro profiles were generally reflected by the in silico scores, which suggests that steric factors play a key role in determining regiospecificity in P450 1A1-mediated oxidations. We have also shown that molecular dynamics simulations may be very useful in determination of product profiles for structurally diverse substrates of P450 enzymes.
Journal of Pharmacology and Experimental Therapeutics, 2010
Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of e... more Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of endogenous catecholamines with ␣ 1 -and ␣ 2 -adrenergic,  2 -adrenergic, and D1, D2, and D3 dopamine receptors. The unique high-affinity interaction of the D4 dopamine receptor subtype with both norepinephrine and dopamine, and the fact that TM5 serine interactions have never been studied for this receptor subtype, led us to investigate the interactions of ligands with D4 receptor TM5 serines. Serine-to-alanine mutations at positions 5.42 and 5.46 drastically decreased affinities of dopamine and norepinephrine for the D4 receptor. The D4-S5.43A receptor mutant had substantially reduced affinity for norepinephrine, but a modest loss of affinity for dopamine. In functional assays of cAMP accumulation, norephinephrine was unable to activate any of the mutant receptors, even though the agonist quinpirole displayed wild-type functional ABBREVIATIONS: TM, transmembrane segment; ABT-724, 2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]-1H-benzimidazole; CP226,269, 5-fluoro-2-{[4-(2-pyridinyl)-1-piperazinyl]methyl}-1H-indole; L-750,667, 3-{[4-(4-iodophenyl)piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine; MIA, methylisobutylamiloride; PD168,077, N-{[4-(2-cyanophenyl)piperazin-1-yl]methyl}-3-methylbenzamide; PNU101,387G, 4-[4-[2-[(1S)-3,4dihydro-1H-isochromen-1-yl]ethyl]piperazin-1-yl]benzenesulfonamide (sonepiprazole); RMSD, root mean square deviation; Ro10-4548, RAC-2Ј-2-hydroxy-3-4-(4-hydroxy-2-methoxyphenyl)-1-piperazinyl-propoxy-acetanilide; Ro10-5824, 5-[(3,6-dihydro-4-phenyl-1(2H)pyridinyl)methyl]-2-methyl-4-pyrimidinamine; Ro20-1724, 4-(3-butoxy-4-methoxyphenyl)methyl-2-imidazolidone; ANOVA, analysis of variance; HBSS, Hanks' balanced salt solution; GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; HEK, human embryonic kidney.
Biochemical Pharmacology, 2004
Human cytochrome P450 1A1 (CYP1A1) and human NADPH-cytochrome P450 reductase were expressed and p... more Human cytochrome P450 1A1 (CYP1A1) and human NADPH-cytochrome P450 reductase were expressed and purified from Spodoptera frugiperda insect cells. A reconstituted enzymatically active system metabolized polyunsaturated fatty acids such as arachidonic (AA) and eicosapentaenoic acid (EPA). CYP1A1 was an AA hydroxylase which oxidizes this substrate at a rate of 650 AE 10 pmol/min/nmol CYP1A1, with over 90% of metabolites accounted for by hydroxylation products and with 19-OH-AA as major product. Epoxyeicosatrienoic acid (EET), mainly 14,15-EET, accounted for about 7% of total metabolites. Unlike rat CYP1A1, the human enzyme exhibited no 20-OH-AA as product. In contrast, with EPA as substrate CYP1A1 was mainly an epoxygenase, oxidizing with over 68% of total metabolites EPA to 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EETeTr). 19-OH-EPA accounted for about 31% of total metabolites. Significantly, the 17,18-olefinic bond of EPA was epoxidized to 17(R),18(S)-EETeTr with nearly absolute regio-and stereoselectivity. Molecular modeling analyses provided rationale for high efficiency of AA hydroxylation at C 19 and its gradual decrease down to C 14 , as well as for the limited EPA 17(S),18(R) epoxidation due to unfavorable enzyme-substrate interactions. The absence of ohydroxylation for both substrates is not due to steric factors, but probably a consequence of different reactivities of o and (o À 1) carbons for hydrogen abstraction. It is suggested that the capacity of human CYP1A1 to metabolize AA and EPA and its inducibility by polycyclic aromatic hydrocarbons may affect the production of physiologically active metabolites, in particular, in the cardiovascular system and other extrahepatic tissues including lung.
Archives of Biochemistry and Biophysics, 2004
Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 ! A... more Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 ! Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy-and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue. Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models.
Molecular Pharmacology, 2006
The imidazopyridine zolpidem (Ambien) is one of the most commonly prescribed sleep aids in the Un... more The imidazopyridine zolpidem (Ambien) is one of the most commonly prescribed sleep aids in the United States . Similar to classic benzodiazepines (BZDs), zolpidem binds at the extracellular N-terminal ␣/␥ subunit interface of the GABA-A receptor (GABAR). However, zolpidem differs significantly from classic BZDs in chemical structure and neuropharmacological properties. Thus, classic BZDs and zolpidem are likely to have different requirements for high-affinity binding to GABARs. To date, three residues-␥2Met57, ␥2Phe77, and ␥2Met130 -have been identified as necessary for high-affinity zolpidem binding (Proc Natl Acad Sci USA 94:8824 -8829, 1997; Mol Pharmacol 52: 874 -881, 1997). In this study, we used radioligand binding techniques, ␥2/␣1 chimeric subunits (), site-directed mutagenesis, and molecular modeling to identify additional ␥2 subunit residues important for high-affinity zolpidem binding. Whereas ␣12 receptors containing only the first 161 amino-terminal residues of the ␥2 subunit bind the classic BZD flunitrazepam with wild-type affinity, zolpidem affinity is decreased ϳ8-fold. By incrementally restoring ␥2 subunit sequence, we identified a seven-amino acid stretch in the ␥2 subunit loop F region (amino acids 186 -192) that is required to confer high-affinity zolpidem binding to GABARs. When mapped to a homology model, these seven amino acids make up part of loop F located at the ␣/␥ interface. Based on in silico zolpidem docking, three residues within loop F, ␥2Glu189, ␥2Thr193, and ␥2Arg194, emerge as being important for stabilizing zolpidem in the BZD binding pocket and probably interact with other loop F residues to maintain the structural integrity of the BZD binding site.
The Dopamine Receptors, 2010
G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins representing the larges... more G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins representing the largest and most universally expressed cell surface receptors and are present in almost all species and in a wide variety of cells. Here we will focus our attention on the catecholamine-binding GPCRs and in particular on the dopamine receptors. The catecholamine-binding GPCRs form a group of rhodopsin-like GPCRs composed of adrenoceptors, which are endogenously activated by epinephrine and norepinephrine, and dopamine receptors. We review the different "molecular switches" involved in GPCR activation and we emphasize the importance of extracellular loop 2 (ECL2) in ligand binding. A better understanding of the functional role of ECL2 can be achieved after the release of the crystal structures of B2AR and rhodopsin, which are consistent with dopamine D2 receptor substituted cysteine accessibility method (SCAM) experimental data. Even though reconstituted GPCR monomers appear sufficient to activate a G protein, in the native setting their dimerization/oligomerization may modulate activation through changes at the dimerization interface or a larger-scale reorientation of the protomers. Therefore, the structural aspects of oligomerization and their importance for receptor activation and signaling are also addressed.
Journal of Pharmacology and Experimental Therapeutics, 2009
We have uncovered a significant allosteric response of the D 2 dopamine receptor to physiological... more We have uncovered a significant allosteric response of the D 2 dopamine receptor to physiologically relevant concentrations of sodium (140 mM), characterized by a sodium-enhanced binding affinity for a D 4 -selective class of agonists and antagonists. This enhancement is significantly more pronounced in a D 2 -V2.61(91)F mutant and cannot be mimicked by an equivalent concentration of the sodium replacement cation N-methyl-D-glucamine. This phenomenon was explored computationally at the molecular level by analyzing the effect of sodium binding on the dynamic properties of D 2 receptor model constructs. Normal mode analysis identified one mode (M 19 ), which is involved in the open/closed motions of the binding cleft as being particularly sensitive to the sodium effect. To examine the consequences for D 2 receptor ligand recognition, one of the ligands, L-745,870 [3-{[4-(4-chlorophenyl) piperazin-1-yl]-methyl}-1H-pyrrolo[2,3-b]pyridine or CPPMA, chlorophenyl-piperazinyl methylazaindole], was docked into conformers along the M 19 trajectory. Structurally and pharmacologically well established ligand-receptor interactions, including the ionic interaction with D3.32(114) and interactions between the ligand aryl moieties and V2.61(91)F, were achieved only in "open" phase conformers.
Molecular Neurobiology, 2007
Receptor subunits in the Cys-loop superfamily assemble to form channels as hom opentamers or hete... more Receptor subunits in the Cys-loop superfamily assemble to form channels as hom opentamers or heteropentamers, expanding functional diversity through modularity. Expression of two or more compatible subunit types can lead to various receptor assemblies or subtypes. However, what may be good for diversity in vivo may be undesirable for the bench scientist, because we often wish to reduce our analyses to a single receptor subtype. By linking two or more subunits, creating tandems or concatamers, we can control stoichiometry and limit expression to exactly one receptor subtype. In this fashion, receptors with mixed subunit subtypes and heterozygous mutations can be separated from a mixture and can be described in detail. However, several recent studies have shown that this may be more easily conceived than accomplished, because several unforeseen problems have arisen. Concatamers can degrade, linkers can sometimes be clipped after or during translation, and one subunit may “loop out” or even become part of a second (now linked) pentamer with different characteristics. Some strategies have been developed to overcome these drawbacks, and the resultant new information that has begun to emerge has revitalized the study of these receptors in heterologous expression systems.
Journal of Neurochemistry, 2009
The D2 dopamine receptor is an important therapeutic target for the treatment of psychotic, agita... more The D2 dopamine receptor is an important therapeutic target for the treatment of psychotic, agitated, and abnormal behavioral states. To better understand the specific interactions of subtype-selective ligands with dopamine receptor subtypes, seven ligands with high selectivity (>120-fold) for the D4 subtype of dopamine receptor were tested on wild-type and mutant D2 receptors. Five of the selective ligands were observed to have 21-fold to 293-fold increases in D2 receptor affinity when three non-conserved amino acids in TM2 and TM3 were mutated to the corresponding D4 amino acids. The two ligands with the greatest improvement in affinity for the D2 mutant receptor [i.e., 3-{[4-(4-iodophenyl) piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine (L-750,667) and 1-[4-iodobenzyl]-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine (RBI-257)] were investigated in functional assays. Consistent with their higher affinity for the mutant than for the wild-type receptor, concentrations of L-750,667 or RBI-257 that produced large reductions in the potency of quinpirole’s functional response in the mutant did not significantly reduce quinpirole’s functional response in the wild-type D2 receptor. In contrast to RBI-257 which is an antagonist at all receptors, L-750,667 is a partial agonist at the wild-type D2 but an antagonist at both the mutant D2 and wild-type D4 receptors. Our study demonstrates for the first time that the TM2/3 microdomain of the D2 dopamine receptor not only regulates the selective affinity of ligands, but in selected cases can also regulate their function. Utilizing a new docking technique that incorporates receptor backbone flexibility, the three non-conserved amino acids that encompass the TM2/3 microdomain were found to account in large part for the differences in intermolecular steric contacts between the ligands and receptors. Consistent with the experimental data, this model illustrates the interactions between a variety of subtype-selective ligands and the wild-type D2, mutant D2, or wild-type D4 receptors.
Drug Metabolism and Disposition, 2003
This article is available online at http://dmd.aspetjournals.org
Journal of Pharmacology and Experimental Therapeutics, 2009
We have uncovered a significant allosteric response of the D 2 dopamine receptor to physiological... more We have uncovered a significant allosteric response of the D 2 dopamine receptor to physiologically relevant concentrations of sodium (140 mM), characterized by a sodium-enhanced binding affinity for a D 4 -selective class of agonists and antagonists. This enhancement is significantly more pronounced in a D 2 -V2.61(91)F mutant and cannot be mimicked by an equivalent concentration of the sodium replacement cation N-methyl-D-glucamine. This phenomenon was explored computationally at the molecular level by analyzing the effect of sodium binding on the dynamic properties of D 2 receptor model constructs. Normal mode analysis identified one mode (M 19 ), which is involved in the open/closed motions of the binding cleft as being particularly sensitive to the sodium effect. To examine the consequences for D 2 receptor ligand recognition, one of the ligands, L-745,870 [3-{[4-(4-chlorophenyl) piperazin-1-yl]-methyl}-1H-pyrrolo[2,3-b]pyridine or CPPMA, chlorophenyl-piperazinyl methylazaindole], was docked into conformers along the M 19 trajectory. Structurally and pharmacologically well established ligand-receptor interactions, including the ionic interaction with D3.32(114) and interactions between the ligand aryl moieties and V2.61(91)F, were achieved only in "open" phase conformers.
G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins representing the larges... more G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins representing the largest and most universally expressed cell surface receptors and are present in almost all species and in a wide variety of cells. Here we will focus our attention on the catecholamine-binding GPCRs and in particular on the dopamine receptors. The catecholamine-binding GPCRs form a group of rhodopsin-like GPCRs composed of adrenoceptors, which are endogenously activated by epinephrine and norepinephrine, and dopamine receptors. We review the different “molecular switches” involved in GPCR activation and we emphasize the importance of extracellular loop 2 (ECL2) in ligand binding. A better understanding of the functional role of ECL2 can be achieved after the release of the crystal structures of B2AR and rhodopsin, which are consistent with dopamine D2 receptor substituted cysteine accessibility method (SCAM) experimental data. Even though reconstituted GPCR monomers appear sufficient to activate a G protein, in the native setting their dimerization/oligomerization may modulate activation through changes at the dimerization interface or a larger-scale reorientation of the protomers. Therefore, the structural aspects of oligomerization and their importance for receptor activation and signaling are also addressed.
Journal of Biomolecular Structure & Dynamics, 2005
Cytochrome P450 1A1 oxidizes a diverse range of substrates, including the procarcinogenic xenobio... more Cytochrome P450 1A1 oxidizes a diverse range of substrates, including the procarcinogenic xenobiotic benzo[a]pyrene (B[a]P) and endogenous fatty acid precursors of prostaglandins, such as arachidonic acid (AA) and eicosapentaenoic acid (EA). We have investigated the extent to which enzyme-substrate interactions govern regio- and stereoselectivity of oxidation of these compounds by using docking and molecular dynamics (MD) simulations to examine the likelihood of substrate oxidation at various sites. Due to structural differences between the substrates analyzed, B[a]P and its diols (planar, rigid), and the fatty acids AA and EA (long, flexible), different docking strategies were required. B[a]P, B[a]P-7,8-diols, (+) 7S,85- and (-) 7R,8R-diols, were docked into the active site of a homology model of P450 1A1 using an automated routine. Affinity (Accelrys, San Diego, CA). AA and EA, on the other hand, required a series of restrained MD simulations to obtain a variety of productive binding modes. All complexes were evaluated by MD-based in silico site scoring to predict product profiles based on certain geometric criteria, such as angle and distance of a given substrate atom from the ferryl oxygen. For all substrates studied, the in vitro profiles were generally reflected by the in silico scores, which suggests that steric factors play a key role in determining regiospecificity in P450 1A1-mediated oxidations. We have also shown that molecular dynamics simulations may be very useful in determination of product profiles for structurally diverse substrates of P450 enzymes.
Journal of Pharmacology and Experimental Therapeutics, 2010
Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of e... more Conserved serines of transmembrane segment (TM) five (TM5) are critical for the interactions of endogenous catecholamines with ␣ 1 -and ␣ 2 -adrenergic,  2 -adrenergic, and D1, D2, and D3 dopamine receptors. The unique high-affinity interaction of the D4 dopamine receptor subtype with both norepinephrine and dopamine, and the fact that TM5 serine interactions have never been studied for this receptor subtype, led us to investigate the interactions of ligands with D4 receptor TM5 serines. Serine-to-alanine mutations at positions 5.42 and 5.46 drastically decreased affinities of dopamine and norepinephrine for the D4 receptor. The D4-S5.43A receptor mutant had substantially reduced affinity for norepinephrine, but a modest loss of affinity for dopamine. In functional assays of cAMP accumulation, norephinephrine was unable to activate any of the mutant receptors, even though the agonist quinpirole displayed wild-type functional ABBREVIATIONS: TM, transmembrane segment; ABT-724, 2-[(4-pyridin-2-ylpiperazin-1-yl)methyl]-1H-benzimidazole; CP226,269, 5-fluoro-2-{[4-(2-pyridinyl)-1-piperazinyl]methyl}-1H-indole; L-750,667, 3-{[4-(4-iodophenyl)piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine; MIA, methylisobutylamiloride; PD168,077, N-{[4-(2-cyanophenyl)piperazin-1-yl]methyl}-3-methylbenzamide; PNU101,387G, 4-[4-[2-[(1S)-3,4dihydro-1H-isochromen-1-yl]ethyl]piperazin-1-yl]benzenesulfonamide (sonepiprazole); RMSD, root mean square deviation; Ro10-4548, RAC-2Ј-2-hydroxy-3-4-(4-hydroxy-2-methoxyphenyl)-1-piperazinyl-propoxy-acetanilide; Ro10-5824, 5-[(3,6-dihydro-4-phenyl-1(2H)pyridinyl)methyl]-2-methyl-4-pyrimidinamine; Ro20-1724, 4-(3-butoxy-4-methoxyphenyl)methyl-2-imidazolidone; ANOVA, analysis of variance; HBSS, Hanks' balanced salt solution; GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; HEK, human embryonic kidney.
Biochemical Pharmacology, 2004
Human cytochrome P450 1A1 (CYP1A1) and human NADPH-cytochrome P450 reductase were expressed and p... more Human cytochrome P450 1A1 (CYP1A1) and human NADPH-cytochrome P450 reductase were expressed and purified from Spodoptera frugiperda insect cells. A reconstituted enzymatically active system metabolized polyunsaturated fatty acids such as arachidonic (AA) and eicosapentaenoic acid (EPA). CYP1A1 was an AA hydroxylase which oxidizes this substrate at a rate of 650 AE 10 pmol/min/nmol CYP1A1, with over 90% of metabolites accounted for by hydroxylation products and with 19-OH-AA as major product. Epoxyeicosatrienoic acid (EET), mainly 14,15-EET, accounted for about 7% of total metabolites. Unlike rat CYP1A1, the human enzyme exhibited no 20-OH-AA as product. In contrast, with EPA as substrate CYP1A1 was mainly an epoxygenase, oxidizing with over 68% of total metabolites EPA to 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EETeTr). 19-OH-EPA accounted for about 31% of total metabolites. Significantly, the 17,18-olefinic bond of EPA was epoxidized to 17(R),18(S)-EETeTr with nearly absolute regio-and stereoselectivity. Molecular modeling analyses provided rationale for high efficiency of AA hydroxylation at C 19 and its gradual decrease down to C 14 , as well as for the limited EPA 17(S),18(R) epoxidation due to unfavorable enzyme-substrate interactions. The absence of ohydroxylation for both substrates is not due to steric factors, but probably a consequence of different reactivities of o and (o À 1) carbons for hydrogen abstraction. It is suggested that the capacity of human CYP1A1 to metabolize AA and EPA and its inducibility by polycyclic aromatic hydrocarbons may affect the production of physiologically active metabolites, in particular, in the cardiovascular system and other extrahepatic tissues including lung.
Archives of Biochemistry and Biophysics, 2004
Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 ! A... more Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 ! Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy-and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue. Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models.
Molecular Pharmacology, 2006
The imidazopyridine zolpidem (Ambien) is one of the most commonly prescribed sleep aids in the Un... more The imidazopyridine zolpidem (Ambien) is one of the most commonly prescribed sleep aids in the United States . Similar to classic benzodiazepines (BZDs), zolpidem binds at the extracellular N-terminal ␣/␥ subunit interface of the GABA-A receptor (GABAR). However, zolpidem differs significantly from classic BZDs in chemical structure and neuropharmacological properties. Thus, classic BZDs and zolpidem are likely to have different requirements for high-affinity binding to GABARs. To date, three residues-␥2Met57, ␥2Phe77, and ␥2Met130 -have been identified as necessary for high-affinity zolpidem binding (Proc Natl Acad Sci USA 94:8824 -8829, 1997; Mol Pharmacol 52: 874 -881, 1997). In this study, we used radioligand binding techniques, ␥2/␣1 chimeric subunits (), site-directed mutagenesis, and molecular modeling to identify additional ␥2 subunit residues important for high-affinity zolpidem binding. Whereas ␣12 receptors containing only the first 161 amino-terminal residues of the ␥2 subunit bind the classic BZD flunitrazepam with wild-type affinity, zolpidem affinity is decreased ϳ8-fold. By incrementally restoring ␥2 subunit sequence, we identified a seven-amino acid stretch in the ␥2 subunit loop F region (amino acids 186 -192) that is required to confer high-affinity zolpidem binding to GABARs. When mapped to a homology model, these seven amino acids make up part of loop F located at the ␣/␥ interface. Based on in silico zolpidem docking, three residues within loop F, ␥2Glu189, ␥2Thr193, and ␥2Arg194, emerge as being important for stabilizing zolpidem in the BZD binding pocket and probably interact with other loop F residues to maintain the structural integrity of the BZD binding site.