Biomedical subjects

Charles W Locuson

Publications and source records attributed to Charles W Locuson.

10 recordsLinked to original sources

PubMed2006

Identification of binding sites of non-I-helix water molecules in mammalian cytochromes p450.

The cytochromes P450 (P450s) enzymes are integral in determining the disposition of many therapeutic compounds. At the molecular level, the details of P450 catalysis are still under investigation, but the importance of water-mediated proton shuttles seems evident in the catalytic cycle as it progresses through various heme iron-oxygen enzyme intermediates. The study of P450-bound waters has been largely restricted to bacterial enzymes that may or may not reflect the location or function of waters in human drug-metabolizing P450s. However, in recent years, 16 structures of mammalian P450s containing crystallographic waters have been deposited in the Protein Data Bank. Described herein is the identification of seven well defined water clusters in mammalian P450s identified by calculating the density of globally aligned waters as reported by Tanner and coworkers [Bottoms CA, White TA, and Tanner JJ (2006) Proteins 64:404-421 (DOI: 10.1002/prot.21014)]. All water binding sites were in or within the immediate vicinity of the active sites of the P450s, but most were not near the conserved I-helix threonine often implicated in P450 catalysis. Therefore, it is possible that some of the water binding sites identified here ultimately determine P450 catalytic efficiency either by working as an extension of the I-helix water network, or by acting in novel proton shuttles that modulate the nonproductive shunting of reactive oxygen species.

Animals↗

PubMed2006

Preparation, characterization, and substrate metabolism of gold-immobilized cytochrome P450 2C9.

The cytochrome P450 enzymes represent an important class of heme-containing enzymes. There is considerable interest in immobilizing these enzymes on a surface so that interactions between a single enzyme and other species can be studied with respect to electron transfer, homodimer or heterodimer interactions, or for construction of biological-based chips for standardizing cytochrome P450 metabolism or for high-throughput screening of pharmaceutical agents. Previous studies have generally immobilized P450 enzymes in a matrix or on a surface. Here, we have attached CYP2C9 to gold substrates such that the resulting construct maintains the ability to bind and metabolize substrates in the presence of NADPH and cytochrome P450 reductase. The activity of these chips is directly dependent upon the linkers used to attach CYP2C9 and to the presence of key molecules in the active site during enzyme attachment. A novel method to detect substrate-enzyme binding, namely, superconducting quantum interference device (SQUID) magnetometry, was used to monitor the binding of substrates. Most significantly, conditions that allow measurable CYP2C9 metabolism to occur have been developed.

Aryl Hydrocarbon Hydroxylases↗

PubMed2006

Heteroactivator effects on the coupling and spin state equilibrium of CYP2C9.

The cytochromes P450 are capable of oxidizing a variety of xenobiotics. Binding of a small molecule heteroactivator to a P450 can alter the coupling of substrate oxidation during P450 catalysis, but the degree to which coupling or shunting via one of the three catalytic cycle branch points is linked to the heteroactivator-modified position of bound substrate is unknown. Using reconstituted CYP2C9, stoichiometric measurements were gathered with three substrates and two classes of heteroactivators to further understand the mechanisms involved in heteroactivation. Heteroactivation of P450 metabolism appeared to involve, but not require, changes in coupling and that increased uncoupling to a specific byproduct like H(2)O(2) does not necessarily correlate to the degree of coupling. In addition, spectroscopy demonstrated that every heteroactivator tested influenced the spin equilibrium of the heme iron even in the presence of saturating substrate suggesting that both substrate proximity and the ability to desolvate the heme can be involved in heteroactivation.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2006

Inhibition of cytochrome P450 3A4 by a pyrimidineimidazole: Evidence for complex heme interactions.

PH-302 inhibits the inducible form of nitric oxide synthase (iNOS) by coordinating with the heme of the monomeric form and preventing formation of the active dimer. Inherent with the mechanism of pharmacology for this compound was the inhibition of cytochrome P450 3A4 (P450 3A4), observed from early ADME screening. Further investigation showed that PH-302 inhibited P450 3A4 competitively with a Ki of approximately 2.0 microM against both midazolam and testosterone hydroxylation in human liver microsomes. As expected, spectral binding analysis demonstrated that inhibition was a result of type II coordination to the P450 heme with the imidazole moiety of PH-302, although only 72% of the maximal absorbance difference was achievable with PH-302 compared to that of the smaller ligand imidazole. Time-dependent inhibition of P450 3A4 by PH-302 was also observed because of metabolite-inhibitory (MI) complex formation via metabolism of the methylenedioxyphenyl group. The profile for time-dependent inhibition in recombinant P450 3A4 was biphasic, and was kinetically characterized by a kinact of 0.08 min-1 and a Ki of 1.2 microM for the first phase (0-1.5 min) and a kinact of 0.06 min-1 and a Ki of 23.8 microM for the second phase (1.5-10 min). Spectral characterization of the PH-302 MI complex demonstrated that formation began to plateau within 3 min, consistent with the kinetic profile of inactivation by PH-302. Meanwhile, spectral evidence for the imidazole-derived type II difference spectrum of PH-302 was captured simultaneously with the formation of the MI complex. The presence of simultaneously operable type II coordination and rapidly saturable MI complex formation with heme by PH-302 indicates the presence of complex heme interactions with this unique molecule. Information from these mechanistic studies adds to our understanding of the nature of P450 3A4 inhibition by PH-302 and provides a potentially useful tool compound for future studies investigating binding interactions in this important drug-metabolizing enzyme.

Benzodioxoles↗

PubMed2005

CYP2C9 genotype-dependent effects on in vitro drug-drug interactions: switching of benzbromarone effect from inhibition to activation in the CYP2C9.3 variant.

The CYP2C9.3 variant exhibits marked decreases in substrate turnover compared with the wild-type enzyme, but little is known regarding the effect this variant form may have on the occurrence of drug-drug interactions. To examine this possibility, the effect of the potent CYP2C9 inhibitor, benzbromarone, was studied with regard to CYP2C9.1- and CYP2C9.3-mediated flurbiprofen metabolism to evaluate whether the variant enzyme exhibits differential inhibition kinetics. Although benzbromarone inhibited CYP2C9.1 activity as expected, CYP2C9.3-mediated flurbiprofen 4'-hydroxylation was activated in the presence of benzbromarone. T1 relaxation studies revealed little change in distances of flurbiprofen protons from the heme iron of either CYP2C9.1 or CYP2C9.3 in the presence of benzbromarone compared with flurbiprofen alone. Spectral binding studies were also performed to investigate whether benzbromarone affected substrate binding, with the addition of benzbromarone having little effect on flurbiprofen-binding affinity in both CYP2C9.1 and CYP2C9.3. Docking studies with the 2C9.1 structure crystallized with a closed active site identified multiple but overlapping subsites with sufficient space for benzbromarone binding in the enzyme when flurbiprofen was positioned closest to the heme. If the closed conformation of 2C9.3 is structurally similar to 2C9.1, as expected for the conservative I359L mutation, then the dynamics of benzbromarone binding may account for the switching of drug interaction effects. In conclusion, the I359L amino acid substitution found in CYP2C9.3 not only reduces metabolism compared with CYP2C9.1 but can also dramatically alter inhibitor effects, suggesting that differential degrees of drug inhibition interactions may occur in individuals with this variant form of CYP2C9.

Aryl Hydrocarbon Hydroxylases↗

PubMed2005

Three-dimensional quantitative structure-activity relationship analysis of cytochromes p450: effect of incorporating higher-affinity ligands and potential new applications.

Recently, two new classes of reversible inhibitors, the benzbromarones (BZBRs) and the N-3 substituted phenobarbitals (PBs), were used to study the active site characteristics of CYP2C9 and 2C19, respectively. Since these ligands are some of the first CYP2C ligands to extend into the low nanomolar K(i) range (K(i) < 100 nM), they were subjected to three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis. Given that BZBRs or the PB ligands bind very tightly, it can be assumed that these structures complement the binding pocket(s) for these enzymes. Thus, the resulting models should output a 3D arrangement of interaction sites predicted to be important for near optimal binding to the CYP2C9 and CYP2C19 enzymes. These predicted interaction regions may then improve the ability to predict drug-drug interactions. The resulting models generated from these new high affinity ligands are discussed, as are novel uses of 3D-QSAR and molecular modeling techniques that may be useful in the study of cytochromes P450 specifically.

Cytochrome P-450 Enzyme System↗

PubMed2004

Charge and substituent effects on affinity and metabolism of benzbromarone-based CYP2C19 inhibitors.

Human cytochrome P450 (CYP) 2C19 is one of the most important CYP2C family members responsible for metabolizing commonly prescribed drugs. This research describes synthetic modifications to benzbromarone (Bzbr) to create the most potent CYP2C19 inhibitor ever reported. The most important features enabling analogues of Bzbr to bind to CYP2C19 with high affinity are low acidity (high pK(a) or nonionizability) and hydrophobic substituents adjacent to the phenol moiety. Though CYP2C19 was known to prefer neutral substrates, the extent was perhaps not realized until the anionic, parent compound Bzbr (K(i) = 3.7 microM) was compared to a less acidic dimethyl analogue (K(i) = 0.033 microM). However, differences in affinity for anionic and neutral Bzbr analogues did not appear to affect the regiospecificity of their metabolism by CYP's 2C19 and 2C9. In addition, some Bzbr analogues were metabolized both on the phenol and benzofuran rings. By using a substrate with a methyl ether in place of the Bzbr phenol, it was shown that some Bzbr analogues must be able to freely reposition after binding and oxidize the more energetically favorable position. Normally, O-demethylation of this methyl ether is favored over benzofuran hydroxylation based on ion current from LC/MS. Deuterium substitution of the methyl ether results in an inverse isotope effect on benzofuran hydroxylation (i.e. increased oxidation of this less favorable site). Likewise, Bzbr-based CoMFA models of CYP2C19 demonstrated no clear preference for any one ligand alignment. This suggests results from this modeling method must be interpreted carefully for each CYP isoform. In summary, Bzbr analogues have demonstrated they can be adapted to other CYP2C enzymes in order to probe isoform-specific properties.

Aryl Hydrocarbon Hydroxylases↗

PubMed2004

Quantitative binding models for CYP2C9 based on benzbromarone analogues.

The cytochrome P450 (CYP) isoforms involved in xenobiotic metabolism are enzymes whose substrate selectivity remains difficult to predict due to wide specificity and dynamic protein-substrate interactions. To uncover the determinants of specificity for cytochrome CYP2C9, a novel library of benzbromarone (bzbr) inhibitors was used to reevaluate its pharmacophore. CoMSIA was used with the bzbr ligands to generate both quantitative binding models and three-dimensional contour plots that pinpoint predicted interactions that are important for binding to 2C9. Since this class of compounds is more potent than any other toward 2C9, the small molecule properties deemed most ideal by the software were used to address protein-ligand interactions using new mutagenesis and structural data. Nine new bzbr analogues provide evidence that specific electrostatic and hydrophobic interactions contribute the most to 2C9's specificity. Three of the new analogues are better isosteres of bzbr that contain bulky groups adjacent to the phenol and have increased pK(a) values. These ligands test the hypothesis that anionic substrates bind with higher affinity to 2C9. Since they have higher affinity than the previous nonacidic analogues, the importance of bulky groups on the phenol ring appears to have been underestimated. CoMSIA models predict that these bulky groups are favorable for their hydrophobicity, while a negative charge is favored at the ketone oxygen rather than the phenol oxygen. The overlap of this ketone with electronegative groups of other 2C9 substrates suggests they act as key positive charge acceptors.

Aryl Hydrocarbon Hydroxylases↗

PubMed2004

Differential roles of Arg97, Asp293, and Arg108 in enzyme stability and substrate specificity of CYP2C9.

CYP2C9 metabolizes a wide range of drugs, many of which are negatively charged at physiological pH. Therefore, it has been thought that complementarily charged amino acid(s) are critically involved in substrate binding. Previous studies have implicated arginine residues at positions 97, 105, and 108 and aspartate at position 293 in the normal catalytic function of the enzyme. To elucidate the role of these amino acids in the substrate specificity of CYP2C9, a series of mutants were constructed and analyzed for functional activity, thermal stability, and ligand binding. Charge-modifying mutations at positions 97, 105, and 293 decreased catalytic activity toward diclofenac, (S)-warfarin, and pyrene in a substrate-independent manner with Arg105 the least, and Arg97 the most, sensitive amino acids in this regard. Decreases in functional activity paralleled thermal instability of the mutants, suggesting that loss of function reflects more generalized structural changes rather than the absence of a specifically charged amino acid at these three positions. The R108H mutant was inactive toward all three substrates because of unexpected nitrogen ligation to the heme. Conversely, the R108F mutant exhibited substrate-dependent catalytic behavior, with almost complete loss of activity toward (S)-warfarin and diclofenac, but preservation of pyrene metabolism. In addition, the R108F mutation abrogated the Type I difference spectra induced by flurbiprofen and benzbromarone, obligate anions at physiological pH. These data identify critical roles for Arg97 and Asp293 in the structural stability of the enzyme and demonstrate a selective role for Arg108 in the binding and metabolism of negatively charged substrates of CYP2C9.

Amino Acid Substitution↗

PubMed2003

A new class of CYP2C9 inhibitors: probing 2C9 specificity with high-affinity benzbromarone derivatives.

Noncovalent forces, other than hydrophobic interactions, are important determinants of substrate bias exhibited by some cytochromes P450. The CYP2C9 pharmacophore is proposed to include either an anionic group or hydrogen bond donor in addition to its hydrophobic groups. By constructing analogs of benzbromarone, evidence supporting the existence of a 2C9 anion-binding site was revealed. A nonsubstituted phenol analog was determined to have a pKa of 8.4 and a Ki of 414 nM whereas those with dihalogenated benzoyl phenols had pKa values between 4.2 to 5.2 and Ki values as low as 1 nM. The nonhalogenated, nonionizable analog is the poorest binder at 796 nM. The Ki range covers around three orders of magnitude with even the weakest binder being a more potent inhibitor than 2C9 substrate phenytoin. Thus, benzbromarone derivatives represent a class of molecules with the potential to reveal more structural details of the 2C9 active site.

Aryl Hydrocarbon Hydroxylases↗