Impact of single nucleotide polymorphisms (R132Q and W120R) on the binding affinity and metabolic activity of CYP2C19 toward some therapeutically important substrates.

Although CYP2C19 is minor human liver enzyme, it is responsible for the metabolism of many clinically important drugs. In this work, CYP2C19 wild type and its SNP mutants (R132Q and W120R) were prepared using over-expression system in E. coli, purified by column chromatography and their biological activities were compared. The enzyme activity toward certain drugs (amitriptyline, imipramine, lansoprazole and omeprazole) was investigated. Resonance Raman and UV-VIS spectroscopies revealed a minimal effect of SNP mutations on the heme structure. However, the mutation greatly affected the drug metabolism activities of CYP2C19. The degree of these effects was dependent on both the mutation and the chemical structure of the substrate. Surprisingly, the affected amino acid residue is located remotely from the heme center. Therefore, the direct effect of the mutation on the metabolic center is excluded. Alternatively, the significant impairment in the drug metabolism of these mutants could be attributed to a decrease in the electron flow to the iron center. Accordingly, understanding the effect of SNPs of CYP2C19, and the extents in which they participate in the drug metabolism, are important pillars that can enhance the therapeutic drugs efficacy and improve the patient outcomes toward the development of patient's tailored medicine.

Correlation of indoleamine-2,3-dioxigenase 1 inhibitory activity of 4,6-disubstituted indazole derivatives and their heme binding affinity.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that acts on the first and rate-limiting step of the tryptophan/kynurenine pathway. Since the pathway is one of the means of cancer immune evasion, IDO1 inhibitors have drawn interest as potential therapeutics for cancers. We found a 4,6-disubstituted indazole 1 as a hit compound that showed both IDO1 inhibitory activity and binding affinity for IDO1 heme. Structural modification of 1 yielded compound 6, whose relatively large substituent at the 4-position and proper size substituent at the 6-position were found to be important for the enhancement of IDO1 inhibitory activity and heme affinity. A series of compounds synthesized in this work were evaluated by in silico docking simulations and by in vitro experiments using a C129Y mutant of the pocket-A of IDO1. Our results revealed that proper substituents at the 6- and 4-positions of the compounds interact with pockets A and B, respectively, and that, in particular, a good fit in pocket-A is important for the compounds' biological activities. Absorption spectral analysis of these compounds showed that they strongly bound to the ferrous heme rather than its ferric heme. Furthermore, we observed that the heme affinities of these compounds strongly correlate with their IDO1 inhibitory activities.

Effect of Drug Combination on Omeprazole Metabolism by Cytochrome P450 2C19 in Helicobacter pylori Eradication Therapy.

Helicobacter pylori (H. pylori) infection is common and can result in gastric and duodenal ulcers, and in some cases, gastric lymphoma and cancer. Omeprazole (OMP)-in combination with clarithromycin (CLR), amoxicillin (AMX), tinidazole (TND), or metronidazole (MET)-is used in double or triple combination therapy for eradication of H. pylori. However, the roles of the drugs other than OMP are not clearly understood. Therefore, in the present study, we aimed to investigate any effects of these drugs on OMP metabolism by wild-type CYP2C19 using spectroscopy and enzyme kinetics. The dissociation constants (Kd) for CYP2C19 with OMP, CLR, AMX, TND, and MET were 8.6, 126, 156, 174, and 249 µM, respectively. The intrinsic clearance of OMP was determined to be 355 mL/min/µmol of CYP2C19. Metabolism of OMP was significantly inhibited by 69, 66, 28, and 40% in the presence of CLR, TND, AMX, and MET, respectively. Moreover, the combination of CLR and TND resulted in 76% inhibition of OMP metabolism, while the combination of AMX and MET resulted in 48% inhibition of OMP metabolism. Both combinations of drugs not only have antibacterial effects, but also enhance the effect of OMP by inhibiting its metabolism by CYP2C19. These results indicate that drug-drug interactions of co-administered drugs can cause complex effects, providing a basis for OMP dose adjustment when used in combination therapy for H. pylori eradication.

Allosteric activation of cytochrome P450 3A4 by efavirenz facilitates midazolam binding.

1. The purpose of this study is to investigate the heteroactivation mechanism of CYP3A4 by efavirenz, which enhances metabolism of midazolam in vivo, in terms of its binding to CYP3A4 with in vitro spectroscopic methods. 2. Efavirenz exhibited a type II spectral change with binding to CYP3A4 indicating a possible inhibitor. Although dissociation constant (K d) was approximated as 520 µM, efavirenz enhanced binding affinity of midazolam as a co-existing drug with an estimated iK d value of 5.6 µM which is comparable to a clinical concentration. 3. Efavirenz stimulated the formation of 1'-hydroxymidazolam, and the product formation rate (V max) concentration-dependently increased without changing the K m. Besides, an efavirenz analogue, [6-chloro-1,4-dihydro-4-(1-pentynyl)-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one] (efavirenz impurity) slightly facilitated the binding affinity of midazolam in a concentration-dependent manner. These results propose that efavirenz affects midazolam-binding via binding to the peripheral site which is apart from the active site of CYP3A4. 4. A molecular dynamics simulation also suggested the bound-efavirenz was repositioned to effector-binding site. As a consequence, our spectroscopic studies clarified the heteroactivation of CYP3A4 caused by efavirenz with a proper affinity to the peripheral site, and we concluded the method can be a useful tool for characterising the potential for drug-drug interactions.

Development of an Enyne Metathesis/Isomerization/Diels-Alder One-Pot Reaction for the Synthesis of a Novel Near-Infrared (NIR) Dye Core.

N-Alkyl-N-allyl-2-alkynylaniline derivatives undergo a tandem ring-closing enyne metathesis/isomerization/Diels-Alder cycloaddition sequence in the presence of a second-generation Grubbs catalyst and dienophiles. In practice, the acyclic enyne in the presence of the ruthenium alkylidene first undergoes ring-closing metathesis to generate cyclic 4-vinyl-1,2-dihydroquinolines; following diene isomerization and then the addition of a dienophile, these ring-closing metathesis products are selectively converted into a 7-methyl-4H-naphtho[3,2,1-de]quinoline-8,11-dione core. Overall, the reaction sequence converts simple aniline derivatives into π-conjugated small molecules, which have characteristic absorption in the near-infrared region, in a single operation through three unique ruthenium-catalyzed transformations.

Membrane anchor of cytochrome P450 reductase suppresses the uncoupling of cytochrome P450.

Cytochrome P450 reductase (CPR) is an important redox partner of microsomal CYPs. CPR is composed of a membrane anchor and a catalytic domain that contains FAD and flavin mononucleotide (FMN) as redox centers and mediates electron transfer to CYP. Although the CPR membrane anchor is believed to be requisite for interaction with CYP, its physiological role is still controversial. To clarify the role of the anchor, we constructed a mutant (Δ60-CPR) in which the N-terminal membrane anchor was truncated, and studied its effect on binding properties, electron transfer to CYP2C19, and drug metabolism. We found that Δ60-CPR could bind to and transfer electrons to CYP2C19 as efficiently as WT-CPR, even in the absence of lipid membrane. In accordance with this, Δ60-CPR could mediate metabolism of amitriptyline (AMT) and imipramine (IMP) in the absence of lipids, although activity was diminished. However, Δ60-CPR failed to metabolize omeprazole (OPZ) and lansoprazole (LPZ). To clarify the reason for this discrepancy in drug metabolism, we investigated the uncoupling reaction of the CYP catalytic cycle. By measuring the amount of H2O2 by-product, we found that shunt pathways were markedly activated in the presence of OPZ/LPZ in the Δ60-CPR mutant. Because H2O2 levels varied among the drugs, we conclude that the proton network in the distal pocket of CYP2C19 is perturbed differently by different drugs, and activated oxygen is degraded to become H2O2. Therefore, we propose a novel role for the membrane anchor as a suppressor of the uncoupling reaction in drug metabolism by CYP.

Development of an anti-claudin-3 and -4 bispecific monoclonal antibody for cancer diagnosis and therapy.

Most malignant tumors are derived from epithelium, and claudin (CLDN)-3 and CLDN-4 are frequently overexpressed in such tumors. Although antibodies have potential in cancer diagnostics and therapy, development of antibodies against CLDNs has been difficult because the extracellular domains of CLDNs are too small and there is high homology among human, rat, and mouse sequences. Here, we created a monoclonal antibody that recognizes human CLDN-3 and CLDN-4 by immunizing rats with a plasmid vector encoding human CLDN-4. A hybridoma clone that produced a rat monoclonal antibody recognizing both CLDN-3 and -4 (clone 5A5) was obtained from a hybridoma screen by using CLDN-3- and -4-expressing cells; 5A5 did not bind to CLDN-1-, -2-, -5-, -6-, -7-, or -9-expressing cells. Fluorescence-conjugated 5A5 injected into xenograft mice bearing human cancer MKN74 or LoVo cells could visualize the tumor cells. The human-rat chimeric IgG1 monoclonal antibody (xi5A5) activated FcγRIIIa in the presence of CLDN-3- or -4-expressing cells, indicating that xi5A5 may exert antibody-dependent cellular cytotoxicity. Administration of xi5A5 attenuated tumor growth in xenograft mice bearing MKN74 or LoVo cells. These results suggest that 5A5 shows promise in the development of a diagnostic and therapeutic antibody for cancers.

Ferric human neuroglobin scavenges superoxide to form oxy adduct.

Neuroglobin (Ngb) is the third member of the vertebrate globin family, and the structure was solved as a typical globin fold with a b-type heme. Although it has been proposed that Ngb could be involved in neuroprotection against oxidative stress, the protective mechanism has not been fully identified yet. In order to clarify functions under hypoxic condition, in this study, we focused on the scavenger activity of human Ngb (hNgb) against superoxide. The activity of hNgb for superoxide was evaluated to be 7.4 µM for IC50, the half maximal inhibitory concentration. The result indicates that hNgb can be an anti-oxidant, and the value was almost the same as that of ascorbic acid. In addition, we characterized oxidation states of a heme iron in superoxide-treated hNgb with spectroscopic measurements. Superoxide-treated hNgb in the ferric form was readily converted to the oxygenated ferrous form, and the result suggested that ferric hNgb could scavenge superoxide by change of an oxidation state in a heme iron. Moreover, mutational experiments were performed, and the each variant mutated at 46 and 55 positions suggested a disulfide bond between Cys46 and Cys55 could be essential to be sensors for oxidative stress with the direct binding of superoxide. As a consequence, we concluded that redox changes of the heme iron and the disulfide bond could regulate neuroprotective functions of hNgb, and it suggests that hNgb can afford protection against hypoxic and ischemic stress in the brain.

Effect of cytochrome P450 2C19 and 2C9 amino acid residues 72 and 241 on metabolism of tricyclic antidepressant drugs.

Although cytochromes P450 2C9 (CYP2C9) and 2C19 (CYP2C19) have 91% amino acid identity, they have different substrate specificities. Previous studies have suggested that several amino acid residues may be involved in substrate specificity. In this study, we focused on the roles of two amino acids, residues 72 and 241. The amino acids in these positions have opposite charges in CYP2C9 and 2C19; the former has lysines in both positions (Lys72 and Lys241), and the latter has glutamic acids (Glu72 and Glu241). Reciprocal mutants for both CYP2C19 and 2C9 were produced, and their metabolic activities and spectroscopic properties were examined using three tricyclic antidepressant (TCA) drugs: amitriptyline, imipramine, and dothiepin. Although CYP2C19 wild-type (WT) had a high metabolic activity for all three drugs, the E72K mutation decreased enzymatic activity by 29-37%, while binding affinities were diminished 2.5- to 20-fold. On the other hand, low activity and low affinity of CYP2C9 WT were recovered notably by K72E mutation. The metabolic activities and binding affinities were minimally affected by CYP2C19 E241K and CYP2C9 K241E mutations. We could also show linear correlations between metabolic activities and binding affinities, and hence we conclude that amino acid residue 72 plays a key role in TCA drug metabolism by limiting the binding affinities of CYP2C19 and CYP2C9.

Comparison of cytochrome p450 mediated metabolism of three central nervous system acting drugs.

The investigation of cytochrome P450 (CYP) mediated metabolism reactions by determination of enzyme kinetic parameters, Michaelis constant (K(m)), maximum reaction velocity (V(max)), and intrinsic clearance (CL(int)) is important aspects in discovery and development of drugs. The kinetic parameters can be used to predict the clearance prior to human administration and for better understanding the mechanism of clearance in vivo. In this study, the metabolic activities of three major hepatic CYP isoforms (2C19, 2D6, and 3A4) were investigated on structurally different central nervous system (CNS) acting drugs, amitriptyline, fluphenazine, and dothiepin. By using our novel in vitro evaluation system, we could compare the kinetic parameters for the metabolism of fluphenazine and dothiepin for the first time. Comparing CL(int) values thus obtained, we concluded that 2C19 could be predominant for metabolic activity on tricyclic antidepressants as expected, but not on phenothiazine-related antipsychotic drugs. Since the metabolism of CNS drugs is susceptible to single nucleotide polymorphisms of human gene, our results suggest that phenothiazine could be an alternative to clinical application of CNS drugs.

Absorption, Fluorescence, and Two-Photon Excitation Ability of 5-Phenylisolidolo[2,1-a]quinolines.

We report on the absorption, fluorescence, and two-photon excitation spectra of a series of 5-phenylisoindolo[2,1-a]quinoline dyes. Depending on the substituents, we observed increasing two-photon absorption cross sections, with values up to 56 GM@973 nm, which are similar to those of the enhanced green fluorescent protein and fluorescein, common fluorescent chromophores.

Investigation on drug-binding in heme pocket of CYP2C19 with UV-visible and resonance Raman spectroscopies.

Cytochrome P450 (CYP) is a class of heme-containing enzymes which mainly catalyze a monooxygenation reaction of various chemicals, and hence CYP plays a key role in the drug metabolism. Although CYP2C19 isoform is a minor hepatic CYP, it metabolizes clinically important drugs such as omeprazole and S­mephenytoin. In this work, the interaction of purified CYP2C19 WT (CYP2C19) with seven drugs (phenytoin, S­mephenytoin, omeprazole, lansoprazole, cimetidine, propranolol, and warfarin) was investigated using spectroscopic methods. The binding of each drug and the induced structural change in the heme distal environment were evaluated. Ferric form of CYP2C19 was revealed to contain a six-coordinate low-spin heme with a water molecule as a sixth ligand in a distal site, and the addition of each drug caused varied minor fraction of five-coordinate heme. It was suggested that the ligated water molecule was partly moved away from the heme distal environment and that the degree of water removal was dependent on the type of drugs. The effect on the coordination was varied with the studied drugs with wide variation in the dissociation constants from 2.6 µM for lansoprazole to 5400 µM for warfarin. Phenytoin and S­mephenytoin showed that binding to CYP2C19 occurred in a stepwise manner and that the coordination of a water molecule was facilitated in the second binding step. In the ferrous CO-bound state, ν(FeCO) stretching mode was clearly observed at 471 cm-1 in the absence of drugs. The Raman line was greatly up-shifted by omeprazole (487 cm-1) and lansoprazole (477 cm-1) but was minimally affected by propranolol, phenytoin, and S­mephenytoin. These results indicate that slight chemical modification of a drug greatly affects the heme distal environments upon binding.

Direct synthesis of dialkylarylvinylsilane derivatives: metathesis of dialkylaryl-iso-propenylsilane and its application to tetracyclic silacycle dye synthesis.

The metathesis of dialkylarylvinylsilane, which has not been accomplished to date, is achieved using dialkylaryl-iso-propenylsilane as a substrate. In addition, we discovered that the reason why the metathesis of a ruthenium carbene complex and dialkylarylvinylsilane is difficult is the formation of a carbide complex.

Binding of cationic bis-porphyrins linked with p- or m-xylylenediamine and their zinc(II) complexes to duplex DNA.

Spectroscopic, viscometric, and molecular docking analysis of binding of cationic bis-porphyrins linked with p- or m-xylylenediamine (H(2)pXy and H(2)mXy) and their zinc(II) complexes (ZnpXy and ZnmXy) to duplex DNA are described. H(2)pXy and H(2)mXy bound to calf thymus DNA (CTDNA) stronger than unichromophoric H(2)TMPyP, and showed exciton-type induced circular dichroism spectra of their Soret bands. The H(2)TMPyP-like units of the metal-free bis-porphyrins did not intercalate into CTDNA, and thus the binding mode is outside binding with intramolecular stacking. ZnpXy showed favorable binding to A.T over G.C region, and should lie in the major groove of A.T region.

Roles of N- and C-terminal domains in the ligand-binding properties of cytoglobin.

Cytoglobin (Cygb) is a member of the hexacoordinated globin protein family and is expressed ubiquitously in rat and human tissues. Although Cygb is reportedly upregulated under hypoxic conditions both in vivo and in vitro, suggesting a physiological function to protect cells under hypoxic/ischemic conditions by scavenging reactive oxygen species or by signal transduction, the mechanisms associated with this function have not been fully elucidated. Recent studies comparing Cygbs among several species suggest that mammalian Cygbs show a distinctly longer C-terminal domain potentially involved in unique physiological functions. In this study, we prepared human Cygb mutants (ΔC, ΔN, and ΔNC) with either one or both terminal domains truncated and investigated the enzymatic functions and structural features by spectroscopic methods. Evaluation of the superoxide-scavenging activity between Cygb variants showed that the ΔC and ΔNC mutants exhibited slightly higher activity involving superoxide scavenging as compared with wild-type Cygb. Subsequent experiments involving ligand titration, flash photolysis, and resonance Raman spectroscopic studies suggested that the truncation of the C- and N-terminal domains resulted in less effective to dissociation constants and binding rates for carbon monoxide, respectively. Furthermore, structural stability was assessed by guanidine hydrochloride and revealed that the C-terminal domain might play a vital role in improving structure, whereas the N-terminal domain did not exert a similar effect. These findings indicated that long terminal domains could be important not only in regulating enzymatic activity but also for structural stability, and that the domains might be relevant to other hypothesized physiological functions for Cygb.

One-pot enyne metathesis/Diels-Alder/oxidation to six-membered silacycles with a multi-ring core: discovery of novel fluorophores.

Polycyclic compounds containing a six-membered silacycle are important. However, we have limited knowledge of the nature of these six-membered silacycles because methodologies for their synthesis remain under-developed. Here, we have developed a one-pot enyne metathesis/Diels-Alder/oxidation methodology for the synthesis of six-membered silacycles. Some of these compounds are novel fluorophores.

Notable deuterium effect on the electron transfer rate of myoglobin.

The electron transfer reaction of wild-type myoglobin at an electrode was significantly facilitated in a D2O buffer as compared with that in an H2O buffer, with k(0)'(H2O)/k(0)'(D2O)= 0.13, while a minimal deuterium kinetic isotope effect on the myoglobin with modification at distal histidine (His-64) was observed.

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket.

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.

Binding of meso-Tetrakis(N-methylpyridinium-4-yl)porphyrin to Double Helical RNA and DNA.RNA Hybrids.

The binding properties of meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (H(2)TMPyP) to RNA and DNA.RNA hybrid duplexes were studied by absorption and circular dichroism (CD) spectra. The duplexes studied were poly(rA).poly(rU), poly(rA).poly(dT), poly(rI).poly(rC), poly(rI).poly(dC), poly(rG).poly(rC), and poly(rG).poly(dC). The hypochromicity (about 40%) and the bathochromic shift (about 15 nm) of the porphyrin Soret absorption band upon binding were quite similar among the duplexes examined. The large bathochromic shift and hypochromicity suggested a significant perturbation in the porphyrin pi electrons upon binding. H(2)TMPyP was found to bind in a single step to poly(rI).poly(rC), poly(rG).poly(rC), and poly(rG).poly(dC) and in a multistep manner to poly(rA).poly(rU), poly(rA).poly(dT), and poly(rI).poly(dC). The induced CD spectra in the visible range suggested that the porphyrin preferred to bind to the RNA duplexes with self-stacking along the polymer surface and to the hybrids with intercalation, at least at higher duplex load. This implied a distinct conformational difference between the RNA duplexes and DNA.RNA hybrids, and a drug molecule is able to recognize the difference. The number of binding sites per base pairs (n), however, was very different among the RNA duplexes examined. We also found that the intensity of the bisignate-induced CD bands is proportional to the n value. This suggested that the transition moments on the neighboring porphyrins are interacting considerably with each other to produce intense induced CD peaks.

Disulfide bonds regulate binding of exogenous ligand to human cytoglobin.

Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands.