Electrochemical Synthesis of the In Human S-oxide Metabolites of Phenothiazine-Containing Antipsychotic Medications.

The tractable preparation of Phase I drug metabolites is a critical step to understand the first-pass behaviour of novel chemical entities (NCEs) in drug discovery. In this study, we have developed a structure-electroactivity relationship (SeAR)-informed electrochemical reaction of the parent 2-chlorophenothiazine and the antipsychotic medication, chlorpromazine. With the ability to dial-in under current controlled conditions, the formation of S-oxide and novel S,S-dioxide metabolites has been achieved for the first time on a multi-milligram scale using a direct batch electrode platform. A potential rationale for the electrochemical formation of these metabolites in situ is proposed using molecular docking to a cytochrome P450 enzyme.

Spectroscopic studies under properties of chlorpromazine conjugated to gold nanoparticles.

Scientific studies have demonstrated that conjugates of anticancer drugs with metal nanoparticles (MeNPs) lead to a more effective deactivation of tumor cells compared to free drugs. Similarly, it has been established that conjugates of antibiotics with MeNPs exhibit higher biocidal activity against bacteria than their unbound counterparts. However, limited information is available regarding conjugates formed from drugs other than anticancer and antibiotics. Therefore, our research aims to develop synthesis methods for conjugates of chlorpromazine (CPZ), a neuroleptic, with gold nanoparticles (AuNPs). CPZ-AuNP conjugates were prepared through a ligand exchange reaction conducted on the surface of quasi-spherical, negatively charged citrate-stabilized TC-AuNPs with an average size of 55 ± 5 nm. UV-vis spectroscopy was employed to determine the stability range of the conjugates under controlled conditions of pH and ionic strength. Based on electrokinetic measurements, it was observed that the zeta potential of CPZ-AuNP conjugates strongly depends on the amount of CPZ adsorbed on the TC-AuNP surface. Additionally, the conjugates exhibited an isoelectric point at pH 8.8. Surface-enhanced Raman spectroscopy (SERS) and surface-enhanced infrared absorption spectroscopy (SEIRA) were employed to elucidate the adsorption structure of CPZ on TC-AuNPs. The interpretation of the spectra was conducted based on the Raman and FTIR spectra of CPZ, along with calculations performed using Density Functional Theory (DFT). The results indicated that CPZ primarily interacts with the TC-AuNP surface through the angularly oriented phenothiazine ring and the propylene bridge. Furthermore, it was demonstrated that the C-N-C fragment is perpendicular to the surface of the TC-AuNP with which it interacts. The findings from this analysis suggest the potential for further research on the use of these conjugates in biomedical applications.

Novel Tetracyclic Azaphenothiazines with the Quinoline Ring as New Anticancer and Antibacterial Derivatives of Chlorpromazine.

Phenothiazine derivatives are widely studied in various fields such as biology, chemistry, and medicine research because of their pharmaceutical effects. The first compound used successfully in the treatment of psychosis was a phenthiazine derivative, chlorpromazine. Apart from its activity in neurons, chlorpromazine has also been reported to display anticancer and antibacterial properties. In this study, we present the synthesis and research on the activity of A549, MDA, MiaPaCa, PC3, and HCT116 cancer cell lines and of S. aureus, S. epidermidis, E. coli, and P. aeruginosa bacterial strains against a series of new tetracyclic chlorpromazine analogues containing a quinoline scaffold in their structure instead of the benzene ring and various substituents at the thiazine nitrogen. The structure of these novel molecules has been determined by 1H NMR, 13C NMR, and HRMS spectral techniques. The seven most active of the twenty-four new chlorpromazine analogues tested were selected to study the mechanism of cytotoxic action. Their ability to induce apoptosis or necrosis in cancer cells was assessed by flow cytometry analysis. The results obtained confirmed the proapoptotic activity of selected compounds, especially in terms of inducing late apoptosis or necrosis in cancer cell lines A549, MiaPaCa-2, and HCT-116. Furthermore, studies on the induction of cell cycle arrest suggest that the new chlorpromazine analogues exert antiproliferative effects by inducing cell cycle arrest in the S phase and, consequently, apoptosis.

Comparative studies on the aggregate formation of synthesized zwitterionic gemini and monomeric surfactants in the presence of the amphiphilic antipsychotic drug chlorpromazine hydrochloride in aqueous solution: an experimental and theoretical approach.

The formation of aggregates, which are widely used in the field of biochemistry and the medical industry, was studied with different compositions of alkyl betaine gemini surfactant (C14Ab) in conjugation with chlorpromazine hydrochloride (CPZ). The results were compared with those of a single-chain zwitterionic surfactant (C12DmCB) of the same type with CPZ. Dynamic light scattering (DLS), confocal laser scanning microscopy (CLSM), and transmission electron microscopy (TEM) methods were used to distinguish the aggregates for the CPZ/C14Ab system in aqueous solutions above a certain mole fraction of the drug CPZ (αCPZ = 0.2). Time-resolved fluorescence decay measurements of acridine orange revealed relative polarity near the head group regions of mixed micelle (CPZ/C14Ab and CPZ/C12DmCB) systems. The hydrophilic environment around the head group regions of the CPZ/C14Ab system was different from that in the case of the CPZ/C12DmCB system. On the other hand, several theoretical models were employed (Clint, Rubingh, Motomura, and SPB) for mixed micellar systems to elucidate the different interaction parameters. Such a systematic study of a zwitterionic gemini amphiphile and its interaction with other amphiphiles and an amphiphilic drug molecule is rare in the literature.

Cyclodextrin complexation studies as the first step for repurposing of chlorpromazine.

The antipsychotic drug chlorpromazine (CPZ) has potential for the treatment of acute myeloid leukemia, if central nervous system side-effects resulting from its passage through the blood-brain barrier can be prevented. A robust drug delivery system for repurposed CPZ would be drug-in-cyclodextrin-in-liposome that would redirect the drug away from the brain while avoiding premature release in the circulation. As a first step, CPZ complexation with cyclodextrin (CD) has been studied. The stoichiometry, binding constant, enthalpy, and entropy of complex formation between CPZ and a panel of CDs was investigated by isothermal titration calorimetry (ITC). All the tested CDs were able to include CPZ, in the form of 1:1, 1:2 or a mixture of 1:1 and 1:2 complexes. In particular, a substituted γ-CD, sugammadex (the octasodium salt of octakis(6-deoxy-6-S-(2-carboxyethyl)-6-thio)cyclomaltooctaose), formed exclusively 1:2 complexes with an extremely high association constant of 6.37 × 109 M-2. Complexes were further characterized by heat capacity changes, one- and two-dimensional (ROESY) nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. Finally, protection of CPZ against photodegradation by CDs was assessed. This was accelerated rather than reduced by complexation with CD. Altogether these results provide a molecular basis for the use of CD in delayed release formulations for CPZ.

Photomutagenicity of chlorpromazine and its N-demethylated metabolites assessed by NGS.

The human genome is constantly attacked by endogenous and exogenous agents (ultraviolet light, xenobiotics, reactive oxygen species), which can induce chemical transformations leading to DNA lesions. To combat DNA damage, cells have developed several repair mechanisms; however, if the repair is defective, DNA lesions lead to permanent mutations. Single-cell gel electrophoresis (COMET assay) is a sensitive and well-established technique for quantifying DNA damage in individual cells. Nevertheless, this tool lacks relationship with mutagenesis. Therefore, to identify errors that give rise to mutations it would be convenient to test an alternative known procedure, such as next generation sequencing (NGS). Thus, the present work aims to evaluate the photomutagenicity of neuroleptic drug chlorpromazine (CPZ), and its N-demethylated metabolites using COMET assay and to test NGS as an alternative method to assess photomutagenesis. In this context, upon exposure to UVA radiation, COMET assay reveals CPZ-photosensitized DNA damage partially repaired by cells. Conversely with this result, metabolites demethylchlorpromazine (DMCPZ) and didemethylchlorpromazine (DDMCPZ) promote extensive DNA-photodamage, hardly repaired under the same conditions. Parallel assessment of mutagenesis by NGS is consistent with these results with minor discrepancies for DDMCPZ. To our knowledge, this is the first example demonstrating the utility of NGS for evaluating drug-induced photomutagenicity.

Investigation of metabolite-protein interactions by transient absorption spectroscopy and in silico methods.

Transient absorption spectroscopy in combination with in silico methods has been employed to study the interactions between human serum albumin (HSA) and the anti-psychotic agent chlorpromazine (CPZ) as well as its two demethylated metabolites (MCPZ and DCPZ). Thus, solutions containing CPZ, MCPZ or DCPZ and HSA (molar ligand:protein ratios between 1:0 and 1:3) were submitted to laser flash photolysis and the ΔAmax value at λ = 470 nm, corresponding to the triplet excited state, was monitored. In all cases, the protein-bound ligand exhibited higher ΔAmax values measured after the laser pulse and were also considerably longer-lived than the non-complexed forms. This is in agreement with an enhanced hydrophilicity of the metabolites, due to the replacement of methyl groups with H that led to a lower extent of protein binding. For the three compounds, laser flash photolysis displacement experiments using warfarin or ibuprofen indicated Sudlow site I as the main binding site. Docking and molecular dynamics simulation studies revealed that the binding mode of the two demethylated ligands with HSA would be remarkable different from CPZ, specially for DCPZ, which appears to come from the different ability of their terminal ammonium groups to stablish hydrogen bonding interactions with the negatively charged residues within the protein pocket (Glu153, Glu292) as well as to allocate the methyl groups in an apolar environment. DCPZ would be rotated 180° in relation to CPZ locating the aromatic ring away from the Sudlow site I of HSA.

Unraveling the Mechanisms Behind the Complete Suppression of Cocaine Electrochemical Signals by Chlorpromazine, Promethazine, Procaine, and Dextromethorphan.

The present work investigates the challenges accompanied by the electrochemical cocaine detection in physiological conditions (pH 7) in the presence of chlorpromazine, promethazine, procaine, and dextromethorphan, frequently used cutting agents in cocaine street samples. The problem translates into the absence of the cocaine oxidation signal (signal suppression) when in a mixture with one of these compounds, leading to false negative results. Although a solution to this problem was provided through earlier experiments of our group, the mechanisms behind the suppression are now fundamentally investigated via electrochemical and liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS) strategies. The latter was used to confirm the passivation of the electrodes due to their interaction with promethazine and chlorpromazine. Electron transfer mechanisms were further identified via linear sweep voltammetry. Next, adsorption experiments were performed on the graphite screen printed electrodes both with and without potential assistance in order to confirm if the suppression of the cocaine signals is due to passivation induced by the cutting agents or their oxidized products. The proposed strategies allowed us to identify the mechanisms of cocaine suppression for each cutting agent mentioned. Suppression due to procaine and dextromethorphan is caused by fouling of the electrode surface by their oxidized forms, while for chlorpromazine and promethazine the suppression of the cocaine signal is related to the strong adsorption of these (nonoxidized) cutting agents onto the graphite electrode surface. These findings provide fundamental insights in possible suppression and other interfering mechanisms using electrochemistry in general not only in the drug detection sector.

Sonochemical synthesis of iron-graphene oxide/honeycomb-like ZnO ternary nanohybrids for sensitive electrochemical detection of antipsychotic drug chlorpromazine.

We report a novel electrochemical sensor for the sensitive and selective determination of the antipsychotic drug chlorpromazine (CPZ) based on the iron (Fe) nanoparticles-loaded graphene oxide (GO-Fe)/three dimensional (3D) honeycomb-like zinc oxide (ZnO) nanohybrid modified screen printed carbon electrode (SPCE). The 3D hierarchical honeycomb-like ZnO was synthesized using a novel aqueous hydrothermal method and the GO-Fe/ZnO nanohybrid was prepared based on an inexpensive and fast sonochemical method using a high-intensity ultrasonic bath (Delta DC200H, 200 W, 40 KHz). Characterizations including scanning electron microscopy, elemental mapping, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and Raman spectroscopy were carried out as part of this work. The electrocatalytic oxidation behavior of CPZ at various electrodes was investigated using the cyclic voltammetry technique, through which the GO-Fe/ZnO modified SPCE was identified as the best performing electrode. The quantitative determination of CPZ was then performed using the differential pulse voltammetry technique. The as-prepared GO-Fe/ZnO/SPCE sensor exhibited a quick and sensitive response towards the oxidation of CPZ with linear concentration ranges from 0.02 to 172.74 µM and 222.48 to 1047.74 µM. The modified SPCE sensor displayed a low detection limit (LOD) of 0.02 µM and a high sensitivity of 7.56 µA µM-1 cm-2. The proposed sensor also showed remarkable operational and storage stability, reproducibility, and repeatability. Furthermore, the practicability of the GO-Fe/ZnO/SPCE sensor has been verified with real sample analysis using commercial antipsychotic CPZ tablets and human urine samples, and adequate recovery has been achieved.

Classics in Chemical Neuroscience: Chlorpromazine.

The discovery of chlorpromazine in the early 1950s revolutionized the clinical treatment of schizophrenia, galvanized the development of psychopharmacology, and standardized protocols used for testing the clinical efficacy of antipsychotics. Furthermore, chlorpromazine expanded our understanding of the role of chemical messaging in neurotransmission and reduced the stigma associated with mental illness, facilitating deinstitutionalization in the 1960s and 1970s. In this review, we will discuss the synthesis, manufacturing, metabolism and pharmacokinetics, pharmacology, structure-activity relationship, and adverse effects of chlorpromazine. In conclusion, we summarize the history and significant contributions of chlorpromazine that have resulted in this potent first-generation antipsychotic maintaining its clinical relevance for nearly 70 years.

Selective adsorption and removal of drug contaminants by using an extremely stable Cu(II)-based 3D metal-organic framework.

The adsorption capacity of three representative pharmaceutical drugs and personal care products (PPCPs) viz. diclofenac sodium (DCF), chlorpromazine hydrochloride (CLF) and amodiaquin dihydrochloride (ADQ), were preliminarily studied using a water-stable Cu(II)-based metal organic framework (MOF) [Cu(BTTA)]n·2DMF (1) (H2BTTA = 1,4-bis(triazol-1-yl)terephthalic acid). We also investigated the factors influencing the adsorption such as concentration, pH, contact time, temperature and dosages. The results show that the adsorption capacity of 1 for DCF (650 mg g-1) from aqueous medium, which is higher in comparison to most of the reported MOFs. While the adsorption of CLF and ADQ are only 67 mg g-1 and 72 mg g-1, respectively. The adsorption isotherm and adsorption kinetics indicated that the adsorption of diclofenac sodium by 1 follows Freundlich model with R2 value of 0.9902 and pseudo-first-order kinetics with correlation coefficient 0.9939 and K1 value of 0.0058 min-1, respectively. Investigations indicate that 1 could become a potential material to adsorb DCF from aqueous medium.

Sertraline, chlorprothixene, and chlorpromazine characteristically interact with the REST-binding site of the corepressor mSin3, showing medulloblastoma cell growth inhibitory activities.

Dysregulation of repressor-element 1 silencing transcription factor REST/NRSF is related to several neuropathies, including medulloblastoma, glioblastoma, Huntington's disease, and neuropathic pain. Inhibitors of the interaction between the N-terminal repressor domain of REST/NRSF and the PAH1 domain of its corepressor mSin3 may ameliorate such neuropathies. In-silico screening based on the complex structure of REST/NRSF and mSin3 PAH1 yielded 52 active compounds, including approved neuropathic drugs. We investigated their binding affinity to PAH1 by NMR, and their inhibitory activity toward medulloblastoma cell growth. Interestingly, three antidepressant and antipsychotic medicines, sertraline, chlorprothixene, and chlorpromazine, were found to strongly bind to PAH1. Multivariate analysis based on NMR chemical shift changes in PAH1 residues induced by ligand binding was used to identify compound characteristics associated with cell growth inhibition. Active compounds showed a new chemo-type for inhibitors of the REST/NRSF-mSin3 interaction, raising the possibility of new therapies for neuropathies caused by dysregulation of REST/NRSF.

Calculating Kinetic Rates and Membrane Permeability from Biased Simulations.

We present a simple approach to calculate the kinetic properties of lipid membrane crossing processes from biased molecular dynamics simulations. We demonstrate that by using biased simulations, one can obtain highly accurate kinetic information with significantly reduced computational time with respect to unbiased simulations. We describe how to conveniently calculate the transition rates to enter, cross, and exit the membrane in terms of the mean first passage times. To obtain free energy barriers and relaxation times from biased simulations only, we constructed Markov models using the dynamic histogram analysis method (DHAM). The permeability coefficients that are calculated from the relaxation times are found to correlate highly with experimentally evaluated values. We show that more generally, certain calculated kinetic properties linked to the crossing of the membrane layer (e.g., barrier height and barrier crossing rates) are good indicators of ordering drugs by permeability. Extending the analysis to a 2D Markov model provides a physical description of the membrane crossing mechanism.

A Potential Therapeutic Target RNA-binding Protein, Arid5a for the Treatment of Inflammatory Disease Associated with Aberrant Cytokine Expression.

BACKGROUND: Infection, tissue damage and aging can cause inflammation with high levels of inflammatory cytokines. Overproduction of inflammatory cytokines often leads to systemic inflammatory response syndrome (SIRS), severe sepsis, and septic shock. However, prominent therapeutic targets have not been found, although the incidence of sepsis is likely to increase annually. Our recent studies indicate that some RNA-binding proteins, which control gene expression of inflammatory cytokines at the post-transcriptional level, may play a critical role in inflammatory diseases such as sepsis. RESULTS: 1) One of the RNA-binding proteins, AT-rich interactive domain-containing 5a (Arid5a) promotes cytokine production through control of mRNA half-lives of pro-inflammatory molecules such as IL-6, STAT3, T-bet, and OX40 in activated macrophages and T cells. Arid5a KO mice are refractory to endotoxin shock, bleomycininduced lung injury, and inflammatory autoimmune disease. 2) Chlorpromazine (CPZ), which is recognized as a psychotic drug, impairs post-transcriptional gene expression of Il6 in LPS-stimulated macrophages: CPZ inhibits the binding activity of Arid5a to the 3'UTR of Il6 mRNA, thereby destabilizing Il6 mRNA possibly through suppression of Arid5a expression. 3) CPZ has strong suppressive effects on cytokine production such as TNF-α in vivo. Mice with treatment of CPZ are resistant to lipopolysaccharide (LPS)-induced shock. CONCLUSION: Thus, Arid5a contributes to the activation of macrophages and T cells through positive control of mRNA half-lives of inflammatory cytokines and its related molecules, which might lead to cytokine storm. Interestingly, Arid5a was identified from an inhibitory effect of CPZ on IL-6 production in macrophages activated by LPS. Therefore, CPZ derivatives or Arid5a inhibitors may have a potential to suppress severe sepsis through control of post-transcriptional gene expression.

The engineered ß-lactoglobulin with complementarity to the chlorpromazine chiral conformers.

Chlorpromazine (CPZ) is a phenothiazine acting as dopamine antagonist. Aside from application in schizophrenia therapy, chlorpromazine is found to be a putative inhibitor of proteins involved in cancers, heritable autism disorder and prion diseases. Four new ß-lactoglobulin variants with double or triple substitutions: I56F/L39A, F105L/L39A, I56F/L39A/M107F or F105L/L39A/M107F changing the shape of the binding pocket were produced and their chlorpromazine binding properties have been investigated by X-ray crystallography, circular dichroism, isothermal titration calorimetry and thermophoresis. The CD spectra and crystal structures revealed that mutations do not affect the protein overall structure but in comparison to WT protein, variants possessing I56F substitution had lower stability while mutation F105L increased melting temperature of the protein. The new variants showed affinity to chlorpromazine in the range 4.2-15.4 × 103 M-1. The CD spectra and crystal structures revealed complementarity of the binding pocket shape, to only one chlorpromazine chiral conformer. The (aR)-CPZ was bonded to variants containing I56F substitution while variants with F105L substitution preferred (aS)-CPZ.

Chemical Synthetic Platform for Chlorpromazine Oligomers That Were Reported as Photo-degradation Products of Chlorpromazine.

A synthetic platform for chlorpromazine (CPZ) oligomers, which could be generated via photo-reaction of CPZ, is essential to promote their biological and structural studies. In this paper, the first synthetic platform for CPZ oligomers is described. A photo-irradiation experiment of CPZ to confirm whether the structure of the CPZ dimer generated by the photo-irradiation was identical to that prepared by our synthetic method is also reported.

A review of drug therapy for sporadic fatal insomnia.

BACKGROUND: Sporadic fatal insomnia (sFI) is a rapid progressive neurodegenerative disease characterised by gradual to perpetual insomnia, followed by dysautonomia, coma and death. 1 The cause of sFI was recently mapped to a mutation in a protein, the prion, found in the human brain. It is the unfolding of the prion that leads to the generation of toxic oligomers that destroy brain tissue and function. Recent studies have confirmed that a methionine mutation at codon 129 of the human Prion is characteristic of sFI. Current treatment slows down the progression of the disease, but no cure has been found, yet. METHODS: We used Molecular Docking and Molecular Dynamics simulation methods, to study the toxic Fatal-Insomnia-prion conformations at local unfolding. The idea was to determine these sites and to stabilise these regions against unfolding and miss-folding, using a small ligand, based on a phenothiazine "moiety". CONCLUSION: As a result we here discuss current fatal insomnia therapy and present seven novel possible compounds for in vitro and in vivo screening.

Discriminative stimulus properties of the atypical antipsychotic amisulpride: comparison to its isomers and to other benzamide derivatives, antipsychotic, antidepressant, and antianxiety drugs in C57BL/6 mice.

RATIONALE: Racemic (RS)-amisulpride (Solian®) is an atypical antipsychotic drug used to treat schizophrenia and dysthymia. Blockade of dopamine D2/D3 and/or serotonin 5-HT7 receptors is implicated in its pharmacological effects. While the (S)-amisulpride isomer possesses a robust discriminative cue, discriminative stimulus properties of (RS)-amisulpride have not been evaluated. OBJECTIVES: The present study established (RS)-amisulpride as a discriminative stimulus and assessed amisulpride-like effects of amisulpride stereoisomers, other benzamide derivatives, and antipsychotic, antidepressant, and anxiolytic drugs. METHODS: Adult, male C57BL/6 mice were trained to discriminate 10 mg/kg (RS)-amisulpride from vehicle in a two-lever food-reinforced operant conditioning task. RESULTS: (RS)-Amisulpride's discriminative stimulus was dose-related, time-dependent, and stereoselective. (S)-Amisulpride (an effective dose of 50% (ED50) = 0.21 mg/kg) was three times more potent than (RS)-amisulpride (ED50 = 0.60 mg/kg) or (R)-amisulpride (ED50 = 0.68 mg/kg). (RS)-Amisulpride generalized fully to the structurally related atypical antipsychotic/antidysthymia drug sulpiride (Sulpor®; ED50 = 7.29 mg/kg) and its (S)-enantiomer (ED50 = 9.12 mg/kg); moderate to high partial generalization [60-75% drug lever responding (%DLR)] occurred to the benzamide analogs tiapride (Tiapridal®) and raclopride, but less than 60% DLR to metoclopramide (Reglan®), nemonapride (Emilace®), and zacopride. Antipsychotic, antidepressant, and antianxiety drugs from other chemical classes (chlorpromazine, quetiapine, risperidone, and mianserin) produced 35-55% amisulpride lever responding. Lastly, less than 35% DLR occurred for clozapine, olanzapine, aripiprazole imipramine, chlordiazepoxide, and bupropion. CONCLUSIONS: (RS)-Amisulpride generalized to some, but not all benzamide derivatives, and it failed to generalize to any other antipsychotic, antidepressant, or antianxiety drugs tested. Interestingly, the (R)-isomer shared very strong stimulus properties with (RS)-amisulpride. This finding was in contrast to findings from Donahue et al. (Eur J Pharmacol 734:15-22, 2014), which found that the (R)-isomer did not share very strong stimulus properties when the (S)-isomer was the training drug.

Chromatographic studies of drug interactions with alpha1-acid glycoprotein by ultrafast affinity extraction and peak profiling.

Interactions with serum proteins such as alpha1-acid glycoprotein (AGP) can have a significant effect on the behavior and pharmacokinetics of drugs. Ultrafast affinity extraction and peak profiling were used with AGP microcolumns to examine these processes for several model drugs (i.e., chlorpromazine, disopyramide, imipramine, lidocaine, propranolol and verapamil). The association equilibrium constants measured for these drugs with soluble AGP by ultrafast affinity extraction were in the general range of 104-106M-1 at pH 7.4 and 37°C and gave good agreement with literature values. Some of these values were dependent on the relative drug and protein concentrations that were present when using a single-site binding model; these results suggested a more complex mixed-mode interaction was actually present, which was also then used to analyze the data. The apparent dissociation rate constants that were obtained by ultrafast affinity extraction when using a single-site model varied from 0.14 to 7.0s-1 and were dependent on the relative drug and protein concentrations. Lower apparent dissociation rate constants were obtained by this approach as the relative amount of drug versus protein was decreased, with the results approaching those measured by peak profiling at low drug concentrations. This information should be useful in better understanding how these and other drugs interact with AGP in the circulation. In addition, the chromatographic approaches that were optimized and used in this report to examine these systems can be adapted for the analysis of other solute-protein interactions of biomedical interest.

Elucidating the role of the pLG72 R30K substitution in schizophrenia susceptibility.

In the human brain, pLG72 interacts with the flavoenzyme d-amino acid oxidase (hDAAO), which is involved in catabolism of d-serine, a co-agonist of N-methyl-d-aspartate receptors (NMDAR). Here, we investigated the wild-type pLG72, the R30K variant associated with schizophrenia susceptibility, and the K62E variant. The protein conformation, oligomeric state, ligand-, and hDAAO-binding properties are only slightly modified by the substitutions. All pLG72 variants inhibit hDAAO and lead to an increase in cellular (d/d+l)-serine. However, the R30K pLG72 is significantly more prone to degradation than the R30 and the K62E variants in a cell system, thus possessing a lower ability to interact/inhibit hDAAO. This links R30K pLG72 with the hyperactivity of hDAAO, the decreased d-serine level, and NMDAR hypofunction observed in schizophrenia-affected patients.