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PubMed1992

Transport of isometamidium (Samorin) by drug-resistant and drug-sensitive Trypanosoma congolense.

The uptake kinetics of a 14C-labelled trypanocidal compound isometamidium chloride (Samorin, RMB Animal Health Ltd, UK) was measured in drug-resistant and drug-sensitive Trypanosoma congolense. It was established that drug uptake was significantly more rapid and quantitatively greater in drug-sensitive parasites. There was clear evidence that drug uptake in both the resistant and sensitive trypanosomes was by a specific, receptor-mediated process. This specific drug transport was energy-dependent, being sensitive to metabolic inhibition with SHAM/glycerol. Significant differences in drug transport were observed which could be correlated with resistance to isometamidium. The optimal pH for drug accumulation was lowered in the resistant trypanosomes; this finding, along with an observed change in specificity for the related compound homidium bromide, suggested that the specific receptor for isometamidium is altered in the resistant trypanosomes, possibly resulting in a reduction in drug uptake. In addition to these alterations in drug uptake, efflux of isometamidium also appears to occur in the resistant trypanosomes. Both a reduction in incubation temperature and metabolic inhibition increased the level of trypanosome-associated isometamidium in the resistant parasites. This was in contrast to observations using drug-sensitive parasites. Furthermore, the addition of calcium flux-modulating agents to the incubation medium also resulted in an increase in accumulation by the resistant parasites.

Animals↗
PubMed2003

Decreased thalamic D2/D3 receptor binding in drug-naive patients with schizophrenia: a PET study with [11C]FLB 457.

The thalamus is a neuroanatomic structure that has reciprocal connections with several brain regions suggested to be involved in the pathophysiology of schizophrenia. Recent studies have reported structural as well as functional abnormalities of the thalamus in schizophrenia. The aim of the present exploratory study was to examine D2/D3 dopamine receptors in the thalamus as well as the anterior cingulate and the frontal and temporal cortices by using the high-affinity radioligand [11C]FLB 457 and positron emission tomography (3D PET) and to explore the data in relation to disease, age and psychopathology. Nine drug-naive patients with schizophrenia and eight control subjects were examined. Regional binding potential (BP) values were calculated using the simplified reference tissue model. The D2/D3 receptor binding was significantly lower in the right medial thalamus in the schizophrenia patients compared to control subjects. In addition, we found a significant negative age effect on the D2/D3 BP in the frontal and temporal cortex for both groups. There was no significant age effect on the D2/D3 BP in the thalamus or in the anterior cingulate. The result provides additional support to the view that the age effect on D2/D3 receptors differ between brain regions. The preliminary finding of low thalamic D2/D3 BP in patients strengthens the hypothesis that the thalamus is a key region in the pathophysiology of schizophrenia.

Adult↗
PubMed2004

Estimation of the time-course of dopamine D2 receptor occupancy in living human brain from plasma pharmacokinetics of antipsychotics.

Although the kinetic profile of antipsychotics at dopamine D2 receptor sites has been suggested to be important for antipsychotic action and dosing schedule, the kinetic profiles of the respective antipsychotic drugs in the brain have not yet been clearly defined. We aimed to estimate the time-course of dopamine D2 receptor occupancy from plasma pharmacokinetics and the apparent in-vivo affinity parameter (ED50; concentration required to induce 50% occupancy). Dopamine D2 receptor occupancies and plasma concentrations of risperidone were measured in five patients with schizophrenia using positron emission tomography with [11C]FLB 457. Measured dopamine D2 occupancies were compared with those estimated from plasma kinetics and in-vivo ED50. The time-course of dopamine D2 receptor occupancy was simulated with altered plasma kinetics or apparent in-vivo affinity parameters of the drug. Mean half-life of dopamine D2 receptor occupancy of risperidone was 80.2 h while that of the plasma concentration was 17.8 h. Dopamine D2 receptor occupancy estimated from plasma pharmacokinetics and in-vivo ED50 was within 1 S.D. of the mean measured occupancy. When the ED50 value was changed to one-tenth and 10-fold, the simulated half-life of receptor occupancy changed to 117.6 h and 27.3 h respectively. Using plasma pharmacokinetics and in-vivo ED50, the time-course of receptor occupancy could be calculated. Simulation of drug kinetics at receptors would provide useful information for the evaluation of antipsychotics.

Adult↗
PubMed2004

High striatal occupancy of D2-like dopamine receptors by amisulpride in the brain of patients with schizophrenia.

The 'atypicality' of the antipsychotic drug, amisulpride, has been attributed to preferential extrastriatal binding. Previous investigations of striatal D2 receptor occupancy by amisulpride revealed conflicting results. The aim of this PET study was to measure the striatal occupancy by amisulpride and to correlate it with the corresponding drug plasma concentrations. Nine amisulpride-treated patients and 12 healthy volunteers serving as controls were studied with PET and [18F]desmethoxyfallypride. Occupancy values and plasma concentrations were nonlinearly fitted to an E max model. Results showed 43-85% (putamen) and 67-90% (caudate) D2-like receptor occupancy. Plasma amisulpride concentrations at the time of tracer injection, but not administered doses, were significantly nonlinearly correlated to occupancy levels (putamen: rS=0.88, p=0.0017; caudate: r S=0.78, p=0.0127). Calculated Emax was similar in both caudate and putamen, but occupancy levels were lower in caudate at lower amisulpride plasma concentrations. Calculated plasma levels to attain 60-80% receptor occupancy ranged from 119 to 474 ng/ml (caudate) and from 241 to 732 ng/ml (putamen). This reveals a broad range of plasma concentrations producing less than 80% striatal receptor occupancy. However, our data show high striatal D2-like receptor occupancies under rising plasma concentrations. Using the full range of recommended amisulpride dosage, striatal occupancies up to 90% can be measured.

Adult↗
PubMedDate not supplied

Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography.

Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.

Alkaline Phosphatase↗
PubMedDate not supplied

Phenylboronic acid-salicylhydroxamic acid bioconjugates. 1. A novel boronic acid complex for protein immobilization.

A chemical affinity system exhibiting antibody-like properties is described. The system exploits bioconjugates with appended phenylboronic acid (PBA) moieties and a support-bound phenylboronic acid complexing reagent derived from salicylhydroxamic acid (SHA) for protein immobilization on a chromatographic support. The structure of the PBA.SHA complex was characterized by 11B NMR and mass spectrometry and compared with complexes derived from model compounds. Protein modification reagents were synthesized from 3-aminophenylboronic acid and utilized to prepare bioconjugates from alkaline phosphatase (AP) and horseradish peroxidase (HRP). AP obtained from one source afforded PBA bioconjugates exhibiting significant loss of enzymatic activity, whereas AP obtained from a second source afforded PBA bioconjugates exhibiting only a modest loss of enzymatic activity. Conversely, HRP afforded PBA bioconjugates exhibiting no loss of enzymatic activity. SHA-modified Sepharose was prepared by reaction of methyl 4-[(6-aminohexanoylamino)methyl]salicylate with CNBr-activated Sepharose 4B, followed by treatment with aqueous alkaline hydroxylamine. PBA-AP and PBA-HRP conjugates were efficiently immobilized on SHA-Sepharose at pH 8.3. PBA-AP conjugates were retained after washing with acidic buffers at pH 6.7, 4.2, and 2.5, whereas PBA-HRP conjugates were retained after washing with buffer at pH 6.7, but were eluted to some extent at and below pH 4.2. The results are interpreted in terms of multivalent interactions involving boronic acid complex formation between the enzyme bioconjugates and immobilized complexing reagent.

Alkaline Phosphatase↗
PubMed2001

Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase.

Peroxidases typically bind their reducing substrates weakly, with K(d) values in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferric horseradish peroxidase isoenzyme C (HRPC) [K(d) = 2.4 microM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provided a useful tool for probing the environment of the peroxidase aromatic-donor-binding site and the distal heme cavity. Knowledge of the underlying thermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isothermal titration calorimetry results of this study on the binding of aromatic hydroxamic acid analogues to ferric HRPC under nonturnover conditions (no H(2)O(2) present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to HRPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. The overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, with the distal Arg38 acting as an "entropic gate keeper". Dramatic enthalpy-entropy compensation is observed for BHA and 2-naphthohydroxamic acid binding to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due to increased flexibility of Arg38 in the complexes at low pH and greater access by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the K(d) values corrected for substrate ionization suggests that enthalpy-entropy compensation is operative over a wide pH range. Examination of the thermodynamics of binding of ring-substituted hyrazides to HRPC reveals that the binding affinities of aromatic donors are highly sensitive to the position and nature of the ring substituent.

Benzamides↗
PubMed2003

New insight into the peroxidase-hydroxamic acid interaction revealed by the combination of spectroscopic and crystallographic studies.

Aromatic hydroxamic acids, such as salicylhydroxamic (SHA) and benzohydroxamic (BHA) acids, are commonly used as probes for studying the active sites of peroxidases. In this paper, we have extended the study of the complexes of Arthromyces ramosus peroxidase (ARP/CIP) with BHA and SHA by analyzing their Raman spectra in solution and in single crystals. The experiments were carried out under various conditions to identify the best experimental conditions, and hence, avoid artifacts deriving from the preparation of the samples or collection of the spectra. The analysis of the data takes also into account the characteristic of the electronic absorption spectra in solution and the crystal structures of the complexes. The results showed small differences between the solution and the crystal phases even though the coordination state can be dramatically affected by the physical or chemical conditions. The greater sensitivity of the spectroscopic technique enabled us to establish the existence of multiple species upon complexation of the protein with the hydroxamic acids that could not be detected by ordinary X-ray crystallography. Furthermore, SHA titration experiments and singular value decomposition analysis of the absorption spectra indicated the presence of two binding sites in the protein, one with a high affinity (K(d) = 1.7 mM), which should correspond to the SHA bound protein as determined by X-ray, and the other with a very low affinity (K(d) > 80 mM) probably located in a non-heme site. This suggests that the heterogeneous titration line shape involves ligand binding to a non-heme site in competition with the canonical heme site. In contrast, the titration profile obtained with the BHA ligand is monophasic, in agreement with all the peroxidases so far studied.

Binding Sites↗
PubMed2004

Crystal structure of the ascorbate peroxidase-salicylhydroxamic acid complex.

Ascorbate peroxidase is a bifunctional peroxidase that catalyzes the H(2)O(2)-dependent oxidation of both ascorbate and various aromatic substrates. The ascorbate binding site was recently identified as being close to the gamma-heme edge [Sharp, K. H., Mewies, M., Moody, P. C. E., and Raven, E. L. (2003)Nat. Struct. Biol. 10, 303-307]. In this work, the X-ray crystal structure of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) in complex with salicylhydroxamic acid (SHA) has been determined to 1.46 A. The SHA molecule is bound close to the delta-heme edge in a cavity that connects the distal side of the heme to the surface of the protein. There are hydrogen bonds between the phenolic hydroxide of the SHA and the main chain carbonyl of Pro132, between the carbonyl oxygen of SHA and the side chain guanadinium group of Arg38, and between the hydroxamic acid group and the indole nitrogen of Trp41. The structure provides the first information about the location of the aromatic binding site in ascorbate peroxidase and, together with our previous data [Sharp, K. H., et al. (2003) Nat. Struct. Biol. 10, 303-307], completes the structural description of the binding properties of ascorbate peroxidase. The mechanistic implications of the results are discussed in terms of our current understanding of how APX catalyzes oxidation of different types of substrates bound at different locations.

Arginine↗
PubMed1996

2.3 A resolution X-ray crystal structure of the bisubstrate analogue inhibitor salicylhydroxamic acid bound to human myeloperoxidase: a model for a prereaction complex with hydrogen peroxide.

The X-ray crystal structure of a salicylhydroxamic acid (SHA) inhibitory complex with human myeloperoxidase (MPO) has been determined at 2.3 A resolution. The aromatic ring of the inhibitor binds to a hydrophobic region at the entrance to the distal heme pocket between heme pyrrole ring D and the side chain of Arg 239. The hydroxamic acid moiety is hydrogen bonded to both the distal histidine 95 and the adjacent glutamine 91 amide group but is not coordinated to the heme iron. SHA binding displaces three water molecules from the distal heme cavity and causes a small shift in the position of a fourth water molecule. Otherwise, there are no significant conformational differences between the active site regions of the complex and the native enzyme. The ability of the three SHA oxygen atoms to closely duplicate the hydrogen-bonding pattern of these three water molecules in the native enzyme is postulated to account for the strong binding of this inhibitor to MPO. The mode of binding of SHA to MPO provides information on the binding sites for aromatic peracid substrates that promote compound I formation as well as aromatic alcohols and amines that carry out single-electron reductions of compound I. Similarities in the hydrogen-bonding patterns of amino acid residues and water molecules in the distal heme pockets of myeloperoxidase and the nonhomologous cytochrome c peroxidase suggest that they may have similar mechanisms of compound I formation. A model is presented for a prereaction complex of myeloperoxidase in which hydrogen peroxide is hydrogen bonded to the distal histidine, as a prerequisite for deprotonation and subsequent binding at the sixth coordination site of the heme iron.

Crystallography, X-Ray↗
PubMed1998

Structure-based design of potent inhibitors of scytalone dehydratase: displacement of a water molecule from the active site.

Scytalone dehydratase (SD) is a molecular target of inhibitor design efforts aimed at protecting rice plants from the fungal disease caused by Magnaporthe grisea. As determined from X-ray diffraction data of an SD-inhibitor complex [Lundqvist et al. (1994) Structure (London) 2, 937-944], there is an extended hydrogen-bonding network between protein side chains, the inhibitor, and two bound water molecules. From models of SD complexed to quinazoline and benztriazine inhibitors, a new class of potent SD inhibitors involving the displacement of an active-site water molecule were designed. We were able to increase inhibitory potency by synthesizing compounds with a nitrile functionality displayed into the space occupied by one of the crystallographic water molecules. Sixteen inhibitors are compared. The net conversion of potent quinazoline and benztriazine inhibitors to cyanoquinolines and cyanocinnolines increased binding potency 2-20-fold. Replacement of the nitrile with a hydrogen atom lowered binding affinity 100-30,000-fold. X-ray crystallographic data at 1.65 A resolution on a SD-inhibitor complex confirmed that the nitrile functionality displaced the water molecule as intended and that a favorable orientation was created with tyrosines 30 and 50 which had been part of the hydrogen-bonding network with the water molecule. Additional data on inhibitors presented herein reveals the importance of two hydrogen-bonding networks toward inhibitory potency: one between Asn131 and an appropriately positioned inhibitor heteroatom and one between a bound water molecule and a second inhibitor heteroatom.

Aminoquinolines↗
PubMed1999

Binding of salicylhydroxamic acid and several aromatic donor molecules to Arthromyces ramosus peroxidase, investigated by X-ray crystallography, optical difference spectroscopy, NMR relaxation, molecular dynamics, and kinetics.

The X-ray crystal structure of the complex of salicylhydroxamic acid (SHA) with Arthromyces ramosus peroxidase (ARP) has been determined at 1.9 A resolution. The position of SHA in the active site of ARP is similar to that of the complex of benzhydroxamic acid (BHA) with ARP [Itakura, H., et al. (1997) FEBS Lett. 412, 107-110]. The aromatic ring of SHA binds to a hydrophobic region at the opening of the distal pocket, and the hydroxamic acid moiety forms hydrogen bonds with the His56, Arg52, and Pro154 residues but is not asscoiated with the heme iron. X-ray analyses of ARP-resorcinol and ARP-p-cresol complexes failed to identify the aromatic donor molecules, most likely due to the very low affinities of these aromatic donors for ARP. Therefore, we examined the locations of these and other aromatic donors on ARP by the molecular dynamics method and found that the benzene rings are trapped similarly by hydrophobic interactions with the Ala92, Pro156, Leu192, and Phe230 residues at the entrance of the heme pocket, but the dihedral angles between the benzene rings and the heme plane vary from donor to donor. The distances between the heme iron and protons of SHA and resorcinol are similar to those obtained by NMR relaxation. Although SHA and BHA are usually considered potent inhibitors for peroxidase, they were found to reduce compound I and compound II of ARP and horseradish peroxidase C in the same manner as p-cresol and resorcinol. The aforementioned spatial relationships of these aromatic donors to the heme iron in ARP are discussed with respect to the quantum chemical mechanism of electron transfer in peroxidase reactions.

Binding Sites↗
PubMed2002

Structural investigations of palladium(II) and platinum(II) complexes of salicylhydroxamic acid.

Complexes of salicylhydroxamic acid (shaH) with palladium(II) and platinum(II) were investigated. The synthesis of [Pt(sha)(2)] was attempted via a number of methods, and ultimately (1)H NMR investigations revealed that salicylhydroxamate would not coordinate to chloro complexes of platinum(II). However, [Pt(sha-H)(PPh(3))(2)] was successfully synthesized and the crystal structure determined (orthorhombic, space group Pca2(1) a = 17.9325(19) A, b = 11.3102(12) A, c = 18.2829(19) A, Z = 4, R = 0.0224). The sha binds via an [O,O] binding mode, in its hydroximate form. In contrast the palladium complex [Pd(sha)(2)] was readily synthesized and crystallized as [Pd(sha)(2)](DMF)(4) in the triclinic space group P(-)1,a = 7.066(1) A, b = 9.842(2) A, c = 12.385(2) A, alpha = 99.213(3)(o), beta = 90.669(3), gamma = 109.767(3)(o), Z = 1, R = 0.037. The unexpected [N,O'] binding mode of the salicylhydroxamate ligand in [Pd(sha)(2)] prompted investigation of the stability of a number of binding modes of salicylhydroxamic acid in [M(sha)(2)] (M = Pd, Pt) by density functional theory, using the B3LYP hybrid functional at the 6-311G* level of theory. Geometry optimizations were carried out for various binding modes of the ligands and their relative energies established. It was found that the [N,O'] mode gave the more stable complex, in accord with experimental observations. Stabilization of hydroxamate binding to platinum is evidently afforded by soft ligands lying trans to them.

Crystallography, X-Ray↗