Use of a vinyl phosphonate analog of ATP as a rotationally constrained probe of the C5′---O5′ torsion angle in ATP complexed to methionine adenosyl transferase

An analog of adenosine 5′-triphosphate (ATP) was synthesized in which the C4′---C5′---O---P^α system is replaced by a trans C4′---CH=CH---P^α system. In the form of 1:1 complexes with Mg, this analog and its counterpart with a C4′---CH₂---CH₂---P^α system were linear competitive inhibitors, with respect to MgATP, of the MAT-II (normal tissue) and MAT-T (hepatoma tissue) forms of rat ATP: -methionine-S-adenosyltransferase (MAT); K_m(ATP)/K_i values ranged from 0.4 to 2.4. 2′-Deoxy-ATP was a weak substrate, K_m(ATP)/K_m = 0.035, of MAT-II and a weak competitive inhibitor, K_m(ATP)/K_i = 0.07, of MAT-T. These findings, together with interactions of the MAT forms with other substrates and inhibitors, indicate that binding of ATP to these transferases is accompanied by little rotation about the C5′---O5′ bond, and that C4′ and P^α are in a trans-type relationship in enzyme-bound ATP.     

Inhibitory Effect of di- and Tripeptidyl Aldehydes on Calpains and Cathepsins

Abstract

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of α-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B < calpain II ≈ calpain I < cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (K_i) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (K_i, 36 nM) and calpain II (K_i, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (K_i, 0.5nM); acetyl-Leu-Leu-Met-H for cathepsin B (K_i, 100nM).     

A rapid assay for assessment of sphingosine kinase inhibitors and substrates

Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-³²P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K_m, and the K_i values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.     

The human carbonic anhydrase isoenzymes I and II inhibitory effects of some hydroperoxides,alcohols, and acetates

The carbonic anhydrases (CAs, EC 4.2.1.1) represent a superfamily of widespread enzymes, which catalyze a crucial biochemical reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. In this study, a series of hydroperoxides, alcohols, and acetates were tested for the inhibition of the cytosolic hCA I and II isoenzymes. These compounds inhibited both hCA isozymes in the low nanomolar ranges. These compounds were good hCA I inhibitors (K_is in the range of 24.93–97.99?nM) and hCA II inhibitors (K_is in the range of 26.04–68.56?nM) compared to acetazolamide as CA inhibitor (K_i: 34.50?nM for hCA I and K_i: 28.93?nM for hCA II).     

Inhibition of Achromobacter Protease I by Lysinal Derivatives

Z-Val-, Z-Pro-, Z-Leu-Leu-, and Z-Leu-Pro-lysinals and BZ-DL-lysinal were chemically synthesized and tested as novel inhibitors for Achromobacter protease I (API), a lysine-specific serine protease. Among the lysinal derivatives tested, Z-Val-lysinal was the most potent competitive inhibitor, its K_i being estimated as 6.5 nM in an esterolytic assay with Tos-Lys-OMe. In an amidolytic assay, Z-Leu-Leu-lysinal was the most potent inhibitor and the apparent mode of inhibition was non-competitive. The K_is of the other lysinal derivatives in both esterolytic and amidolytic assays were more than 10³ times lower than that of leupeptin. Z-Val-lysinol, lacking the aldehyde group, was a poor competitive inhibitor. These results suggest that acyl-, acylaminoacyl-, and acylpeptidyllysinals function as a transition-state inhibitor for Achromobacter protease I.     

Purification and kinetic properties of ATP: citrate oxaloacetate lyase from rat brain.

Abstract— A method for a partial purification of ATP:citrate oxaloacetate lyase from rat brain is described. The Lineweaver–Burk plots of velocity vs citrate concentration are biphasic in the presence of fixed concentrations of MgCl₂. Therefore two values of K_m, corresponding to low and high concentrations of citrate, can be determined. When MgCl₂ is added in equimolar concentrations with citrate, a monophasic plot with one K_m of 0.13 mm is obtained. The K_m value for MgATP^2- was independent of citrate concentration, being equal to 0.40–0.43 mm. The K_m for CoA was 0.0007 mm. ADP and P_i are competitive inhibitors with respect to ATP. K_i for MgADP⁻ is equal to 0.13 mm. dl -isocitrate and cis-aconitate are partially competitive inhibitors with respect to citrate with K_i values of 5.8 and 4.8 mm, respectively. α-Ketoglutarate and pyruvate are noncompetitive inhibitors with respect to ATP and citrate, with K_i values equal to 9 and 45 mm, respectively. The physiological significance of these effectors for the regulation of citrate lyase activity in brain is discussed.     

ATP-Induced Inhibition of Na+, K+, Cl− Cotransport in Madin-Darby Canine Kidney Cells: Lack of Involvement of Known Purinoceptor-Coupled Signaling Pathways

P_2U/2Y-receptors elicit multiple signaling in Madin-Darby canine kidney (MDCK) cells, including a transient increase of [Ca²⁺] _i , activation of phospholipases C (PLC) and A₂ (PLA₂), protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). This study examines the involvement of these signaling pathways in the inhibition of Na⁺,K⁺,Cl⁻ cotransport in MDCK cells by ATP. The level of ATP-induced inhibition of this carrier (∼50% of control values) was insensitive to cholera and pertussis toxins, to the PKC inhibitor calphostin C, to the cyclic nucleotide-dependent protein kinase inhibitors, H-89 and H-8 as well as to the inhibitor of serine-threonine type 1 and 2A phosphoprotein phosphatases okadaic acid. ATP led to a transient increase of [Ca²⁺]_i that was abolished by a chelator of Ca²⁺ _i , BAPTA. However, neither BAPTA nor the Ca²⁺ ionophore A231287, or an inhibitor of endoplasmic reticulum Ca²⁺-pump, thapsigargin, modified ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. An inhibitor of PLC, U73122, and an inhibitor of MAPK kinase (MEK), PD98059, blocked ATP-induced inositol-1,4,5-triphosphate production and MAPK phosphorylation, respectively. However, these compounds did not modify the effect of ATP on Na⁺,K⁺,Cl⁻ cotransport activity. Inhibitors of PLA₂ (AACOCF₃), cycloxygenase (indomethacin) and lypoxygenase (NDGA) as well as exogenous arachidonic acid also did not affect ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Inhibition of the carrier by ATP persisted in the presence of inhibitors of epithelial Na⁺ channels (amiloride), Cl⁻ channels (NPPB) and Na⁺/H⁺ exchanger (EIPA) and was insensitive to cell volume modulation in anisosmotic media and to depletion of cells with monovalent ions, thus ruling out the role of other ion transporters in purinoceptor-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport. Our data demonstrate that none of the known purinoceptor-stimulated signaling pathways mediate ATP-induced inhibition of Na⁺,K⁺,Cl⁻ cotransport and suggest the presence of a novel P₂-receptor-coupled signaling mechanism. Received: 29 July 1998/Revised: 19 October     

Molecular insights into human monoamine oxidase (MAO) inhibition by 1,4-naphthoquinone: evidences for menadione (vitamin K3) acting as a competitive and reversible inhibitor of MAO

Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic and exogenous amines and its inhibitors have therapeutic value for several conditions including affective disorders, stroke, neurodegenerative diseases and aging. The discovery of 2,3,6-trimethyl-1,4-naphthoquinone (TMN) as a nonselective and reversible inhibitor of MAO, has suggested 1,4-naphthoquinone (1,4-NQ) as a potential scaffold for designing new MAO inhibitors. Combining molecular modeling tools and biochemical assays we evaluate the kinetic and molecular details of the inhibition of human MAO by 1,4-NQ, comparing it with TMN and menadione. Menadione (2-methyl-1,4-naphthoquinone) is a multitarget drug that acts as a precursor of vitamin K and an inducer of mitochondrial permeability transition. Herein we show that MAO-B was inhibited competitively by 1,4-NQ (K_i = 1.4 μM) whereas MAO-A was inhibited by non-competitive mechanism (K_i = 7.7 μM). Contrasting with TMN and 1,4-NQ, menadione exhibited a 60-fold selectivity for MAO-B (K_i = 0.4 μM) in comparison with MAO-A (K_i = 26 μM), which makes it as selective as rasagiline. Fluorescence and molecular modeling data indicated that these inhibitors interact with the flavin moiety at the active site of the enzyme. Additionally, docking studies suggest the phenyl side groups of Tyr407 and Tyr444 (for MAO-A) or Tyr398 and Tyr435 (for MAO-B) play an important role in the interaction of the enzyme with 1,4-NQ scaffold through forces of dispersion as verified for menadione, TMN and 1,4-NQ. Taken together, our findings reveal the molecular details of MAO inhibition by 1,4-NQ scaffold and show for the first time that menadione acts as a competitive and reversible inhibitor of human MAO.     

Development and exploitation of CK2 inhibitors

A number of quite specific and fairly potent inhibitors of protein kinase CK2, belonging to the classes of condensed polyphenolic compounds, tetrabromobenzimidazole/triazole derivatives and indoloquinazolines are available to date. The structural basis for their selectivity is provided by a hydrophobic pocket adjacent to the ATP/GTP binding site, which in CK2 is smaller than in the majority of other protein kinases due to the presence of a number of residues whose bulky side chains are generally replaced by smaller ones. Consequently a doubly substituted CK2 mutant V66A,I174A is much less sensitive than CK2 wild type to these classes of inhibitors. The most efficient inhibitors both in terms of potency and selectivity are 4,5,6,7-tetrabromo-1H-benzotriazole, TBB (K_i = 0.4 μM), the TBB derivative 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, DMAT (K_i = 0.040 μM), the emodin related coumarinic compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2-one, NBC (K_i = 0.22 μM) and the indoloquinazoline derivative ([5-oxo-5,6-dihydroindolo-(1,2a)quinazolin-7-yl]acetic acid), IQA (K_i = 0.17 μM). These inhibitors are cell permeable as judged from ability to block CK2 in living cells and they have been successfully employed, either alone or in combination with CK2 mutants refractory to inhibition, to dissect signaling pathways affected by CK2 and to identify the endogenous substrates of this pleitropic kinase. By blocking CK2 these inhibitors display a remarkable pro-apoptotic efficacy on a number of tumor derived cell lines, a property which can be exploited in perspective to develop antineoplastic drugs.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

A graphical method for determining inhibition constants

A new simple graphical method is described for the determination of inhibition type and inhibition constants of an enzyme reaction without any replot. The method consists of plotting experimental data as (V–v)/v versus the inhibitor concentration at two or more concentrations of substrate, where V and v represent the maximal velocity and the velocity in the absence and presence of inhibitor with given concentrations of the substrate, respectively. Competitive inhibition gives straight lines that converge on the abscissa at a point where [I]?=??K_i. Uncompetitive inhibition gives parallel lines with the slope of 1/K’_i. For mixed type inhibition, the intersection in the plot is given by [I]?=??K_i and (V–v)/v?=??K_i/K’_i in the third quadrant, and in the special case where K_i?=?K’_i (noncompetitive inhibition) the intersections occur at the point where [I]?=??K_i and (V–v)/v?=??1. The present method, the “quotient velocity plot,” provides a simple way of determining the inhibition constants of all types of inhibitors.     

The effect of antimycin A on cytochromes b561, b566, and their relationship to ubiquinone and the iron-sulfer centers S-1 (+N-2) and S-3.

Three nuclear RNA polymerases and one poly(A) polymerase were isolated from the yeast, Saccharomyces cerevisiae. The ability of cordycepin triphosphate to inhibit each was determined. RNA polymerase II was significantly more sensitive to this compound than the other polymerases. RNA polymerase I was relatively insensitive, being inhibited less than 20% by 40 μm cordycepin triphosphate. The calculated apparent K_i values of RNA polymerases II and III and poly(A) polymerase were, respectively, 0.3, 3.0, and 4.6 μm. Inhibition was competitive with regard to ATP. These data do not support the idea that, in yeast, poly(A) addition to preformed RNA in vivo is the primary site of cordycepin action.     

Acyl coenzyme A inhibition of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase: a comparison of the TPN and DPN linked reactions

The inhibitory effects of ATP, coenzyme A, and acetyl, malonyl, and oleyl derivatives of coenzyme A on the TPN and DPN dependent activities of Leuconostoc glucose-6-phosphate dehydrogenase are compared. At pH 7.8, 24°, saturating levels of DPN or TPN, and inhibitor concentrations of 2–4 mM only ATP has an appreciable effect on the TPN dependent reaction, but all were potent inhibitors of the DPN dependent reaction. Oleyl coenzyme A was the most effective (K_i ～ 0.15 mM against glucose-6-phosphate) while acetyl coenzyme A was least effective (K_i ～ 1.0 mM). A possible regulatory role of this inhibition in fatty acid synthesis is suggested.     

Kinetic and regulatory properties of pyruvate kinase isozymes from flight muscle and fat body of the cockroach,Periplaneta americana

Summary Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withK _m values of 0.27 and 0.04 mM, respectively.K _m for Mg²⁺ was 0.60 mM andK _a for K⁺ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentK _i values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theK _m for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P₂ did not activate the enzyme but reversed inhibitions by ATP and arginine-P.Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S_0.5=0.32 mM andn _H=1.43.K _m values for ADP and Mg²⁺ were 0.30 and 0.80 mM, respectively with aK _a for K⁺ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S_0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S_0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P₂ with an apparentK _a of 1.5 M. Fructose-1,6-P₂ at 0.1 mM reduced S_0.5 for P-enolpyruvate to 0.05 mM andn _H to 1.0.Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P₂ is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowK _m for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P₂.     

Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential

Triterpenoids are in the focus of scientific interest, and they were evaluated for many pharmacological applications among them their ability to act as inhibitors of cholinesterases. These inhibitors are still of interest as drugs that improve the life quality of patients suffering from age-related dementia illnesses especially of Alzheimer’s disease. Herein, we prepared several derivatives of ursolic and oleanolic acid and screened them in Ellman’s assays for their ability to inhibit acetylcholinesterase and/or butyrylcholinesterase, and for each of the active compounds the type of inhibition was determined. As a result, several compounds were shown as good inhibitors for acetylcholinesterase and butyrylcholinesterase even in a micromolar range. An ursolic acid derived hydroxyl-propinyl derivative 10 was a competitive inhibitor for butyrylcholinesterase with an inhibition constant of K_i = 4.29 μM, and therefore being twice as active as gold standard galantamine hydrobromide. The best inhibitor for acetylcholinesterase, however, was 2-methyl-3-oxo-methyl-ursoloate (18), acting as a mixed-type inhibitor showing K_i = 1.72 µM and K_i′ = 1.28 μM, respectively.     

Synthesis and inhibitory properties of some carbamates on carbonic anhydrase and acetylcholine esterase

A series of carbamate derivatives were synthesized and their carbonic anhydrase I and II isoenzymes and acetylcholinesterase enzyme (AChE) inhibitory effects were investigated. All carbamates were synthesized from the corresponding carboxylic acids via the Curtius reactions of the acids with diphenyl phosphoryl azide followed by addition of benzyl alcohol. The carbamates were determined to be very good inhibitors against for AChE and hCA I, and II isoenzymes. AChE inhibition was determined in the range 0.209–0.291?nM. On the other hand, tacrine, which is used in the treatment of Alzheimer’s disease possessed lower inhibition effect (K_i: 0.398?nM). Also, hCA I and II isoenzymes were effectively inhibited by the carbamates, with inhibition constants (K_i) in the range of 4.49–5.61?nM for hCA I, and 4.94–7.66?nM for hCA II, respectively. Acetazolamide, which was clinically used carbonic anhydrase (CA) inhibitor demonstrated K_i values of 281.33?nM for hCA I and 9.07?nM for hCA II. The results clearly showed that AChE and both CA isoenzymes were effectively inhibited by carbamates at the low nanomolar levels.     

Chemical Composition of Roots Flemingia philippinensis and Their Inhibitory Kinetics on Aromatase

Aromatase is the key enzyme responsible for catalyzing the conversion of C₁₉ steroids to estrogens. Its inhibitors are widely used in breast cancer therapy. The CH₂Cl₂ partition of a crude ethanolic extract from the roots of Flemingia philippinensis showed potent inhibitory activity of aromatase. The constituents of the extract were analyzed and identified by liquid chromatography tandem mass spectrometry. Five purified prenylated isoflavones were evaluated for aromatase inhibition and their IC₅₀ values ranged between 2.98 and 58.08 μm . In kinetic studies, all tested compounds behaved as reversible competitive inhibitors and their K_i values were calculated by Dixon plots. The most potent inhibitor (6,8‐diprenylorobol) had a K_i value of 1.42 μm . Furthermore, using UPLC and LC/MS, 6,8‐diprenylorobol was proven to be present in the native roots in high quantities.     

Identification of peptide inhibitors of penicillinase using a phage display library

There is a constant need to identify novel inhibitors to combat β-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM⁻¹) and binding affinity (K_D = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV–vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a K_i of 9.22 μM and a K_i′ of 33.12 μM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of β-lactamases and development of novel inhibitors of β-lactamases.     

Modulation of gating of a metabolically regulated,ATP-dependent K+ channel by intracellular pH in B cells of the pancreatic islet

Summary Membrane-permeant weak acids and bases, when applied to the bath, modulate the resting membrane potential and the glucose-induced electrical activity of pancreatic B cells, as well as their insulin secretion. These substances alter the activity of a metabolite-regulated. ATP-sensitive K⁺ channel which underlies the B-cell resting potential. We now present several lines of evidence indicating that the channel may be directly gated by pH _i . (1) The time course of K⁺(ATP) channel activity during exposure to and washout of NH₄Cl under a variety of experimental conditions, including alteration of the electrochemical gradient for NH₄Cl entry and inhibition of the Na _o ⁺ H _i ⁺ exchanger, resembles the time course of pH _i measured in other cell types that have been similarly treated. (2) Increasing pH _o over the range 6.25–7.9 increases K⁺(ATP) channel activity in cell-attached patches where the cell surface exposed to the bath has been permeabilized to H⁺ by the application of the K⁺/H⁺ exchanger nigericin. (3) Increasing pH _i over a similar range produces similar effects on K⁺(ATP) channels in inside-out excised patches exposed to small concentrations of ATP _i . The physiological role of pH _i in the metabolic gating of this channel remains to be explored.     

Inhibitors of Glutathione Reductase as Potential Antimalarial Drugs Kinetic Cooperativity and Effect of Dimethyl Sulphoxide on Inhibition Kinetics

Abstract

We have developed inhibitors of glutathione reductase that improve on the inhibition of literature lead compounds by up to three orders of magnitude. Thus, analogues of Safranine O and menadione were found to be strong, reversible inhibitors of yeast glutathione reductase. Safranine O exhibited partial, uncompetitive inhibition with K_i and α values of 0.5 mM and 0.15, respectively. Thionine O was a partial (hyperbolic) uncompetitive inhibitor with K_i and α values of 0.4μM and 0.15, respectively. LY83583 and 2-anilino-l,4-naphthoquinone also showed (hyperbolic) partial, uncompetitive inhibition with micromolar K_i values. For Nile Blue A a model for two-site binding with (parabolic) uncompetitive inhibition fitted the data with a K_i value of 11 μM and a kinetic cooperativity between the sites of 0.12, increased to 0.46 by pre-incubation of the enzyme and Nile Blue A in the presence of glutathione disulphide. Analysis of the effects of preincubation on the kinetics and cooperativity indicated the possibility of a slow conformational change in the homodimeric enzyme, the first such indication of kinetic cooperativity in the native enzyme to our knowledge. Further evidence of conformational changes for this enzyme came from studies of the effects of dimethyl sulphoxide which indicated that this co-solvent, which at low concentrations has no apparent effect on initial velocities under normal assay conditions, induced a slow conformational change in the enzyme. Thionine O, Nile Blue A and LY83583 were redox-cycling substrates producing superoxide ion, detectable by means of cytochrome c reduction, but leading to no loss of glutathione reductase activity, under aerobic or anaerobic conditions. The water-soluble Safranine analogues Methylene Blue, Methylene Green, Nile Blue A and Thionine O (5 mg/kg i.p. x 5) were effective antimalarial agents in vivo against P. berghei, but their effect was small and a higher dose (50mg/kg i.p. x 1) was toxic in mice. Comparison was made with human glutathione reductase and its literature-reported interactions with several tricyclic inhibitors as studied by X-ray diffraction. It is possible that the conformational changes detected in the present study from alterations in detailed kinetic inhibition mechanisms may shed light on information transfer through the glutathione reductase molecule from the dimer interface ligand pocket to the active-site.