Discovery of novel and potent benzhydryl-tropane trypanocides highly selective for Trypanosoma cruzi.

A benzhydryl tropinone oxime that is potently toxic to Trypanosoma cruzi has been previously identified. An SAR investigation determined that no part of the original compound was superfluous and all early SAR probes led to significant drops in activity. The only alteration that could be achieved without loss of activity was replacement of the aryl chloride substituent with chloro homologues. This led to the discovery of a trifluoromethyl-containing analogue with an EC(50) against T. cruzi of 30 nM and a cytotoxicity selectivity index of over 1000 relative to rat skeletal myoblast L-6 cells.

Substituted glycals as probes of glycosidase mechanisms.

D-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis beta-glucosidase in order to probe structure/function relationships in this enzyme. D-Glucal is shown to be a substrate (kcat = 2.3 min-1, Km = 0.85 mM) undergoing hydration with stereospecific protonation from the alpha-face to yield 2-deoxy-beta-D-glucose. 1-Methyl-D-glucal surprisingly serves as only a poor substrate (kcat = 0.056 min-1, Km = 57 mM), also undergoing protonation from the alpha-face. 2-Fluoro-D-glucal, however is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak (Ki = 24 mM) inhibitor. Similar behaviour was seen with almond beta-glucosidase and yeast alpha-glucosidase and for the interaction of 2-fluoro-D-galactal with Escherichia coli beta-galactosidase. A series of of alpha, beta-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis beta-glucosidase. Of these only 1-nitro-D-glucal functioned as a time dependent, irreversible inactivator (ki = 0.011 min-1, Ki = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed multiple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors (Ki = 3-96 mM).

Probing the ionization state of substrate alpha-D-glucopyranosyl phosphate bound to glycogen phosphorylase b.

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

A continuous fluorescence-based assay for the human high-molecular-weight cytosolic phospholipase A2.

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the purified recombinant cPLA2 was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA2 by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A2 and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A2 activity of cPLA2, also inhibited the 7-HCEase activity. A study of the 7-HCEase activity demonstrated that when 7-hydroxycoumarinyl gamma-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by cPLA2 at a rate comparable to that of an arachidonyl-containing phospholipid substrate and with an identical reaction progress curve. In the presence of phospholipid vesicles, the cPLA2-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA2-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA2 in the absence of calcium and other lipids.

Identification of Cys139 and Glu207 as catalytically important groups in the active site of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (EC 5.3.3.2) catalyzes the antarafacial [1.3] allylic rearrangement of isopentenyl diphosphate (IPP) to its electrophilic allylic isomer dimethylallyl diphosphate (DMAPP). Active-site thiols at C138 and C139 were recently identified by covalent modification using active-site-directed irreversible inhibitors [Street, I. P., & Poulter, C. D. (1990) Biochemistry 29, 7531-7538; Lu, X. J., Christensen, D. J., & Poulter, C. D. (1992) Biochemistry 31, 9955-9960]. Kinetic studies were conducted with site-directed mutants of IPP isomerase (IPPIase) to evaluate the roles of these amino acids. C138S and C138V mutants were active catalysts with V/K values only 10-fold lower than that of wild-type IPPIase. In contrast, the C139S mutant was a poor catalyst, and the C139A and C139V mutants were inactive. Treatment of the C139S mutant with 3-(fluoromethyl)-3-butenyl diphosphate, an electrophilic active-site-directed irreversible inhibitor, resulted in inactivation of the enzyme by covalent modification of E207. The E207Q and E207V mutants were inactive, suggesting a role for the E207 carboxylate moiety in catalysis.

NMR structural studies of the tight complex between a trifluoromethyl ketone inhibitor and the 85-kDa human phospholipase A2.

Arachidonyl trifluoromethyl ketone (AACOCF3) is a slow- and tight-binding inhibitor of the human cytosolic phospholipase A2 (cPLA2) [Street et al. (1993) Biochemistry 32, 5935]. 19F and 13C NMR experiments have been carried out to elucidate the structure of the cPLA2.AACOCF3 complex. One mole of AACOCF3 per mole of enzyme is tightly bound in the active site while excess molar equivalents of the inhibitor associate loosely and nonspecifically with hydrophobic regions of the protein. Incubation of the cPLA2.AACOCF3 complex with a 10-fold molar excess of a structurally related inhibitor allows the slow dissociation of the enzyme-inhibitor complex to be followed with 19F NMR. These results establish that the bound inhibitor is in slow exchange with the free ligand and that inhibition of the cPLA2 by AACOCF3 is not due to irreversible modification of the protein. AACOCF3 labeled with 13C at the carbonyl position was used to determine the nature of the bound inhibitor species. A comparison of the 13C NMR chemical shift value obtained from labeled enzyme-inhibitor complex (delta c 101.0 ppm) with the chemical shift values obtained from model compounds suggests that the enzyme-bound inhibitor species is a charged hemiketal. These results are very similar to those obtained previously with alpha-chymotrypsin and a peptidyl trifluoromethyl ketone inhibitor [Liang, T.-C., & Abeles, R. H. (1987) Biochemistry 26, 7603] and, by analogy with the serine proteases, a structural model for the cPLA2.AACOCF3 complex is proposed.

Human group II phospholipase A2 expressed in Trichoplusia ni larvae--isolation and kinetic properties of the enzyme.

Human secreted synovial fluid/platelet-type group II phospholipase A2 (sPLA2) was expressed in Trichoplusia ni (cabbage looper) larvae and cultured Sf9 insect cells by infection with a recombinant baculovirus. Active sPLA2, with correct N-terminal proteolytic processing, was not secreted by Sf9 cells in culture. The enzyme was isolated from their homogenate without any need for refolding or renaturation of the protein. The enzyme was extracted from the 5000g pellet with 1 M KBr and isolated by chromatography on a cation exchange column followed by reverse-phase chromatography on a Butyl Aquapore column. The yield of active enzyme (25 micrograms/g insect) was comparable to yields obtained in CHO cells or Escherichia coli by other investigators. The recombinant enzyme had the correct N-terminal sequence, expected molecular weight, and reacted with antisera raised against peptides inferred from the cDNA sequence of the natural enzyme. Monoclonal antibodies were raised against the recombinant sPLA2 and they permitted the isolation of the natural enzyme from human serum by immunoaffinity. The recombinant sPLA2 showed a preference for substrate vesicles with a net negative charge. The baculovirus expression system provided active sPLA2 that can be produced economically in insects, purified simply, had well-defined kinetic properties, and should be useful in studies of inflammatory disorders.

Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2.

A trifluoromethyl ketone analogue of arachidonic acid in which the COOH group is replaced with COCF3 (AACOCF3) was prepared and found to be a tight- and slow-binding inhibitor of the 85-kDa cytosolic human phospholipase A2 (cPLA2). Enzyme inhibition was observed when AACOCF3 was tested in assays using either phospholipid vesicles or phospholipid/Triton X-100 mixed micelles. The fact that the inhibition developed over several minutes in both assays establishes that AACOCF3 inhibits by direct binding to the enzyme rather than by decreasing the fraction of enzyme bound to the substrate interface. From the measured values of the inhibitor association and dissociation rate constants, an upper limit of the equilibrium dissociation constant for the Ca(2+).AACOCF3.PLA2 complex of 5 x 10(-5) mole fraction was obtained. Thus, detectable inhibition of cPLA2 by AACOCF3 occurs when this compound is present in the assay at a level of one inhibitor per several thousand substrates. Arachidonic acid analogues in which the COOH group is replaced by COCH3, CH(OH)CF3, CHO, or CONH2 did not detectably inhibit the cPLA2. The arachidonyl ketones AACOCF2CF3 and AACOCF2Cl were found by 19F NMR to be less hydrated than AACOCF3 in phospholipid/Triton X-100 mixed micelles, and compared to AACOCF3 these compounds are also weaker inhibitors of cPLA2. In keeping with the fact that cPLA2 displays substrate specificity for arachidonyl-containing phospholipids, the arachidic acid analogue C19H39COCF3 is a considerably less potent inhibitor compared to AACOCF3.(ABSTRACT TRUNCATED AT 250 WORDS)

Interfacial catalysis and production of a high ratio of leukotriene A4 to 5-HPETE by 5-lipoxygenase in a coupled assay with phospholipase A2.

The activity of purified 5-lipoxygenases (5-LO) shows a requirement for the presence of phosphatidylcholine or other lipids, in addition to Ca2+ and ATP. The enzymatic activity of purified human 5-lipoxygenase was dependent on the ratio of arachidonic acid to phospholipids rather than on the bulk arachidonic acid concentration, suggesting that the concentration of substrate at the lipid-water interface is important for the rate of the 5-LO reaction. Enzyme activity was also examined using vesicles of dimyristoylphosphatidylmethanol/1-palmitoyl-2-arachidonoylphosp hat idylcholine. Using PLA2 to release arachidonic acid from phospholipid, the ratio of leukotriene A4 (detected as trans-LTB4 isomers) to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) accumulated depended on the 5-LO concentration and was relatively independent of the amount of PLA2. The ratio of leukotriene A4 to 5-HPETE production increased with the amount of 5-LO (1-15 micrograms/ml) to reach values (> 10) similar to those observed with ionophore-challenged human leukocytes. The data are consistent with the catalysis of 5-LO occurring at the surface of phospholipid vesicles with the 5-HPETE product being re-utilized by the LTA4 synthase reaction of 5-lipoxygenase under conditions of limiting arachidonic acid availability.

Inactivation of a beta-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate: a detailed investigation.

The inactivation of glycosidases by 2-deoxy-2-fluoroglycosides has been shown previously to occur via the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucopyranosyl enzyme intermediate [Withers, S. G., & Street, I. P. (1988) J. Am. Chem. Soc. 110, 8551]. Further characterization of this process with Agrobacterium beta-glucosidase is described, and the range of glycosides engaging in this behavior is examined. Inactivation is shown to be accompanied by the release of a stoichiometric "burst" of aglycon, thereby providing a new class of active site titration agents for glycosidases. The rate of inactivation is shown to be very strongly dependent on the leaving group ability of the aglycon, the slowest inactivator studied (p-nitrophenyl2-deoxy-2-fluoro-beta-D-glucopyranoside) yielding only partial inactivation due to turnover of the intermediate becoming competitive with its formation. Such turnover of the intermediate is shown to be greatly accelerated by transglycosylation to a suitable glycoside bound in the aglycon site, resulting in the release of a disaccharide product which was isolated and characterized. The pH dependences of both the formation and the hydrolysis of the 2-deoxy-2-fluoroglycosyl-enzyme closely resemble those of each step for normal catalysis, indicating that the same catalytic groups are involved in both processes. A model system for the partial "steady-state" inactivation observed previously [Withers, S. G., Rupitz, K., & Street, I. P. (1988) J. Biol. Chem. 263, 7929] with certain other glycosidases was established by incubating the enzyme with an inactivator known to undergo relatively rapid transglycosylation in the presence of various concentrations of a suitable reactivator.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of enzymatic activity of phospholipases A2 by minocycline and doxycycline.

Extracellular phospholipases A2 play an important role in articular and extra-articular inflammatory processes. Secretory non-pancreatic phospholipase A2 (PLA2) has been implicated in the pathogenesis of articular inflammation in rheumatoid arthritis, whereas pancreatic PLA2 contributes to the tissue damage associated with acute pancreatitis. Since in experimental models lipophilic tetracyclines such as minocycline and doxycycline are antiinflammatory, we examined their effects on PLA2 activity using two assay systems in vitro. We found that minocycline and to a lesser degree doxycycline were markedly inhibitory to both pancreatic and non-pancreatic PLA2. Using [14C]oleic acid labeled Escherichia coli membrane phospholipids as substrate, the IC50 values for minocycline and doxycycline were 3.6 x 10(-5) M (18 micrograms/mL) and 0.98 x 10(-4) M (47 micrograms/mL), respectively. In a scooting mode assay using the synthetic phospholipid 1-palmitoyl-2-(10-pyrenedecanoyl)-3-L-phosphatidylmethanol as substrate, IC50 values for minocycline were 5 microM (2.47 micrograms/mL) for non-pancreatic PLA2 and 8 microM (3.95 micrograms/mL) for pancreatic PLA2. Addition of excess calcium up to 50 mM did not reverse the inhibitory activity of tetracyclines. We conclude that lipophilic tetracyclines inhibit PLA2, probably by interaction with the substrate, and may be a useful adjunct in the therapy of inflammatory conditions in which PLA2 is implicated pathogenetically.

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase: construction of a high-level heterologous expression system for the gene from Saccharomyces cerevisiae and identification of an active-site nucleophile.

Isopentenyldiphosphate:dimethylallyldiphosphate isomerase (IPP isomerase) is an enzyme in isoprene metabolism which catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks for the pathway. The gene encoding IPP isomerase has recently been isolated from Saccharomyces cerevisiae [Anderson, M. S., Muehlbacher, M., Street, I.P., Proffitt, J., & Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175]. A heterologous expression system was constructed for the gene and used to overexpress IPP isomerase in Escherichia coli. In transformants carrying the expression vector, IPP isomerase activity was increased by over 100,000-fold relative to that of the untransformed host strain. The overexpressed enzyme constitutes 30-35% of the total soluble cell protein and can be purified to homogeneity in two steps. Recombinant IPP isomerase was indistinguishable from that purified from yeast. 3-(Fluoromethyl)-3-butenyl diphosphate (FIPP) is a specific active-site-directed inhibitor of IPP isomerase from Claviceps purpurea [Muehlbacher, M., & Poulter, C. D. (1988) Biochemistry 27, 7315-7328]. Inactivation of yeast IPP isomerase by FIPP was active-site-directed, and inhibition resulted in formation of a stoichiometric enzyme-inhibitor complex. The site of covalent attachment in the enzyme-inhibitor complex was determined by inactivating IPP isomerase with [4-3H]FIPP, followed by digestion of the labeled enzyme with trypsin and purification of the resulting radioactive peptides by reversed-phase high-performance liquid chromatography. The primary site of attachment was Cys-139.

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase. An improved purification of the enzyme and isolation of the gene from Saccharomyces cerevisiae.

Isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase) is an enzyme in the isoprenoid biosynthetic pathway which catalyzes the interconversion of the primary five-carbon homoallylic and allylic diphosphate building blocks. We report a substantially improved procedure for purification of this enzyme from Saccharomyces cerevisiae. An amino-terminal sequence (35 amino acids) was obtained from a highly purified preparation of IPP isomerase. Oligonucleotide probes based on the protein sequence were used to isolate the structural gene encoding IPP isomerase from a yeast lambda library. The cloned gene encodes a 33,350-dalton polypeptide of 288 amino acids. A 3.5-kilobase EcoRI fragment containing the gene was subcloned into the yeast shuttle vector YRp17. Upon transformation with plasmids containing the insert, a 5-6-fold increase in IPP isomerase activity was seen in transformed cells relative to YRp17 controls, confirming the identity of the cloned gene. This is the first reported isolation of the gene for IPP isomerase.

Fluorinated and deoxygenated substrates as probes of transition-state structure in glycogen phosphorylase.

A series of deoxyfluoro- and deoxy-alpha-D-glucopyranosyl phosphates have been tested as substrates of rabbit muscle glycogen phosphorylase b. All are found to be utilized by the enzyme, but at substantially reduced rates. Values of Vm/Km for these analogues range from 10(2) to 10(5) times lower than that for the parent substrate. The large rate reductions are suggested to arise from a combination of intrinsic electronic effects and poorer binding of these substrates at the transition state. The data provide substantial evidence for an oxocarbonium-ion-like transition state. They also provide estimates of the strengths of hydrogen bonds to individual sugar hydroxyls at the transition state of the reaction. Further, comparison of such data with those obtained for glucose analogues binding as inhibitors to T-state phosphorylase suggests that these two glucose subsites are essentially identical; thus, the glucose pocket remains intact during the conformational transition associated with activation of the enzyme.

2-Deoxy-2-fluoro-D-glycosyl fluorides. A new class of specific mechanism-based glycosidase inhibitors.

Mechanism-based glycosidase inhibitors are of considerable use in studies of enzyme mechanism, in studies of glycoprotein processing, and possibly therapeutically in control of sugar uptake. This paper describes a new general approach to mechanism-based inactivation of glycosidases which involves trapping a covalent glycosyl enzyme intermediate. This is achieved by use of 2-deoxy-2-fluoro-D-glycosyl fluorides, for which the rate of hydrolysis of the fluoroglycosyl enzyme intermediate is extremely slow, resulting in accumulation of the intermediate. Eleven different glycosidases were tested with their corresponding 2-deoxy-2-fluoro-D-glycosyl fluorides. Eight of the eleven were inactivated, four of them according to pseudo first-order kinetics and four according to a more complex kinetic scheme. The specificity of these inhibitors was investigated by assaying for inhibition of one enzyme with four different 2-deoxy-2-fluoro-D-glycosyl fluorides. Large differences in inactivation rate were observed which paralleled previously observed substrate specificities.

Hydrogen bonding and specificity. Fluorodeoxy sugars as probes of hydrogen bonding in the glycogen phosphorylase-glucose complex.

The affinities of a large number of deoxy and fluorodeoxy sugars for the glucose binding site in glycogen phosphorylase have been measured, and polarities and relative strengths of the hydrogen bonds at each position have been predicted on the basis of these data. Comparison with the recently refined X-ray crystal structure of the phosphorylase-glucose complex shows a generally good correlation between predicted and observed bond strengths, vindicating this approach to the evaluation of hydrogen bonding. Estimates of the net contributions of hydrogen bonds of different types (neutral-neutral and neutral-charged) are essentially identical with those obtained by a complementary approach on the tyrosyl tRNA synthetase-substrate complex [Fersht, A. R., Shi, J. P., Knill-Jones, J., Lowe, D. M., Wilkinson, A. J., Blow, D. M., Brick, P., Cortes, P., Waye, M. M. Y., & Winter, G. (1985) Nature (London) 314, 235-238]. The carbohydrate binding site structure determined is compared with that recently determined for the arabinose binding protein.