Joan MacKinnon Mansour

Some phosphonic acid analogs as inhibitors of pyrophosphate-dependent phosphofructokinase, a novel target in Toxoplasma gondii

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was identified previously in Toxoplasma gondii as the only kinase that phosphorylates fructose-6-P to fructose-1,6-bisP. Since such an enzyme is not present in mammals, it was considered to be a good target for prospective selective inhibitors of the parasite. We have examined the effects of several phosphonic acid derivatives, analogs of pyrophosphate, on PPi-PFK activity, as well as on the replication of T. gondii in human foreskin fibroblast (HFF) cells. The most active compound in inhibiting PPi-PFK was tetrasodium carbonyldiphosphonate. Several bisphosphonates and related arylhydrazones showed inhibition of the enzyme, but with higher IC50 values. Although several phosphonoacetic acid derivatives also inhibited PPi-PFK, as a group they were less potent than the bisphosphonate derivatives. Comparison among the structures of various inhibitors and their effects against PPi-PFK indicates that a carbonyl (C=O) or amino (C=N) group between two phosphoryl moieties is associated with more potent enzyme inhibiton. Tetrasodium carbonyldiphosphonate did not show a significant effect against replication of T. gondii cells, probably because, as a charged molecule, it could not cross the cell membrane to reach the intracellular parasite. Tetraisopropyl carbonyldiphosphonate 2,4-dinitrophenylhydrazone showed some selective inhibitory effect against replication of the parasite in the HFF cells and protected the mammalian cells from damage by T. gondii. The results indicate that carbonyldiphosphonic acid is a good prototype compound that is amenable to chemical manipulation, which, in turn, may optimize selective inhibition of T. gondii PPi-PFK and increase accessibility to the intracellular parasite.

The dual effects of aluminum as activator and inhibitor of adenylate cyclase in the liver fluke Fasciola hepatica.

The liver fluke, Fasciola hepatica, has a very active adenylate cyclase which can be stimulated by NaF or by serotonin and guanine nucleotides. Micromolar amounts of AlCl3 augment the activation by F-. In contrast, when the enzyme is activated with serotonin and guanine nucleotides, AlCl3 inhibits the activation. Aluminum also inhibits the activation by forskolin. Gallium mimics the effects of aluminum.

Chemotherapeutic targets in parasites: contemporary strategies.

Parasitic infections are the most prevalent of human diseases. Parasites’ effective evasion of their hosts’ immune defenses and their complex physiology and life cycles make them especially resistant to attack by chemotherapeutic agents. Researchers continue to face the challenge of designing drugs to successfully counteract them. Chemotherapeutic Targets in Parasites analyzes the critical metabolic reactions and structural features essential for parasite survival and advocates the latest molecular and biochemical strategies with which to identify effective antiparasitic agents. An introduction to the early development of parasite chemotherapy is followed by an overview of biophysical techniques and genomic and proteomic analyses. Several chapters are devoted to specific types of chemotherapeutic agents and their targets in malaria, trypanosomes, leishmania, and amitochondrial protists. Chapters on helminths include metabolic, neuromuscular, microtubular, and tegumental targets. Emphasized throughout is the design of drugs that are more selective and less toxic than those used in the past. A comprehensive discussion of selective targets in parasites for new drugs is long overdue. This up-to-date book will be especially relevant to medical and clinical researchers and to graduate students in parasitology, pharmacology, medicine, microbiology, and biochemistry.

Phosphodiesterase in the liver fluke, fasciola hepatica

Abstract Phosphodiesterase was found in homogenates of the liver fluke, Fasciola hepatica , and was distributed between a supernatant and particulate fraction after centrifugation at 2000 g . Mg 2+ was necessary for enzyme activity; Ca 2+ in the presence of Mg 2+ did not affect enzyme activity. Enzyme kinetics followed the Michaelis-Menten model with a K m of 8 μM for cAMP and 300 μM for cGMP as the substrate. The most potent inhibitor tested was 1-ethyl-4-(isopropylidenehydrazino)-1 H -pyrazolo- (3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HC1 (SQ 20009) which had a K i of 26 μM. The K i for isobutyl methyl xanthine (IBMX) was 45 μM; for 6,7 dimethyl-4 ethylquinazoline (Quazodine) 75 μM; papaverine. 100 μM; theophylline, 550 μM; and for caffeine or D -lysergic acid diethylamide (LSD), 800 μM. The effects on fluke motility of these phosphodiesterase inhibitors were tested. All phosphodiesterase inhibitors except caffeine stimulated the rhythmical movement of the flukes. None of the inhibitors tested significantly increased the endogenous cAMP concentrations of fluke heads. IBMX potentiated the rise in endogenous cAMP caused by 5-hydroxytryptamine (5-HT) but SQ 20009, LSD, and papaverine prevented it. The latter results could not be explained on the basis of phosphodies-terase inhibition, but might be attributed to interference with the stimulation of adenylate cyclase by 5-HT.

GTP-binding proteins associated with serotonin-activated adenylate cyclase in Fasciola hepatica

The liver fluke Fasciola hepatica has serotonin (5-hydroxytryptamine) receptors that function through a transmembrane signalling system requiring GTP which activates adenylate cyclase (ATP pyrophosphate-lyase (cyclising), EC 4.6.1.1). Non-hydrolysable GTP analogs and NaF activate adenylate cyclase in membrane particles of these organisms. The nature of GTP-binding proteins in these membranes was studied using bacterial toxins and photoaffinity labelling. Treatment of membrane particles from flukes with cholera toxin increased basal adenylate cyclase activity, but markedly decreased activation by serotonin, non-hydrolysable GTP analogs, and NaF. [32P]ADP-ribosylation by cholera toxin or photoaffinity labelling with [32P]-8-N3GTP identified a 53 kDa protein and a 45 kDa protein which appeared to be similar to the forms of the alpha-subunit of the GTP-binding protein associated with adenylate cyclase in mammals. Treatment of membrane particles by pertussis toxin did not significantly change basal adenylate cyclase activity and did not change the stimulation of cyclase by activators. A 43 kDa protein which was [32P]ADP-ribosylated by either cholera or pertussis toxin, depending on the conditions used, and photoaffinity labelled by [32P]-8-N3GTP may be part of the transmembrane signalling system in the liver flukes.

Identification of GTP-binding proteins in Fasciola hepatica and Schistosoma mansoni by immunoblotting

Both the liver fluke Fasciola hepatica and the blood fluke Schistosoma mansoni have GTP-binding proteins which are part of the trans-membrane signalling system. These proteins require GTP in order to interact with the catalytic subunit of adenylate cyclase. The technique of immunoblotting was used in order to distinguish the GTP-binding proteins Gs, Gi, and Go. Immunoblotting was carried out using antisera prepared against peptides deduced from bovine cDNA clones specific for alpha or beta subunits of known G proteins. A 41-kDa Gs alpha has been identified in S. mansoni and a 42.5-kDa Gs alpha in F. hepatica. A 41-kDa Go alpha was found in both parasites. A 36-kDa G beta was identified in both parasites using antiserum made against bovine transducin.

Purification, kinetics and inhibition by antimonials of recombinant phosphofructokinase from Schistosoma mansoni

We reported before on the cloning of a cDNA encoding S. mansoni PFK. In the present investigation we established optimal conditions for expression of the enzyme in insect cells with high yield. The recombinant PFK was purified to homogeneity. Kinetic properties of the pure enzyme were studied with respect to its two substrates, Fru-6-P and ATP, and were compared with properties of mammalian PFK. ATP inhibited the parasite enzyme only at concentrations higher than those which inhibited mammalian muscle PFK. Saturation curves for Fru-6-P showed typical cooperative kinetics. AMP, cAMP and Fru-2,6-bisP activated the enzyme causing reduced apparent Km for Fru-6-P and an increase in maximal activity. Both ATP inhibition and cooperative kinetics for Fru-6-P occur at both pH 6.9 and 8.2. This is a distinct difference from the mammalian enzyme which shows these kinetic properties only at neutral or slightly acidic pH, but not at an alkaline pH. Recombinant PFK is more sensitive to inhibition by the trivalent antimonials, antimony potassium tartrate and Stibophen, than is the mammalian heart muscle enzyme. The inhibition is at least partially antagonized by the sulfhydryl protective reagent, dithiothreitol.

Fluoride and guanosine nucleotide activated adenylate cyclase from Fasciolahepatica: Reconstitution after inactivation

Abstract Detergent solubilized extracts from the liver fluke, Fasciola hepatica , contain adenylate cyclase activity which can be persistently activated by NaF or guanosine-5′-(3-0-thio) triphosphate. Treatment with heat or N-ethylmaleimide inactivated the cyclase. Activity can be partially reconstituted by mixing inactivated preparations with solubilized enzyme that has not been activated. The results provide a means to assay for adenylate cyclase components that are necessary for activation by NaF or the guanine nucleotides and may help in further resolution and purification of the components necessary for cyclase activity.

Effect of some phosphodiesterase inhibitors on adenylate cyclase from the liver fluke, Fasciola hepatica

Abstract The present investigation was prompted by our previous finding that in the liver fluke, Fasciola hepatica , some phosphodiesterase inhibitors, instead of potentiating the rise in endogenous cAMP caused by 5-hydroxytryptamine (5-HT), antagonized it. Papaverine, 1-ethyl-4-(isopropylidenehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HCl (SQ 20009), 6,7-dimethyl-4 ethyl-quinazoline (Quazodine) and caffeine inhibited 5-HT-activated adenylate cyclase from particulate fractions of the liver fluke. This may explain their in vivo antagonism to the 5-HT-mediated rise in endogenous cAMP levels. Isobutylmethylxanthine (IBMX), which did not antagonize the 5-HT effect in vivo , did not inhibit 5-HT-activated adenylate cyclase in fluke particles. None of the above compounds inhibited the NaF-activated adenylate cyclase. Kinetic studies showed that inhibition of 5-HT-activated adenylate cyclase by papaverine or SQ 20009 was not competitive with the substrate, ATP, or with GTP. While high levels of 5-HT decreased the degree of inhibition by papaverin and SQ 20009. the kinetics of inhibition does not appear to be strictly competitive.

Chemotherapeutic Targets in Parasites: Contents

Parasitic infections are the most prevalent of human diseases. Parasites’ effective evasion of their hosts’ immune defenses and their complex physiology and life cycles make them especially resistant to attack by chemotherapeutic agents. Researchers continue to face the challenge of designing drugs to successfully counteract them. Chemotherapeutic Targets in Parasites analyzes the critical metabolic reactions and structural features essential for parasite survival and advocates the latest molecular and biochemical strategies with which to identify effective antiparasitic agents. An introduction to the early development of parasite chemotherapy is followed by an overview of biophysical techniques and genomic and proteomic analyses. Several chapters are devoted to specific types of chemotherapeutic agents and their targets in malaria, trypanosomes, leishmania, and amitochondrial protists. Chapters on helminths include metabolic, neuromuscular, microtubular, and tegumental targets. Emphasized throughout is the design of drugs that are more selective and less toxic than those used in the past. A comprehensive discussion of selective targets in parasites for new drugs is long overdue. This up-to-date book will be especially relevant to medical and clinical researchers and to graduate students in parasitology, pharmacology, medicine, microbiology, and biochemistry.