ABSTRACT
FMRFamide-related peptides are widespread among the Nematoda. Among them is a family of extended PNFLRFamide peptides encoded on the flp-1 peptide precursor gene in Caenorhabditis elegans. The most studied peptide from this series is SDPNFLRFamide (PF1). Each residue in this peptide was sequentially substituted with either alanine or the corresponding d-isomer of the native amino acid in order to define structure-function relationships in this peptide using an Ascaris suum muscle tension assay. In general, substitutions in the N-terminal tetrapeptide had only minor consequences for efficacy, while substitutions in the C-terminal tetrapeptide caused more dramatic changes. Such substitutions typically markedly diminished efficacy, but d-isomer substitution at either position 5 (Phe) or 6 (Leu) converted the inhibitory activity of the prototype into excitation. In addition, it has been evident that KPNFLRFamide and SDPNFLRFamide, though encoded on flp-1 and sharing a PNFLRFamide hexapeptide, act through different receptors. KPNFLRFamide directly gates a chloride channel in A. suum muscle cells, while SDPNFLRFamide acts through nitric oxide synthase to open K+ channels in the same tissue. The use of K+ channel blockers and nitric oxide synthase inhibitors in electrophysiological experiments employing A. suum muscle membranes allowed the unambiguous conclusion that the N-terminal lysine is absolutely required for activation of the chloride channel and excludes interaction with the SDPNFLRFamide receptor.
Subject(s)
Ascaris suum/drug effects , FMRFamide/pharmacology , Helminth Proteins/pharmacology , Animals , Ascaris suum/physiology , Chloride Channels/metabolism , FMRFamide/chemistry , Helminth Proteins/chemistry , Ion Channel Gating/physiology , Membrane Potentials/drug effects , Muscle Contraction/drug effects , Muscle, Skeletal/drug effects , Peptide Fragments/pharmacology , Potassium Channels/metabolism , Structure-Activity RelationshipABSTRACT
Oxindole alkaloids in the paraherquamide/marcfortine family exhibit broad-spectrum anthelmintic activity that includes drug-resistant strains of nematodes. Paraherquamide (PHQ), 2-deoxoparaherquamide (2DPHQ), and close structural analogs of these compounds rapidly induce flaccid paralysis in parasitic nematodes in vitro, without affecting adenosine triphosphate (ATP) levels. The mechanism of action of this anthelmintic class was investigated using muscle tension and microelectrode recording techniques in isolated body wall segments of Ascaris suum. None of the compounds altered A. suum muscle tension or membrane potential. However, PHQ blocked (when applied before) or reversed (when applied after) depolarizing contractions induced by acetylcholine (ACh) and the nicotinic agonists levamisole and morantel. These effects were mimicked by the nicotinic ganglionic blocker mecamylamine, suggesting that the anthelmintic activity of PHQ and marcfortines is due to blockade of cholinergic neuromuscular transmission. The effects of these compounds were also examined on subtypes of human nicotinic ACh receptors expressed in mammalian cells with a Ca2+ flux assay. 2DPHQ blocked nicotinic stimulation of cells expressing alpha3 ganglionic (IC50 approximately 9 microm) and muscle-type (IC50 approximately 3 microm) nicotinic cholinergic receptors, but was inactive at 100 microm vs. the alpha7 CNS subtype. PHQ anthelmintics are nicotinic cholinergic antagonists in both nematodes and mammals, and this mechanism appears to underlie both their efficacy and toxicity.
Subject(s)
Anthelmintics , Cholinergic Antagonists , Indolizines , Receptors, Cholinergic/drug effects , Spiro Compounds , Animals , Anthelmintics/chemistry , Anthelmintics/toxicity , Ascaris suum , Cholinergic Antagonists/chemistry , Cholinergic Antagonists/toxicity , Female , Indolizines/chemistry , Indolizines/toxicity , Membrane Potentials/drug effects , Muscles/drug effects , Nematoda , Spiro Compounds/chemistry , Spiro Compounds/toxicity , Structure-Activity RelationshipABSTRACT
Three distinct chemical classes for the control of gastrointestinal nematodes are available: benzimidazoles, imidazothiazoles, and macrocyclic lactones. The relentless development of drug resistance has severely limited the usefulness of such drugs and the search for a new class of compounds preferably with a different mode of action is an important endeavor. Marcfortine A (1), a metabolite of Penicillium roqueforti, is structurally related to paraherquamide A (2), originally isolated from Penicillium paraherquei. Chemically the two compounds differ only in one ring; in marcfortine A, ring G is six-membered and carries no substituents, while in paraherquamide A, ring G is five-membered with methyl and hydroxyl substituents at C14. Paraherquamide A (2) is superior to marcfortine A as a nematocide. 2-Desoxoparaherquamide A (PNU-141962, 53) has excellent nematocidal activity, a superior safely profile, and is the first semi-synthetic member of this totally new class of nematocides that is a legitimate candidate for development. This review describes the chemistry, efficacy and mode of action of PNU-141962.
