ABSTRACT
Calcium-dependent protein kinases play a crucial role in intracellular calcium signaling in plants, some algae and protozoa. In Plasmodium falciparum, calcium-dependent protein kinase 1 (PfCDPK1) is expressed during schizogony in the erythrocytic stage as well as in the sporozoite stage. It is coexpressed with genes that encode the parasite motor complex, a cellular component required for parasite invasion of host cells, parasite motility and potentially cytokinesis. A targeted gene-disruption approach demonstrated that pfcdpk1 seems to be essential for parasite viability. An in vitro biochemical screen using recombinant PfCDPK1 against a library of 20,000 compounds resulted in the identification of a series of structurally related 2,6,9-trisubstituted purines. Compound treatment caused sudden developmental arrest at the late schizont stage in P. falciparum and a large reduction in intracellular parasites in Toxoplasma gondii, which suggests a possible role for PfCDPK1 in regulation of parasite motility during egress and invasion.
Subject(s)
Adenine/analogs & derivatives , Antimalarials/pharmacology , Cyclohexylamines/pharmacology , Gene Expression Regulation, Enzymologic/genetics , Malaria/parasitology , Plasmodium falciparum/enzymology , Protein Kinases/drug effects , Protein Kinases/genetics , Protozoan Proteins/antagonists & inhibitors , Adenine/chemistry , Adenine/pharmacology , Adenine/therapeutic use , Animals , Antimalarials/chemistry , Antimalarials/therapeutic use , CHO Cells , Cell Line , Cell Proliferation/drug effects , Cricetinae , Cricetulus , Cyclohexylamines/chemistry , Cyclohexylamines/therapeutic use , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , HeLa Cells , Humans , Life Cycle Stages/drug effects , Malaria/drug therapy , Malaria/immunology , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Molecular Weight , Movement/drug effects , Oligonucleotide Array Sequence Analysis/methods , Parasitic Sensitivity Tests , Plasmodium falciparum/growth & development , Protein Kinases/physiology , Protozoan Proteins/genetics , Protozoan Proteins/physiology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Small Molecule Libraries , Stereoisomerism , Structure-Activity Relationship , Tissue DistributionABSTRACT
The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.
Subject(s)
Cysteine/metabolism , Disulfides/metabolism , Ion Channel Gating/drug effects , Noxae/pharmacology , Transient Receptor Potential Channels/agonists , Transient Receptor Potential Channels/metabolism , Acrolein/analogs & derivatives , Acrolein/chemistry , Acrolein/metabolism , Acrolein/pharmacology , Animals , Cysteine/chemistry , Disulfides/chemistry , Dithiothreitol/pharmacology , Electric Conductivity , Ethyl Methanesulfonate/analogs & derivatives , Ethyl Methanesulfonate/chemistry , Ethyl Methanesulfonate/metabolism , Ethyl Methanesulfonate/pharmacology , Humans , Mice , Mustard Plant/chemistry , Mustard Plant/metabolism , Noxae/chemistry , Noxae/metabolism , Pain/chemically induced , Pain/physiopathology , Plant Oils/chemistry , Plant Oils/metabolism , Plant Oils/pharmacology , Transient Receptor Potential Channels/chemistryABSTRACT
[reaction: see text] (S)-S-(2-Cyclohexenyl) N,N-diisopropylmonothiocarbamate [(-)-(S)-8] was deprotonated by sec-butyllithium/TMEDA to form a configurationally stable lithium compound (S)-9, which is the first example of a new class of alpha-thio-substituted organolithium compounds with improved properties. It is regioselectively alkylated by alkyl halides with complete stereoinversion to form the monothiocarbamates (+)-10 which afford highly enantioenriched tertiary 2-cyclohexene-1-thiols (+)-6 on reductive cleavage.
