4-Arylhydrazinylidene-5-(polyfluoroalkyl)pyrazol-3-ones (4-AHPs) were found to be obtained by the regiospecific cyclization of 2-arylhydrazinylidene-3-(polyfluoroalkyl)-3-oxoesters with hydrazines, ...by the azo coupling of 4-nonsubstituted pyrazol-5-oles with aryldiazonium chlorides or by the firstly discovered acid-promoted self-condensation of 2-arylhydrazinylidene-3-oxoesters. All the 4-AHPs had an acceptable ADME profile. Varying the substituents in 4-AHPs promoted the switching or combining of their biological activity. The polyfluoroalkyl residue in 4-AHPs led to the appearance of an anticarboxylesterase action in the micromolar range. An NH-fragment and/or methyl group instead of the polyfluoroalkyl one in the 4-AHPs promoted antioxidant properties in the ABTS, FRAP and ORAC tests, as well as anti-cancer activity against HeLa that was at the Doxorubicin level coupled with lower cytotoxicity against normal human fibroblasts. Some Ph-N-substituted 4-AHPs could inhibit the growth of
bacteria at MIC 0.9 μg/mL. The possibility of using 4-AHPs for cell visualization was shown. Most of the 4-AHPs exhibited a pronounced analgesic effect in a hot plate test in vivo at and above the diclofenac and metamizole levels except for the ones with two chlorine atoms in the aryl group. The methylsulfonyl residue was proved to raise the anti-inflammatory effect also. A mechanism of the antinociceptive action of the 4-AHPs through blocking the TRPV1 receptor was proposed and confirmed using in vitro experiment and molecular docking.
CO2 inhalation is currently the most common method of euthanasia for laboratory rats and mice, and it is often used for further terminal blood sampling for clinical biochemical assays. Lately, this ...method has been criticized due to animal welfare issues associated with some processes that develop after CO2 inhalation. The stress reaction and the value of the clinical laboratory parameters significantly depend on the used anesthetics, method, and the site of blood sampling. Especially in small rodents, an acute terminal state followed by a cascade of metabolic reactions that can affect the studied biochemical profile may develop and cause unnecessary suffering of animals. The aim of this study was to compare the stability of biochemical parameters of outbred Sprague Dawley rats and CD-1 mice serum collected after CO2 inhalation or the intramuscular injection of tiletamine–zolazepam–xylazine (TZX). The serum content of total protein and albumin, cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotr ansferase (ALT), alkaline phosphatase (ALP), total bilirubin, and creatinine was decreased by the injection of TZX in comparison with CO2 inhalation. In addition, the levels of calcium, phosphates, chlorides and potassium were lowered by TZX vs. CO2 administration, while the level of sodium increased. Finally, the level of the majority of serum clinical biochemical parameters in rats and mice tend to be overestimated after CO2 inhalation, which may lead to masking the possible effect of anti-inflammatory drugs in animal tests. Injection anesthesia for small rodents with TZX is a more feasible method for terminal blood sampling, which also reduces the suffering of animals.
Chemoselective O‐alkylation of 1‐aryl‐3‐polyfluoroalkylpyrazol‐5‐oles under basic conditions resulted in a series of 5‐alkoxypyrazoles (26 derivatives). They showed an acceptable ADME profile (in ...silico) and can be considered as drug‐like. In experiments in vivo (CD‐1 mice), it was found that the obtained compounds do not have toxic properties at a dose of more than 150 mg/kg (for most compounds at a dose of >300 mg/kg, and for lead compounds – >600 mg/kg). 22 Compounds from this series demonstrated from moderate to high analgesic effects (28–104 % at 1 h and 37–109 % at 2 h after administration) in vivo in the hot plate test (SD rats, 15 mg/kg, intraperitoneal (ip)). The lead compound was 4‐(1‐phenyl‐3‐(trifluoromethyl)pyrazol‐5‐yloxy)butan‐1‐ol, which not only increased the latent period in the hot plate test by 103 % at both measurement points but also showed a pronounced analgesic effect under conditions of capsaicin‐induced nociception (CD‐1 mice, 15 mg/kg, ip). According to molecular modeling, all synthesized compounds can interact with the TRPV1 ion channel. This biological target was confirmed in in vitro experiments on Chinese hamster ovary cells expressing rTRPV1. 5‐Alkoxypyrazoles were partial agonists of the TRPV1 ion channel in various degree, and the most active was the same pyrazole as in in vivo tests.
Chemoselective O‐alkylation of 1‐aryl‐3‐polyfluoroalkylpyrazol‐5‐oles under basic conditions results in a series of 5‐alkoxypyrazoles (26 derivatives) having promising antinociceptive activity. They are partial agonists of the TRPV1 ion channel in various degrees. The most active compound is 4‐(1‐phenyl‐3‐(trifluoromethyl)pyrazol‐5‐yloxy)butan‐1‐ol.
Previous studies have shown that the unusually long S5-P linker lining
human ether a-go-go related gene’s (hERG’s) outer vestibule is critical for its channel function: point mutations at high-impact ...positions here can interfere with the inactivation process and, in many cases, also reduce the pore’s K
+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG’s outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG’s pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG’s outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore.
Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for ...efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K
+
-conducting pore. It is based on
Escherichia coli
spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch–clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the “mix and read” mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.
Figure
The scheme of a fluorescent analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K
+
-conducting pore.
Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full ...amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a-c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.
P2X3 purinoreceptors expressed in mammalian sensory neurons play a key role in several processes, including pain perception. From the venom of the Central Asian spider Geolycosa sp., we have isolated ...a novel peptide, named purotoxin‐1 (PT1), which is to our knowledge the first natural molecule exerting powerful and selective inhibitory action on P2X3 receptors. PT1 dramatically slows down the removal of desensitization of these receptors. The peptide demonstrates potent antinociceptive properties in animal models of inflammatory pain. ANN NEUROL 2010;67:680–683
A new marine guanidine-containing compound, pulchranin A (1) was isolated from the marine sponge Monanchora pulchra. Its structure was elucidated by detailed NMR and MS analyses. The Mosher’s method ...did not allow determination of the absolute configuration of the carbinol center in 1, but after conversion of 1 into the bis-MTPA esters of 1,3-diol 2, the same method was successfully used to solve the problem. Compound 1 was active as an inhibitor of the TRPV1 receptor with an EC50 value of 41.2μM.
Modulation of P2X3 receptors by spider toxins Kabanova, Natalia V.; Vassilevski, Alexander A.; Rogachevskaja, Olga A. ...
Biochimica et biophysica acta,
November 2012, 2012-Nov, 2012-11-00, Letnik:
1818, Številka:
11
Journal Article
Recenzirano
Odprti dostop
Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. ...Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3–4-fold decrease in the IC50 dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.
The spider toxin PT1 enhances the high affinity desensitization of the mouse P2X3 receptor. (A) Time evolution of mP2X3 currents in control (o), in the presence of 50 nM PT1 (●), and in the presence 40 nM CTP and 50 nM PT1 (▲). (B) mP2X3 current elicited by 100 μM CTP versus ambient CTP concentration in control (▲) and in the presence of 50 nM PT1 (Δ). Display omitted
► Peptides, purotoxin-1 and purotoxin-2, were isolated from the venom of the spider Geolycosa sp. ► The sensitivity of recombinant P2X3 receptors to the spider toxins was studied. ► Purotoxin-1 and purotoxin-2 slowed down inactivation of P2X3 currents. ► The spider toxins shifted an inhibitory curve for P2X3 high-affinity desensitization.
The ether-Ã -go-go -related gene (erg) K + channels are known to be crucial for life in Caenorhabditis elegans (mating), Drosophila melanogaster (seizure), and humans (LQT syndrome). The erg genes ...known to date ( erg1 , erg2 , and erg3 ) are highly expressed in various areas of the rat and mouse central nervous system (CNS), and ERG channel blockers alter
firing accommodation. To assign physiological roles to each isoform, it is necessary to design pharmacological strategies
to distinguish individual currents. To this purpose, we have investigated the blocking properties of specific peptide inhibitors
of hERG1 channels on the human and rat isoforms. In particular, we have tested ErgTx1 (from the scorpion Centruroides noxious ), BeKm-1 (from the scorpion Buthus eupeus ), and APETx1 (from the sea anemone Anthopleura elegantissima ). Because these peptides had different species-specific effects on the six different channels, we have also carried out a
biophysical characterization of hERG2 and hERG3 channels that turned out to be different from the rat homologs. It emerged
that APETx1 is exquisitely selective for ERG1 and does not compete with the other two toxins. BeKm-1 discriminates well among
the three rat members. ErgTx1 is unable to block hERG2, but blocks rERG2 and has the lowest K D for hERG3. BeKm-1 and ErgTx1 compete for hERG3 but not for rERG2 blockade. Our findings should be helpful for structure-function
studies and for novel CNS ERG-specific drug design.
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