Inhibition of metalloprotease botulinum serotype A from a pseudo-peptide binding mode to a small molecule that is active in primary neurons.

An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (K(i) = 330 nM) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institute's Open Repository. Four of the inhibitors possess K(i) values ranging from 3.0 to 10.0 microM. Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomal-associated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 microM.

Brain acetylcholinesterase activity in botulism-intoxicated mallards.

Brain acetylcholinesterase (AChE) activity in captive-reared mallards (Anas platyrhynchos) that died of botulism was compared with euthanized controls. AChE levels for both groups were within the range reported for normal mallards, and there was no significant difference in mean AChE activity between birds that ingested botulism toxin and died and those that did not.

[State of coagulation hemostasis and fibrinolysis processes in dynamics of rapidly progressing forms of botulinal intoxication]. / Sostoianie protsessov koaguliatsionnogo gemostaza i fibrinoliza v dinamike bystroprogressiruiushchei formy botulinicheskoi intoksikatsii.

Phase disturbances of coagulation blood potential were revealed in experiments on white rats in dynamics of rapidly progressing form of botulinic intoxication, intoxication being caused by intraperitoneal injection of type C toxin. In preclinical period of intoxication activation of procoagulant and anticoagulant parts of hemostasis system, as well as fibrinolysis system, was noted. Similar shifts were revealed in the developmental period of generalized pareses of skeletal musculature. Only in the terminal stage of intoxication insufficiency of mechanism in formation of prothrombinase activity developed by simultaneous activation of anticoagulant mechanism and fibrinolysis system.

[Possibilities of pharmacologic correction of Na, K-ATPase activity in the spinal cord in botulism]. / O vozmozhnostiakh farmakologicheskoi korrektsii aktivnosti Na, K-ATF-azy spinnogo mozga v dinamike botulinicheskoi intoksikatsii.

The experiments on white rats have shown that gutimin is capable of reactivating Na, K-ATPase of the synaptosomes of the jugular spinal cord in type C botulinic intoxication. Serotonin prevented Na, K-ATPase activity inhibition only in preclinical period of intoxication. Parmidin injection did not prevent suppression of Na, K-ATPase activity either in preclinical period or in skeletal muscle paresis.

[Changes in Na, K-ATPase activity in subcellular fractions of the lumbar region of the spinal cord in botulism and the possibility of their pharmacological correction]. / Izmeneniia aktivnosti Na, K-ATFazy subkletochnykh fraktsii poiasnichnogo otdela spinnogo mozga v dinamike botulinicheskoi intoksikatsii i vozmozhnosti ikh farmakologicheskoi korrektsii.

Inhibition of Na, K-ATPase in fractions of unpurified synaptosomes and of heavy microsomes from lumbar part of spinal cord was found in dynamics of botulinic intoxication of the C type in rats. Both competitive and noncompetitive types of inhibition of the enzymatic activity were found in kinetic studies. Inhibition of transport functions across biological membranes caused by botulinic toxin was shown to have a reversible character. The inhibition might be prevented within the early periods of botulinic intoxication by administration of serotonin and guanidine hydrochloride.

[Effect of botulinum toxin on the activity of transport ATPases in biological membranes]. / O vliianii botulinicheskogo toksina na aktivnost' transportnykh ATFaz biologicheskikh membran.

Activity of transport ATPases was studied in erythrocyte membranes and synaptosomal fraction of cervical department of spinal cord obtained from rats in dynamics of botulinic C intoxication Na+, K+-ATPase was inhibited by the competitive type in the synaptosomal brain fraction at the preclinical period of intoxication and by the noncompetitive type at the step of skeletal muscle paresis. In erythrocyte membranes activity of Na+, K+-ATPase was inhibited by the mixed type at the preclinical period of intoxication and the enzymatic activity was inhibited by the noncompetitive type at the step of skeletal muscle paresis. The Na+, K+-ATPase from biological membranes was reactivated by unithiol and nicotinamide in dynamics of intoxication. The toxin was shown to activate Mg2+-ATPase in brain synaptosomal fraction.

[Morphological and histochemical reactions of an organism to the administration of Clostridium botulinum toxin. IX. Reactions of the neurons of the abducens nerve nucleus and of the Darkshevich's nucleus to the administration of C. botulinum type B toxin]. / Morfologicheskaia i gistokhimicheskaia kharakteristika reaktsii organizma na vvedenie toksina Clostridium botulinum. Soobshchenie IX. Reaktsiia neironov iadra otvodiashchego nerva i iadra Darkshevicha na vvedenie toksina C. botulinum tipa B.

Pathomorphological and histochemical study carried out in guinea pigs at the stage of paralysis of the limbs after the oral administration of C. botulinum toxin has revealed the presence of morpho-metabolic changes pertaining to phase 2 of the development of dystrophic processes in the neurons of the nucleus of the abducent nerve and Darkshevich's nucleus. At the myasthenic stage the development of dystrophic processes in the neurons of Darkshevich's nucleus has been found to reach phase 3. The study indicates that C. botulinum toxin possesses different tropism in respect of the neurons of the oculomotor nuclei; this fact correlates with the clinical picture of botulism.

[Alterations in activity of the ATPase systems in subcellular fractions of brain and spinal cord in the preclinical period of botulinum poisoning]. / K voprosu ob izmenenii aktivnosti ATF-aznykh sistem subkletochnykh fraktsii golovnogo i spinnogo mozga v doklinicheskii period botulinicheskoi intoksikatsii.

Under conditions of the C type botulinic intoxication of rats blockade of cholinergic synaptic transmission and development of paralytic syndrome followed the distinct alteration in activity of the ATPase systems in various subcellular fractions of brain and spinal cord. This suggests that the toxin affects the various intracellular metabolic reactions related directly or indirectly to active transmembrane transport of ions.

[Activity of erythrocyte membrane Na,K-ATPase in rats with experimental botulism]. / Sostoianie aktivnosti Na, K - ATF-azy membran éritrotsitov krys pri botulinicheskoi intoksikatsii.

The effect of type C botulinum toxin on Na, K, Mg-ATPase activities of erythrocyte membranes of white rats was studied in experiments in vivo and in vitro. The activity of Na, K, Mg-ATPase was found to be markedly inhibited in the preclinical period of poisoning, 2 hours after intraperitoneal injection of the toxin. In this case Mg-ATPase activity noticeably increased. In the presence of the development of a grave paralytic syndrome one day after intraperitoneal injection of the toxin, the activity of Na, K-ATPase of the erythrocyte membrane remained decreased as was the case in the preclinical period of poisoning, whereas the activity of Mg-ATPase returned to normal. The experiments in vitro with preincubation of erythrocyte membranes with botulinum toxin in the concentrations corresponding to the mean calculated ones in the experiments in vivo demonstrated inhibition of Na, K-ATPase. The magnitude of Mg-ATPase activity remained virtually unchanged in all the modifications of the experiments with boiled and native botulinum toxin. The in-vivo experiments with intraperitoneal injection of glutathione and unithiol to the pretreated animals attested to normalization of Na, K-ATPase in the preclinical period of poisoning, with this normalization being brought about by unithiol. In the in-vitro experiments with addition of unithiol or glutathione into the incubation medium, each of the donators of sulphhydryl groups prevented Na, K-ATPase inhibition with botulinum toxin.

[Changes in the acetylcholine--cholinesterase system in the dynamics of experimental boltulin poisoning]. / Izmenenie sistemy atsetilkholin--kholinésteraza v dinamike éksperimental'noi botulinicheskoi intoksikatsii

A study was made of the effect of botulin, type B, on the acetylcholine-cholinesterase system. High doses of the toxin increased the level of acetylcholine in the peripheral blood nervous system, as well as in the central nervous system. Injection of DLM of the toxin results in elevation of the acetylcholine level in the peripheral nervous system. Cholinesterase activity was practically unchanged.

[Morphological and histochemical characteristics of the reaction of the body to Cl. botulinum toxin administration. III. The cellular reaction of the diaphragmatic nerve nucleus to the administration of Cl. botulinum type B toxin]. / Morfologicheskaia i gistokhimicheskaia kharakteristika reaktsii organizma na vvedenie toksina C. botulinum. Soobshchenie III. Reaktsiia kletok iadra diafragmal'nogo nerva na vvedenie toksina Cl. botulinum tipa B

On the appearance in the animals (guinea pigs) of paralysis of the limbs and myasthenia after the administration of Cl. botulinum, type B, toxin, there was seen a considerable vascular hyperemia of the spinal cord, and in the neurons of the phrenic nerve nucleus there developed dystrophic-necrotic processes coursing with a marked swelling, hyperchromasia and tigrolysis. As revealed histochemically, at this stage of the botulin intoxication the neurons of the phrenic nerve nucleus displayed metabolic changes expressed in the altered activity of succinic dehydrogenase, acid phosphatase and cholinesterase.

Effects of botulinum toxin on the distribution of succinate dehydrogenase and phosphorylase in fast and slow skeletal muscles of the mouse.

A sublethal quantity of botulinum toxin was injected into the muscles of one leg in mice. The histochemical localization of succinate dehydrogenase (SD) and phosphorylase (P) in soleus and gastrocnemius was studied at intervals of from one week to 15 months after the injection of toxin. In normal mice the fibres of soleus are rich in SD and poor in P, but in the superficial part of gastrocnemius the majority of fibres are rich in P and poor in SD. After the injection of toxin the muscles became paralysed and atrophied for several weeks during which time the fibres of gastrocnemius lost their staining reactivity for P and the fibres of soleus showed a weakening of the SD reaction. With the recovery from the effects of the toxin the muscle fibres increased in size and in P activity. Recovery commenced sooner in soleus than in gastrocnemius and for several weeks soleus fibres showed a stronger reaction for P than normal, but with longer survival the enzyme reactions in soleus became normal. Recovery in gastrocnemius began about six weeks after the injection of toxin. Many muscle fibres in gastrocnemius became much enlarged while others remained atrophied. Many fibres gave a strong histochemical reaction for both enzymes. These abnormalities in gastrocnemius seemed to be permanent.

Failure to inhibit in vitro or in vivo acetycholinesterase with botulinum toxin type A.

An attempt has been made to replicate an earlier finding that type A botulinum toxin can inhibit the in vitro activity of acetylcholinesterase. Two methods of enzyme assay were employed, but with neither technique were we able to reproduce the finding of in vitro enzyme inhibition. In fact, an examination of the data from the previous report leads us to question the possibility of the observations that were given. Furthermore, an investigation was carried out to determine if botulinum toxin can exert an inhibiting effect on acetylcholinesterase that is situated in the biological tissue. The answer again is negative. The experimental observations, coupled with several mathematical computations, do not support the notion that botulinum toxin is an acetylcholinesterase inhibitor.