A modification of neurotoxin RTX-III from Radianthus macrodactylus actinin with acetic anhydride

Upon modification of neurotoxin RTX-III at amino groups with [3H]acetic anhydride, four monoacetyl and four diacetyl derivatives have been obtained. Acetylation of the N-terminal amino group led to 12-fold decrease of toxicity in mice. Monoderivatives of the toxin with either Lys or two out of three C-terminal Lys residues modified showed a 2-fold drop in toxicity as compared with the native RTX-III. Diacetylation caused a 30 to 35-fold decrease in toxicity, the N-terminal amino group being modified in all the derivatives. As assessed by circular dichroism method, the modification of amino groups, except for N-terminal one, affected secondary structure of the toxin. The data suggest the N-terminal amino group to be essential for toxicity of RTX-III.     

Role of arginine and histidine residues in the biological activity of botulinic neurotoxin A

Treatment of botulinic neurotoxin A with cyclohexanedione demonstrated that modification of 5 to 10 arginine residues does not change the neurotoxin toxicity, while after modification of 15-20 arginine residues the toxicity is decreased by 40-50% of the original value. Butanedione exerts a stronger detoxicating effect on neurotoxin than cyclohexanedione. The molecular conformation of the modified toxin derivatives and their precipitability upon interaction with antisera against toxin and toxin fragments does not change thereby. The non-toxic derivatives of toxin containing 40 modified arginine residues possess a partial serological affinity for the original toxin in a reaction with antiserum against toxin but do not interact with the antifragment sera. The molecular conformation of these preparations is changed considerably. It is assumed that one or two arginine residues are located near the toxic site of the neurotoxin molecule and are also components of its antigenic determinants. Modification of histidine residues in the neurotoxin molecule by diethylpyrocarbonate is accompanied by a decrease of its toxicity. An additional 10% toxicity is revealed upon modification of 11-13 histidine residues. The molecular conformation of the modified derivatives of neurotoxin and their precipitability do not change thereby. It is probable that 1 or 2 histidine residues are located at or near the toxic site. The data obtained suggest that histidine residues are not localized in antigenic determinants of the neurotoxin molecule.     

Chemical modification of an antineoplastic antibiotic bleomycetin by the C-end fragment]

Bleomycetin, an antitumor antibiotic, was subjected to chemical modification by the C-end fragment i.e. the residue of 3-[(4-aminobutyl)amino]propylamine (spermidine++) with acylation, carbamoylation and reducing alkylation, which yielded its new semisynthetic derivatives. The use of physicochemical methods showed that the chemical modification involved the primary and secondary amino groups++ of spermidine++ and gave rise to N,N'-diacyl, N,N'-dicarbamoyl and N,N'-dialkyl bleomycetins. The biological properties of the derivatives, i.e. their cytotoxic activity, acute and pulmonary toxicities were studied. The transformation of bleomycetin by the C-end fragment lowered the antibiotic toxicity and was believed to be a promising approach to modifying its molecule.     

Studies on the status of lysine residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.

Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulphonic acid (TNBS), and two major trinitrophenylated (TNP) derivatives, TNP-1 and TNP-2, were separated by h.p.l.c. TNP-1 contained only one TNP group on Lys-6 and showed a marked decrease in enzymic activity, but still retained 45% of the lethal toxicity. Both Lys-6 and Lys-65 were modified in TNP-2, and modification of Lys-65 caused a further reduction of the lethal toxicity to 12.6%. However, the antigenicity of both TNP-1 and TNP-2 remained unchanged. The reactivity of Lys-6 and Lys-65 toward TNBS was greatly enhanced by Ca2+ and dihexanoyl-lecithin, suggesting that the two Lys residues are not directly involved in the binding of Ca2+ and substrate. The modified derivatives retained their affinity for Ca2+, indicating that Lys-6 and Lys-65 did not participate in the Ca2+ binding. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated PLA2 are almost the same as those of native PLA2. These results indicate that Lys-6 and Lys-65 are important for the biological activities of PLA2, and incorporation of a bulky TNP group on Lys-6 and Lys-65 might give rise to a distortion of the active conformation of PLA2.     

Production and antineoplastic activity of new asymmetric azines on the rubomycin base

New alkulidene hydrazones of rubomycin (daunorubicin) with the linear or branched chain of the carbon atoms were studied: rubomycin 13-(hexylidene-2")-hydrazone, rubomycin 13-(heptylidene-3")-hydrazone and rubomycin 13-(4"-methylpentylidene-2")-hydrazone. Alkylidene hydrazones of the formamidine derivatives were also studied: 13:cyclohexylidene hydrazone of 3'-desamino-3'-dimethylformamidine rubomycin and 13-(5"-oxypentyliden-2") hudrazone of 3'-desamino-3'-dimethylformamidine rubomycin. The latter two alkylidene hydrazones were modified twice. It was found that after a single intravenous administration to tumor-free mice the new substance had the same or lower toxicity as compared to that of rubomycin. Antitumor activity of the substances against lymphosarcoma LIO-I was studied comparatively with that of the initial rubomycin. It was shown that the molecule modification at C-13, as well as simultaneous modification at C-13 and the sugar amino group resulted in lowering of the antitumor activity in comparison to that of the starting rubomycin.     

Structure-toxicity relationships of the eremophilane phomenone and PR-toxin

The structure-toxicity relationships of phomenone were studied on cuttings and seedlings of tomato, and on larvae of brine shrim. Four derivatives prepared by chemical modification of phomenone were tested in comparison with PR-toxin. The toxicity to tomato cuttings (wilting and necroses of leaflets) appeared to be dependent on the integrity of the phomenone molecule, as any structural modification markedly reduced or completely abolished its phytotoxicity. By contrast, the toxicity to tomato seedlings (growth inhibition of shoots and rootlets) and to brine shrimp (larvae mortality) suggested a role for the epoxy rings in eremophilane molecules, which was enhanced by acetylation, as demonstrated by the progressive loss of activity for the sequence PR-toxin, phomenone and 9-methoxy- 6,7-de-epoxyphomenone.     

Synthesis,reactivity and biological activity of N(4)-boronated derivatives of 2′-deoxycytidine

By seeking new stable boron-containing nucleoside derivatives, potential BNCT boron delivery agents, a novel synthetic approach was tested, aimed at a boron attachment via a single bond to an aliphatic carbon of sp³ hybridization. The latter allowed successful modification of deoxycytidine in the reaction with 2-(iodomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane of the deoxynucleoside amino group. For new compounds, detailed NMR, LDI HRMS (Laser Desorption/Ionization High-Resolution Mass Spectrometry) analyses along with in vivo phosphorylation studies, toxicity assays and DFT modelling are presented.     

Discovery of novel 3,6-disubstituted 2-pyridinecarboxamide derivatives as GK activators

A novel class of 3,6-disubstituted 2-pyridinecarboxamide derivatives was designed based on X-ray analysis of the 2-aminobenzamide lead class. Subsequent chemical modification led to the discovery of potent GK activators which eliminate potential toxicity concerns associated with an aniline group of the lead structure. Compound 7 demonstrated glucose lowering effect in a rat OGTT model.     

Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy

The hemolysis of red blood cells and muscle damage results in the release of the heme proteins myoglobin, hemoglobin, and free heme into the vasculature. The mechanisms of heme toxicity are not clear but may involve lipid peroxidation, which we hypothesized would result in mitochondrial damage in endothelial cells. To test this, we used bovine aortic endothelial cells (BAEC) in culture and exposed them to hemin. Hemin led to mitochondrial dysfunction, activation of autophagy, mitophagy, and, at high concentrations, apoptosis. To detect whether hemin induced lipid peroxidation and damaged proteins, we used derivatives of arachidonic acid tagged with biotin or Bodipy (Bt-AA, BD-AA). We found that in cells treated with hemin, Bt-AA was oxidized and formed adducts with proteins, which were inhibited by α-tocopherol. Hemin-dependent mitochondrial dysfunction was also attenuated by α-tocopherol. Protein thiol modification and carbonyl formation occurred on exposure and was not inhibited by α-tocopherol. Supporting a protective role of autophagy, the inhibitor 3-methyladenine potentiated cell death. These data demonstrate that hemin mediates cytotoxicity through a mechanism which involves protein modification by oxidized lipids and other oxidants, decreased respiratory capacity, and a protective role for the autophagic process. Attenuation of lipid peroxidation may be able to preserve mitochondrial function in the endothelium and protect cells from heme-dependent toxicity.     

Activation of a cyclic nucleotide phosphodiesterase and of a protein kinase by chemically modified calmodulin

1. Several calmodulin derivatives prepared by chemical modification of lysine residues were tested using bovine heart cyclic nucleotide phosphodiesterase and wheat germ calmodulin-dependent protein kinase. 2. The effect of chemical modification on the activation capacity of calmodulin for the two studied enzymes was different. 3. This was particularly noticeable in the case of alkylated derivatives which exhibited a higher affinity than native calmodulin towards phosphodiesterase but a lower affinity towards protein kinase. 4. The efficiency of these derivatives (maximal activation) was higher than that of native calmodulin in relation with the protein kinase.     

Synthesis and pharmacological evaluation of amide conjugates of NSAIDs with L-cysteine ethyl ester, combining potent antiinflammatory and antioxidant properties with significantly reduced gastrointestinal toxicity

The synthesis and pharmacological evaluation of a series of amide derivatives of NSAIDs with L-cysteine ethyl ester is described. The novel derivatives are potent antiinflammatory, antioxidant and hypocholesterolemic-hypolipidemic agents, while they demonstrate considerably reduced gastrointestinal toxicity. This molecular modification may offer a general route to safer antiinflammatory agents, potentially suitable for chronic use in conditions such as neurodegenerative disorders.     

Role of lysine and tryptophan residues in the biological activity of toxin VII (Ts gamma) from the scorpion Tityus serrulatus.

Toxin VII (TsVII), also known as Ts gamma, is the most potent neurotoxin in the venom of the Brazilian scorpion Tityus serrulatus. It has been purified to homogeneity using a new fast and efficient method. Chemical modification of TsVII with the tryptophan-specific reagent o-nitrophenylsulfenyl chloride yielded three modified derivatives (residues Trp39, Trp50 and Trp54). Acetylation of TsVII mostly generated the monoacetylated Lys12 derivative. No side reactions were detected, as indicated by endoproteinase Lys-C peptide mapping, Edman degradation and electrospray mass spectrometry. Circular dichroism and fluorimetric measurements showed that none of the chemical modifications altered the overall structure of the derivatives. The acetylation of Lys12 or the sulfenylation of Trp39 or Trp54 led to a loss of both toxicity in mice and apparent binding affinity for rat brain and cockroach synaptosomal preparations. Sulfenylation of Trp50, however, moderately affected the toxicity of TsVII in mice and had almost no effect on its binding properties. A 3-dimensional model of TsVII was constructed by homology modeling. It suggests that the most reactive residues (Lys12 and Trp39 and Trp54) are all important in the functional disruption of neuronal sodium channels by TsVII, and are close to each other in the hydrophobic conserved region.     

Synthesis of [3β-acetoxy-urs-12-en-28-oyl]-1-monoglyceride and investigation on its anti tumor effects against BGC-823

Ursolic acid (UA) as the leader compound was designed to prepare a series of derivatives (three novel compounds UA-1a, UA-1b and UA-2) by modification at the C3 and C28 positions. Their chemical structures were confirmed by IR, (1)H NMR and MS. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823 and HT-29 by the MTT assay. The novel derivative UA-1a, [3β-acetoxy-urs-12-en-28-oyl]-1-monoglyceride showed significant anti-growth ability against the assayed cancer cell lines, particularly against BGC-823, while low cytotoxicity to human normal gastric cell line GES-1. Further investigation revealed that UA-1a could induce apoptotic events of the treated BGC-823 cells, such as comet-like DNA bend, sub-G0/G1 phase accumulation and phosphatidylserine externalization. The activity of Caspase-3 was found to be up-regulated, while the expression of Bcl-2 and Survivin were down-regulated in UA-1a treated cells. UA-1a might trigger the death of BGC-823 cells by inducing apoptosis via the mitochondria pathway. UA-1a exerted stronger ability than Taxol to retard tumor growth in nude mice without leaving apparent toxicity to the hosts. The experimental data suggested that UA-1a would have a therapeutic potential in the treatment of gastric cancer.     

Tryptophan 110, a residue involved in the toxic activity but not in the enzymatic activity of notexin

We prepared two derivatives of notexin, a phospholipase A2 from Notechis scutatus scutatus venom, by modifying the protein with 2-nitrophenylsulfenylchloride, a tryptophan-specific reagent. One derivative was modified at both tryptophans 20 and 110 whereas the other was modified at tryptophan 20. Evidence based on circular dichroic analysis and antigenicity towards a notexin-specific monoclonal antibody indicated that derivatization at both tryptophans did not affect the tertiary structure of notexin. Concomitant modification of tryptophans 20 and 110 induced a marked decrease in the capacity of notexin to kill mice and to block neuromuscular transmission in the chick biventer cervicis preparation, whereas selective modification at tryptophan 20 had no effect on the lethal properties of notexin. This implies that the decrease in the lethal properties of notexin after derivatization was due to modification at tryptophan 110. However, the diderivatized notexin retained full enzymatic activity, implying that neither tryptophan 20 and tryptophan 110 are involved in the catalytic function of the molecule. We conclude that notexin harbours two functional sites. One of them corresponds to the enzymatic site, whereas the other, which includes tryptophan 110, provides specific toxic characteristics to notexin. By reference to previous crystallographic studies, the relative spatial positions of elements involved in toxicity and the catalytic site, we propose a possible orientation of notexin with respect to its putative membrane-bound target.     

Modification of tetrodotoxin with succinic anhydride

With an aim toward the synthesis of physiologically active derivatives of the powerful neurotoxin tetrodotoxin and the use of such derivatives in biochemical studies of the molecular makeup of the tetrodotoxin binding site in nerve, the modification of tetrodotoxin with succinic anhydride has been examined. A new toxin derivative, 11-succinylanhydrotetrodotoxin, is described which contains a remote carboxylic acid grouping suitable for subsequent modification or attachment to an affinity column. High voltage electrophoresis and radiochemical techniques served to establish the purity of the derivative and its relative toxicity, namely, 0.4% that of tetrodotoxin (frog nerve assay). Structure assignment was made on the basis of the FT 100 MHz nmr spectrum.     

Tryptophan environment in adenosine deaminase. I. Enzyme modification with N-bromosuccinimide in the presence of adenosine and EHNA analogues

Adenosine deaminase from bovine cerebral hemisphere (white and gray matter) and spleen was treated with N-bromosuccinimide, a reagent known to oxidize selectively tryptophan residues in proteins. Spectrally observable tryptophan modification was accompanied by enzyme inactivation. Tsow graphics revealed that two Trps are essential for the activity of enzyme from both tissues. Enzyme inhibitors and substrate analogues, derivatives of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine, were able to protect Trp against modification, and this effect correlated in general with the enzyme activity protection. In the presence of adenosine deaza analogues (the noninhibitor tubercidin among them) only two Trps were modified in the fully inactivated enzyme. In the presence of EHNA and its deaza analogues, full inactivation of the enzyme was accompanied by the modification of four Trps. The obtained data confirm the previous hypothesis about the presence on the enzyme of different binding sites for adenosine and EHNA derivatives that are responsible for the different effects on the enzyme conformation elicited by the corresponding derivatives. Moreover, these data allow us to suggest that Trp residues, still unidentified by X-ray analysis, are essential for the functioning of the enzyme.     

Synthesis and in vivo diuretic activity of some novel pyrimidine derivatives

A series of 1,6-dihydropyrimidine-2-amine derivatives and 1,6-dihydropyrimidine-2-thiol derivatives were synthesized by the reaction of substituted 1,3-diphenylprop-2-en-1-one (chalcones) with guanidine hydrochloride and thiourea, respectively. All the synthesized compounds were in good agreement with elemental and spectral data. The synthesized compounds were screened in vivo for diuretic activity. The four compounds 2d, 2e, 3d and 3e, which showed moderate to good diuretic activity, were evaluated for their toxicity studies and none of the compounds showed any toxicity of the liver as compared with control. However, compounds 3e and 3d showed diuretic properties more than that of standard (acetazolamide) and were long acting. Overall, compound 3e, 6-(2,6-dichlorophenyl)-4-(pyridin-2-yl)-1,6-dihydropyrimidine-2-thiol, was found to be the most promising candidate of the series.     

Concept of “Binary Oligonucleotide Reagent”

Abstract

A new strategy for site-directed chemical modification of NA is described. NA-target-driven autoligation reaction between two oligonucleotide derivatives with N-(2-chloroethyl)-N-(p-formylphenyl)-N-propyl-N-3 -ydeneamino and 4-carbohydrazidephenyl groups at their opposing termini results in the NA-target modification, which is several times more effective than modification by one of the derivatives.     

Synthesis of Valproic Acid Amides of a Melatonin Derivative,a Piracetam and Amantadine for Biological Tests

Three new amide derivatives of valproic acid have been synthesized and characterized by spectrophotometric studies. The rationale for the preparation of such agents has been based on the observation that chemical combination of the anticonvulsant pharmacophore, valproic acid with amine moieties produces more effective and less toxic amides. The amine components selected in this work also exhibit neuroactivity with the prospect of these agents being biologically active in controlling not just seizures and but also possessing neuroprotective properties. We report here the synthesis and properties of the valproylamides of 5-methoxytryptamine, related to melatonin (1), of N-substituted 2-pyrrolidinone related to piracetam (2), and of adamantylamine related to amantadine (3). In preliminary tests these compounds showed low toxicity and a variety of anticonvulsive properties, including a delay in onset of activity. These compounds and their derivatives are now available to be tested additionally for control of subclinical seizures, enhancement of cognition, behavior modification and alleviation of symptoms and disorders due to neuronal damage.