Screening Snake Venoms for Toxicity to Tetrahymena Pyriformis Revealed Anti-Protozoan Activity of Cobra Cytotoxins.

Snake venoms possess lethal activities against different organisms, ranging from bacteria to higher vertebrates. Several venoms were shown to be active against protozoa, however, data about the anti-protozoan activity of cobra and viper venoms are very scarce. We tested the effects of venoms from several snake species on the ciliate Tetrahymena pyriformis. The venoms tested induced T. pyriformis immobilization, followed by death, the most pronounced effect being observed for cobra Naja sumatrana venom. The active polypeptides were isolated from this venom by a combination of gel-filtration, ion exchange and reversed-phase HPLC and analyzed by mass spectrometry. It was found that these were cytotoxins of the three-finger toxin family. The cytotoxins from several cobra species were tested and manifested toxicity for infusorians. Light microscopy revealed that, because of the cytotoxin action, the infusorians' morphology was changed greatly, from teardrop-like to an almost spherical shape, this alteration being accompanied by a leakage of cell contents. Fluorescence microscopy showed that the fluorescently labelled cytotoxin 2 from cobra N. oxiana was localized mainly at the membrane of killed infusorians, indicating that cytotoxins may kill T. pyriformis by causing membrane rupture. This work is the first evidence of the antiprotozoal activity of cobra venom cytotoxins, as demonstrated by the example of the ciliate T. pyriformis.

Design of targeted B cell killing agents.

B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.

Hydrogen-bonding versus pi-pi stacking interactions in dipyrido[f,h]quinoxaline-6,7-dicarbonitrile and 6,7-dicyanodipyrido[f,h]quinoxalin-1-ium chloride dihydrate.

The solvent-free title compound, C(16)H(6)N(6), is an aromatic derivative of phenanthroline with an extended pi system. It exhibits a remarkable pi-pi columnar stacking in the crystal structure, with interplanar distances of 3.229 (3) and 3.380 (3) A, the shorter spacing being between the two molecules within the asymmetric unit. Adjacent units along the stacked arrays are rotated in-plane with respect to one another by approximately 120 degrees . The hydrochloride derivative, C(16)H(7)N(6)(+).Cl(-).2H(2)O, in which one of the phenanthroline N atoms has been protonated, crystallized as a dihydrate. The supramolecular organization in this compound is characterized by continuous hydrogen bonding between the component species, yielding two-dimensional hydrogen-bonded networks. This study demonstrates the high significance of the pi-pi stacking interactions in the solvent-free aromatic system and how they can be undermined by introducing hydrogen-bonding capacity into the ligand.

Supramolecular interaction patterns in the zinc(II) dichloride and tin(IV) tetrachloride complexes with dipyrido[f,h]quinoxaline-6,7-dicarbonitrile.

This study characterizes the supramolecular synthons that dominate the intermolecular organization of the title compounds, namely dichloridobis(dipyrido[f,h]quinoxaline-6,7-dicarbonitrile)zinc(II), [ZnCl(2)(C(16)H(6)N(6))(2)], (I), and tetrachlorido(dipyrido[f,h]quinoxaline-6,7-dicarbonitrile)tin(IV), [SnCl(4)(C(16)H(6)N(6))], (II), in their respective crystal structures. Molecules of (I) are located on 2(b) axes of rotational symmetry. Their crystal packing is stabilized mostly by pi-pi stacking and dipole-dipole attractions between the organic ligand fragments of inversion-related neighbouring species, as well as by weak intermolecular C-H...N hydrogen bonds. On the other hand, Cl...pi and N...pi interactions seem to direct the crystal packing in (II), which is unusual in the sense that its aromatic fragments are not involved in pi-pi stacking. Molecules of (II) are located on m(b) planes of mirror symmetry. This study confirms the diverse structural chemistry of this organic ligand, which can be involved in a wide range of supramolecular interactions. These include effective coordination to various metal ions via the phenathroline N-atom sites, pi-pi stacking and pi...halogen contacts through its extended pi-system, and hydrogen bonding and N...pi interactions through its nitrile groups. The competing natures of the latter make it difficult to predict a priori the preferred supramolecular motif that may form in a given structure.

Hydrogen-bonding and pi-pi stacking interactions in aquachloridobis(1,10-phenanthroline)cobalt(II) chloride dichloridobis(1,10-phenanthroline)cobalt(II) hexahydrate.

The title compound, [CoCl(C(12)H(8)N(2))(2)(H(2)O)]Cl.[CoCl(2)(C(12)H(8)N(2))(2)].6H(2)O, is the first example of a new 1:1 cocrystal of the octahedral [CoCl(2)(phen)(2)] and [CoCl(phen)(2)(H(2)O)](+).Cl(-) complexes (phen is 1,10-phenanthroline). The latter form heterochiral dimers held by strong pi-pi stacking interactions via their phenathroline ligands, which confirms that pi stacking is an important and reliable synthon in supramolecular design. In addition, the crystal structure is networked by H(2)O...H(2)O, H(2)O...Cl(-) and H(2)O...Cl hydrogen bonds, which interconnect the different units of the cobalt complexes.

Remarkable pi-pi stacking of dipyrido[f,h]quinoxaline-6,7-dicarbonitrile in its ethanol solvate.

The title compound, C16H6N6 x C2H6O, is an ethanol solvate of an aromatic phenanthroline-based flat ligand. The latter exhibits a remarkable pi-pi stacking in the crystal structure, with interplanar distances of 3.27 and 3.40 A, which directs the columnar organization of the ligands. The ethanol solvent molecule is located in channels between these columns, being hydrogen bonded to one of the N-atom sites of the phenanthroline fragment.

Selective inhibition of the interaction of C1q with immunoglobulins and the classical pathway of complement activation by steroids and triterpenoids sulfates.

Since undesirable activation of the complement system through the classical pathway is associated with tissue damage and other pathologic proinflammatory consequences at ischemia/reperfusion injury, autoimmune diseases, and rejection of allo- and xenografts, creation of selective inhibitors of the classical pathway leaving the alternative pathway intact is of great importance. Classical pathway is triggered by binding of its recognizing unit, protein C1q, to a number of targets like antibodies, pentraxins, apoptotic cells, and others. In order to obtain inhibitors blocking the first step of the classical cascade, synthesis of sulfates of steroids (Delta(5)-3beta-hydroxycholenic, Delta(5)-3beta-hydroxyetiocholenic, deoxycholic, and cholic acids) and triterpenoids (betulin, 20,29-dihydro-20,29-dichloromethylenbetulin, betulinic, ursolic, and oleanolic acids) has been performed. Testing of the compounds in classical pathway inhibition assay has displayed derivatives of triterpenoid betulin (betulin disulfate and betulinic acid sulfate) to be the most potent inhibitors. Further studies of the two compounds established that their activity to inhibit the classical pathway had been due to their capability to block the interaction of C1q with antibodies. Betulin disulfate and betulinic acid sulfate have shown weak inhibition of the alternative route of activation, what makes them promising inhibitors for the selective suppression of the classical complement pathway at the earliest possible level as well as perspective agents for blocking the interaction of C1q with its other targets.

Naja melanoleuca cobra venom contains two forms of complement-depleting factor (CVF).

Two forms of complement-depleting cobra venom factor (CVFm1 and CVFm2), possessing molecular masses of 142.6 kDa (CVFm1) and 143.1 kDa (CVFm2), according to MALDI mass-spectrometry, were isolated from the Naja melanoleuca cobra venom. As shown by polyacrylamide gel electrophoresis in the presence of SDS, both forms similarly to factor from the Naja kaouthia cobra venom (CVFk) consist of three polypeptide chains with molecular masses of about 70, 50, and 30 kDa, the two large subunits being glycosylated. As determined by MALDI mass-spectrometry, 30 kDa subunits of CVFm1 and CVFm2 have considerably different finger-prints of tryptic digests that suggests differences in their amino acid sequences. A study of activity in vivo has shown no significant differences in C3 consumption by CVFm1, CVFm2 and CVFk in mouse blood. However, as shown by an immunoassay method, they differ in their ability to activate the complement system via C3 conversion, the ratio of these activities for CVFm1:CVFm2:CVFk being 2.5:1.6:1. Kinetic studies using a hemolytic test showed that complement depletion by CVFm1 is faster than that by CVFm2. Thus, for the first time the presence in a single venom of two forms of CVF differing by both amino acid sequence and biological activity has been shown.

Inhibition of classical pathway of complement activation with negative charged derivatives of bisphenol A and bisphenol disulphates.

In order to obtain strong inhibitors of classical pathway of complement activation the low weight negative charged compounds have been investigated. On the basis of bisphenol A anionic derivatives with one or two carboxylic, sulphate and phosphate groups the critical role of negative charged groups for complement-inhibiting activity has been established. It was determined that two sulphate or phosphate groups in the molecule provide the most inhibiting effect. At the next stage a set of bisphenol disulphates of varying structures has been synthesized and investigated. Bulky hydrophobic groups (cyclohexyliden, fluorenyliden, anthronyliden) at the central part of the bisphenol molecule it was found to increase complement-inhibiting activity markedly. The replacement of the ortho-positions to the charged group by halogens or alkyl groups (allyl, propyl) increases the inhibiting effect. It was showed by ELISA that several compounds studied interact with C1q, C1r /C1s components of complement. For the set of bisphenol disulphates the QSAR equation with hydrophobic coefficient and electronic parameters has been formulated. Both hydrophobic and electrostatic interactions it was established to have a great significance for the inhibition of classical pathway of complement activation.