Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites.

Diverse ligands of the muscle nicotinic acetylcholine receptor (nAChR) are used as muscle relaxants during surgery. Although a plethora of such molecules exists in the market, there is still a need for new drugs with rapid on/off-set, increased selectivity, and so forth. We found that pyrroloiminoquinone alkaloid Makaluvamine G (MG) inhibits several subtypes of nicotinic receptors and ionotropic γ-aminobutiric acid receptors, showing a higher affinity and moderate selectivity toward muscle nAChR. The action of MG on the latter was studied by a combination of electrophysiology, radioligand assay, fluorescent microscopy, and computer modeling. MG reveals a combination of competitive and un-competitive inhibition and caused an increase in the apparent desensitization rate of the murine muscle nAChR. Modeling ion channel kinetics provided evidence for MG binding in both orthosteric and allosteric sites. We also demonstrated that theα1 (G153S) mutant of the receptor, associated with the myasthenic syndrome, is more prone to inhibition by MG. Thus, MG appears to be a perspective hit molecule for the design of allosteric drugs targeting muscle nAChR, especially for treating slow-channel congenital myasthenic syndromes.

A new type of thrombin inhibitor, noncytotoxic phospholipase A2, from the Naja haje cobra venom.

Thrombin is a key enzyme in the blood coagulation cascade and is also involved in carcinogenesis; therefore, its inhibitors are of fundamental and clinical importance. Snake venoms are widely used as sources of proteins that affect blood coagulation. We have isolated a new protein, called TI-Nh, from the Naja haje cobra venom. TI-Nh is a mixed-type inhibitor of thrombin (K(i) of 72.8 nM for a synthetic peptide substrate) and effectively inhibits thrombin-induced platelet aggregation with an IC(50) value of 0.2 nM. At concentrations up to approximately 50 nM, at which the thrombin-clotting time is substantially prolonged, TI-Nh exerts no detectable effects on both the intrinsic and extrinsic pathways of the coagulation cascade. It does not hydrolyze either fibrinogen or thrombin. Although TI-Nh bears structural features typical of group IB phospholipases A(2) (PLA(2)s), it possesses relatively weak enzymatic activity and is nontoxic to PC12 cells at concentrations up to 15 microM. Nevertheless, TI-Nh evokes neurite outgrowth in these cells at a concentration of approximately 1 microM, similar to cytotoxic snake PLA(2)s with strong enzymatic activity. TI-Nh is the first thrombin inhibitor found in the venom of the Elapidae snake family, and it is the first phospholipase shown to inhibit thrombin.

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification.

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Participation of chloroplasts in plant apoptosis.

Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN(-)-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O2 were shown to prevent the CN(-)-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN- effect. The light-dependent activation of CN(-)-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.