Novel TFTs affecting signal transduction
The most fascinating biological activity of TFTs discovered during the last decade is their capacity to interact with acid-sensing ion channels (ASICs). ASICs are proton activated and Na+-selective ion channels, widely distributed throughout the peripheral and central nervous systems (CNS) in vertebrates. ASICs take part in an array of physiological processes, from synaptic plasticity and neurodegeneration to pain sensation. Therefore, the finding of new regulatory modes for these proteins opens up a new avenue of research for pain management, as well as for addiction or fear.
The new mambalgins class of TFTs has been characterized as potent, rapid and reversible inhibitors of ASICs, based on studies with the protein from African black mamba (Dendroaspis polylepis) venom. The mambalgins are composed of 57 amino acids and eight cysteine residues, and have about 50% amino acid sequence identity to other snake TFTs. While mambalgins were found to be nontoxic in mice, they were found to exert a potent analgesic effect, as strong as that of morphine but causing much less tolerance than morphine and no respiratory distress. Pharmacological studies showed that mambalgins produce their analgesic effect through the blockade of heteromeric channels containing ASIC1a and ASIC2a subunits in CNS and of channels including ASIC1b subunit in nociceptors. Mambalgins were also shown to inhibit heteromeric channels including ASIC1a and ASIC1b subunits, homomeric rodent and human ASIC1a channels and homomeric rodent ASIC1b channels, the IC50s being in the range from 11 nmol/L to 252 nmol/L[22,23].
The structure of an ASIC1a–mambalgin-1 complex was determined by cryoelectron microscopy at a resolution of 5.4 Å. The data obtained showed that mambalgin-1 binds precisely to the thumb domain of ASIC1a but not to the acid-sensing pocket, as suggested earlier. However, mambalgin-1 binding induced conformational changes in the thumb domain of the channel, which may disturb the sensing of an acidity in ASIC1a. The structural data obtained might provide a structural basis for further development of ASIC modulators.
No less significant than the discovery of mambalgins was the finding of TFTs that interact with ionotropic GABA receptors (GABAA). Almost simultaneously, three research groups found that snake TFTs were able to bind GABAA receptors[26-28]. Thus, two TFTs, called micrurotoxin 1 (MmTX1) and 2 (MmTX2), were isolated from Costa Rican coral snake (Micrurus mipartitus) venom and sequenced. It was shown that at subnanomolar concentrations MmTX1 and MmTX2 increased receptor affinity for the agonist by binding to allosteric site, and thus potentiated opening and macroscopic desensitization of the receptor. The authors suggested that at the molecular level, the α+/β− subunit interface might be involved in toxin action. When injected into mouse brain, both toxins evoked seizures against the background of reduced basal activity. The discovery of toxins enhancing GABAA receptor sensitivity to agonist established a new class of ligands for this receptor family.
In 2006, it was shown that α-Bgt, a classical blocker of α7 and muscle-type nAChRs, binds to and blocks GABAA receptors containing the interface of β3/β3 subunit. No effects were observed for α-Bgt on heterooligomeric GABAA receptors which contain α-, β- and γ-subunits or α-, β- and δ-subunits. However, recently, two research groups independently showed that α-Bgt and some other TFTs could bind to recombinant and native GABAA receptors[27,28]. Both electrophysiology experiments and fluorescent measurements with α-Bgt coupled to Alexa-Fluor 555 revealed the highest toxin affinity to α2β2γ2 receptor subtype. GABA reduced fluorescent labeling by α-Bgt, suggesting that the α-Bgt binding site overlaps the GABA binding site at the interface of β/α subunits.
Binding at the β/α subunit interface was demonstrated for the long-chain α-neurotoxin α-CTX, and this toxin interacted more efficiently with the GABAA receptor than α-Bgt. Electrophysiology experiments showed mixed competitive and noncompetitive α-CTX action, with highest affinity of this toxin being to the α1β3γ2 receptor (IC50 236 nmol/L). Other receptor subtypes were inhibited less potently, as follows: α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ. Among the several TFTs studied, the long α-neurotoxins Ls III (Laticauda semifasciata) and neurotoxin I (Naja oxiana) as well as the nonconventional toxin WTX (Naja kaouthia) interacted with the GABAA receptor. These data demonstrate that GABAA receptors are a target for diverse TFTs, including the very well-studied α-Bgt and α-CTX.
Among the vast variety of TFTs there is a class of toxins that interact with mAChRs, which are G-protein coupled receptors (GPCRs). For many years, the mAChRs were thought to be the only GPCRs affected by TFTs; however, over the last decade, several TFTs capable of interacting with other GPCRs, namely adrenoreceptors of different types, were reported.
It was shown that muscarinic toxin α (MTα) was a more potent antagonist for the α2B-adrenoceptor than for mAChR. MTα inhibited the α2B-adrenoceptor, but did not affect the α2A-, α2C-, α1A- or α1B-adrenoceptors. In ligand binding experiments, MTα superseded the radioligand efficiently (IC50 3.2 nmol/L) and decreased the maximum binding without any influence on the radioligand affinity, demonstrating a noncompetitive inhibition mode. The study of another MT, MTβ, showed nonselective low affinity interaction with the five muscarinic receptor subtypes. Study of the toxin CM-3 (having undefined biological function to date) and MTβ demonstrated high efficacy for α-adrenoceptors and particularly a subnanomolar affinity for the receptor of α1A-subtype. Both toxins were isolated more than 20 years ago from the venom of the African mamba Dendroaspis polylepis[33,34]. No or very weak affinity of these toxins were found for muscarinic receptors in the work of Blanchet et al.
Targeted searches for toxins interacting with α-adrenoceptors have yielded novel information in the last decade. The interactions of fractions obtained from green mamba (Dendroaspis angusticeps) venom with α1-adrenoceptors were tested in binding experiments using 3H-prazosin as a radioligand. A new TFT inhibitor, AdTx1 (renamed later as ρ-Da1a), comprising 65 amino acid residues with four disulfide bridges, was found. ρ-Da1a showed subnanomolar affinity with Ki of 0.35 nmol/L and demonstrated high specificity for the human adrenoceptor of α1A-subtype. Interestingly, the biological activity profile of ρ-Da1a appeared very similar to those of MTβ and CM-3; however, these latter two toxins interacted more potently than ρ-Da1a with α1B- and α1D-adrenoceptor subtypes. ρ-Da1a was, thus, characterized as a specific and selective peptide inhibitor for the α1A-adrenoceptor, acting as a potent relaxant of smooth muscle.
Using a similar targeted screening approach, but with application of 3H-rauwolscine as a radioligand, the effects of venom fractions obtained from green mamba on α2-adrenoceptors from rat brain synaptosomes were studied. A novel TFT, ρ-Da1b, comprising 66 amino acid residues with four disulfide bridges was isolated. It inhibited binding of 3H-rauwolscine to the three α2-adrenoceptor subtypes by 80% with affinity in the range of 14-73 nmol/L and with Hill coefficient of about unity. Furthermore, calcium imaging experiments on human α2A-adrenoceptors expressed in mammalian cells showed that ρ-Da1b was an antagonist of this adrenoceptor type.
The structural scaffold of aminergic TFTs that are known to interact with various α-adrenergic, muscarinic and dopaminergic receptors was used to generate experimental toxins with new functions. Specifically, the ancestral protein resurrection methodology was applied to identify the functional substitutions that might happen during evolution, and then utilize them for molecular design. Six variants of ancestral toxin (AncTx, 1-6) were generated, and their biological activity was studied. AncTx1 was found to be the toxin possessing to date the highest selectivity to α1A-adrenoceptor. AncTx5 was the strongest inhibitor for α2-adrenoceptor of the three subtypes. The toxin ρ-Da1a affinities for the α1- and α2C-adrenoceptor subtypes were modulated most strongly by amino acids at positions 28, 38 and 43 in the evolutionary pathway. Thus, this molecular engineering study represents the first successful attempt to engineer more potent aminergic TFTs.
Among the snake venoms, the mamba ones are unique in their variety of toxins affecting signal transductions. A multitude of toxins capable of disturbing the different stages of cholinergic and adrenergic (see above paragraphs) transmission have been isolated from these venoms. Several toxins affecting voltage-gated ion channels have been isolated as well. The very recently discovered TFT Tx7335, in eastern green mamba Dendroaspis angusticeps venom, interacts with the KcsA potassium channel. The unusual structure of this toxin was discussed above. Interestingly, Tx7335 is a channel activator but not an inhibitor, as evidenced by its ability to increase in a dose-dependent mode both mean open times and open probabilities of KcsA incorporated in artificial bilayers; yet, the Tx7335 binding site on KcsA is distinct from that of the canonical pore-blocker toxins. The authors of this study suggested that the toxin allosterically reduced inactivation of KcsA that results in increase of potassium flow through the channel.
Blue coral snake Calliophis bivirgatus belong to the Elapidae family of snakes the neurotoxic venoms of which typically produce the flaccid paralysis. However it was shown that the C. bivirgatus venom uniquely produced spastic paralysis. The toxin producing this paralysis was isolated and called calliotoxin (protein name: δ-elapitoxin-Cb1a). Although calliotoxin is a TFT, it has low amino acid sequence similarity to the other known toxins. It comprises 57 amino acid residues with four disulfide bridges in the classical scaffold. Biological activity studies using HEK293 cells heterologously expressing NaV1.4 showed that the voltage-dependence of channel activation was shifted to more hyperpolarized potentials by calliotoxin. It inhibited inactivation and produced significant ramp currents. These data conformed with profound effects of calliotoxin on contractile force in preparation of isolated skeletal muscle. Thus, calliotoxin represents a functionally novel class of TFTs and is the first activator of voltage-gated sodium channel purified from snake venoms.
Novel TFTs affecting blood coagulation
TFTs affecting blood coagulation are not so numerous as those affecting signal transduction. Nevertheless, a new member of the TFT family that is capable of influencing different stages of blood coagulation appeared recently. TFTs inhibiting both primary and secondary hemostasis have been reported.
Primary hemostasis involves platelets, which immediately form a plug at the site of injury. A novel TFT which inhibits the human platelet aggregation process in a dose-dependent manner was purified from cobra Naja kaouthia venom and named KT-6.9. KT-6.9 was shown to inhibit platelet aggregation induced by adenosine diphosphate (ADP), thrombin and arachidonic acid but not by collagen and ristocetin. It was 25-times more active than the antiplatelet drug clopidogrel. Based on the data showing significant inhibition (70%) of the platelet aggregation induced by ADP, the authors suggested toxin binding to ADP receptors located on the platelet surface.
As for secondary hemostasis, two TFTs capable of inhibiting the extrinsic tenase complex (ETC) were purified from the venom of African ringhals cobra Hemachatus haemachatus[42,43]. ETC activates conversion of factor X (FX) to factor Xa (FXa) and represents an important target for the development of novel anticoagulants. A novel TFT anticoagulant, ringhalexin (the ringhals extrinsic tenase complex inhibitor) was shown to inhibit FX activation with an IC50 of 123.8 nmol/L. As an inhibitor of mixed type, on chick biventer cervicis muscle preparations ringhalexin manifested an irreversible weak neurotoxicity. The amino acid sequence of ringhalexin is 94% identical to that of NTL2, an uncharacterized neurotoxin-like protein from Naja atra. X-ray crystallography of ringhalexin revealed a typical three-finger structure stabilized by four conserved disulfide bridges.
Another novel anticoagulant TFT from Hemachatus haemachatus venom, called exactin, can specifically and potently inhibit the activation of FX by ETC (IC50 116.49 nmol/L), similar to ringhalexin. It is also a mixed-type inhibitor of ETC and weakly inhibits FX activation by intrinsic tenase complex (IC50 4.05 µmol/L) and prothrombin activation by prothrombinase complex (IC50 17.66 µmol/L). In contrast to other TFT anticoagulants that are structurally similar to snake cytotoxins, exactin manifests structural similarity to postsynaptic neurotoxins. It also has 82% identity to the weak toxin CM1b from H. haemachatus venom and 58% identity to a number of Ophiophagus hannah neurotoxins, including the Ω-neurotoxin Oh9-1 discussed above.
Novel TFTs with unexpected biological activities
The last decade has also seen the discovery of several new TFTs possessing quite unusual biological activities.
TFTs, being structurally well defined, thermally stable and resistant to proteolysis, are very good subjects for directed evolution. When a randomization scheme was applied to α-neurotoxin amino acid residues in the loops involved in binding with nAChRs, followed by the cDNA display screening method, new modulators of the interleukin-6 receptor (IL-6R) were obtained. The proteins obtained possessed nanomolar affinity and high specificity for IL-6R. The IL-6-dependent cell proliferation assay revealed both antagonists and agonists in the protein pool. Application of the size minimization procedure resulted in proteins with the molecular mass of about one-third of the original toxin; no significant loss of activities was observed. Moreover, the loops important for function were identified. In another work by the same group, directed evolution was applied to produce a trypsin inhibitor based on the TFT scaffold. The DNA sequences converged after seven rounds of selection. The recombinant proteins obtained were good inhibitors s of trypsin (Ki of 33-450 nmol/L). Three groups of proteins had Ki values close to those of soybean trypsin inhibitor and bovine pancreatic trypsin inhibitor. Two proteins inhibited chymotrypsin and kallikrein as well. The authors suggested that the technique developed may be widely applied for the targeted generation of different regulatory molecules based on the TFT motif.
Studies of a TFT cardiotoxin showed a quite unexpected effect on insulin secretion. The fractions of cobra Naja kaouthia venom obtained by combination of ultrafiltration and reversed-phase high-performance liquid chromatography were screened for insulinotropic activity using the rat INS-1E β-cell line. Only one fraction of the total 22 obtained induced secretion of insulin from the INS-1E cells with no influence on cell integrity and viability. Liquid chromatography-tandem mass spectrometry analysis revealed that this fraction represented the cardiotoxin-I (CTX-I) isolated earlier from Naja kaouthia venom. Analysis of the isolated CTX-I toxin in INS-1E cells showed that its insulin stimulation ability persisted even in the absence of glucose. In contrast to typical cobra cardiotoxin, CTX-I did not induce direct hemolysis of human erythrocytes and showed no potent vasoconstriction capability. Based upon this toxin, a truncated analogue [Lys(52)CTX-I(41-60)] was obtained by structure-guided modification. This analogue showed insulinotropic activity similar to CTX-I and appeared to exert its action through Kv channels. As such, it may serve as a basis for the design of new therapeutic agents for the treatment of type 2 diabetes (Table 1).
A new paradoxical TFT, nakoroxin, was isolated from the cobra Naja kaouthia venom. Nakoroxin belongs to the group of orphan TFTs (group “XX”), the biological activities of which are practically unknown. Nakoroxin was not cytotoxic to rat pheochromocytoma PC12 cells nor to human lung carcinoma HT1080 cells. It did not inhibit the binding of α-Bgt to α7 or muscle-type nAChRs, but potentiated the binding of α-Bgt to the acetylcholine-binding protein from Lymnaea stagnalis. The reason for this unusual property of nakoroxin is not clear.
Another quite interesting TFT, actiflagelin, was isolated from cobra Walterinnesia aegyptia venom by combination of reverse-phase and ion-exchange chromatography. Actiflagelin activated in vitro motility of sperm from OF1 male mice. The amino acid sequence established by Edman sequencing combined with tandem mass spectrometry analyses showed that the protein comprised 63 amino acid residues with five disulfide bonds, the pattern of which corresponded to that of nonconventional toxins. Actiflagelin had a noticeable homology to bucandin, a nonconventional toxin from Bungarus candidus venom. The authors suggested that the protein found may have therapeutic potential for cases of infertility when the problem is related to the sperm motility.