From Synthetic Fragments of Endogenous Three-Finger Proteins to Potential Drugs

Kryukova, Elena V.; Egorova, N. S.; Kudryavtsev, Denis S.; Lebedev, Dmitry; Spirova, Ekaterina N.; Zhmak, Maxim N.; Garifulina, Aleksandra; Kasheverov, Igor E.; Utkin, Yuri N.; Tsetlin, Victor I.

doi:10.3389/fphar.2019.00748

Cited by 22 publications

(27 citation statements)

References 57 publications

(79 reference statements)

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“…Though the exact target of cardiotoxins are not identified, they are known to induce cytolysis of cardiomyocytes, erythrocytes and also prevent platelet aggregation [10, 35]. Proteins targeting nAChRs will be useful in developing drugs for neurodegenerative diseases and as analgesics [36]. As an example, cobratoxin from Naja kaouthia was tested for neuropathic pain in rats and has shown to have antinociceptive effect [37].…”

Section: Discussionmentioning

confidence: 99%

Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats

Vanuopadath

Dileepkumar²,

Nair

et al. 2021

Preprint

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Venom proteome profiling is important to understand the toxicology and treatment of persons poisoned by animal venoms. An in depth understanding of the pharmacological mechanisms induced by venom toxins could help in the discovery of novel drug molecules. In the current study, we aimed to delineate the venom toxins of Indian cobra (Naja naja) from the Western Ghats of India through SDS-PAGE and reversed-phase HPLC followed by Q-TOF LC-MS/MS analysis, incorporating PEAKS and Novor assisted de novo sequencing methodologies. A total of 143 proteins distributed across 17 different enzymatic and non-enzymatic venom protein families were identified. The de novo analysis exclusively yielded 59 peptides representing 28 venom protein families. Among these, glutathione peroxidase and endonuclease were reported for the first time in Indian cobra venom. Immunological cross-reactivity of cobra venom assessed using Indian polyvalent antivenoms suggested that VINS showed better EC50 (2.48 μg/mL) values than that of PSAV (6.04 μg/mL) and Virchow (6.03 μg/mL) antivenoms. Also, immunoaffinity chromatography performed using VINS antivenom indicated that it failed to detect few low molecular mass proteins (<10 kDa) that include three-finger toxins, phospholipase A2s and kunitz- type serine protease inhibitors. Taken together, the present study enabled a large-scale characterization of the venom proteome of Naja naja that offers valuable insights on the possible pharmacological mechanisms and future therapeutic potential of hitherto unexplored snake venom constituents.

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Section: Discussionmentioning

confidence: 99%

Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats

Vanuopadath

Dileepkumar²,

Nair

et al. 2021

Preprint

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“…The biological activity of fragments of several other Ly6 proteins, namely, their ability to compete with radioactive α-Bgt for binding to different nAChRs and such their models as AChBPs and ligand-binding domains of individual subunits, as well as the ability of these fragments to inhibit currents in certain nAChR subtypes, was described by us in detail earlier [ 10 ]. The present study confirmed the micromolar affinity of fragments of the central loop II of the Lynx1 protein towards the T. californica nAChR (IC 50 values were in the range 1.1–7.9 μM), showing that the inhibitory activity was higher than that of the full-size protein for the same receptor or AChBPs from L. stagnalis and Aplysia californica [ 25 ] and does not strongly depend on whether the fragment is linear or cyclized ( Figure 1 A).…”

Section: Discussionmentioning

confidence: 99%

“…Linear peptides were prepared by solid-phase peptide synthesis using Fmoc/t-butyl strategy on tritylchloride-polystyrene resin (Intavis, Cologne, Germany) mostly as in [ 10 ]. Polypeptide chain assembly was performed on Syro I automatic peptide synthesizer (MultiSynTech AG, Witten, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…In these studies, we used radiolabelled α-Bgt ( 125 I-α-Bgt) with specific radioactivity of 500 Ci/mmol prepared as described in [ 27 ]. Competition experiments with Lynx1 fragments (compounds (1)–(3)) were performed mostly as described in [ 10 ]. Briefly, the membranes from T. californica ray electric organ (0.6 nM of α-bungarotoxin binding sites) were incubated in 50 μL of binding buffer (20 mM Tris-HCl buffer, pH 8.0, containing 1 mg/mL BSA) for 90 min with different concentrations of the Lynx1 peptides, followed by an additional 5-min incubation with 0.5 nM of 125 I-α-Bgt.…”

Section: Methodsmentioning

confidence: 99%

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Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models

et al. 2020

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Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three β-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.

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“…Preparative purification was carried out as described previously 21 using a Gilson HPLC system (333/334 pump with 215 liquid handler) equipped with a YMC Triart 10um 30 × 150 mm column and UV detection at 210 and 280 nm. Peptides elution was achieved by addition of a H 2 O-acetonitrile in the 10-55% gradient with 0.1% v/v CF3COOH.…”

Section: Synthesis Of Hap and Its Analogs Solid-phase Synthesis Utilmentioning

confidence: 99%

Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide

Kudryavtsev

Tabakmakher

Budylin

et al. 2020

Sci Rep

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Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the c-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAchRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAp-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAp analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions. α-Neurotoxins are snake venom proteins serving as accurate tools in research on nicotinic acetylcholine receptors (nAChRs) (see reviews 1-3). Their valuable feature is the capacity to distinguish certain nAChRs subtypes: short-chain α-neurotoxins bind only to the muscle-type nAChRs, while the long ones, such as α-bungarotoxin (αBgt) and α-cobratoxin (αCtx), are blocking muscle-type nAChRs and the neuronal ones containing α7, α9 and α9/α10 subunits 4-6. The spatial structure of all α-neurotoxins consists of three β-structural loops, giving them the name of "three-finger" proteins (with the abbreviations 3FP or TFPs), their arrangement being maintained by 4 disulfide bridges in the short and by 5 disulfides in the long α-neurotoxins. Interestingly, the TFP family embraces not only numerous α-neurotoxins, but also diverse snake venom proteins acting specifically on different targets (ion channels, G-protein coupled receptors, enzymes) or, in the case of cytotoxins, just disrupting cell membranes (see 7). The interest to α-neurotoxins in recent years was invigorated by the discovery that some

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From Synthetic Fragments of Endogenous Three-Finger Proteins to Potential Drugs

Cited by 22 publications

References 57 publications

Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats

Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats

Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models

Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide

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