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Collection of King Cobra venom. (A) Milking of venom by inducing the snake to bite through a film-covered clean container. (B) Freshly milked venom with its bright golden color. (C) Venom powder in different shades of yellow obtained through lyophilization for long-term storage.
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Abstract King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, sou...
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Context 1
... Cobra venom can be readily collected by inducing the snake to bite through a film covering a clean container, or, by directly placing its fangs over the edge of a collecting device. The venom of King Cobra is typically golden yellow in color, often viscous, and lyophilization yields powder with comparable coloration in different shades of yellow which is indicative of the presence of a flavin adenine dinucleotide (FAD)-containing flavoenzyme, L-amino acid oxidase (LAAO) (Figure 6 [47,48], snake venom metalloproteinases (SVMP) [49], cysteinerich secretory proteins (CRiSP) [50], phospholipases A 2 (PLA 2 s) [51], L-amino acid oxidases (LAAOs) [52], Kunitz-type serine protease inhibitors (KSPIs) [53], cobra venom factors (CVFs) [54] and ohanin (vespryn) [55]. The global protein profile of King Cobra venom was only available with the advent of proteomics, which attempted to unveil the variety and abundances of all toxins (proteins) in the venom comprehensively. ...
Context 2
... Cobra venom is a rich source of snake venom LAAOthe intense yellow coloration of the venom reflects the high content of FAD-containing LAAO in the venom ( Figure 6). LAAO was reported to constitute close to 6% of total venom proteins in the Malaysian King Cobra venom proteome [57]. ...
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... was reported to constitute close to 6% of total venom proteins in the Malaysian King Cobra venom proteome [57]. In the venom proteome of Indonesian specimen, the enzyme was either undetected [58] or present at only 0.5% of total venom proteins [56], although the Indonesian King Cobra venom too exhibits strong LAAO enzymatic activity [21], and shows yellow coloration ( Figure 6). The substantial amount of LAAO in the Malaysian King Cobra venom [57] supported its strong enzymatic activity [21], and suggests the involvement of this enzyme in the development of local tissue damage (cytotoxic activity) and pain in King Cobra envenomation [78]. ...
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... It is known that the venom from the cobra snake that belongs to Elapidae family induces neurotoxic (through nicotine acetyl choline receptors) and cytotoxic activity 36,37 The NAM being discussed here with ML tool has trained the software with neuro and cytotoxic phenotype patterns acquired from treated in vitro platform as part of the training the model exercise. The embedded AI picks signals with confidence against the control patterns to detect toxicity-induced cellular phenotypes (Supplementary Figure 1&2) in the assay system while the derived IC50 and potency values reflect the measurable score. ...
Polyvalent antivenom potency tested for every batch and at four different stages of production is a regulatory requirement to be followed by the industry. Antivenom manufacturers have been following the gold standard test methodology for estimating median effective dose in mice for ages. Here, we report a non-animal New Approach Methodology that aligns with 3Rs in animal testing agenda, leveraging in vitro human stem cell technology for recreating microphysiological system complemented with process automation, Artificial Intelligence and Machine Learning digital workers’ effective utilization in the assay system to measure Naja naja snake antivenom potency. In vitro neutralization performed on specially configured human Microphysiological System acquired phenotype data sets at 20X magnification were analyzed against benchmark panels in the trained prediction model while the Artificial Intelligence predicted median effective dose value of venom-antivenom mixture was 3.9μL, showing a potency value of 2.04 mg recorded as the readout. This method adopted in the antivenom producer’s workflow will reduce reliance on mice-based testing and showcases potential for acceptance of robust alternative strategy to traditionally practiced protocols.
... The high abundance of short neurotoxins is consistent with the venom's high lethality in mice (Tan et al., 2021b; and the rapid onset of severe neuromuscular paralysis reported in clinical envenoming (Watt et al., 1988). Envenoming by O. hannah manifests similar sign of neurotoxicity (descending flaccid paralysis) while the action is mediated primarily by long-chain alpha-neurotoxins (LαNTX) (Tan et al., 2021a;Tan et al., 2015a;Tan et al., 2020), which constitute close to ~40% of the venom proteome (Tan et al., 2015a). To reverse the neurotoxicity, specific antivenom is needed to neutralize the principal neurotoxins (SαNTX or LαNTX) of the respective species. ...
Snakebite envenoming is an important neglected tropical disease. Antivenom supply, however, remains limited in many parts of the world. This study aimed to examine the protein composition, immunoreactivity and neutralization efficacy of a new antivenom product (VINS Philippine Elapid Antivenoms, VPEAV) developed for the treatment of snakebite envenoming caused by the Philippine Cobra (Naja philippinensis), Samar Cobra (Naja samarensis) and King Cobra (Ophiophagus hannah). Size-exclusion chromatography, sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and tandem mass spectrometry showed that VPEAV consisted of F(ab)'2 (∼90% of total antivenom proteins) with minimal protein impurities. Indirect ELISA showed varying immunoreactivity of VPEAV toward the different venoms (EC50 = 4-16 μg/ml), indicating distinct venom antigenicity between the species. In mice, the neutralization potency of VPEAV against the King Cobra venom was moderate (potency, P = 2.6 mg/ml, defined as the amount of venom completely neutralized per unit volume of antivenom). The potency was significantly lower against the N. philippinensis and N. samarensis venoms (P = 0.18-0.30 mg/ml), implying a higher dose may be needed for effective neutralization of the Naja venoms. Together, the findings suggest the potential and limitation of VPEAV in neutralizing the venom toxicity of the three Philippine elapid snakes.
... Since Ophiophagus hannah (King Cobra) venom has been shown to be highly selective for snake (Coelognathus radiata) alpha-1 [11], we hypothesized that the prey-specific toxicity of O. hannah is highly correlated with specific amino acids within the snake-prey alpha-1 orthosteric site [32]. To investigate this hypothesis, we used a well-validated novel biolayer interferometry assay [33,34] to screen the venoms of four different types of King Cobras (Cambodian, Javan, Malaysian, and Thai) using native and mutant orthosteric sites. ...
... This supports the idea of the geographical variation of venom composition and venom toxicity. Recent quantitative proteomic studies on Malaysian and Javanese King Cobra venoms showed that the crude King Cobra venom of the Javanese locale contains a comparatively higher amount of alpha-neurotoxic 3FTxs, which accounts for 64.2% of total venom proteins with ≥20 subtypes [32]. Conversely, the Malaysian King Cobra venom showed a lower percentage of 3FTxs, which makes up 43% of their total venom proteins with 13 subtypes [32]. ...
... Recent quantitative proteomic studies on Malaysian and Javanese King Cobra venoms showed that the crude King Cobra venom of the Javanese locale contains a comparatively higher amount of alpha-neurotoxic 3FTxs, which accounts for 64.2% of total venom proteins with ≥20 subtypes [32]. Conversely, the Malaysian King Cobra venom showed a lower percentage of 3FTxs, which makes up 43% of their total venom proteins with 13 subtypes [32]. The results obtained from our present study, thus, further reveal a piece of evidence for the compositional variation of King Cobra venom linked with geographical distribution. ...
Snake venom is an adaptive ecological trait that has evolved primarily as a form of prey subjugation. Thus, the selection pressure for toxin diversification is exerted by the prey's physiological targets, with this pressure being particularly acute for specialist feeders, such as the King Cobra species, all of which are snake-prey specialists. However, while extensive research has been undertaken to elucidate key amino acids that guide toxin structure-activity relationships, reciprocal investigations into the specific sites guiding prey-lineage selective effects have been lacking. This has largely been due to the lack of assay systems amenable to systematic amino acid replacements of targeted proteins in the prey's physiological pathways. To fill this knowledge gap, we used a recently described approach based upon mimotope peptides corresponding to the orthosteric site of nicotinic acetylcholine receptor alpha-1 subunits, a major binding site for snake venom neurotoxins that cause flaccid paralysis. We investigated the venoms of four different types of King Cobra (Cambodian, Javan, Malaysian, and Thai). This approach allowed for the determination of the key amino acid positions in King Cobra snake prey that are selectively bound by the toxins, whereby replacing these amino acids in the snake-prey orthosteric site with those from lizards or rats resulted in a significantly lower level of binding by the venoms, while conversely replacing the lizard or rat amino acids with those from the snake at that position increased the binding. By doing such, we identified three negatively charged amino acids in the snake orthosteric site that are strongly bound by the positively charged neurotoxic three-finger toxins found in King Cobra venom. This study, thus, sheds light on the selection pressures exerted by a specialist prey item for the evolution of lineage-selective toxins.
... Electrophoretic profiles of the chromatographic fractions of all four venoms showed the proteins eluted from 55 to 140 min were of low molecular weight (MW) (<15 kDa), accounting for >92% of the total venom proteins based on the AUC (area under the curve) of the respective venoms. Minor proteins were eluted in the later course of the fractionation, where heterogeneous mixtures of low and moderate MW proteins (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) were eluted between 140 and 150 min, while high MW proteins (>40 kDa) eluted between 155 and 170 min (Figure 1). ...
The Equatorial Spitting Cobra (Naja sumatrana) is a medically important venomous snake species in Southeast Asia. Its wide geographical distribution implies potential intra-specific venom variation, while there is no species-specific antivenom available to treat its envenoming. Applying a protein-decomplexing proteomic approach, the study showed that three-finger toxins (3FTX), followed by phospholipases A2 (PLA2), were the major proteins well-conserved across N. sumatrana venoms of different locales. Variations were noted in the subtypes and relative abundances of venom proteins. Of note, alpha-neurotoxins (belonging to 3FTX) are the least in the Penang specimen (Ns-PG, 5.41% of total venom proteins), compared with geographical specimens from Negeri Sembilan (Ns-NS, 14.84%), southern Thailand (Ns-TH, 16.05%) and Sumatra (Ns-SU, 10.81%). The alpha-neurotoxin abundance, in general, correlates with the venom’s lethal potency. The Thai Naja kaouthia Monovalent Antivenom (NkMAV) was found to be immunoreactive toward the N. sumatrana venoms and is capable of cross-neutralizing N. sumatrana venom lethality to varying degrees (potency = 0.49–0.92 mg/mL, interpreted as the amount of venom completely neutralized per milliliter of antivenom). The potency was lowest against NS-SU venom, implying variable antigenicity of its lethal alpha-neurotoxins. Together, the findings suggest the para-specific and geographical utility of NkMAV as treatment for N. sumatrana envenoming in Southeast Asia.
... These enzymes (typically of the P-III subtype) can be found in diverse forms at relatively high abundance in elapid venoms albeit not conforming to the phenotype shown by member species within a genus or a closely related clade. Examples of elapids with SVMP-rich venoms include B. flaviceps (this study), King Cobra (Ophiophagus hannah) (Tan et al., 2022a), Snouted Cobra (Naja annulifera) , and Asiatic coral snakes (Calliophis bivirgata and Calliophis intestinalis) (Tan et al., 2016a;Tan et al., 2019c). Comparatively, other enzymes such as AChE and LAAO are more conserved, less diversified, and constitute a minor portion of the venom proteome. ...
The Red-headed Krait (Bungarus flaviceps) is a medically important venomous snake species in Southeast Asia, while there is no specific antivenom available for its envenoming. This study investigated the venom composition through a decomplexation proteomic approach, and examined the immunoreactivity as well as cross-neutralization efficacy of two hetero-specific krait antivenoms, Bungarus candidus Monovalent Antivenom (BcMAV) and Bungarus fasciatus Monovalent Antivenom (BfMAV), against the venom of B. flaviceps from Peninsular Malaysia. A total of 43 non-redundant proteoforms belonging to 10 toxin families were identified in the venom proteome, which is dominated by phospholipases A2 including beta-bungarotoxin lethal subunit (56.20 % of total venom proteins), Kunitz-type serine protease inhibitors (19.40 %), metalloproteinases (12.85 %) and three-finger toxins (7.73 %). The proteome varied in quantitative aspect from the earlier reported Indonesian (Sumatran) sample, suggesting geographical venom variation. BcMAV and BfMAV were immunoreactive toward the B. flaviceps venom, with BcMAV being more efficacious in immunological binding. Both antivenoms cross-neutralized the venom lethality with varying efficacy, where BcMAV was more potent than BfMAV by ~13 times (normalized potency: 38.04 mg/g vs. 2.73 mg/g, defined as the venom amount completely neutralized by one-gram antivenom protein), supporting the potential utility of BcMAV for para-specific neutralization against B. flaviceps venom.
