In 2017, the World Health Organisation officially classified snakebite as a neglected tropical disease (NTD). During a snakebite, venom – a specialised toxic secretion is injected into the victim. Globally, snakebites affect up to 2.7 million people annually, leading to estimated 81,000 to 138,000 deaths yearly.¹ Survivors of snakebites often face long-term consequences, with the burden of premature death and disability measured at 6.07 Disability-Adjusted Life Years (DALYS).² Prevention and treatment of snakebites can be achieved through education, improved access to antivenom, and timely medical intervention by trained healthcare workers, effective governance, and related measures. Conventional surveillance systems reveal that India records the highest number of snakebite-related fatalities, accounting for half of all global cases and contributing to 2.97 million DALYs. Around 70% of these fatalities occur in eight states – Bihar, Jharkhand, Madhya Pradesh, Odisha, Uttar Pradesh, Andhra Pradesh (including Telangana), Rajasthan, and Gujarat, which together account for more than half of India’s population. Among these, Odisha is a critical focus of India’s Avoidable Death Network (ADN) Hub, as snakebite remains a significant and pressing public health concern in the state.³

The interest in PLA₂, LAAO, hyaluronidase, and metalloproteinase is driven by their critical contributions to the lethality of snake venom. Their mechanisms of action reveal how venom can rapidly immobilize or kill prey while also offering insights into physiological processes that can be harnessed for therapeutic purposes. Their study provides opportunities for improving antivenoms and exploring novel medical applications based on their lethal properties. The major venom proteins present in different snake toxin families are PLA₂‚ SVMP, SVSP, and three-finger proteins.^4-6 SVSPs cause a wide range of effects, including coagulation activation, inflammation, and tissue damage. Some SVSPs can interact with other venom proteins to enhance their activity. Other proteins in the snake venom include LAAOs, Kunitz peptides, disintegrins, NP, cysteine-rich secretory proteins, and C-type lectins.

Some of the snake venom proteins have potential for drug development but face challenges in being toxic and storage instability. Some venom proteins resemble human proteins, offering targeted treatment possibilities. Lack of detailed structure-function studies limits therapeutic use and large-scale production. Current antivenoms are broad-spectrum and often miss specific toxins. Integrating venomics data can help create more precise and effective antivenoms. Research centered on exploring the chemistry and understanding the relationship between protein structure and function. Proteins and peptides found in snake venom, as well as the exploration of their potential therapeutic applications, have gained considerable attention in biomedical research.⁷ Recent venom-based drugs currently on the market or in clinical trials have been reviewed.^6,8 Though these review articles provide valuable information, the significance of snake venom proteins as therapeutic agents can be better exploited if the sequence and structure correlations of these proteins, especially in relation to the human proteins, are completely understood. This review examines the similarities in amino acid sequences and structures between major snake venom proteins and human proteins. Further, challenges encountered during the development of drugs from venom proteins and the therapeutic potentials of the various venom proteins for the treatment of different diseases are discussed. To provide a holistic view, we have also included some basic information about the venom proteins, especially the venom composition and the chemistry of selected enzymatic toxins, which will benefit the readers. This study will provide insight into the use of enzymatic toxins as a substitute for non-functional human proteins occurring during various disease states.

The complex chemistry of Snake venom
Although there are notable exceptions and considerable species-level variability, certain toxic proteins play a key role in the effects of snake venom following a bite. 3FTx and PLA₂‚ are the major components of elapid venoms, and comprise about 65%-70% of the total proteins of the venom proteome. Whereas, snakes from the Dendroaspis genus (mambas) and various Australian snakes exhibit significant deviations in the composition and function of these toxins. Mambas’ venoms lack PLA₂, while venoms of Australian snakes have a notably low content (<6%) of 3FTx. On average, 6% of elapid venom is made up of SVMPs,^9,10 snake venom serine proteases (SVSPs),^11,12 and L-amino acid oxidases (LAAOs).^13-15 About 5% of elapid venom is made of Kunitz-type peptides,^12,16 a group of serine protease inhibitors recognized by the presence of the Kunitz domain fold that are specific K⁺-channel blockers. Mambas are extremely rich in Kunitz-type peptides. Approximately 10%-12% of the proteins are mostly non-toxic proteins, with highly variable composition^11,17-19 and the reason for the occurrence of many of these proteins in snake venom is not fully known.

The venoms of viperid snakes encompass toxins from nine distinct protein families, such as PLA₂‚ SVMPs, SVSPs, C-type lectins (CTLs), LAAOs, Disintegrins, Three-Finger Toxins, Kunitz-type peptides, CRISPs families.^20-22 Again, there are numerous exceptions and considerable variability in toxin content in venoms within each species and subspecies of snakes. PLA₂‚^10,11 SVMP,^9-11,19 and SVSP^11,23 toxins are prevalent in the majority of species, accounting for an average of 70% of the total venom proteome. Although viperid PLA₂‚ share higher sequence homology with the neurotoxic PLA₂‚ found in elapids, the majority of viperid PLA₂‚ exhibit myotoxic effects. LAAOs,^13-15 C-type lectins and C-type lectin-like proteins,²⁴ and natriuretic peptides,^6,17 are other toxins present in smaller quantities (4%-7%) (Figure 1), in the Viperid family. Reports indicate that snake venom toxins act synergistically. The pathophysiology of snakebite envenomation is determined by the combination and relative proportions of various toxins present in the venom.^2,25 Hemotoxicity, myotoxicity, cardiotoxicity, neurotoxicity, and cytotoxicity are the results of snakebite envenomation. Further, these four major toxic proteins phospholipase A₂‚ (PLA₂), snake venom metalloproteases (SVMPS), snake venom hyaluronidase and L-amino acid oxidase (LAAO) from snake venom have been reported to have both physiological effects and therapeutic potential in humans. The intricate interplay between the components of snake venom and their varied concentrations among species underpins their diverse pathophysiological effects. The detailed chemistry of venom proteins not only elucidates the mechanisms of envenomation but also highlights their promise as a source of novel drugs. Combining insights from venom composition with molecular studies of individual toxins bridges the gap between understanding venom pathology and harnessing its benefits for human health.

Key Toxic Proteins in Snake Venom: Chemistry and Mechanisms
Snake venom phospholipase
Phospholipase A (PLA₂) enzymes cause a range of pathological effects. PLA₂‚ exerts distinct effects on the nervous and cardiovascular systems through high-affinity interactions with specific receptors, a mechanism that is independent of its enzymatic function. Once attached to the target, they produce pharmacological effects with or without catalytic activity.²⁶ PLA₂‚ in snake venom cause neurotoxicity, thus damaging the neurons at the presynaptic cleft. Bulk of pre-synaptic neurotoxins derived from snake venom discovered so far are protein complexes containing PLA₂. When they bind to receptors or lipid domains in the motor neuron plasma membrane at the neuromuscular junction, they induce changes in membrane activity. This modification in membrane potential facilitates the influx of Ca²⁺ from the extracellular environment. The resulting changes in membrane permeability enhance the exocytosis of synaptic vesicles (Figure 2).²⁷

Myotoxic svPLA₂‚ generally possess aspartic acid at position 49, which is important for their enzymatic activity. Asp49-PLA₂‚ are catalytically active, but Lys49 homologs exhibit either weak catalytic activity or are catalytically inactive. Additionally, substitutions such as Ser49, Arg49, Asn49, or Gln49²⁸ have been identified and are known to alter catalytic activity, thereby influencing the toxic effects of this venom protein. Asp49 PLA₂‚ hydrolyzes phospholipids to release lysophospholipids, which leads to the skeletal muscle necrosis by directly disrupting membrane stabilization and/or indirectly altering membrane biophysics.²⁹ On the other hand, Lys49 PLA₂‚ myotoxins do not have catalytic activity and exist as homodimers.³⁰ The C-terminal regions (residues 115-129) of these peptides are rich in basic, aromatic, and hydrophobic amino acids, which are responsible for their myotoxic effects.^31,32 Site-directed mutagenesis studies have identified Tyr117, Arg118, Tyr119, Lys122, and Phe125 as key residues significantly influencing myotoxicity.³³

Venom protein PLA₂‚ variants can broadly be divided into D49 acidic PLA₂‚ (Asp-49), S49 PLA₂‚ (Ser-49), and K49 basic PLA₂‚ (Lys-49 replacing Asp-49). Basic PLA₂‚ homologs, whose amino acid residues, such as K49 and S49 PLA₂‚ are involved in different Ca²⁺ independent functions, but they are still catalytically inactive.³⁴ Among these, K49 basic PLA₂‚ exhibits higher cytotoxicity compared to D49 acidic PLA₂. Generally, acidic PLA₂‚ has a higher IC₅₀ value than basic PLA₂, indicating lower cytotoxic potency.

S49 PLA₂‚ variants, identified in the venom of saw-scaled vipers (Echis sp.), exhibit enzyme activity that is independent of calcium ions and exhibits higher toxicity to cells in comparison to K49 PLA₂‚ (IC₅₀ = 2.5-12.2 µM).³⁵ However, K49 PLA₂‚ demonstrates greater lipolytic activity compared to S49 PLA₂.³⁵

Snake venom metalloproteases (SVMPs)
SVMPs account for 30%-60% of the snake venom in the Viperidae family and are categorized into three distinct classes (P-I, P-II and P-III) mainly depending on their organization of the protein domain and therefore their functional variation. All classes of SVMPs contain the zinc ion-binding domain, which is involved in enzymatic activity. P-I SVMPs exclusively possess the zinc ion-binding domain and exhibit molecular weight range of 20 to 30 kDa. However, P-II and P-III SVMPs contain an additional domain that confers additional functional characteristics to these proteins. P-II SVMPs contain a disintegrin-like domain and have a molecular weight of 30-60 kDa, whereas P-III SVMPs, have a molecular weight in the range of 50 to 70 kDa, are composed of a cysteine-rich and disintegrin-like domain. Some of them have a quaternary structure, whereas a disulfide bond of a smaller lectin type C subunit is linked to the P-III SVMP subunit.³⁶ These snake venom metalloproteases activate prothrombin, defibrinogenesis, and coagulation X factor, which results in fibrinolytic stimulation.^37,38 They contribute actively to the breakdown of the extracellular matrix and induce an inflammatory response.³⁹ Snake venom metalloproteases have the potential to act as thrombolytic agents. For example, a fibrinolytic enzyme isolated from Agkistrodon contortrix has potent fibrinolytic activity (Figure 3). Alfimeprase recombinant form is employed in the treatment of abnormal blood clot formation by efficiently promoting thrombolysis in case of acute limb ischemia.⁴⁰

Snake venom hyaluronidase
Hyaluronidases are a diverse group of enzymes found in various organisms, primarily responsible for cleaving hyaluronic acid, a glycosaminoglycan molecule of high molecular weight. Some of these enzymes can also degrade other glycosaminoglycans, although at a slower rate.⁴¹ In snake venom, hyaluronidases function as spreading factors during envenomation, when hyaluronic acid present in the extracellular matrix of the local tissues gets broken down into smaller molecules. These venom hyaluronidases are classified as neutral endo-β-N-acetyl-D-hexosaminidases, with a different range of molecular weight from 33 kDa to 110 kDa. They display structural diversity due to post-translational modifications or truncation of transcripts. The translated proteins typically consist of 440 to 450 amino acids.⁴² Snake venom hyaluronidase facilitates the breakdown of internal glycoside bonds in acidic muco-polysaccharides and decreases the integrity and viscosity of connective tissues. As the hyaluronic acid barrier is ruptured, other venom components penetrate the tissues effectively. The breakdown of HA in the extracellular matrix enhances membrane permeability, making tissues more susceptible to injected fluids.⁴³

L-amino acid oxidase (LAAO)
The homodimeric flavoenzyme LAAO (EC 1.4.3.2) contains covalently linked flavin adenine dinucleotide and renders the snake venom its characteristic yellow colour (FADs). Each LAAO subunit has a molecular weight of 50-70 kDa and 110-159 kDa in its dimeric state.^44,45 The three major domains of the LAAO are substrate binding, FAD binding, and helical domains. It has been reported that the substrate-binding domain contains 7 mixed β-pleated sheets.⁴⁶ The enzyme engages with amino acid residues (serving as substrates) and oxygen to create a ternary complex, subsequently catalyzing the reduction of flavin semiquinone. During the reductive half-reaction, the FAD cofactor undergoes re-oxidation, generating hydrogen peroxide in the process, as described by Kang.¹⁸

The structural and functional resemblance of snake venom proteins to human counterparts allows them to interact with human physiological systems with high specificity. These similarities explain their potent effects in envenomation and their emerging therapeutic applications. Snake venom proteins exploit conserved biochemical pathways in humans, such as membrane dynamics (PLA₂), matrix remodelling (SVMPs), redox processes (LAAOs), and tissue permeability (hyaluronidases). Understanding the chemistry of venom proteins and their mimicry of human proteins aids in repurposing them for medical use, such as anti-inflammatory agents (PLA₂), thrombolytics (SVMPs), cancer therapeutics (LAAOs), and drug delivery systems (hyaluronidases). Snake venom proteins often exhibit enhanced efficiency and potency compared to their human analogues, providing a blueprint for designing synthetic or recombinant therapeutics. By linking the chemistry of these toxins to their similarities with human proteins, researchers can harness venom proteins’ dual roles-mitigating their toxic effects while leveraging their therapeutic potential.

Similarities between snake venom toxins and human proteins
As indicated earlier, understanding the sequence and structural similarities of the selected snake venoms and the human counterparts will be of high value for exploiting the therapeutic benefits of snake venom proteins. Investigating the similarity of human proteins with snake venom proteins will be of potential help to design the model compounds for targeting human diseases and to develop enzyme-based replacement therapy for the enzyme-deficient disorder or diseases in humans.

Phospholipase A₂ (PLA₂)
Sequence analysis reveals that human pancreatic phospholipase A₂‚ (accession number 3ELO_A) has the highest sequence similarity with svPLA₂‚ of Naja naja species (UniProt accession number P15445) with 98% query coverage, 48.03% identity and 69.4% similarity (Figure 4). However, the lowest sequence similarity is with group IIF secretory phospholipase A‚ isoform X1 (Homo sapiens) (accession number XP_011540257.1) with 63% query coverage, 38.67% identity and 48.0% similarity. However, snake venom PLA₂‚ (svPLA₂) enzymes share significant structural and functional similarities with human PLA₂‚ such as high catalytic activity, specificity towards phospholipids, and pro-inflammatory responses.⁴⁷ The domain structure of snake venom PLA₂‚ varies between different species, but generally consists of a calcium-binding loop, a helical domain, and a catalytic domain. The calcium-binding loop stabilizes the reactive intermediate,⁴⁸ while the helical domain stabilizes the enzyme structure and contains residues involved in substrate recognition. The amino acids in the catalytic domain are highly conserved and are responsible for phospholipid hydrolysis, and are highly specific to phospholipids with fatty acid chains of 16-20 carbon atoms.²⁶ In comparison, human PLA₂‚ also consists of a calcium-binding loop, a helical domain, and a catalytic domain, but has greater structural complexity and diversity due to alternative splicing, post-translational modifications, and the presence of multiple isoforms with different tissue expression patterns and substrate specificities. Human PLA₂‚ is classified into several subfamilies, including cytosolic PLA₂ (cPLA₂), calcium-independent PLA₂ (iPLA₂), secreted PLA₂‚ (sPLA₂), and platelet-activating factor acetyl-hydrolase (PAF-AH).⁴⁹ There are differences in the amino acid sequence and 3D structure that confer distinct biochemical and pharmacological properties to these enzymes.⁵⁰ For example, some svPLA₂‚ enzymes have a myotoxic or neurotoxic effect, while human PLA₂‚ is involved in the regulation of various physiological mechanisms such as inflammation, lipid metabolism, and cell signalling. With the identification of diverse targeted delivery systems with high specificity, the enzyme replacement therapeutic approaches are highly feasible to combat the pathophysiological effects related to the underexpression of PLA₂.

Metalloproteinase
Sequence analysis indicates that ADAM metallopeptidase domain 28 (Homo sapiens) (accession number XP_011542671.1) has the highest sequence similarity with snake venom metalloprotease of Naja atra species (UniProt accession number D3TTC2) with 96% query coverage, 43.16% identity and 58.5% similarity (Figure 5). Lowest sequence similarity is with ADAM metallopeptidase domain 9 (meltrin gamma), the isoform CRA_a (H. sapiens) (accession number EAW63283.1) with 68% query coverage, 36.74% identity and 54.0% similarity. Snake venom metalloproteases (SVMPs) and human metalloproteases (HMPs) are part of the broader metalloproteinase family, characterized by the presence of a zinc ion in the enzyme’s active site. In Class P-I SVMPs, alongside the catalytic domain, there is a disintegrin domain responsible for inhibiting platelet aggregation. Class P-II SVMPs feature a cysteine-rich domain that facilitates binding to the extracellular matrix, while Class P-III SVMPs encompass both a disintegrin-like domain and a cysteine-rich domain. In contrast, human HMPs are divided into six groups based on their domain structure. These metalloproteases play crucial roles in the regulation of various physiological mechanisms such as tissue regeneration and wound healing. However, their dysregulation has been implicated in various pathological conditions, including cancer and inflammatory diseases. Similarly, SVMPs contribute to the pathogenesis of oedema, inflammation, myonecrosis, skin damage, and the onset of cardiovascular failure.^22,51,52

Hyaluronidase
Sequence analysis indicates that hyaluronidase 3 and hyalurono-glucosaminidase 3 of Homo sapiens (accession number KAI2529754.1) has the highest sequence similarity with snake venom hyaluronidase of Ophiophagus hannah species (UniProt accession number V8PG63) with 82% query coverage, 48.65% identity and 63.0% similarity (Figure 6). Lowest sequence similarity is with hyaluronidase 2, partial (Homo sapiens) (accession number KAI2529781.1) with 11% query coverage, 32.08% identity and 47.8% similarity. Hyaluronidases are enzymes that break the glycosidic bond between glucuronic acid and N-acetylglucosamine to degrade hyaluronic acid, a significant component of the extracellular matrix. Despite playing a crucial role in the dissemination and toxicity of venom, snake venom hyaluronidases (svHA) have received less attention in comparison to exploring the human hyaluronidase enzyme.⁵³ Human and snake venom hyaluronidases both have conserved amino acid residues in the catalytic domain. The catalytic domain of hyaluronidases is classified as a glycoside hydrolase family 56 (GH56) domain and has a characteristic (β/α) 8 barrel fold. A non-catalytic linker found in the link domain of human hyaluronidase (hHA) is thought to be involved in protein-protein interactions and the regulation of enzyme activity. However, snake venom hyaluronidases are simple molecules and do not possess a separate link domain, but the catalytic domain contains surface-exposed loops that may participate in substrate binding. Some svHA also contain a C-terminal disintegrin-like domain, such as the hyaluronidase from the venom of the snake Bothrops asper, which may be involved in binding to integrins and other extracellular matrix proteins. Despite the similarities between the catalytic domains of human and snake venom hyaluronidases, there are also differences in their biochemical properties and substrate specificities.^54,55 For example, snake venom hyaluronidases often exhibit broader substrate specificity, degrading not only hyaluronic acid but also chondroitin sulfates, whereas human hyaluronidases are more selective. Additionally, venom hyaluronidases are generally more heat-stable and function effectively across a wider pH range compared to their human counterparts.

L-amino oxidase
Sequence analysis indicates that L-amino oxidase isoform 1 precursor and interleukin 4 induced 1 of Homo sapiens (accession number NP_690863.1) has more sequence similarity with snake venom L-amino oxidase of Ophiophagus hannah species (UniProt accession number P81383) with 96% query coverage, 37.17% identity and 56.5% similarity (Figure 7). Although the alignment with peroxisomal N(1)-acetyl-spermine/spermidine oxidase isoform 4 (Homo sapiens) (NP_997011.1) shows relatively high similarity (64.2%) within the aligned region, it spans only 56% of the query sequence. Snake venom LAAOs are typically monomeric proteins with molecular weights ranging from 50-140 kDa. They are known to contain two or more domains. N-terminal flavin adenine dinucleotide (FAD)-binding domain and a C-terminal catalytic domain are the two constitutive domains in these proteins. The catalytic domain is more variable and can vary in length and sequence composition, whereas the FAD-binding domain is highly conserved and crucial for enzyme function. Some snake venom LAAOs also contain additional domains, such as a C-type lectin-like domain or a disintegrin-like domain, which may contribute to their distinct biological activity and toxicity. LAAOs in humans are expressed in a variety of tissues, including the placenta, kidney, and liver. The molecular weight of human LAAOs is approximately 140 kDa and is a homo-dimeric protein. They contain three domains: N-terminal signal peptide, a middle domain, and a C-terminal FAD-binding domain. The middle domain is more variable and contributes to the substrate specificity and impacts the catalytic efficiency of the enzyme.¹⁵

The structural and functional similarities between venom proteins and human counterparts underpin their dual applications in venomics. The resemblance of venom proteins to human enzymes and receptors enables their direct application in drug development. For instance, PLA₂‚ interaction with lipid membranes inspires anti-inflammatory and anti-cancer strategies. Understanding these similarities helps predict venom protein interactions in the human body, enabling targeted inhibition of toxic effects while preserving therapeutic potential. By addressing these gaps, venomics can fully leverage the similarities between snake venom and human proteins to optimize drug discovery and improve antivenom therapies. While many venom proteins show promise as drug candidates, their inherent toxicity issues limit their clinical application. There is a lack of detailed structure-function studies and scalable production methods for venom-derived drugs. Traditional antivenoms are broad-spectrum but lack efficacy against specific venom components. There is limited integration of venomics data into antivenom production to achieve species-specific or toxin-specific efficacy.

Harnessing Snake venom proteins for biomedical breakthroughs
Interestingly, the same properties that make venom harmful in envenomation hold significant promise for developing innovative therapies. Snake venom proteins offer significant benefits due to their relatively low molecular mass. Their compact size makes them easier to be synthesized, both biologically and chemically, allowing for large-scale production. The simple structure of these proteins, in terms of their secondary and tertiary conformations, facilitates a clearer understanding of their roles and interactions, making them valuable templates for drug design. Furthermore, the flexible loops within snake venom proteins grant those unique biological activities and therapeutic potential. These loops can interact with a wide range of targets, adding versatility and efficacy, and they can be customized for specific binding with high precision, making them useful for molecular recognition and modulation. Since these proteins lack complex post-translational modifications, production, characterization, and application are simplified, reducing both complexity and costs. This simplicity also makes it easier to study their structure-function relationships and biological activities, offering insights into their mechanisms of action. There has been a mixed information in literature regarding the thermodynamic stability of the snake venom proteins. Such instability also has been pointed out to be a deterrent for the production of snake antivenom. Therefore, a clear investigation on the thermodynamic stability of different snake venom proteins should be taken up. This necessitates the production of mutant proteins with significant stability. Stable proteins are more likely to behave consistently and predictably, which is crucial for drug discovery and diagnostic applications.

Snake venom proteins for human therapeutics
Antibacterial activity
Toxins from various Viperid and Elapid species exhibit bactericidal effects. The basic PLA₂‚ extracted from Bothrops marajeonsis showed no inhibitory effect against Pseudomonas aeruginosa or Staphylococcus aureus,⁵⁶ while the acidic PLA₂‚ isolated from Bothrops erythromela inhibits the growth of Gram-positive bacteria but does not affect Gram-negative bacteria.⁵⁷ In contrast, basic PLA₂‚ derived from Daboia russellii pulchella demonstrated enhanced bactericidal activity against Gram-positive bacteria, while showing less impact on Gram-negative bacteria.⁵⁸ The bactericidal effect of PLA₂, especially the basic form, has been linked to its ability to disrupt bacterial membrane integrity.⁵⁹ Although catalytic activity is not the sole determinant of bactericidal effect, there appears to be a correlation between hemolytic and bactericidal activity of D. russellii pulchella PLA₂.⁵⁸ Additionally, p-bromophenacyl bromide (p-BPP) not only helps in reducing enzyme activity but also helps in destabilizing the cell wall of the bacteria.⁵⁹

Crotoxin A and B (PLA₂‚ -CA and PLA₂‚ -CB) from Crotalus durissus terrificus exhibit antibacterial activity against Ralstonia solanacearum.⁶⁰ Likewise, crotoxin B from C. durissus terrificus and daboiatoxin from Daboia russelli show inhibitory effect against two strains of Burkholderia pseudomallei (TES and KHW).⁶¹ While some PLA₂‚ variants display bactericidal activity against various range of bacteria, including Gram-positive and Gram-negative.^62,63 others specifically target Gram-positive bacteria.⁶⁴ This suggests a complex relationship between the bactericidal effects of PLA₂‚ and its sequence and structure.

L-amino acid oxidases (LAAOs) also exhibit antibacterial activity.^56,65-68 It has been reported that Bothrops leucurus venom can cause a dose-dependent inhibition of S. aureus growth, with a MIC value of 25 µg/ml. SvLAAOs from Crotalus adamanteus and Bothrops asper show antibacterial activity against S. aureus and Proteus mirabilis, similar to SvLAAOs from Bothrops venoms.^56,69,70 Additionally, Pseudomonas aeruginosa and Escherichia coli growth are inhibited by svLAAO from Bothrops pirajai venom.⁷⁰ Peptides derived from svLAAO showed enhanced antibacterial effects compared to the whole protein, suggesting that smaller peptide fragments may be promising candidates for novel peptide-based antibiotics.⁷¹ The bactericidal mechanism of svLAAOs is generally attributed to the induction of oxidative stress in bacterial cells, leading to disruption and increased permeability of the plasma membrane, ultimately causing cell death. This process is associated with the presence of hydrogen peroxide in the reaction medium.⁷²

The three-finger toxin (3FTx) family member, gamma toxin from the cobra Naja nigricollis venom, increases the membrane permeability of both Gram-positive and Gram-negative bacteria, causing bactericidal effects. Its physical interaction with primary membrane proteins demonstrates direct molecular activity on the components of Gram-negative and Gram-positive bacteria, namely lipopolysaccharide (LPS) and lipoteichoic acid (LTA), respectively. Disrupting the LPS layer and inhibiting LTA production leads to bactericidal effects.⁷³ Cardiotoxin 3 from venom of Naja naja atra, another 3FTx family member, shows a similar mode of action but is more effective against S. aureus than E. coli.⁷⁴

Lectins and their homologs, like BlL from Bothrops leucurus venom, also have antibacterial properties. BlL and a protein from Bothrops jararacussu venom inhibit S. aureus by interfering with biofilm formation.⁷⁵ The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for S. aureus are 31.5 µg/ml and 500 µg/ml, respectively, while for Bacillus subtilis, the corresponding values are 125 µg/ml and 250 µg/ml.⁷⁶

Antimicrobial peptides (AMPs) are a critical part of the innate immune defense on human skin. Two main categories of AMPs found in humans are defensins and cathelicidins, many of which are effective against Staphylococci.⁷⁷ These peptides help to eliminate bacteria from neutrophil phagosomes through phagocytosis. Like humans, snakes also produce cathelicidins, which are smaller cationic AMPs (cAMPs). These peptides are highly effective against various bacteria, enveloped viruses, and fungi.⁷⁸ Ophiophagus hannah cathelicidin (OH-CATH) and its analogues exhibit potent antibacterial and moderate hemolytic effects.⁷⁹ They are more effective than commonly used natural antibiotics against Acinetobacter species, including multi-drug-resistant Acinetobacter baumannii (MRAB) and methicillin-resistant S. aureus (MRSA).⁸⁰ AMPs rich in cysteine demonstrate broad antibacterial activity, with their positively charged surface and flexible shape facilitating interaction with the negatively charged components of bacterial membranes, thus enhancing their antibacterial effects.⁷⁸ CoaTx-II, a small peptide derived from Crotalus oreganus abyssus based on the primary structure of Lys49 PLA₂, shows antibacterial activity against drug-resistant clinical isolates. The peptide’s charged and aromatic amino acids are crucial for its interaction with bacterial cell membranes⁸¹ (Table 1).

Table (1):
Antibacterial activity of snake venom protein against different bacteria with their proposed active components

Analgesic and antinociceptive activity
Controlling pain with the best and most appropriate drugs is still a challenge in healthcare systems.⁸² Researchers have discovered that PLA₂‚ derived from snake venom exhibits efficacy in alleviating pain and inflammation in animal models experiencing arthritis and neuropathic pain.⁸³ Distinct mechanism of action and potent analgesic effects exhibited by the isolated neurotoxin, PLA₂‚ and myotoxin from snake venom make them potential therapeutic drugs for pain treatment.⁸⁴ Several studies have highlighted the analgesic potential of various snake venom toxins. For example, κ-bungarotoxin has been shown to exhibit significant analgesic effects in animal models.⁸⁵ Crotoxin has also demonstrated efficacy in treating neuropathic pain and inflammation.⁵⁵ These peptides work by rapidly and irreversibly inhibiting specific subtypes of neuronal acid-sensing ion channels (ASICs) found in the central nervous system (CNS).⁸⁶ ASICs are typically activated by protons, which induce pain sensations in a reversible manner. By blocking these channels, these toxins provide an analgesic effect in both acute and chronic inflammatory pain conditions.⁸⁷

Anti-arthritic and anti-inflammatory activity
Various animal models are being utilized to examine the pathophysiological effects of snake venom toxins on arthritis. For instance, the venom of Naja kaouthia has been shown to induce significant changes in arthritis biomarkers in mouse models, including alterations in paw and ankle diameter, urinary markers such as hydroxyproline and glucosamine, biochemical markers like acid and alkaline phosphatase, the molecular marker IL-10, and liver antioxidant parameters such as catalase and glutathione.⁸⁸ Additionally, the anti-arthritic and anti-inflammatory effects of the NN-32 peptide, derived from Indian cobra venom, were evaluated using mouse models of Freund’s complete adjuvant (FCA)-induced arthritis and carrageenan-induced inflammation. The results demonstrated that NN-32 peptide treatment significantly reduced physical and urinary parameters, serum enzyme levels, and cytokine levels compared to the arthritic control group. Furthermore, NN-32 peptide treatment effectively alleviated carrageenan-induced inflammation in rats, suggesting that this cytotoxic NN-32 protein possesses both anti-arthritic and anti-inflammatory properties.⁸⁹

The prolonged anti-inflammatory effects observed with crude Crotalus durissus terrificus venom are attributed to crotoxin, an isoform of PLA₂‚ by activating the formyl peptide receptors, which acts as a major factor in enhancing the anti-inflammatory effects.⁷⁶ In a related study, a single dose of Crotalus durissus terrificus venom (CdtV) results in prolonged effect of altering the inflammatory response with changes in the major symptoms such as paw edema and migration of the cells observed in the mice, where inflammation is induced by carrageenan compound similar to the effects observed with the crude venom.⁹⁰

Anti-cancer activity
Multiple research has stated that snake venom protein PLA₂‚ and LAAO possess anti-cancer property by inducing apoptosis mechanism leading to cell death, altering the cell cycle, reducing the cell growth to G0 and G1 phase, and further stops the proliferation of the cells, thereby helping in controlling the cancer metastasis in the body. Both apoptosis and necrosis are observed in cells treated with these enzymes. The cytotoxicity of PLA₂ is mainly attributed to its C-terminal region, which interacts with the cell membrane. In contrast, LAAO produces significant amounts of H₂O₂ through its enzymatic activity, leading to cell death by accumulating the reactive oxygen species (ROS) in the cells. Along with LAAO, some disintegrins are also known to induce apoptosis. The disintegrin derived from Naja naja venom has been reported to suppress the proliferation of MCF-7 (IC₅₀ = 2.5 ± 0.5 µg/mL), A549 (3.5 ± 0.5 µg/mL), and HepG2 (3 ± 0.5 µg/mL) cell lines.⁹¹ Additionally, some compounds extracted from the snake venom toxins are reported to possess anti-melanoma activity. The possible mechanism of action for anti-melanoma activity might be because of the highly specificity of the disintegrins for disrupting the integrity integrins.⁹²

PLA₂‚ and LAAO also demonstrate anticoagulant activity, presenting potential applications in cancer treatment, particularly in addressing venous thromboembolism frequently seen in cancer patients. Nevertheless, the precise mechanism by which these enzymes induce cell death remains unclear, particularly their interactions with death receptors, is not well understood and requires further investigation. Ensuring the selectivity of enzymes towards cancer cells is essential, as non-cancer cells are less sensitive to their cytotoxic effects. These enzymes exhibit stronger cytotoxic activity in cancer cells than in normal cells, making them promising candidates for potential use as chemotherapeutic agents. It is believed that these enzymes promote oxidative stress. PLA₂‚ during lipolysis, can generate ROS, while LAAO produces H₂O₂ both of which contribute to cell death¹³ (Table 2, Figure 8). 

Table (2):
Anticancer agents from snake venom proteins

Anti-viral activity
Recent studies have highlighted the antiviral effects of various snake venom components.^93,94 Non-cytotoxic fractions of Cdt venom have shown anti-viral activity against the measles virus, inhibiting its replication in Vero cells at concentrations of 0.1 µg/mL and 100 µg/mL.⁹⁵ Naja nigricollis venom has demonstrated in controlling the viral load in the human erythrocytes which are infected with the Sendai virus. Virus-infected cells exhibited tenfold higher susceptibility, by lysing the two among five venom toxins selected for the study, with 4 identified cytotoxins from Naja nigricollis venom showing that virus-infected cells were ten times more vulnerable to cytotoxic effects than healthy cells.⁹³ Additionally, LAAO from Bothrops jararaca has been shown to inhibit the viral growth of dengue virus, therefore possessing antiviral properties. Another study states that the cells infected with dengue type 3 virus (DENV-3) had reduced viral load after treatment with LAAO compared to untreated cells.⁷⁰

The venom of Naja siamensis contains an oxidized derivative of alpha-toxins and immunokines, which suppresses the lymphocytes infection caused by HIV and Feline Immunodeficiency Virus (FIV). There is a significant resemblance between the amino acid sequences of long-chain neurotoxins protein present in Naja siamensis and Bungarus multicinctus venoms and a short sequence in the HIV-1 gp120, suggesting that these molecules may compete for the same binding sites.^96,97 Additionally, a metalloprotease inhibitor isolated from Trimeresurus stejnegeri venom has the potential to block protease enzymes and prevent the formation of new virus particles.

PLA₂‚ from snake venom prevents HIV- 1 replication in primary human leukocytes by blocking viral entry before the virus uncoats and releases proteins from the viral capsid.^98,99

LAAO from venoms of Crotalus atrox, Pseudechis australis, and Trimeresurus stejnegeri showed dose-dependent inhibition of HIV-1 infection and replication by blocking the p24 antigen.^44,100,101 Free radicals like H₂O₂, produced during LAAO activity, contribute to the antiviral effects by preventing HIV infection and replication, though H₂O₂ interaction with catalases may reduce its antiviral activity.¹⁰¹ Snake venoms from Crotalus adamanteus, Oxyuranus microlepidotus, Bungarus candidus, Hydrophis cyanocinctus, Naja naja, Notechis ater, Naja sumatrana, and Naja kaouthia have also been reported to exhibit anti-HIV activity.^61,102,103

HIV entry requires CD4 and a coreceptor, primarily CCR5 or CXCR4, which determine viral tropism and disease progression. Discoveries of coreceptor roles and CCR5 mutations have provided key insights into HIV biology and therapeutic targets.¹⁰⁴

PLA₂‚ from snake venom prevents HIV-1 replication in primary human leukocytes by blocking viral entry before the virus uncoats and releases proteins from the viral capsid.^98,99,104 Anti-HIV activity has been demonstrated by synthetic peptides derived from PLA₂‚ including p3bv peptide, which blocks HIV-1 attachment to T cells by binding to the CXCR4 receptor, thereby preventing virus entry.¹⁰⁵ Crotoxin, a phospholipase isolated from Crotalus durissus terrificus venom, has been shown to inhibit HIV in vitro.¹⁰⁶ Chemokines and their derivatives can compete with the HIV-1 gp120 for binding to receptors like CXCR4, effectively suppressing HIV replication.^104,107

PLA₂ from snake venom has antiviral potency against SARS-CoV-2, particularly the dimeric form. On the other hand, dimeric PLA₂ catalytic activity determines its virucidal and antiviral properties. The unique ability of PLA₂ is to inactivate the virus from spreading infection.¹⁰⁸ Batroxobin is a safe drug that is used extensively for perioperative bleeding and is effective for a variety of illnesses, such as deep vein thrombosis and pulmonary embolism.¹⁰⁹ This provides the opportunity to introduce Batroxobin, which is isolated from Bothrops atrox venom and catalyzes the conversion of fibrinogen to fibrin (Pefakit Reptilase Time; Pentapharm), as part of an anti-COVID-19 therapeutic strategy aimed at preventing the deadly pulmonary embolism that can lead to severe SARS-CoV-2 infections.¹¹⁰

Obstacles encountered in the drug development journey
The most common application of animal toxins is as pharmacological tools for target validation. Although there have been many instances of success, the availability of authorized compounds possessing significant pharmacological properties derived from animal venoms remains limited. Challenges in developing animal toxin-based drugs are commonly linked to gaps in fundamental research, preclinical assessment, and clinical trials (Figure 9). Additionally, a lot of the difficulties encountered during the different phases of drug development are not been sufficiently documented in the scientific literature because the information about compounds that are in the development stage and for which the development is halted due to many internal problems, as they are protected by intellectual property rights.¹¹¹

During the initial phase of research
Studies utilizing animal toxins are not simple tasks because several concerns need to be overcome. Genomic analysis and cDNA library are the choices which can be explored to strengthen the research.¹¹² Recent studies have shown that these toxic molecules can be obtained by utilising organoid technology. However, this is still a challenging and daunting task.¹¹³

Second phase of research (preclinical phase)
The pharmaceutical applications of drugs formulated from snake venom toxins have been extensively investigated due to their diverse range of biological activities. These toxins showed shown significant activity in areas such as anticoagulation, anti-thrombosis, antimicrobial activity, analgesic properties, and potential for anti-tumour treatments. However, several limitations and challenges need to be resolved before using snake venom toxins as drugs. Selectivity, mode of action, formulation, stability, and cost of production are some constraints with toxin-based drugs.¹¹⁴ The selectivity of snake venom toxins may have off-target effects due to their non-specific binding to other proteins and enzymes, which can result in unwanted side effects. Therefore, the development of toxin-based drugs is a major challenge for achieving high selectivity for the target protein or enzyme. Understanding the mechanism of action of toxin-based drugs is vital in developing pharmaceuticals reliant on their biological activity. Nevertheless, it is challenging to determine the exact mechanism of action and the complexity of the interactions between toxins and their target molecules, which often requires extensive research and experimentation. Another challenge in the development of drugs based on snake venom toxins is that the size of the molecules is often large, complex, and unstable. However, compared to humans, these have better physicochemical properties. The purification, storage, and delivery of these drugs entail a multifaceted process that demands meticulous attention to factors like pH, temperature, and stability. The stability of snake venom toxins is a crucial factor in the development of drugs based on toxins. These molecules limit their therapeutic efficacy unless targeted delivery systems are chosen, are prone to degradation and can be rapidly eliminated from the body. Therefore, appropriate drug delivery systems should be developed. The cost of production of snake venom toxin-based drugs is also a major challenge. The extraction and purification process of toxins is both time-consuming and expensive, making it challenging to produce these molecules on a large scale, often rendering it infeasible. Purification also introduces complexity, as many of the proteins are bound to each other, especially when they are being purified under native conditions. Cloning and expression of certain snake venom proteins can help to overcome the problems associated with purification.^115,116 Despite the obstacles faced, there have been notable advancements in drug development through the utilization of snake venom toxins. For example, the snake venom derived Echistatin,¹¹⁷ has been used as an antiplatelet agent, and extensive research has been conducted on crotoxin, a constituent extracted from the venom toxins of the South American rattlesnake Crotalus durissus terrificus, because of its potential antitumor properties. To advance the formulation of snake venom toxin-oriented drugs and enhance their effectiveness and safety, additional research is required to tackle the associated challenges.²¹

Final phase of research (clinical trial)
The standard method for assessing certain drug-related issues involves assessing the efficacy, safety and the long-term effects. However, at times the studies lack access to specific population, especially children, pregnant women and elderly people.¹¹⁸ Therefore, the developmental process needs to address these constraints and identify which drugs need more epidemiological research. Electronic health care records for post-marketing and comparing the safety of the medication can be utilized to overcome these limitations.¹¹⁹ In addition, snake venom toxin-based drugs face regulatory challenges, particularly in countries where there is limited experience with these types of drugs. Moreover, the regulatory process for approving drugs is lengthy and not economical. There are also ethical considerations related to the applications of snake venom toxins in clinical trials, and obtaining venom, even for research purposes, is becoming a cumbersome process. The challenge also includes concerns about exposing patients to unnecessary risks or about the use of animal testing in the formulation of new and suitable drugs.¹²⁰ Due to these constraints, despite the high therapeutic potential of snake venom proteins, the transition from the laboratory to bedside is very slow and challenging. Drugs, which are in the phase of trials and being approved by the Food and Drug Administration (FDA) are listed in Table 3.

Table (3):
Food and Drug Administration (FDA) approved snake venom-based drugs

Snake venom proteins represent a promising candidate for human therapeutics, with active compounds showing significant effects in treating conditions such as cardiovascular diseases, cancer, and pain management. Notable compounds include Bradykinin Potentiating Peptides and snake venom metalloproteinases, which are being explored for their medicinal properties.

Snake venom proteins offer significant benefits due to their relatively low molecular mass. Regulatory challenges and scalability issues further hinder progress. Integrating nanotechnology into this process offers promising solutions, enabling improved drug delivery, enhanced stability, and targeted action while minimizing side effects. Nanocarriers such as liposomes, nanoparticles, and dendrimers can optimize venom-derived therapeutics, overcoming conventional barriers. This innovative approach not only enhances efficacy but also reduces the risks associated with venom-based drugs, marking a significant advancement in addressing critical challenges in modern drug development.

Integrating nanotechnology in drug development
Nanotechnology has become a promising approach in the development and formulation of drug, offering improved effectiveness and reduced adverse effects when compared to conventional therapies. Through use of nanoscale drug delivery systems, nanoparticle technology alters the kinetics, distribution, and release of drugs, providing several benefits, such as increased patient compliance, lower healthcare costs, and enhanced drug efficacy while reducing toxicity.¹²¹ Various nanoparticle formulation approaches have been investigated to improve the targeted delivery of venom peptides. This approach is gaining momentum due to its ability to reduce systemic toxicity and improve therapeutic efficacy. For instance, the use of nanoparticle systems that efficiently destroy tumor cells directed by immune cells to play an important role in preventing the tumors development. Recent studies have focused on the development and exploration of nanoparticle-conjugated venom peptides to enhance therapeutic efficacy and reduce toxicity.¹²²

Establishing the efficacy, potency, and safety of enzymatic toxins is crucial before they can be developed into chemotherapeutic agents. Targeted delivery approaches, such as nanoparticles formulation or conjugating with the ligands or production of monoclonal/polyclonal antibodies, need to be explored to recognize cancer cells. NN-32 purified toxin and toxin conjugating with nanogold GNP-NN-32 demonstrates remarkable cytotoxic potential against MCF-7 and MDA-MB-231 cell lines, effective interaction with dose and duration exposure.¹²³

Formulation of gold nanoparticles with Naja kaouthia cytotoxin 1 (NKCT1) have a synergistic effect that reduces the required dosage and course of action of the NKCT1. This controlled release of NKCT1 to target cells via GNP also increases the cytotoxic effect by two to threefold and minimizes the toxic effects of NKCT1. Due to its ability to induce cell cycle arrest, promote apoptosis, and regulate nuclear fragmentation, conjugation has shown high antileukemic action. GNP-NKCT1 treatment supports the emerging concept that conjugating nanoparticles may prove advantageous for leukemia, as GNP-NKCT1 regulates various biochemical pathways by inhibiting transcriptional and translational levels.¹²⁴ Chitosan nanoparticles encapsulated with Echis carinatus snake venom result in increased efficacy compared to traditional adjuvant systems and also enhance the stability of the snake venom proteins in physiological systems.¹²⁵ In another study, the apoptotic action of venom from Walterinnesia aegyptia (WEV) on breast cancer cells using free WEV and WEV formulated with silver nanoparticles has been able to inhibit the cell proliferation in a dose-dependent manner, with Walterinnesia aegyptia venom silver nanoparticle conjugate (WEV-NP) exhibiting greater efficacy and substantially enhancing the anticancer properties of WEV, according to WEV conjugated with silica nanoparticles (WEV-NP).¹²⁶ In a recent study, CdtV (rattlesnake) was used to develop a fibrin sealant. This enzyme, which resembles thrombin, can convert fibrinogen into fibrin, which can naturally gel and is non-toxic, non-immunogenic, and biodegradable. For the formulation of fibrin sealant, multi-layered carbon nanotubes were utilized and nanohydroxyapatites for potential application in bone regeneration.¹²⁷ The utilization of targeted delivery systems in employing snake venom toxins as therapeutic agents is crucial due to their ability to enhance specificity and minimize side effects on normal cells.