Insights into the antiviral activity of phospholipases A₂ (PLA₂s) from snake venoms

Abstract

1. Viral diseases: a public health problem

Viruses are associated to several endemic diseases, including Enterovirus [1], HPV (Human papillomavirus) [2], HIV (Human immunodeficiency virus) [3] and HSV (Herpes simplex virus) [4], as well as in outbreaks as Ebola virus, ZIKV (Zika virus), Influenza virus, YFV (Yellow Fever virus), DENV (Dengue virus) and, currently, the SARS-CoV-2 (Severe Acute Respiratory Coronavirus 2) [[5], [6], [7], [8], [9]]. Most of the reported outbreaks since 1980 were related to virus infections [10], which are still a global burden for public health and economy. In addition, due to their genetic diversity, viruses are able to infect a wide range of hosts that can result in host jumps after zoonotic contacts [11,12].

Pandemics caused by viruses are usually severe and can claim up to million lives, as shown during the pandemic of H1N1 in 1918 [13], H1N1 ‘swine flu’ in 2009 [14] and Coronavirus Disease 2019 (COVID-19) [15], that infected 4,993,470 people and caused 327,738 deaths until May 22, 2020 in worldwide according to World Health Organization. Furthermore, the global incidence of dengue has grown dramatically in recent decades. It is estimated that 100 to 400 million cases of dengue occur annually worldwide [16].

Viral infection depends on the successful replication into the host cells [17,18]. In general, the replicative cycle starts by the viral particle attaching to specific receptors in the surface of host cells that triggers the viral entry by endocytosis (non-enveloped or enveloped virus), membrane fusion (enveloped virus) and direct penetration [[19], [20], [21]]. After internalization, the capsid is released into the cytoplasm, allowing viral genome uncoating [22], which is replicated to produce copies of the genome and translated to viral proteins. In the endoplasmic reticulum (ER) and Golgi complex, the viral structure is assembled, matured and then forwarded to host cell membrane, where the progeny of virus particles is released [23].

Currently, specific antiviral drugs and vaccines are not sufficient to control emerging and reemerging viral diseases [24,25]. Thus, the discovery of novel antiviral drugs is mandatory. In general, antiviral therapy is the only approach to specifically treat viral infections, abrogating viral replicative cycle [26]. However, due to the high genetic variability, viruses can rapidly acquire resistance to antiviral treatment, especially RNA viruses [[27], [28], [29]]. Furthermore, antiviral therapy and the prolonged treatment can cause several adverse effects, including gastrointestinal effects, fatigue, headache, neuropathy and liver toxicity [[30], [31], [32]]. In addition, there are no antivirals to all diseases and the only course is supportive therapy and, thereby, numerous innovative drugs have been developed from natural prototypes such as aspirin (anti-inflammatory) and morphine (analgesic) [33].

In this way, a diversity of compounds isolated from natural sources has set grounds for further advances in drug development against various diseases [34]. Among them, many drugs based on snake venoms were approved by the FDA or are involved in preclinical or clinical trials for a variety of therapeutic applications [[35], [36], [37], [38], [39]]. The development of snake venom-derived drugs gained a significant improve since the discovery of bradykinin-potentiating peptides (BPP) isolated from the Brazilian arrowhead viper (Bothrops jararaca) venom, which allowed the development of captopril, an inhibitor of the angiotensin-converting enzyme that is widely used against hypertensive process [40,41]. Besides that, other snake venom-derived drugs have been found in clinical use, such as tirofiban and eptifibatide (antiplatelet agents) [42,43], batroxobin, moojenin and vivostat (anticoagulant agents) [[44], [45], [46], [47]]. Other drugs which comprise molecules from snake venom as scaffolds are also being explored in preclinical studies [48].

Due to this therapeutic potential, snake venom toxins have been widely explored for the discovery of new bioactive compounds and stand out as an alternative source for therapeutics for a variety of diseases, including life-threatening viral illnesses [[49], [50], [51], [52]].

2. Phospholipases A₂ from snake venom

Phospholipases (EC 3.1.) family is widely distributed in nature and includes hydrolase enzymes, which are essential for phospholipid metabolism and for the regulation of membrane lipids, membrane composition, signaling, digestion, and inflammation [53]. These proteins are classified into four major families (A, B, C and D) based on the site cleaved in the phospholipid molecule [54,55].

Among phospholipases family, the Phospholipases A₂ (PLA₂s) are the most studied group [53,54]. These enzymes hydrolyze 2-acyl ester bond to 2-sn phospholipids, releasing free fatty acid and lysophospholipids [56,57]. The free fatty acids (arachidonic acid) can be converted into eicosanoids (prostaglandins, thromboxanes, prostacyclins and leukotrienes), which are associated to a range of physiological and pathological effects, such as inflammation and platelet activation. In addition, the lysophospholipids are also related to a variety of physiological roles in cell signaling [53,58].

PLA₂s are classified into six groups: cytosolic (cPLA₂), Ca(²⁺)-independent (iPLA₂), platelet-activating factor acetylhydrolase (PAF-AH), lysosomal PLA₂ (LyPLA₂), adipose specific PLA₂ (AdPLA₂) and secretory PLA2 (sPLA₂) [53]. In addition, the sPLA₂s are divided into the following groups: IA, IB, IIA, IIB, IIC, IID, IIE, IIF, III, V, IX, X, XIA, XIB, XII, XIII and XIV [53,54]. PLA₂s from snake venom belong to the group of secreted type of enzymes (sPLA₂s) and can be classified into the structural group IB (in Elapidae snake venoms), which exhibits homology to the mammalian pancreatic juice PLA₂, and also into the group IIA (in Viperidae snake venoms), that is homologous to the mammalian ‘inflammatory’ PLA₂ [59]. Although the PLA₂s family is more frequent in snake venom, recent proteome studies have demonstrated that Phospholipases B (PLB) can also be found in snake venom [[60], [61], [62]].

sPLA₂s are proteins with molecular mass of about 14 kDa, pH optimum at 7 and share a conserved catalytic mechanism based on a His/Asp dyad using Ca²⁺ as an essential cofactor for the catalytic activity. Group II of sPLA₂s presents an extended C-terminal segment (5–7 amino acids) [63,64] and is subdivided into two main subgroups, depending on the amino acid residue at position 49 in the protein primary structure. Aspartate (Asp49 or D49) sPLA₂s are enzymatically active, while lysine (Lys49 or K49) sPLA₂s present no enzymatic activity [[65], [66], [67]]. However, there are further variants, as the serine (Ser49), asparagine (Asn49) or arginine (Arg49) [[68], [69], [70]].

Lys49 PLA₂s are devoid of catalytic activity due to their inability to bind Ca²⁺, a key cofactor for PLA₂ activity. Although the lack of enzymatic activity, the Lys49 PLA₂ homologues have shown to display toxicity, especially myotoxicity [67]. The toxicity of Lys49 proteins can be related to a cluster of cationic and hydrophobic/aromatic amino acid residues located at the C-terminal region of these toxins [71,72].

Therefore, the cytotoxicity of sPLA₂ is probably mediated by the interaction between the C-terminal region and the plasma membrane [73,74]. Moreover, the PLA₂ effects can be mediated through the integrins and other receptors, such as vascular endothelial growth factor receptor-2 (VEGFR-2), and M–Type receptors [[75], [76], [77]]. Recently, it was demonstrated that the cell surface nucleolin interacts with and internalizes the PLA₂-like Bothrops asper myotoxin-II, which is responsible to mediate its toxic activity [78].

sPLA₂s from snake venom can act on cell membranes of specific tissues inducing several pharmacological actions such as myotoxicity, neurotoxicity, cardiotoxicity, platelet aggregation activation or inhibition, hypotension, edema among others [57,79]. In this scenario, these proteins have emerged as a potential therapeutic model, since numerous studies have focused on their microbicidal [80], antitumor [[81], [82], [83]], antiangiogenesis [84], antiparasitic [[85], [86], [87]] and antiviral activities [51].

The development of efficient antiviral therapies has become a global health emergency. In this sense, several researches have demonstrated the antiviral activity of sPLA₂s from snake venom against human viruses, including DENV, YFV, HCV and others [[88], [89], [90], [91], [92]]. Hence, the present review aimed to summarize the sPLA₂s from snake venom that were previously described to possess antiviral activity, highlighting the mechanisms of action of sPLA₂s against different stages of virus replication cycle (Table 1 ).

3. sPLA₂s from snake venom with antiviral effects

4. Proposed antiviral mechanism of sPLA₂s from snake venom

Findings from the current literature about the antiviral activity of toxins (Table 1) are heterogeneous, since authors developed a variety of assays/models using different sPLA₂s and viruses. The virucidal model corresponds to the strategy in which the toxins act directly on the virus particles before infecting the cell monolayer; in the pre-infection model, the uninfected monolayers are previously treated with toxins before viral infection; and in the post-infection model, cell monolayers are adsorbed with the virus followed by toxin treatment. In this sense, many studies raised the following questions: in which stages of virus replication are the sPLA₂s able to interfere? Does the antiviral action of sPLA₂s depend on their catalytic activity? Based on this, we summarize a possible model of antiviral action mediated by sPLA₂s from snake venom.

As discussed above, sPLA₂s have demonstrated to be potent antiviral inhibitors by interfering in different stages of virus replicative cycle as entry steps, replication and release (Fig. 1 ). Current studies have reported that the antiviral action of sPLA₂s on steps of viral cycle can occur through a direct action upon viral particle and/or by an interaction with virus or host cell components.

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Fig. 1

Schematic representation of the mechanisms of action of sPLA₂s from snake venom on viral replicative cycle. sPLA₂s, which possess antiviral activity, are indicated in early and/or late stages of the viral life cycle: entry, replication and release. Mt-I and Mt-II (Bothrops asper), BthTX-I (Bothrops jararacussu), crotoxin, PLA₂-CB and PLA₂-IC (Crotalus durissus terrificus), CM-II-sPLA₂ and Nmm_CMIII (Naja mossambica mossambica), nigexine (Naja nigricollis), taipoxin (Oxyuranus scutellatus), BlK-PLA₂ and BlD-PLA₂ (Bothrops leucurus) are demonstrated.

Regarding the virucidal activity, studies have shown that both catalytically-active sPLA₂s (crotoxin, PLA₂-IC, PLA₂-CB, Mt-I and CM-II-sPLA₂) and catalytically-inactive sPLA₂s (Mt-II and BthTX-I) indicated virucidal activity preferentially against enveloped viruses, such as DENV (serotypes 1, 2 and 3), YFV, ROCV, OROV, MAYV, HCV, JEV, MERS-CoV, SINV, FLUAV, SeV, VSNJV, HIV-1 and HSV-1 [[88], [89], [90],92,114].

It is proposed that the potent virucidal activity of sPLA₂s against enveloped viruses is likely associated with the ability that catalytically-active sPLA₂s have to cleave glycerophospholipids in the virus lipid envelope, and it is reasonable to propose that sPLA₂s also present domains that are capable to interact with viral envelope components, which could lead to viral envelope disruption, hence resulting in exposure of the viral content (viral inactivation) and compromising the early stages of viral replication. Additionally, Muller and colleagues [88,89], through a steric and electrostatic analysis of the interaction of PLA₂-CB with the DENV envelope lipid bilayer, showed that PLA₂-CB probably accesses the DENV lipid bilayer through the pores found on each of the twenty 3-fold vertices in the E protein shell on the DENV surface, which would allow the glycerophospholipid cleavage on the virus envelope and destabilization of the E proteins. Interestingly, it has been demonstrated that the structural organization and lipid composition of viral envelope may influence the antiviral efficiency of some sPLA₂s, suggesting that the virucidal mechanism mediated by sPLA₂s is specific [92].

Independent studies have revealed that sPLA₂s such as crotoxin, PLA₂-IC, PLA₂-CB, Bl-PLA₂ and BthTX-I are also able to dramatically impact the entry, replication and release of viruses by targeting host cell components [[88], [89], [90], [91]]. To gain insights into these viral cycle stages, it was demonstrated that Nmm_CMIII, taipoxin and nigexine prevented the intracellular release of HIV-1 Gap p24 proteins from the viral capsid (inhibition virus entry) by a direct binding to membrane receptors of host cells [107]. In addition, PLA₂-CB was able to disrupt HCV replication probably by an interference in lipid metabolism of host cell [90].

It was demonstrated through the use of both specific sPLA₂ inhibitors and the catalytically-inactive sPLA₂s that the antiviral effect of the major tested catalytically-active sPLA₂s, such as crotoxin, PLA₂-IC, PLA₂-CB, BlD-PLA₂, Mt-I and CM-II-sPLA₂ is significantly higher when presented their functional catalytic site to sPLA₂s with no enzymatic activity (BthTX-I, BlK-PLA₂ and Mt-II) [88,91,92,114].

In order to corroborate with the data from the current literature, we performed docking simulations between the sPLA₂s from Bothrops asper venom and three phospholipids found in the DENV envelope, which are 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) and 1-Palmitoyl-2-oleoylphosphatidylserine (POPS) [118]. The molecular docking simulations were done by using the 3D crystal structure of Mt-I (PDB: 5TFV) and Mt-II (PDB ID 4YV5) retrieved from Protein Database (https://www.rcsb.org/).

We simulated the interaction in the enzymatic site of sPLA₂s with palmitoyl phospholipids (head or complete structure) using AutoDock Vina software [119]. The predicted affinity between sPLA₂s and palmitoyl phospholipids (head or complete) was similar (Table 2 ). Concerning to the interaction with the phospholipids head, Mt-I showed a higher affinity to POPC, POPE and POPS (−5.1, −4.7 and −5.0 kcal/mol, respectively) related to Mt-II (Table 2). When we simulated the docking with the complete palmitoyl phospholipid molecule, Mt-I also has a higher affinity than Mt-II (Table 2).

Although we do not observe a strong difference in the affinity between Mt-I and Mt-II for palmitoyl phospholipids, it is possible to note structural variation in the enzymatic site of these two toxins. Compared to Mt-II, the enzymatic site of Mt-I (Fig. 2A) is more suitable due to a smaller aspartic acid radical group. The van der Waals radii volume of aspartic acid is 91 and hence it is more prominent, while the lysine has a volume of 135, and this results in less space in enzymatic site entrance in Mt-II (Fig. 2B). This difference could create an enzymatic site more restricted to palmitoyl phospholipid entrance/binding and be partially responsible for the absence of enzymatic activity in Mt-II [120]. In addition, the enzymatic activity of Mt-I can be attributed to highly conserved catalytic site formed by the amino acid residues His48, Asp49, Tyr52 and Asp99. Asp49 coordinates the hydrolysis reaction of phospholipids together with the residues of the Ca2+ binding loop, essential in the catalytic activity of PLA₂s. The substitution of lysine residue at the same position affects the ability of this protein to bind to Ca^2+, resulting in the absence of catalytic activity [92].

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Fig. 2

Docking simulations between sPLA₂s and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). Mt-I (A) and Mt-II (B) are showed as surface and the enzymatic site is colored. The amino acids from the enzymatic site are H48 in red, D49 (Mt-I) or K49 (Mt-II) in blue, and Y52 in green. The POPC is showed as wire structure. The aspartic acid has a smaller volume than lysine, which may result in a less open entrance in Mt-II (panel B) compared with Mt-I (panel A). H: histidine; D: aspartate; K: lysine; Y: tyrosine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Despite the stronger antiviral activity is associated with the enzymatic activity, the antiviral mechanism of sPLA₂s does not depend exclusively on their catalytic site, since Lys49 sPLA₂s and inhibited catalytically-active sPLA₂s were also able to show antiviral effects, suggesting that sPLA₂s may possess different mechanisms of action. However, additional studies with different Lys49 from snake venom are required to better characterize the antiviral potential of this protein class.

Functional and structural studies have described that the activity of Lys49 PLA₂s from snake venom toward cell membranes in myotoxic mechanism involves an allosteric transition, and the participation of two independent interaction sites with the target membrane [67,72,[121], [122], [123]]. The action of Lys49 PLA₂s is related to a cluster of cationic and hydrophobic/aromatic amino acid residues located at the C-terminal region of this toxin. These two conserved regions in most Lys49-PLA₂s are designed by “cationic membrane-docking site” (MDoS), which are formed by the strictly conserved C-terminal residues (Lys115 and Arg118), eventually aided by other cationic and exposed residues such as Lys20, Lys80, Lys122 and Lys127; and the “hydrophobic membrane-disruption site” (MDiS) formed by residues of Leu121 and Phe125. The key step for protein activation is the binding of a fatty acid at the hydrophobic channel, which leads to allosteric transition and structure stabilization exposing MDoS and MDiS to the membrane, following by the insertion of the MDiS region from both monomers into the target membrane. This penetration disrupts the lipid bilayer, causing alterations in the membrane permeability, highlighted by a prominent influx of ions (i.e., Ca²⁺ and Na⁺), and eventually, irreversible intracellular alterations and cell death [123].

According to myotoxic mechanism of Lys49 PLA₂s from viperid snake venoms, it is proposed that the fatty acids which are important to protein activation may come from membrane phospholipid hydrolysis by catalytic PLA₂s (Asp49), highlighting the synergism between Asp49 PLA₂s and Lys49 PLA₂ in snake envenomation [124]. In this way, the antiviral effects of the Lys49 PLA₂s from snake venom, showed in this review, may be associated to fatty acids from the catalytic activity of cytosolic PLA₂ (cPLA₂) from virus lipid envelope, once it was demonstrated that enzymatic activity of the cPLA₂ is required for replication of various virus [[125], [126], [127]]. Muller and colleagues [126] showed that the pharmacological inhibition of a cellular phospholipase, cPLA₂, using a specific small-molecule inhibitor, drastically reduces coronavirus RNA synthesis and, as a consequence, protein accumulation and the production of infectious virus progeny. In addition, cPLA₂ activity was shown to be critically involved in the production of infectious progeny of HCV and DENV [128].

5. Concluding remarks: sPLA₂s as a possible useful tool for the development of antiviral compounds

The present review highlighted that PLA₂s from snake venom have become valuable as pharmacological tools and/or therapeutic approaches due to their extremely high specificity and potent activity against microbial infection. Regarding to antiviral properties, we highlighted the following remarks: (i) the antiviral effects of sPLA₂s can be mediated by either a dependent or independent catalytic mechanism; (ii) sPLA₂s-antiviral effects are more evident against enveloped virus; (iii) sPLA₂s promoted the blockage of viral entry into host cells by the direct action on the viral particle, resulting in glycerophospholipids cleavage and destabilization of viral envelope proteins; (iv) the structural organization, physicochemical composition, and the curvature and fluidity of viral envelope may influence in the antiviral efficiency of some sPLA₂s; (v) sPLA₂s promoted the blockage of entry, replication and release of virus probably by the interference on the host cell components.

The structure and function of sPLA₂s from snake venom have been widely explored. Homology studies with sPLA₂s have demonstrated highly conserved regions in these proteins capable of disrupting the integrity of membranes and provoking many pharmacological effects. Despite extensive studies on sPLA₂s in over decades, there is few of them focusing on mechanistic aspects of the antiviral activities and to date are limited to in vitro and in silico models. It is important to note that these sPLA₂s showed damage effects in vivo, such as myotoxicity and inflammation. Thus, further in vivo studies for attesting antiviral effects of sPLA₂s need to be addressed to investigate their safety, toxicity and pharmacokinetics. Taken together, new structural and functional studies with sPLA₂s are essential to discover new relevant motifs responsible for the antiviral activities that would allow the future use of these proteins or peptides for the design of antiviral drugs, capable of ensuring more stability and targeting the specific site of action.

Acknowledgements

References