Antimicrobial Compounds from Skin Secretions of Species That Belong to the Bufonidae Family

Skin secretions of toads are a complex mixture of molecules. The substances secreted comprise more than 80 different compounds that show diverse pharmacological activities. The compounds secreted through skin pores and parotid glands are of particular interest because they help toads to endure in habitats full of pathogenic microbes, i.e., bacteria, fungi, viruses, and protozoa, due to their content of components such as bufadienolides, alkaloids, and antimicrobial peptides. We carried out an extensive literature review of relevant articles published until November 2022 in ACS Publications, Google Scholar, PubMed, and ScienceDirect. It was centered on research addressing the biological characterization of the compounds identified in the species of genera Atelopus, Bufo, Duttaphrynus, Melanophryniscus, Peltopryne, Phrynoidis, Rhaebo, and Rhinella, with antibacterial, antifungal, antiviral, and antiparasitic activities; as well as studies performed with analogous compounds and skin secretions of toads that also showed these activities. This review shows that the compounds in the secretions of toads could be candidates for new drugs to treat infectious diseases or be used to develop new molecules with better properties from existing ones. Some compounds in this review showed activity against microorganisms of medical interest such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Coronavirus varieties, HIV, Trypanosoma cruzi, Leishmania chagasi, Plasmodium falciparum, and against different kinds of fungi that affect plants of economic interest.


Compounds with Antibacterial Activity
After antibiotic misuse or overuse, it is almost inevitable that bacteria will become resistant to antibiotics. This kind of situation demands the development of new antiinfective drugs. The pharmaceutical industry attempts to meet this need through the timely redesign and modification of existing antibiotics and the development of new ones [31].
Research on the therapeutic potential of extracts and compounds isolated from true toads has intensified recently. Some studies support using secretions from bufonids in common treatments for bacterial diseases [32]. The water-soluble secretion of the parotid gland and the methanolic extract of the skin of the Sudanese toad Duttaphrynus melanostictus showed antibiotic activity against Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, Staphylococcus aureus, and Staphylococcus epidermidis; however, no action was found against methicillin-resistant S. aureus [33]. Sales et al. [34] observed that combining antibiotics of the aminoglycoside and β-lactam groups with the glandular secretion of Rhinella jimi led to a significant reduction in the minimal inhibitory concentration (MIC) value, mainly in strains of E. coli, Pseudomonas aeruginosa, and S. aureus. This indicated that components of the venom of R. jimi have significant pharmaceutical value in combination with available antibiotics. Other studies evaluated the antibiotic capacity of secretions from the dorsal skin of Phrynoidis asper extracted with deionized water, and demonstrated their action against the Gram-positive pathogens S. aureus and Bacillus subtilis with MIC of 12.25 ± 0.4 and 25 ± 1.3 µg/mL, respectively [35]. On the other hand, Thirupathi et al. [36] observed antibacterial activity from skin secretions from the Indian toad Bufo melanostictus on E. coli, S. aureus, P. vulgaris, and K. pneumoniae through the Kirby-Bauer Test.
In the Cundinamarca forest region of Colombia, Rhinella toads are used to treat erysipelas, an infection caused by the multi-drug-resistant bacterium Streptococcus pyogenes [37]. The Rhinella marina toad from the Brazilian Amazon has also been used to treat patients with erysipelas. Additionally, analysis of the crude extract and fractions of secretions from R. marina showed biological activity against S. aureus , P. aeruginosa, and E. coli [32]. In northeastern Brazil, the skin and fatty tissue of Rhinella jimi are used to treat some infections, such as, sore throat, flu, cough, and earache [13]. Bufadienolides are the most abundant compounds in the toads' cutaneous secretions [38,39]. Their basic structure consists of a steroid nucleus of 17 carbon atoms arranged in four rings, and an α-pyrone ring at the C17-position, like in marinobufagin and telocinobufagin. It is important to mention that chemical modifications at various steroid molecule positions can generate remarkable differences in their antimicrobial activity (Figure 1). Cunha Filho et al. [40] used extraction with chloroform to isolate two bufadienolides, marinobufagin and telocinobufagin, from skin secretions of the Brazilian toad Bufo rubescens. The antibacterial capacity of both molecules was evaluated against E. coli ATCC 25922 and S. aureus ATCC 25213 and showed activities better than amoxicillin, imipenem, and trimethoprim antibiotics, especially against the Gram-positive bacteria (Table 1). Further, these authors determined that the two molecules can promote and increase cardiac contraction force. Both bufadienolides have been identified in toads of the genera Atelopus [41], Rhinella, Rhaebo [42], and Peltopryne [43].
The increasing spread of resistant bacteria has led to the constant search for bioactive compounds. The study of amphibian antimicrobial peptides (AMPs) has been employed as a folk remedy to treat bacterial infectious diseases. One group of these AMPs are cathelicidins, identified in the skin secretion of the Asian toad Bufo bufo gargarizans. Two sequences coding for these peptides were found through a skin transcriptomic analysis; they were chemically synthesized and named BG-CATH37 and BG-CATH(5-37). The first is formed by 37 amino acids, the second by 32, and both are rich in glycine and arginine residues. The antibacterial activity of these peptides was evaluated against E. coli, S. aureus, P. aeruginosa, and against aquatic bacteria (Aeromorus hydrophia, Vibrio harveyi, Vibrio vulnificus, Vibrio splendidus, Lysogenic bacterium, and Streptococcus iniae), these cathelicidins showed weak antimicrobial activity against human pathogenic bacteria (MIC > 200 µg/mL), but they showed strong antimicrobial activities against aquatic bacteria (MIC 3.125-40 µg/mL) from their habitat [44]. Other antibacterial peptides isolated from this toad were buforin I and buforin II (Table 1) [45]. These AMPs are from the histone family are composed of 39 and 21 amino acid residues, respectively, and can adopt a secondary structure of an alpha helix ( Figure 2). The parent peptide buforin I was found to have a high degree of sequence similarity to the N-terminal segment of the histone H2A DNA-binding protein, leading to the assumption that this AMP is structurally similar to histone H2A and would have similar antibacterial effects to buforin II [46]. Buforin I showed antimicrobial activity against a wide range of microorganisms, including the Gram-positive, Bacillus subtilis, S. aureus, Streptococcus mutans, Streptococcus pneumoniae, Pseudomonas putida; the Gram-negative bacteria E. coli, S. typhimurium, Serratia sp., and the fungi Candida albicans, Cryptococcus neoformans, and Saccharomyces cerevisiae [45]. Subsequently, Roshanak et al. [47] demonstrated that this peptide has an antibacterial effect on the most resistant and sensitive microbial strains, Staphylococcus salivarius and Clostridium perfringens, with a minimum inhibitory concentration (MIC) of 4 and 16 µg/mL, respectively. Furthermore, buforin I inhibited the formation of biofilms. Additionally, the thermal stability of the peptide in plasma at different temperatures was evaluated, and hemolytic and cytotoxic assays were performed, indicating that this peptide has the pharmaceutical potential to be used as an antibiotic in infectious diseases (Table 1). S. aureus (4 µg/mL), S. pneumoniae (4 µg/mL), B. subtilis (2 µg/mL), S. mutans (2 µg/mL), P. putida (2 µg/mL), E. coli (4 µg/mL), Serratia spp. (4 µg/mL), S. typhimurium (1 µg/mL). S. mutans (2 μg/mL), P. putida (2 μg/mL), E. coli (4 μg/mL), Serratia spp. (4 μg/mL), S. typhimurium (1 μg/mL).   Meanwhile, buforin II, a peptide derived from the digestive system with endoproteinase Lys-C, retains the residues from Thr16 to Lys 36 of buforin I, and is more than 12 times more potent than buforin I [48]. A proline hinge region in buforin II is essential for inducing membrane permeation in E. coli [46,49]. It has been suggested that buforin II inhibits cellular processes by interfering with DNA and RNA metabolism [50].

Compounds with Antifungal Activity
Skin secretions of many toads contain peptides with antifungal activity. These peptides are stored in a granular gland located mainly in the skin of the dorsal region. Most amphibian species produce one or more peptides with potent activity against fungi [7].
The skin secretions of Leptophryne cruentata show antifungal activity against Trichophyton mentagrophytes; forty microliters caused an inhibition halo of 14.5 ± 2.9 mm diameter [51]. Furthermore, the skin secretions of Rhinella icterica showed antifungal activity against Candida krusei [28]. The bufadienolides arenobufagin, gamabufotalin, and telocinobufagin were identified in the skin secretions of the toad Anaxyrus boreas. These compounds inhibited the growth of the Batrachochytrium dendrobatidis strain, and their MIC were 12.9, 50.0, and 14.3 μM, respectively [7]. These bufadienolides were also identified in Rhinella horribilis [52]. The discovery of these compounds, effective against these fungal strains, will allow the development of therapeutic agents that treat infections caused in humans and other animals [53]. One alkaloid called dehydrobufotenine was found in the skin secretions of the toad Bufo arenarum. It showed fungicidal activity against phytopathogenic fungi that affect plants of economic interest. Further, forty-five analogs of dehydrobufotenine were synthesized and evaluated for their activity. Compounds 6, 16c, 16d, 16h, 16j, and 19 showed similar or higher activity than the control carbendazim at 50 μg/mL. Dehydrobufotenine and most of its analogues showed higher activity against Alternaria solani (5-400%) than the controls chlorothalonil and carbendazim at 50 μg/mL. Their fungicidal activity is shown in Table 2. Meanwhile, buforin II, a peptide derived from the digestive system with endoproteinase Lys-C, retains the residues from Thr16 to Lys 36 of buforin I, and is more than 12 times more potent than buforin I [48]. A proline hinge region in buforin II is essential for inducing membrane permeation in E. coli [46,49]. It has been suggested that buforin II inhibits cellular processes by interfering with DNA and RNA metabolism [50].

Compounds with Antifungal Activity
Skin secretions of many toads contain peptides with antifungal activity. These peptides are stored in a granular gland located mainly in the skin of the dorsal region. Most amphibian species produce one or more peptides with potent activity against fungi [7].
The skin secretions of Leptophryne cruentata show antifungal activity against Trichophyton mentagrophytes; forty microliters caused an inhibition halo of 14.5 ± 2.9 mm diameter [51]. Furthermore, the skin secretions of Rhinella icterica showed antifungal activity against Candida krusei [28]. The bufadienolides arenobufagin, gamabufotalin, and telocinobufagin were identified in the skin secretions of the toad Anaxyrus boreas. These compounds inhibited the growth of the Batrachochytrium dendrobatidis strain, and their MIC were 12.9, 50.0, and 14.3 µM, respectively [7]. These bufadienolides were also identified in Rhinella horribilis [52]. The discovery of these compounds, effective against these fungal strains, will allow the development of therapeutic agents that treat infections caused in humans and other animals [53]. One alkaloid called dehydrobufotenine was found in the skin secretions of the toad Bufo arenarum. It showed fungicidal activity against phytopathogenic fungi that affect plants of economic interest. Further, forty-five analogs of dehydrobufotenine were synthesized and evaluated for their activity. Compounds 6, 16c, 16d, 16h, 16j, and 19 showed similar or higher activity than the control carbendazim at 50 µg/mL. Dehydrobufotenine and most of its analogues showed higher activity against Alternaria solani (5-400%) than the controls chlorothalonil and carbendazim at 50 µg/mL. Their fungicidal activity is shown in Table 2.

Compounds with Antiviral Activity
Infectious diseases caused by viruses have impacted human life throughout evolutionary history and certainly in the adaptation of many civilizations. Molecular phylogenies support the hypothesis that some "modern" viruses, such as herpes or human papillomavirus (HPV), have co-evolved in association with some human ethnic groups and other vertebrate lineages and that these were dispersed around the world due to migration. The emergence or re-emergence of new diseases caused by viruses such as influenza, dengue, or hepatitis can be explained by the accelerated growth of populations and the density of human settlements, where the viruses were able to establish and persist, giving rise to the first epidemics [54].
In the last two years, the COVID-19 pandemic caused by the SARS-CoV-2 outbreak resulted in more than 50 million official cases and countless deaths. Due to this, many researchers focused on searching for alternatives to diagnose and treat this virus and others, such as influenza A (H1N1 and H5N1), HIV, Chikungunya, Zika, and Hepatitis C and B [55].
The importance of research on disease-causing viruses lies in the global public health problem and economic burden caused by the ability of viruses to transmit from nature to humans, triggering unpredictable outbreaks. The current strategy for managing viral diseases consists, on the one hand, of vaccines, whose development is usually slow, long, laborious, and costly; and on the other hand, of broad-spectrum antiviral treatments, an effective option to reduce human-to-human transmission [56]. For this reason, the bioactive components of toad skin secretions represent an important source for the development of new therapeutic agents that help in the treatment of various viral diseases.
Bufalin and cinobufagin are bufadienolides isolated from the toad skin secretion of the Bufo bufo gargarizan. Both are pharmacologically active components of huachansu or cinobufacini, a preparation of traditional Chinese medicine, which has been used as a treatment against hepatitis B virus (HBV). The activity of cinobufacini, bufalin, and cinobufagin was examined against HBV. The three of them were tested in the human cell line HepG2.2.15 at different concentrations, where all showed HBV secretion antigen inhibition activity at high concentrations (Table 3). Unlike bufalin and cinobufagin, the results demonstrated that cinobufacini exhibited higher activity against HBV at low concentrations. This was attributed to the specific inhibition of HBV messenger RNA expression [17,57,58].
In another study, bufalin and resibufogenin were studied for their effects against Epstein-Barr virus (EBV) or human herpesvirus 4, one of the most common viruses in humans. Both bufadienolides showed inhibitory activity on early activation of EBV antigen. In addition, bufalin showed in vivo inhibitory activity in two-stage carcinogenesis tests on mouse cutaneous papilloma caused by human papillomavirus (HPV) [59] and inhibition of rhinovirus [60].
Bufotenine is an alkaloid isolated from the secretions of Rhinella jimi. This compound was tested against rabies virus, which is responsible for a deadly zoonotic disease that affects more than 150 countries causing about 55,000 deaths annually. The study showed that bufotenine could inhibit the penetration of the rabies virus into mammalian cells through an apparent mechanism of competition for the nicotinic acetylcholine receptor. However, bufotenine is not presented as a proposal for drug development due to its hallucinogenic, cytotoxic, and psychotropic effects [61].
In 2021, Jin and coworkers tried to find broad-spectrum antiviral drugs to treat coronavirus infections. They assessed the antiviral activity of some cardiotonic steroids (bufadienolides) found in skin secretions of the toad species Bufo bufo gargarizans. The activity was tested against MERS-CoV, SARS-CoV, and SARS-CoV2 coronavirus varieties (Table 3). In the study, the authors identified antiviral activity. The IC 50 (Table 3). Resinobufogenin showed low activity, and, sadly, bufalin and cinobufagin showed lethal activity in mice after intraperitoneal administration of 10 mg/kg/day in a 5-day repeated dose toxicity test. Cinobufotalin showed lethal activity in rats after 1mg/kg IV injection during pharmacokinetic studies. Telocinobufagin was the best candidate as a therapeutic drug for COVID-19 among the bufadienolides tested [16].
Further, the components of toad skin secretions can act against viruses that affect plants, as in the case of dehydrobufotenine (Figure 1). This alkaloid was found in skin secretions from Bufo arenarum. It was selected by Tian et al. [27] who designed and synthesized dehydrobufotenine analogs, with the goal of evaluating their biological activity. Of the forty-five synthesized analogs, compounds 12 and 17 showed~50% higher antiviral activity against Tobacco mosaic virus (TMV) than dehydrobufotenine. Remarkably, the potency of these molecules was higher than ningnanmycin, which is perhaps the most effective antiplant-virus agent (ningnanmycin inhibitory effect of 50-60% over TMV at 500 µg/mL).
Compound 12 presented evidence of virus-assembly inhibition (Table 3) [27]. So, these two analogs could be considered new antiviral candidates.

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At 500 µg/mL showed 5-6% higher inactivating, curative, and protective effects against TMV than the control ningnanmycin and 20-21% higher effects than the control ribavirin.

Compounds with Antiparasitic Activity
A parasite is an "organism that lives on or in a host organism and gets its food from or at the expense of its" host [62]. The parasites use vectors such as mosquitos to reach their host. Some of these organisms cause diseases that have been associated with poverty [63] and tropical conditions [64]. Therefore warm weather in developing countries favors an increase in the case number, leading to deaths and other health problems [65].
Death due to parasitic diseases went down from 2000 to 2019. Still, in 2020, due to the SARS-CoV-2 pandemic, only malaria was determined to increase by 12%, estimating 627,000 deaths caused by the Plasmodium vivax parasite [64].
Chagas disease is caused by the parasite Trypanosoma cruzi, which is widespread in America (mainly rural zones of Latin America), affecting approximately 6-7 million people. The symptoms are nonspecific or asymptomatic in the acute phase of the infection. Patients suffer heart or digestive diseases only when the chronic stage is reached [28].
Drugs used in parasite-infection treatment are mainly formed by nitrogen rings, such as pyrimidine and imidazole, other substituents with nitrogen, and sulfhydryl groups are also common. There are antiparasitic drugs with broad spectra, such as albendazole and mebendazole; the majority are indicated against special cases, such as the use of mefloquine, which is a drug used against malaria produced by P. falciparum, a chloroquine-resistant strain, or suramin which is used on Trypanosoma brucei rhodesiense infection treatment [66].
For T. cruzi, benznidazole is a drug indicated for treating Chagas disease, especially in patients in the acute phase when the parasite is still in the blood. On the other hand, nifurtimox can be used in the acute phase or the chronic phase [67].
Toad venom comprises chemical compounds such as proteins, peptides, steroidal bufadienolides, alkaloids, and others [68]. The genus Bufo was divided into two genera in South America, Rhinella and Rhaebo. The secretions from nine different species (eight of Rhinella and one of Rhaebo) were analyzed biochemically, founding molecules in common. But, the authors realized that related species living in different habitats secrete distinct molecules, so they proposed that biological features influence skin secretion [68].
Telocinobufagin and hellebrigenin were isolated from Rhinella jimi and tested against Leishmania chagasi and T. cruzi (Table 4). Both bufadienolides inhibited the growth of the promastigotes from Leishmania, while hellebrigenin showed activity against trypomastigotes of T. cruzi. These molecules did not show cytotoxicity at 200 µg/mL, so that result is a promising for developing antiparasitic agents [28].
The skin secretion of the toads R. marina and R. guttatus were evaluated for their antiplasmodial activity against the Plasmodium falciparum W2 strain and showed a mortality above 70% against the parasite. Banfi et al. [69] isolated the compounds bufalin, dehydrobufotenin, marinobufotoxin, and marinobufagin of R. marina toads and evaluated their action in vitro on P. falciparum viability, showed values of IC 50 of 1.329, 3.88, 5.31, and 1.557 µg/mL, respectively (Table 4). Although the bufalin was more active, only dehydrobufotenin demonstrated high selectivity for the parasites when analyzed in a cytotoxicity assay. Hence, it is a good candidate for the design of new therapeutic agents. On the other hand, secretions of Rhinella marina showed biological activity against Leishmania guyanensis and Leishmania braziliensis [32].
Meanwhile, ten bufadienolides isolated from Rhinella alata exhibited anti-trypanosomal activity, where the ranges of IC 50 were between 1.329 and 14.343 µg/mL on Trypanosoma cruzi in an in vitro assay ( Table 4). The anti-trypanosomal activity revealed that an arginyldiacid at C-3 and an OH group at C-14 in the structure of bufadienolides were essential for the bioactivity observed.

Discussion
The skin secretions of the toads Bufo bufo gargarizans and Bufo melanostictus have been used in Chinese traditional medicine to treat bacterial infections, abdominal pain, and diarrhea [70]. These pharmacological properties are due to substances the secretions contain: bufadienolides, alkaloids, toxins, peptides, and proteins. In this work, we performed a review of the compounds with antimicrobial activity. The bufadienolides such as marinobufagin and telocinobufagin from Bufo rubescens presented lower antibacterial activity than the peptides buforins, especially against medically important bacteria such as E. coli, S. aureus, S. pneumoniae, and S. typhimurium. Buforin II showed better activity than buforin I. Although both peptides are rich in positive residues, the distribution of these in the alpha helix is crucial to induce bacterial killing [71]. In the case of buforin II, these positive residues are mainly distributed on the hydrophilic side. In this, the presence of arginine residues provides capabilities to interact with the negatively charged phospholipid headgroups of bacterial membranes. The studies with buforin II only included preclinical assays despite showing antibacterial activities similar to the derived peptide of magainin, called MSI-78, which was evaluated in Phase IIbAII clinical trials and demonstrated effectiveness in the treatment of impetigo, a skin infection caused by group A Streptococcus and S. aureus [72]. Buforins and cathelicidins could be used as parent peptides to design some analogs to increase their antimicrobial activity and prepare formulations of use to treat skin infections caused by bacteria.
Bufadienolides, also called cardiotonic steroids, contain a 6-membered lactone ring in the C17 position of the steroid nucleus (Figure 1) and have a broad spectrum of antimicrobial activities. Telocinobufagin and marinobufagin showed moderate antibiotic activity against E. coli (64 and 16 µg/mL) and low activity against S. aureus (128 µg/mL). Arenobufagin, gamabufotalin and marinobufagin showed medium antifungal activity (MIC 12.9, 50, 14.3 µg/mL, respectively). Dehydrobufotenine and some of its analogs showed better antifungal activity against Alternaria solani than the controls chlorothalonil and carbendazim.
The bufadienolides secreted from the skin of Anaxyrus boreas present an important opportunity for biology, since they present activity against the Batrachochytrium dendrobatidis strain, which affects the skin of many amphibians and has shown resistance to conventional treatment. This fungus has caused the extinction or near extinction of more than 200 species of amphibians, radically reprogramming ecosystems around the planet [7]. Alkaloids synthesized from the secretion of the toad Bufo arenarum showed potent fungicidal activity, surpassing fungicides such as chlorothalonil and carbendazim. Of all the synthesized components, it was found that compound 16h was the main candidate to be a broadspectrum fungicide [27].
On the other hand, telocinobufagin is one of the most interesting bufadienolides, it showed high activity against the MERS-CoV and SARS-CoV-2 coronavirus variants, and against L. chagasi, and, most importantly, it caused low cytotoxicity. Telocinobufagin is one of the crucial bufadienolides to consider a new medicine. In this review, we revised that slight differences in the molecules can cause significant changes in biological activities. There are still different compounds that have not been analyzed yet.
Steroidal compounds show high activity against parasites; 15 bufadienolides obtained from toad secretions demonstrated good activity (IC 50 126.2 to 1.32 µg/mL), but only 7 demonstrated viability in the therapeutic window. Commercial drugs used in treating parasites usually have hydrophobic areas that help in transportation across lipid barriers and contain amino hetero-substituted structures such as triazole and benzimidazole compounds. SAR-guided studies of aryl methylamino steroids showed steroid moieties as essential in biological activity to cross membrane barriers, and 3-OH and 17-OH substitution as essential to disable side effects of steroid hormones [73] like those observed in skin secretions of toads in this review.

Conclusions
In this review, we showed that skin secretions of toads are a source of biomolecules with diverse antimicrobial activities. In the secretions, bufadienolides are a diverse group of molecules. Some possess high antimicrobial activity but were reported to have toxic effects in mice, such as bufalin and cinobufagin. Telocinobufagin had moderate antifungal activity, high antiviral activity (against MERS-CoV, SARS-CoV, and SARS-CoV-2), and low toxicity. This molecule is a candidate for a new antiviral drug or could be a base for developing better antiviral agents.
Buforin II had high antibacterial activity killing Gram-negative and Gram-positive bacteria. This AMP is a clear candidate for a new antibiotic and could be used to treat skin and stomach infections. On the other hand, Buforin II could be used as a food preservative.
The study on dehydrobufotenine analogs was a clear example of designing new molecules with better properties ; compounds 6, 16c, 16d, 16h, 16j, and 19 increased their antifungal activity compared to dehydrobufotenine. They exhibited similar or higher activity than the commercial antifungals chlorothalonil and carbendazim. Furthermore, compounds 12 and 17 achieved higher activity that ningnanmycin, which may be the most effective anti-plant virus agent. Compounds 12 and 17 are therefore new antiviral candidates.
Up to date, 644 species in the Bufonidae family have been described, but the secretion of many of them has not been studied yet. We think there are still many antimicrobial compounds that can help us fight multiple diseases caused by microorganisms.