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Amino acid–derived defense metabolites from plants: A potential source to facilitate novel antimicrobial development

Open AccessPublished:February 18, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100438
      For millennia, humanity has relied on plants for its medicines, and modern pharmacology continues to reexamine and mine plant metabolites for novel compounds and to guide improvements in biological activity, bioavailability, and chemical stability. The critical problem of antibiotic resistance and increasing exposure to viral and parasitic diseases has spurred renewed interest into drug treatments for infectious diseases. In this context, an urgent revival of natural product discovery is globally underway with special attention directed toward the numerous and chemically diverse plant defensive compounds such as phytoalexins and phytoanticipins that combat herbivores, microbial pathogens, or competing plants. Moreover, advancements in “omics,” chemistry, and heterologous expression systems have facilitated the purification and characterization of plant metabolites and the identification of possible therapeutic targets. In this review, we describe several important amino acid–derived classes of plant defensive compounds, including antimicrobial peptides (e.g., defensins, thionins, and knottins), alkaloids, nonproteogenic amino acids, and phenylpropanoids as potential drug leads, examining their mechanisms of action, therapeutic targets, and structure–function relationships. Given their potent antibacterial, antifungal, antiparasitic, and antiviral properties, which can be superior to existing drugs, phytoalexins and phytoanticipins are an excellent resource to facilitate the rational design and development of antimicrobial drugs.

      Keywords

      Abbreviations:

      AMR (antimicrobial resistance), CHIKV (chikungunya virus), CRP (cysteine-rich peptide), DENV (dengue virus), HCV (hepatitis C virus), HLP (hevein-like peptide), IAV (influenza A virus), IBV (infectious bronchitis virus), JEV (Japanese encephalitis virus), MAPK (mitogen-activated protein kinase), MRSA (methicillin-resistant Staphylococcus aureus), NPAA (nonproteinaceous amino acid), PA (phytoalexin), PP (phenylpropanoid), ROS (reactive oxygen species), WNV (West Nile virus), ZIKV (Zika virus)
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      ,
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      ). These amino acid–derived defense compounds represent privileged scaffolds, which evolved to bind biological targets, and can therefore provide a rich resource for the development of antimicrobials. Here, we describe the biosynthesis of selected amino acid–derived small molecules and peptides, and their potential in the development of antimicrobials, namely, antibacterial, antifungal, anti-parasitic, and antiviral therapeutics, by focusing on structural and mechanistic aspects.

      Antimicrobial peptides

      Plants produce a variety of defensive antimicrobial peptides, many of which are cysteine-rich peptides (CRPs), such as cyclotides, defensins, knottins, snakins, and thionins (
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      Overview on plant antimicrobial peptides.
      ,
      • Tam J.P.
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      Antimicrobial peptides from plants.
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      ,
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      ). Here, we confine the discussion of the biomedical applications of compounds with a molecular mass ≤7 kDa and thereby exclude small defense proteins such as puroindolines and lipid transfer proteins. A summary of the activities of different antimicrobial peptide classes is shown in Table 1. Antimicrobial peptides are considered especially good drug leads (
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      Natural antimicrobial peptides as Inspiration for design of a new generation antifungal compounds.
      ).
      Table 1A summary of selected examples of the in vitro and in vivo antimicrobial activity of plant-derived AMPs discussed in this article
      AMP familyProminent examples (plant sources)Activity (putative mechanism)Type of testing, target pathogen/infection, referenceAvailable production methods
      CyclotidesCyO2 (Viola odorata)Antibacterial (membrane binding), antifungal (membrane disruption, spore penetration), antiviral (disruption of viral integrity, pore formation in infected cells)Animal tests, S. aureus wound infections (
      • Fensterseifer I.C.
      • Silva O.N.
      • Malik U.
      • Ravipati A.S.
      • Novaes N.R.
      • Miranda P.R.
      • Rodrigues E.A.
      • Moreno S.E.
      • Craik D.J.
      • Franco O.L.
      Effects of cyclotides against cutaneous infections caused by Staphylococcus aureus.
      ); Mammalian cell culture, HIV-1 (
      • Gerlach S.L.
      • Chandra P.K.
      • Roy U.
      • Gunasekera S.
      • Göransson U.
      • Wimley W.C.
      • Braun S.E.
      • Mondal D.
      The membrane-active Phytopeptide cycloviolacin O2 simultaneously targets HIV-1-infected cells and infectious viral particles to potentiate the efficacy of antiretroviral drugs.
      )
      Chemical, chemoenzymatic, heterologous
      DefensinsRsAFP2 (Raphanus sativus)Antifungal (reactive oxygen species, elevated septin and ceramide, apoptosis induction; targets cell wall and membrane sphingolipids)Animal tests (prophylactic), Candida spp. (
      • Tavares P.M.
      • Thevissen K.
      • Cammue B.P.
      • François I.E.
      • Barreto-Bergter E.
      • Taborda C.P.
      • Marques A.F.
      • Rodrigues M.L.
      • Nimrichter L.
      In vitro activity of the antifungal plant defensin RsAFP2 against Candida isolates and its in vivo efficacy in prophylactic murine models of candidiasis.
      )
      Chemical, heterologous
      ThioninsCaThi (Capsicum annuum)Antibacterial (membrane disruption), antifungal (membrane disruption, apoptosis, reactive oxygen species)In vitro, bacteria and Candida spp. (
      • Taveira G.B.
      • Mathias L.S.
      • da Motta O.V.
      • Machado O.L.
      • Rodrigues R.
      • Carvalho A.O.
      • Teixeira-Ferreira A.
      • Perales J.
      • Vasconcelos I.M.
      • Gomes V.M.
      Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts.
      ,
      • Taveira G.B.
      • Carvalho A.O.
      • Rodrigues R.
      • Trindade F.G.
      • Da Cunha M.
      • Gomes V.M.
      Thionin-like peptide from Capsicum annuum fruits: Mechanism of action and synergism with fluconazole against Candida species.
      ,
      • Taveira G.B.
      • Mello É.
      • Carvalho A.O.
      • Regente M.
      • Pinedo M.
      • de La Canal L.
      • Rodrigues R.
      • Gomes V.M.
      Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani.
      ,
      • Taveira G.B.
      • Mello É.
      • Souza S.B.
      • Monteiro R.M.
      • Ramos A.C.
      • Carvalho A.O.
      • Rodrigues R.
      • Okorokov L.A.
      • Gomes V.M.
      Programmed cell death in yeast by thionin-like peptide from Capsicum annuum fruits involving activation of caspases and extracellular H+ flux.
      )
      Chemical, heterologous
      KnottinsAs1 (Alstonia scholaris)Antiviral (inhibits viral spike protein and maturation protein)Mammalian cell culture, influenza B virus (
      • Nguyen P.Q.
      • Ooi J.S.
      • Nguyen N.T.
      • Wang S.
      • Huang M.
      • Liu D.X.
      • Tam J.P.
      Antiviral cystine knot α-amylase inhibitors from Alstonia scholaris.
      )
      Chemical, heterologous
      Snakin-like peptidesSnakin-Z (Zizyphus jujube)Antibacterial and antifungal (membrane disruption by pore formation)Mammalian cell culture, S. aureus (
      • Daneshmand F.
      • Zare-Zardini H.
      • Ebrahimi L.
      Investigation of the antimicrobial activities of Snakin-Z, a new cationic peptide derived from Zizyphus jujuba fruits.
      )
      Chemical, heterologous
      α-Hairpinin-like peptidesEcAMP1 (Echinochloa crus-galli)Antifungal (binding cell wall carbohydrates and membrane lipids)In vitro, Fusarium spp. (
      • Nolde S.B.
      • Vassilevski A.A.
      • Rogozhin E.A.
      • Barinov N.A.
      • Balashova T.A.
      • Samsonova O.V.
      • Baranov Y.V.
      • Feofanov A.V.
      • Egorov T.A.
      • Arseniev A.S.
      • Grishin E.V.
      Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli).
      )
      Chemical, heterologous
      Luffin P1 (Luffa cylindrica)Antiviral (binds the rev response element)Mammalian cell culture, HIV-1 (
      • Ng Y.M.
      • Yang Y.
      • Sze K.H.
      • Zhang X.
      • Zheng Y.T.
      • Shaw P.C.
      Structural characterization and anti-HIV-1 activities of arginine/glutamate-rich polypeptide Luffin P1 from the seeds of sponge gourd (Luffa cylindrica).
      )
      Hevein-like peptides(Pereskia bleo)Antifungal (chitin assembly inhibition, membrane disruption)Mammalian cell culture, C. albicans and C. tropicalis (
      • Loo S.
      • Kam A.
      • Xiao T.
      • Tam J.P.
      Bleogens: Cactus-Derived anti-Candida cysteine-rich peptides with three different precursor Arrangements.
      )
      Chemical, heterologous

      Cyclotides

      Plants from the seemingly unrelated Cucurbitaceae, Fabaceae, Rubiaceae, Solanaceae, and Violaceae families produce antimicrobial and insecticidal “mini-proteins,” known as cyclotides (
      • de Veer S.J.
      • Kan M.W.
      • Craik D.J.
      Cyclotides: From structure to function.
      ), which represent the best-known plant antimicrobial peptides and display activity against multiple groups of pathogens. Antimalarial activity has been reported from cyclotide-rich extracts of Oldenlandia affinis (Rubiaceae), a West African medicinal plant (
      • Nworu C.S.
      • Ejikeme T.I.
      • Ezike A.C.
      • Ndu O.
      • Akunne T.C.
      • Onyeto C.A.
      • Okpalanduka P.
      • Akah P.A.
      Anti-plasmodial and anti-inflammatory activities of cyclotide-rich extract and fraction of Oldenlandia affinis (R. & S.) D.C. (Rubiaceae).
      ). Various cyclotides from the sweet violet (Viola odorata) demonstrated broad-spectrum antibacterial and antifungal activities, with low inhibitory concentrations against E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica, S. aureus, and the fungi Candida albicans (
      • Pränting M.
      • Lööv C.
      • Burman R.
      • Göransson U.
      • Andersson D.I.
      The cyclotide cycloviolacin O2 from Viola odorata has potent bactericidal activity against Gram-negative bacteria.
      ,
      • Strömstedt A.A.
      • Park S.
      • Burman R.
      • Göransson U.
      Bactericidal activity of cyclotides where phosphatidylethanolamine-lipid selectivity determines antimicrobial spectra.
      ), Fusarium graminearum (
      • Parsley N.C.
      • Kirkpatrick C.L.
      • Crittenden C.M.
      • Rad J.G.
      • Hoskin D.W.
      • Brodbelt J.S.
      • Hicks L.M.
      PepSAVI-MS reveals anticancer and antifungal cycloviolacins in Viola odorata.
      ), and Fusarium oxysporum (
      • Slazak B.
      • Kapusta M.
      • Strömstedt A.A.
      • Słomka A.
      • Krychowiak M.
      • Shariatgorji M.
      • Andrén P.E.
      • Bohdanowicz J.
      • Kuta E.
      • Göransson U.
      How does the sweet violet (Viola odorata L.) `ht pathogens and pests – cyclotides as a comprehensive plant host defense system.
      ). In Gram-negative bacteria, the interaction with phosphatidylethanolamine-lipids appears to determine species selectivity (
      • Strömstedt A.A.
      • Park S.
      • Burman R.
      • Göransson U.
      Bactericidal activity of cyclotides where phosphatidylethanolamine-lipid selectivity determines antimicrobial spectra.
      ). In mouse models, the cyclotide cycloviolacin 2 limits subcutaneous S. aureus infections in surgical wounds without toxicity to monocytes while stimulating immune cell phagocytosis (
      • Fensterseifer I.C.
      • Silva O.N.
      • Malik U.
      • Ravipati A.S.
      • Novaes N.R.
      • Miranda P.R.
      • Rodrigues E.A.
      • Moreno S.E.
      • Craik D.J.
      • Franco O.L.
      Effects of cyclotides against cutaneous infections caused by Staphylococcus aureus.
      ).
      Several cyclotides have also been explored for their antiviral properties especially toward HIV (
      • Gustafson K.
      • Sowder II, R.
      • Henderson L.
      • Parsons I.
      • Kashman Y.
      • Cardellina II, J.
      • McMahon J.
      • Buckheit Jr., J.
      • Pannell L.
      • Boyd M.
      Circulins A and B. Novel human immunodeficiency virus (HIV)-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia.
      ). The cyclotide cycloviolacin 2 induces pore formation in HIV-infected T cells and monocytes, disrupting viral integrity and improving the efficacy of antiretroviral drugs (
      • Gerlach S.L.
      • Chandra P.K.
      • Roy U.
      • Gunasekera S.
      • Göransson U.
      • Wimley W.C.
      • Braun S.E.
      • Mondal D.
      The membrane-active Phytopeptide cycloviolacin O2 simultaneously targets HIV-1-infected cells and infectious viral particles to potentiate the efficacy of antiretroviral drugs.
      ,
      • Henriques S.T.
      • Huang Y.H.
      • Rosengren K.J.
      • Franquelim H.G.
      • Carvalho F.A.
      • Johnson A.
      • Sonza S.
      • Tachedjian G.
      • Castanho M.A.
      • Daly N.L.
      • Craik D.J.
      Decoding the membrane activity of the cyclotide kalata B1: The importance of phosphatidylethanolamine phospholipids and lipid organization on hemolytic and anti-HIV activities.
      ). It is important to note that cyclotide cycloviolacin 2 is effective at a nanomolar concentration, which is considered a safe dose for preclinical animal testing for HIV (
      • Troeira Henriques S.
      • Craik D.J.
      Cyclotide structure and function: The role of membrane binding and Permeation.
      ). Initial tests in murine models with intravenous cyclotide cycloviolacin 2 administered at < 2 mg/kg could not find any appreciable toxicity or hemolysis (
      • Burman R.
      • Svedlund E.
      • Felth J.
      • Hassan S.
      • Herrmann A.
      • Clark R.J.
      • Craik D.J.
      • Bohlin L.
      • Claeson P.
      • Göransson U.
      • Gullbo J.
      Evaluation of toxicity and antitumor activity of cycloviolacin O2 in mice.
      ,
      • Kumar N.
      • Chahroudi A.
      • Silvestri G.
      Animal models to achieve an HIV cure.
      ).
      Cyclotides usually range in size from 25 to 40 amino acids and feature a unique head-to-tail macrocyclic structure containing cystine knots that confer proteolytic stability (
      • Craik D.J.
      • Daly N.L.
      • Bond T.
      • Waine C.
      Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif.
      ,
      • Tam J.P.
      • Lu Y.A.
      • Yang J.L.
      • Chiu K.W.
      An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides.
      ). They tolerate high sequence variation in the nonconserved cysteine residues and can pass through membranes, a useful quality for oral formulations. This class of antimicrobial peptides are also candidates for the modulation of protein–protein interactions, and their potential in drug development has gained attention in recent years (
      • Camarero J.A.
      • Campbell M.J.
      The potential of the cyclotide scaffold for drug development.
      ). Cyclotides of the Möbius and bracelet types contain well-defined hydrophilic and hydrophobic patches, leading to an amphiphilic property similar to that of classical antimicrobial peptides (
      • Troeira Henriques S.
      • Craik D.J.
      Cyclotide structure and function: The role of membrane binding and Permeation.
      ). However, the variation of these hydrophobic patches differs among individual cyclotides, resulting in different membrane-binding properties for each (
      • Wang C.K.
      • Colgrave M.L.
      • Ireland D.C.
      • Kaas Q.
      • Craik D.J.
      Despite a conserved cystine knot motif, different cyclotides have different membrane binding modes.
      ). Owing to their short peptide lengths, cyclotides are amenable to synthesis and bioengineering efforts, which has accelerated development of synthetic analogs as antivirals (
      • Thongyoo P.
      • Roqué-Rosell N.
      • Leatherbarrow R.J.
      • Tate E.W.
      Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides.
      ). In addition to the chemical routes for the synthesis of cyclotides, large-scale heterologous production is reasonably achievable, since the enzymes involved in their cyclization are characterized (
      • Rehm F.B.H.
      • Jackson M.A.
      • De Geyter E.
      • Yap K.
      • Gilding E.K.
      • Durek T.
      • Craik D.J.
      Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization.
      ).

      Defensins

      Members of this group of antimicrobial peptides are usually of 45 to 54 amino acids and positively charged CRPs. They have eight Cys residues with four disulfide linkages stabilizing their triple-stranded β-sheet and α-helical regions (
      • Fant F.
      • Vranken W.
      • Broekaert W.
      • Borremans F.
      Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR.
      ,
      • Almeida M.S.
      • Cabral K.M.
      • Kurtenbach E.
      • Almeida F.C.
      • Valente A.P.
      Solution structure of Pisum sativum defensin 1 by high resolution NMR: Plant defensins, identical backbone with different mechanisms of action.
      ). Defensins are widely distributed in plant families, including many crops where they accumulate in a tissue-specific manner (
      • Benko-Iseppon A.M.
      • Galdino S.L.
      • Calsa T.
      • Kido E.A.
      • Tossi A.
      • Belarmino L.C.
      • Crovella S.
      Overview on plant antimicrobial peptides.
      ,
      • Terras F.R.
      • Schoofs H.M.
      • De Bolle M.F.
      • Van Leuven F.
      • Rees S.B.
      • Vanderleyden J.
      • Cammue B.P.
      • Broekaert W.F.
      Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.
      ,
      • Lay F.T.
      • Anderson M.A.
      Defensins--components of the innate immune system in plants.
      ). Defensins bind sphingolipids (
      • Poon I.K.h.
      • Baxter A.A.
      • Lay F.T.
      • Mills G.D.
      • Adda C.G.
      • Payne J.A.
      • Phan T.K.
      • Ryan G.F.
      • White J.A.
      • Veneer P.K.
      • van der Weerden N.L.
      • Anderson M.A.
      • Kvansakul M.
      • Hulett M.D.
      Phosphoinositide-mediated oligomerization of a defensin induces cell lysis.
      ), a promising target for a newer generation of antifungals (
      • Rollin-Pinheiro R.
      • Singh A.
      • Barreto-Bergter E.
      • Del Poeta M.
      Sphingolipids as targets for treatment of fungal infections.
      ). Sphingolipids are widely distributed in eukaryotes, including fungi, but are rarer and less diverse in bacteria (
      • Hannich J.T.
      • Umebayashi K.
      • Riezman H.
      Distribution and functions of sterols and sphingolipids.
      ). This might explain why most defensins have antifungal rather than antibacterial properties. Of importance, some plant defensins, such as DmAMP1, HsAFP1, and RsAFP2, were shown to have increased activity against clinical pathogens such as Aspergillus flavus, C. albicans, Candida krusei, and Fusarium solani compared with commonly used azole-derived antifungals (
      • Thevissen K.
      • Kristensen H.H.
      • Thomma B.P.
      • Cammue B.P.
      • François I.E.
      Therapeutic potential of antifungal plant and insect defensins.
      ).
      The rice (Oryza sativa) defensin OsAFP1 kills C. albicans by inducing apoptosis and targeting cell-wall components; mutational analysis suggests that about 10 residues at the N and C termini are important for this activity (
      • Ochiai A.
      • Ogawa K.
      • Fukuda M.
      • Ohori M.
      • Kanaoka T.
      • Tanaka T.
      • Taniguchi M.
      • Sagehashi Y.
      Rice defensin OsAFP1 is a new drug candidate against human pathogenic fungi.
      ). The defensin PsD1 from pea (Pisum sativum) inhibits growth of several species by interacting with sphingolipids on the fungal envelope and permeabilizing cell membranes leading to growth arrest (
      • Almeida M.S.
      • Cabral K.M.
      • Zingali R.B.
      • Kurtenbach E.
      Characterization of two novel defense peptides from pea (Pisum sativum) seeds.
      ). Of interest, recombinant protein analysis showed that addition of four extra amino acids at the N terminus decreased the activity of PsD1 against Aspergillus niger and F. solani by 5-fold, but not against Neurospora crassa. This suggests that defensins are not merely cytotoxic and instead target distinct biological functions. In fact, analysis of the mode of action of the plant defensin NaD1 showed that the presence of the fungal cell wall is essential for the antifungal effect (
      • van der Weerden N.L.
      • Hancock R.E.
      • Anderson M.A.
      Permeabilization of fungal hyphae by the plant defensin NaD1 occurs through a cell wall-dependent process.
      ). Defensins engineered to have species-specific activity would potentially be able to target pathogenic fungi while preserving beneficial fungi.
      Some defensins such as HsAFP1 can also impair the fungal cell cycle independently from their antifungal activity (
      • Struyfs C.
      • Cools T.L.
      • De Cremer K.
      • Sampaio-Marques B.
      • Ludovico P.
      • Wasko B.M.
      • Kaeberlein M.
      • Cammue B.P.A.
      • Thevissen K.
      The antifungal plant defensin HsAFP1 induces autophagy, vacuolar dysfunction and cell cycle impairment in yeast.
      ) and have broad-spectrum fungicidal properties via distinct modes of action. The radish (Raphanus sativus) defensin, RsAFP2, was shown to bind fungal glucosylceramide sphingolipids but not those from plant or humans (
      • Thevissen K.
      • Warnecke D.C.
      • François I.E.
      • Leipelt M.
      • Heinz E.
      • Ott C.
      • Zähringer U.
      • Thomma B.P.
      • Ferket K.K.
      • Cammue B.P.
      Defensins from insects and plants interact with fungal glucosylceramides.
      ). Most promising, RsAFP2 was also shown to be prophylactically effective in vivo in mouse models against candidiasis (
      • Tavares P.M.
      • Thevissen K.
      • Cammue B.P.
      • François I.E.
      • Barreto-Bergter E.
      • Taborda C.P.
      • Marques A.F.
      • Rodrigues M.L.
      • Nimrichter L.
      In vitro activity of the antifungal plant defensin RsAFP2 against Candida isolates and its in vivo efficacy in prophylactic murine models of candidiasis.
      ). Recent work shows that it does not induce membrane permeabilization but instead triggers reactive oxygen species (ROS) production (
      • Aerts A.M.
      • François I.E.
      • Meert E.M.
      • Li Q.T.
      • Cammue B.P.
      • Thevissen K.
      The antifungal activity of RsAFP2, a plant defensin from raphanus sativus, involves the induction of reactive oxygen species in Candida albicans.
      ), increased septin and ceramide levels (
      • Thevissen K.
      • de Mello Tavares P.
      • Xu D.
      • Blankenship J.
      • Vandenbosch D.
      • Idkowiak-Baldys J.
      • Govaert G.
      • Bink A.
      • Rozental S.
      • de Groot P.W.
      • Davis T.R.
      • Kumamoto C.A.
      • Vargas G.
      • Nimrichter L.
      • Coenye T.
      • et al.
      The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans.
      ), and ultimately apoptosis without caspase activation (
      • Aerts A.M.
      • Carmona-Gutierrez D.
      • Lefevre S.
      • Govaert G.
      • François I.E.
      • Madeo F.
      • Santos R.
      • Cammue B.P.
      • Thevissen K.
      The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans.
      ). RsAFP2-mediated fungal inhibition can synergize with the antifungal drug caspofungin preventing C. albicans biofilm formation (
      • Vriens K.
      • Cools T.L.
      • Harvey P.J.
      • Craik D.J.
      • Braem A.
      • Vleugels J.
      • De Coninck B.
      • Cammue B.P.
      • Thevissen K.
      The radish defensins RsAFP1 and RsAFP2 act synergistically with caspofungin against Candida albicans biofilms.
      ). C. albicans biofilms tolerate common antifungals as well as the human immune system extremely well (
      • Silva S.
      • Rodrigues C.F.
      • Araújo D.
      • Rodrigues M.E.
      • Henriques M.
      Candida species biofilms' antifungal resistance.
      ), making the synergistic biofilm inhibition an important advancement.

      Thionins

      This class of antimicrobial peptides contains positively charged CRPs ∼5 kDa in size. Their structure consists of antiparallel α-helices and a double-stranded β-sheet with three to four disulfide bridges. They are classified into five groups of α-/β-thionins with high homology and previously included the superficially similar γ-thionins, now known as defensins (
      • Nawrot R.
      • Barylski J.
      • Nowicki G.
      • Broniarczyk J.
      • Buchwald W.
      • Goździcka-Józefiak A.
      Plant antimicrobial peptides.
      ,
      • Stec B.
      Plant thionins--the structural perspective.
      ). Most thionins have a groove between the α-helices and β-sheets with a conserved Tyr residue and may lead to cell lysis through membrane leakage (
      • Majewski J.
      • Stec B.
      X-ray scattering studies of model lipid membrane interacting with purothionin provide support for a previously proposed mechanism of membrane lysis.
      ). CaThi is a thionin isolated from the fruit of jalapeño (Capsicum annuum, Solanaeceae) and active against both fungi and bacteria (
      • Taveira G.B.
      • Mathias L.S.
      • da Motta O.V.
      • Machado O.L.
      • Rodrigues R.
      • Carvalho A.O.
      • Teixeira-Ferreira A.
      • Perales J.
      • Vasconcelos I.M.
      • Gomes V.M.
      Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts.
      ). Of interest, although CaThi caused membrane disruption in six Candida species, nuclear localization and ROS production were observed only in C. tropicalis (
      • Taveira G.B.
      • Carvalho A.O.
      • Rodrigues R.
      • Trindade F.G.
      • Da Cunha M.
      • Gomes V.M.
      Thionin-like peptide from Capsicum annuum fruits: Mechanism of action and synergism with fluconazole against Candida species.
      ). It also exhibited synergistic effects with the common azole antifungal fluconazole, making F. solani susceptible to low concentrations of the antifungal (
      • Taveira G.B.
      • Mello É.
      • Carvalho A.O.
      • Regente M.
      • Pinedo M.
      • de La Canal L.
      • Rodrigues R.
      • Gomes V.M.
      Antimicrobial activity and mechanism of action of a thionin-like peptide from Capsicum annuum fruits and combinatorial treatment with fluconazole against Fusarium solani.
      ) and inhibiting all six Candida species tested (
      • Taveira G.B.
      • Carvalho A.O.
      • Rodrigues R.
      • Trindade F.G.
      • Da Cunha M.
      • Gomes V.M.
      Thionin-like peptide from Capsicum annuum fruits: Mechanism of action and synergism with fluconazole against Candida species.
      ). It was shown to induce apoptosis in C. tropicalis by caspase and pH imbalance–related mechanisms (
      • Taveira G.B.
      • Mello É.
      • Souza S.B.
      • Monteiro R.M.
      • Ramos A.C.
      • Carvalho A.O.
      • Rodrigues R.
      • Okorokov L.A.
      • Gomes V.M.
      Programmed cell death in yeast by thionin-like peptide from Capsicum annuum fruits involving activation of caspases and extracellular H+ flux.
      ).

      Knottins

      These AMPs contain three disulfide linkages, whereby a pair of disulfides form a loop through which the third disulfide bond passes, creating a heat-stable and protease-resistant structure known as an inhibitor cystine knot (
      • Craik D.J.
      • Daly N.L.
      • Waine C.
      The cystine knot motif in toxins and implications for drug design.
      ). Knottins often possess protease inhibitory activities at nanomolar concentrations and occur in the seeds of several plants, such as MJTI I and II in the garden four o'clock (Mirabilis jalapa), MCoTI-III in bitter gourd (Momordica cochinchinensis), EETI-III in squirting cucumber (Ecballium elaterium), and SOTI-III in spinach (Spinacia oleracea) (
      • Favel A.
      • Mattras H.
      • Coletti-Previero M.A.
      • Zwilling R.
      • Robinson E.A.
      • Castro B.
      Protease inhibitors from Ecballium elaterium seeds.
      ,
      • Heitz A.
      • Hernandez J.F.
      • Gagnon J.
      • Hong T.T.
      • Pham T.T.
      • Nguyen T.M.
      • Le-Nguyen D.
      • Chiche L.
      Solution structure of the squash trypsin inhibitor MCoTI-II. A new family for cyclic knottins.
      ,
      • Kowalska J.
      • Pszczoła K.
      • Wilimowska-Pelc A.
      • Lorenc-Kubis I.
      • Zuziak E.
      • Ługowski M.
      • Łegowska A.
      • Kwiatkowska A.
      • Sleszyńska M.
      • Lesner A.
      • Walewska A.
      • Zabłotna E.
      • Rolka K.
      • Wilusz T.
      Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: Isolation, characterization and chemical synthesis.
      ). Cystine knot α-amylase inhibitors, which are approximately 30 amino acid–long knottins produced by the amaranthaceae and apocynaceae families, contain one or more cis-proline bonds (
      • Chagolla-Lopez A.
      • Blanco-Labra A.
      • Patthy A.
      • Sánchez R.
      • Pongor S.
      A novel alpha-amylase inhibitor from amaranth (Amaranthus hypocondriacus) seeds.
      ,
      • Svensson B.
      • Fukuda K.
      • Nielsen P.K.
      • Bønsager B.C.
      Proteinaceous alpha-amylase inhibitors.
      ,
      • Nguyen P.Q.
      • Wang S.
      • Kumar A.
      • Yap L.J.
      • Luu T.T.
      • Lescar J.
      • Tam J.P.
      Discovery and characterization of pseudocyclic cystine-knot α-amylase inhibitors with high resistance to heat and proteolytic degradation.
      ). Cystine knot α-amylase inhibitors–type knottins from the blackboard tree Alstonia scholaris called alstotides were demonstrated to be cell-permeable inhibitors of the infectious bronchitis virus (IBV) and dengue virus (
      • Nguyen P.Q.
      • Ooi J.S.
      • Nguyen N.T.
      • Wang S.
      • Huang M.
      • Liu D.X.
      • Tam J.P.
      Antiviral cystine knot α-amylase inhibitors from Alstonia scholaris.
      ). One of the alstotides, As1, was shown to rapidly bind and block the function of the IBV spike (S) protein, which drives viral fusion with the cell membrane. This activity is reduced when the N terminus is blocked by biotinylation (
      • Nguyen P.Q.
      • Ooi J.S.
      • Nguyen N.T.
      • Wang S.
      • Huang M.
      • Liu D.X.
      • Tam J.P.
      Antiviral cystine knot α-amylase inhibitors from Alstonia scholaris.
      ) highlighting the importance of the N terminus and its neighboring residues for the antiviral activity of alstotides. Pull-down assays show that As1 also binds to the IBV M protein involved in budding and maturation, thereby suggesting that its antiviral effects occur via the engagement of multiple targets.
      Antifungal activities have been reported for the knottin peptides Mj-AMP1 from M. jalapa and PAFP-S from the pokeweed Phytolacca americana (
      • Cammue B.P.
      • De Bolle M.F.
      • Terras F.R.
      • Proost P.
      • Van Damme J.
      • Rees S.B.
      • Vanderleyden J.
      • Broekaert W.F.
      Isolation and characterization of a novel class of plant antimicrobial peptides form Mirabilis jalapa L. seeds.
      ,
      • Gao G.-H.
      • Liu W.
      • Dai J.-X.
      • Wang J.-F.
      • Hu Z.
      • Zhang Y.
      • Wang D.-C.
      Solution structure of PAFP-S:  A new knottin-type Antifungal peptide from the seeds of Phytolacca americana.
      ). MJ-AMP-1 and Mj-AMP2 from M. jalapa also inhibit Gram-positive bacteria but are ineffective against Gram-negative bacteria (
      • Cammue B.P.
      • De Bolle M.F.
      • Terras F.R.
      • Proost P.
      • Van Damme J.
      • Rees S.B.
      • Vanderleyden J.
      • Broekaert W.F.
      Isolation and characterization of a novel class of plant antimicrobial peptides form Mirabilis jalapa L. seeds.
      ). The solved structure of PAFP-S reveals the presence of an extended hydrophobic patch composed of both aromatic and aliphatic amino acids with neighboring cationic and hydrophobic residues; this amphiphilic character is considered to be the basis of its antifungal property (
      • Gao G.-H.
      • Liu W.
      • Dai J.-X.
      • Wang J.-F.
      • Hu Z.
      • Zhang Y.
      • Wang D.-C.
      Solution structure of PAFP-S:  A new knottin-type Antifungal peptide from the seeds of Phytolacca americana.
      ). As their three conserved disulfide bonds can generate 15 different isomers and because the proper folding of CRPs is required for their activity, it is challenging to mass produce knottins in their proper configuration (
      • Kintzing J.R.
      • Cochran J.R.
      Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles.
      ). An approach to overcome this constraint utilizes selenocysteine residues to form diselenide bonds at lower redox potentials than cysteine disulfides, which substitute for the cysteine pairs and induce cross-linking of the remaining cysteines (
      • Gowd K.H.
      • Yarotskyy V.
      • Elmslie K.S.
      • Skalicky J.J.
      • Olivera B.M.
      • Bulaj G.
      Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.
      ,
      • Walewska A.
      • Zhang M.M.
      • Skalicky J.J.
      • Yoshikami D.
      • Olivera B.M.
      • Bulaj G.
      Integrated oxidative folding of cysteine/selenocysteine containing peptides: Improving chemical synthesis of conotoxins.
      ). Furthermore, heterologous expression systems with bacteria have also been developed to facilitate the production of knottins (
      • Klint J.K.
      • Senff S.
      • Saez N.J.
      • Seshadri R.
      • Lau H.Y.
      • Bende N.S.
      • Undheim E.A.
      • Rash L.D.
      • Mobli M.
      • King G.F.
      Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli.
      ,
      • Moore S.J.
      • Cochran J.R.
      Engineering knottins as novel binding agents.
      ).

      Snakin-like peptides

      Snakins are CRPs with up to 12 cysteines, usually wound or infection induced and studied most in potato (Solanum tuberosum) (
      • Segura A.
      • Moreno M.
      • Madueño F.
      • Molina A.
      • García-Olmedo F.
      Snakin-1, a peptide from potato that is active against plant pathogens.
      ,
      • Berrocal-Lobo M.
      • Segura A.
      • Moreno M.
      • López G.
      • García-Olmedo F.
      • Molina A.
      Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection.
      ). Orthologs are found in several plants such as Arabidopsis thaliana, castor bean, common daisy, petunia, strawberry, and tomato (
      • Shi L.
      • Gast R.T.
      • Gopalraj M.
      • Olszewski N.E.
      Characterization of a shoot-specific, GA3- and ABA-regulated gene from tomato.
      ,
      • Herzog M.
      • Dorne A.-M.
      • Grellet F.
      GASA, a gibberellin-regulated gene family from Arabidopsis thaliana related to the tomato GAST1 gene.
      ,
      • Ben-Nissan G.
      • Weiss D.
      The petunia homologue of tomato gast1: Transcript accumulation coincides with gibberellin-induced corolla cell elongation.
      ,
      • Kotilainen M.
      • Helariutta Y.
      • Mehto M.
      • Pollanen E.
      • Albert V.A.
      • Elomaa P.
      • Teeri T.H.
      GEG participates in the regulation of cell and organ shape during corolla and carpel development in Gerbera hybrida.
      ). The fruits of jujube (Zizyphus jujuba; Rhamnaceae) contain a cationic antimicrobial peptide called Snakin-Z, with activity against S. aureus and well tolerated by blood cells (
      • Daneshmand F.
      • Zare-Zardini H.
      • Ebrahimi L.
      Investigation of the antimicrobial activities of Snakin-Z, a new cationic peptide derived from Zizyphus jujuba fruits.
      ). Snakins produced in heterologous systems, such as E. coli, the yeast Pichia pastoris, and baculovirus-infected insect cells (
      • Almasia N.I.
      • Molinari M.P.
      • Maroniche G.A.
      • Nahirñak V.
      • Barrios Barón M.P.
      • Taboga O.A.
      • Vazquez Rovere C.
      Successful production of the potato antimicrobial peptide Snakin-1 in baculovirus-infected insect cells and development of specific antibodies.
      ), retain their antibacterial and antifungal activities (
      • Kovalskaya N.
      • Hammond R.W.
      Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins.
      ,
      • Mao Z.
      • Zheng J.
      • Wang Y.
      • Chen G.
      • Yang Y.
      • Feng D.
      • Xie B.
      The new CaSn gene belonging to the snakin family induces resistance against root-knot nematode infection in pepper.
      ,
      • Herbel V.
      • Schäfer H.
      • Wink M.
      Recombinant production of snakin-2 (an antimicrobial peptide from tomato) in E. coli and analysis of its bioactivity.
      ,
      • Kuddus M.R.
      • Rumi F.
      • Tsutsumi M.
      • Takahashi R.
      • Yamano M.
      • Kamiya M.
      • Kikukawa T.
      • Demura M.
      • Aizawa T.
      Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris.
      ). Heterologously produced Snakin-2 from Solanum lycopersicum inhibits F. solani (
      • Herbel V.
      • Schäfer H.
      • Wink M.
      Recombinant production of snakin-2 (an antimicrobial peptide from tomato) in E. coli and analysis of its bioactivity.
      ) and is also reported to be active against Bacillus subtilis, E. coli, and Saccharomyces cerevisiae (
      • Herbel V.
      • Wink M.
      Mode of action and membrane specificity of the antimicrobial peptide snakin-2.
      ). The antimicrobial activity of snakins is suggested to be from an unspecific pore formation mechanism that leads to cell aggregation (
      • Herbel V.
      • Wink M.
      Mode of action and membrane specificity of the antimicrobial peptide snakin-2.
      ).
      Both native and recombinant forms of the snakin-like peptide, PdSN1, isolated from the South American tree Peltophorum dubium (Fabaceae) and subsequently produced heterologously in E. coli, displayed antifungal properties against A. niger and C. albicans (
      • Rodríguez-Decuadro S.
      • Barraco-Vega M.
      • Dans P.D.
      • Pandolfi V.
      • Benko-Iseppon A.M.
      • Cecchetto G.
      Antimicrobial and structural insights of a new snakin-like peptide isolated from Peltophorum dubium (Fabaceae).
      ). The structural analysis showed that PdSN1 possesses a helix–turn–helix motif, which is stable under varying combinations of disulfide bonding, including when all the 12 cysteines are reduced. This suggests that the disulfide bonding is dispensable for its antimicrobial activity (
      • Rodríguez-Decuadro S.
      • Barraco-Vega M.
      • Dans P.D.
      • Pandolfi V.
      • Benko-Iseppon A.M.
      • Cecchetto G.
      Antimicrobial and structural insights of a new snakin-like peptide isolated from Peltophorum dubium (Fabaceae).
      ). In addition to the membrane disruption, the helix–turn–helix motif is often found in DNA-binding proteins, and combined with its positive electrostatic potential, PdSN1 may bind to DNA and interfere with microbial gene expression (
      • Rodríguez-Decuadro S.
      • Barraco-Vega M.
      • Dans P.D.
      • Pandolfi V.
      • Benko-Iseppon A.M.
      • Cecchetto G.
      Antimicrobial and structural insights of a new snakin-like peptide isolated from Peltophorum dubium (Fabaceae).
      ). The phenomenon is proposed as the structural basis for the mode of action of defensins as a whole (
      • Lay F.T.
      • Anderson M.A.
      Defensins--components of the innate immune system in plants.
      ).

      α-Hairpinin-like peptides

      Members of the hairpinin family contain the unique (XnC1X3C2XnC3X3C4Xn) motif forming a characteristic helix–loop–helix structure, known as the α-hairpin (
      • Duvick J.P.
      • Rood T.
      • Rao A.G.
      • Marshak D.R.
      Purification and characterization of a novel antimicrobial peptide from maize (Zea mays L.) kernels.
      ,
      • Oparin P.B.
      • Mineev K.S.
      • Dunaevsky Y.E.
      • Arseniev A.S.
      • Belozersky M.A.
      • Grishin E.V.
      • Egorov T.A.
      • Vassilevski A.A.
      Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides.
      ,
      • Slavokhotova A.A.
      • Rogozhin E.A.
      • Musolyamov A.K.
      • Andreev Y.A.
      • Oparin P.B.
      • Berkut A.A.
      • Vassilevski A.A.
      • Egorov T.A.
      • Grishin E.V.
      • Odintsova T.I.
      Novel antifungal α-hairpinin peptide from Stellaria media seeds: Structure, biosynthesis, gene structure and evolution.
      ). The α-hairpinin EcAMP1 from kernels of barnyard grass, Echinochloa crusgalli, is active against several fungal and bacterial genera. Of interest, all Fusarium species tested showed substantial sensitivity to EcAMP1 (
      • Nolde S.B.
      • Vassilevski A.A.
      • Rogozhin E.A.
      • Barinov N.A.
      • Balashova T.A.
      • Samsonova O.V.
      • Baranov Y.V.
      • Feofanov A.V.
      • Egorov T.A.
      • Arseniev A.S.
      • Grishin E.V.
      Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli).
      ) through induced apoptosis (
      • Vasilchenko A.S.
      • Yuryev M.
      • Ryazantsev D.Y.
      • Zavriev S.K.
      • Feofanov A.V.
      • Grishin E.V.
      • Rogozhin E.A.
      Studying of cellular interaction of hairpin-like peptide EcAMP1 from barnyard grass (Echinochloa crusgalli L.) seeds with plant pathogenic fungus Fusarium solani using microscopy techniques.
      ). On the other hand, Sm-AMP-X, from chickweed (Stellaria media) seed is active against A. niger but is not effective on Fusarium spp. (
      • Slavokhotova A.A.
      • Rogozhin E.A.
      • Musolyamov A.K.
      • Andreev Y.A.
      • Oparin P.B.
      • Berkut A.A.
      • Vassilevski A.A.
      • Egorov T.A.
      • Grishin E.V.
      • Odintsova T.I.
      Novel antifungal α-hairpinin peptide from Stellaria media seeds: Structure, biosynthesis, gene structure and evolution.
      ), opening up possibilities for narrow-spectrum antifungal development of the hairpinins. Furthermore, the structural elements important for the activity of EcAMP1 are two α-helices and a small cluster of positively charged amino acids, which together interact with negatively charged fungal cell wall carbohydrates, as well as fungal cell membrane lipids like sphingolipids or ergosterols (
      • Rogozhin E.
      • Zalevsky A.
      • Mikov A.
      • Smirnov A.
      • Egorov T.
      Characterization of Hydroxyproline-containing hairpin-like antimicrobial peptide EcAMP1-Hyp from barnyard grass.
      ). The hairpinin family has diverse biological activity and includes members with trypsin-inhibiting (
      • Conners R.
      • Konarev A.V.
      • Forsyth J.
      • Lovegrove A.
      • Marsh J.
      • Joseph-Horne T.
      • Shewry P.
      • Brady R.L.
      An unusual helix-turn-helix protease inhibitory motif in a novel trypsin inhibitor from seeds of Veronica (Veronica hederifolia L.).
      ) and ribosome-inactivating properties (
      • Li F.
      • Yang X.X.
      • Xia H.C.
      • Zeng R.
      • Hu W.G.
      • Li Z.
      • Zhang Z.C.
      Purification and characterization of Luffin P1, a ribosome-inactivating peptide from the seeds of Luffa cylindrica.
      ). Hairpinins with ribosome-binding activity are of interest for the development of antiviral treatments. For instance, Luffin P1 from sponge gourd (Luffa cylindrica) inhibits the replication and transportation of HIV (
      • Ng Y.M.
      • Yang Y.
      • Sze K.H.
      • Zhang X.
      • Zheng Y.T.
      • Shaw P.C.
      Structural characterization and anti-HIV-1 activities of arginine/glutamate-rich polypeptide Luffin P1 from the seeds of sponge gourd (Luffa cylindrica).
      ).

      Hevein-like peptides

      Hevein is a chitin-binding antifungal protein from the rubber tree Hevea brasiliensis (
      • Van Parijs J.
      • Broekaert W.F.
      • Goldstein I.J.
      • Peumans W.J.
      Hevein: An antifungal protein from rubber-tree (Hevea brasiliensis) latex.
      ). The active portion of the protein is a shorter peptide 43 amino acids long and generated following co- and posttranslational processing (
      • Lee H.I.
      • Broekaert W.F.
      • Raikhel N.V.
      • Lee H.
      Co- and post-translational processing of the hevein preproprotein of latex of the rubber tree (Hevea brasiliensis).
      ). Peptides with sequences similar to that of hevein have been identified in a wide range of plants (
      • Slavokhotova A.A.
      • Shelenkov A.A.
      • Andreev Y.A.
      • Odintsova T.I.
      Hevein-like antimicrobial peptides of plants.
      ). The species Eucommia ulmoides Oliv (Eucommiaceae family) is used in Chinese herbal medicine and produces hevein-like peptides (HLPs) with activity against F. oxysporum and F. solani (
      • Huang R.H.
      • Xiang Y.
      • Liu X.Z.
      • Zhang Y.
      • Hu Z.
      • Wang D.C.
      Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv.
      ). Two mechanisms have been uncovered for the antifungal activity of HLPs; interference with chitin assembly (
      • Nielsen K.K.
      • Nielsen J.E.
      • Madrid S.M.
      • Mikkelsen J.D.
      Characterization of a new antifungal chitin-binding peptide from sugar beet leaves.
      ) and disruption of the fungal cell membrane by ionic interactions (
      • Van den Bergh K.P.
      • Proost P.
      • Van Damme J.
      • Coosemans J.
      • Van Damme E.J.
      • Peumans W.J.
      Five disulfide bridges stabilize a hevein-type antimicrobial peptide from the bark of spindle tree (Euonymus europaeus L.).
      ,
      • Van den Bergh K.P.
      • Van Damme E.J.
      • Peumans W.J.
      • Coosemans J.
      Ee-CBP, a hevein-type antimicrobial peptide from bark of the spindle tree (Euonymus europaeus L.).
      ). The molecules known as bleogens from Pereskia bleo (Cactaceae) are HLPs; one of these, pB1, contains the cystine-knot disulfide motif, β-sheets, and a motif containing four loops. It is antifungal with low micromolar minimum inhibitory concentrations against C. albicans and C. tropicalis, while showing no cytotoxicity toward mammalian cells (
      • Loo S.
      • Kam A.
      • Xiao T.
      • Tam J.P.
      Bleogens: Cactus-Derived anti-Candida cysteine-rich peptides with three different precursor Arrangements.
      ). The seeds of wheat Triticum kiharae produces a 10-Cys peptide, which inhibits F. oxysporum and F. solani through chitinase activity (
      • Odintsova T.I.
      • Vassilevski A.A.
      • Slavokhotova A.A.
      • Musolyamov A.K.
      • Finkina E.I.
      • Khadeeva N.V.
      • Rogozhin E.A.
      • Korostyleva T.V.
      • Pukhalsky V.A.
      • Grishin E.V.
      • Egorov T.A.
      A novel antifungal hevein-type peptide from Triticum kiharae seeds with a unique 10-cysteine motif.
      ). The medicinal plants of the Ginseng group (genus Panax) contain a novel class of HLPs, peptides rich in cysteine and glycine, called ginsentides, containing a pseudocyclic structure that confers heat and proteolytic degradation resistance (
      • Tam J.P.
      • Nguyen G.K.T.
      • Loo S.
      • Wang S.
      • Yang D.
      • Kam A.
      Ginsentides: Cysteine and glycine-rich peptides from the Ginseng family with unusual disulfide Connectivity.
      ). Recently, the gymnosperm Ginkgo biloba was shown to produce proline-rich acid-stable HLPs called gingkotides, which inhibit A. niger and F. oxysporum, and bioinformatic analysis suggests that gingkotide-like HLPs are ubiquitous throughout gymnosperms (
      • Wong K.H.
      • Tan W.L.
      • Serra A.
      • Xiao T.
      • Sze S.K.
      • Yang D.
      • Tam J.P.
      Ginkgotides: Proline-Rich hevein-like peptides from gymnosperm.
      ).

      Other antimicrobial peptides

      Among the best understood CRPs outside of the categories discussed above are the cationic 6- to 8-Cys peptides ToAMP1, ToAMP2, and ToAMP3, produced by the flowers of the common dandelion Taraxacum officinale (
      • Astafieva A.A.
      • Rogozhin E.A.
      • Odintsova T.I.
      • Khadeeva N.V.
      • Grishin E.V.
      • Egorov T.A.
      Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.
      ). All three peptides have antifungal activities against A. niger and antibacterial activities against B. subtilis, whereas ToAMP3 also inhibits F. oxysporum (
      • Astafieva A.A.
      • Rogozhin E.A.
      • Odintsova T.I.
      • Khadeeva N.V.
      • Grishin E.V.
      • Egorov T.A.
      Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.
      ). The ToAMPs display unusual spacing between the cysteines and form a separate class of CRPs found so far only in T. officinale (
      • Astafieva A.A.
      • Rogozhin E.A.
      • Odintsova T.I.
      • Khadeeva N.V.
      • Grishin E.V.
      • Egorov T.A.
      Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.
      ). The seeds of the wax gourd, Benincasa hispida, produce a cationic peptide called hispidalin, which inhibits the fungus A. flavus and the bacteria B. cereus, E. coli, P. aeruginosa, S. aureus, and S. enterica at concentrations comparable with commercially available drugs (
      • Sharma S.
      • Verma H.N.
      • Sharma N.K.
      Cationic bioactive peptide from the seeds of Benincasa hispida.
      ). Active hispidalin has been heterologously produced in P. pastoris and shown to have protease stability and low hemolytic toxicity even at 300 μg/ml (
      • Meng D.M.
      • Li W.J.
      • Shi L.Y.
      • Lv Y.J.
      • Sun X.Q.
      • Hu J.C.
      • Fan Z.C.
      Expression, purification and characterization of a recombinant antimicrobial peptide Hispidalin in Pichia pastoris.
      ). Several bean species produce trypsin-resistant defensive peptides in their seeds; for example, vulgarinin produced by haricot beans (Phaseolus vulgaris) is fungicidal against F. oxysporum (
      • Wong J.H.
      • Ng T.B.
      Vulgarinin, a broad-spectrum antifungal peptide from haricot beans (Phaseolus vulgaris).
      ), inhibits several bacteria such as Bacillus megaterium, B. subtilis, Mycobacterium phlei, and Proteus vulgaris, and also inhibits the HIV reverse transcriptase (
      • Wong J.H.
      • Ng T.B.
      Vulgarinin, a broad-spectrum antifungal peptide from haricot beans (Phaseolus vulgaris).
      ).

      Unusual amino acids and derivatives

      Hundreds of amino acids not involved in peptide synthesis are produced in the plant kingdom. Instead, these amino acids are used for defensive functions such as deterring herbivores, pests, and pathogens or for allelopathy (
      • Vranova V.
      • Rejsek K.
      • Skene K.
      • Formanek P.
      Non-protein amino acids: Plant, soil and ecosystem interactions.
      ). Although many of these nonproteinaceous amino acids (NPAAs) display toxicity to animals, they show promise for their anticancer or neuroprotective effects and several studies have explored their antimicrobial properties against human pathogens (
      • Bell E.A.
      Nonprotein amino acids of plants: Significance in medicine, nutrition, and agriculture.
      ).
      Mimosine (also known as leucenol or β-[N-(3-hydroxy-4-pyridone)]-aminopropionic acid; Figure 1), produced by the seeds, leaves, and roots of several Fabaceae (
      • Renz J.
      Uber das mimosin.
      ,
      • Adams R.
      • Cristol S.J.
      • Anderson A.A.
      • Albert A.A.
      The structure of leucenol. I.
      ,
      • Bickel A.
      • Wibaut J.
      On the structure of Leucaenine (leucaenol) from Leucaena glauca Bentham.
      ), has potent activity against the dermatophytic fungi Trichophyton rubrum and Trichophytum tonsurans (
      • Anitha R.
      • Jayavelu S.
      • Murugesan K.
      Antidermatophytic and bacterial activity of mimosine.
      ). Pea (P. sativum) seedlings produce β-(3-isoxazolin-5-on-2-yl)-alanine (βIA; Fig. 1) (
      • Schenk S.
      • Werner D.
      β-(3-isoxazolin-5-on-2-yl)-alanine from Pisum: Allelopathic properties and antimycotic bioassay.
      ), which has broad-spectrum antifungal activity, including against S. cerevisiae (
      • Schenk S.
      • Werner D.
      β-(3-isoxazolin-5-on-2-yl)-alanine from Pisum: Allelopathic properties and antimycotic bioassay.
      ,
      • Schenk S.
      • Lambein F.
      • Werner D.
      Broad antifungal activity of beta-isoxazolinonyl-alanine, a non-protein amino acid from roots of pea (Pisum sativum L.) seedlings.
      ). Aside from their biological activities as free amino acids, some NPAAs are incorporated into larger molecules. m-Tyrosine (Fig. 1) is produced by many grasses as an herbicide (
      • Bertin C.
      • Weston L.A.
      • Huang T.
      • Jander G.
      • Owens T.
      • Meinwald J.
      • Schroeder F.C.
      Grass roots chemistry: meta-tyrosine, an herbicidal nonprotein amino acid.
      ), and macrocycles containing m-Tyrosine have been developed as viral protease inhibitors (
      • Chen K.X.
      • Njoroge F.G.
      • Pichardo J.
      • Prongay A.
      • Butkiewicz N.
      • Yao N.
      • Madison V.
      • Girijavallabhan V.
      Design, synthesis, and biological activity of m-tyrosine-based 16- and 17-membered macrocyclic inhibitors of hepatitis C virus NS3 serine protease.
      ). Ornithine, nicotinic acid (Fig. 1), anthranilic acid, and some β-hydroxy amino acids are also precursors for several classes of alkaloid compounds (
      • Dey P.
      • Kundu A.
      • Kumar A.
      • Gupta M.
      • Lee B.
      • Bhakta T.
      • Dash S.
      • Kim H.
      Analysis of alkaloids (indole alkaloids, isoquinoline alkaloids, tropane alkaloids).
      ,
      • Tuenter E.
      • Exarchou V.
      • Apers S.
      • Pieters L.
      Cyclopeptide alkaloids.
      ).
      Figure thumbnail gr1
      Figure 1Nonprotein amino acids (NPAAs) with anti-infective properties. Mimosine, leucenol or β-[N-(3-hydroxy-4-pyridone)]-aminopropionic acid (antifungal), β-(3-isoxazolin-5-on-2-yl)-alanine or βIA (antifungal), m-Tyrosine (part of antiviral molecules), nicotinic acid (part of bioactive alkaloids), l-canavanine (antibacterial), and azetidine-2-carboxylic acid, l-Aze, or A2C (part of antibacterial and antifungal molecules).
      l-Canavanine (Fig. 1), produced by leguminous plants such as Medicago sativa, interferes with quorum sensing in root-colonizing soil bacteria (
      • Keshavan N.D.
      • Chowdhary P.K.
      • Haines D.C.
      • González J.E.
      L-Canavanine made by Medicago sativa interferes with quorum sensing in Sinorhizobium meliloti.
      ), likely through inhibiting bacterial arginine deiminase. Arginine deiminase is absent in humans, and it provides beneficial traits to bacterial pathogens making it an attractive antibacterial and antiparasitic drug target (
      • Li L.
      • Li Z.
      • Chen D.
      • Lu X.
      • Feng X.
      • Wright E.C.
      • Solberg N.O.
      • Dunaway-Mariano D.
      • Mariano P.S.
      • Galkin A.
      • Kulakova L.
      • Herzberg O.
      • Green-Church K.B.
      • Zhang L.
      Inactivation of microbial arginine deiminases by L-canavanine.
      ,
      • Billard-Pomares T.
      • Clermont O.
      • Castellanos M.
      • Magdoud F.
      • Royer G.
      • Condamine B.
      • Fouteau S.
      • Barbe V.
      • Roche D.
      • Cruveiller S.
      • Médigue C.
      • Pognard D.
      • Glodt J.
      • Dion S.
      • Rigal O.
      • et al.
      The arginine deiminase Operon is responsible for a Fitness Trade-Off in extended-spectrum-β-Lactamase-producing strains of Escherichia coli.
      ). Azetidine-2-carboxylic acid or A2C (Fig. 1) contains an unusual four-membered heterocycle, produced by lily of the valley (Convallaria majalis) (
      • Fowden L.
      Azetidine-2-carboxylic acid: A new cyclic imino acid occurring in plants.
      ) and beet (Beta vulgaris) (
      • Rubenstein E.
      • Zhou H.
      • Krasinska K.M.
      • Chien A.
      • Becker C.H.
      Azetidine-2-carboxylic acid in garden beets (Beta vulgaris).
      ). A2C is an analog of both proline and alanine and activated by both human prolyl-and alanyl-tRNA synthetases, leading to misincorporation in proteins and protein toxicity (
      • Song Y.
      • Zhou H.
      • Vo M.-N.
      • Shi Y.
      • Nawaz M.H.
      • Vargas-Rodriguez O.
      • Diedrich J.K.
      • Yates J.R.
      • Kishi S.
      • Musier-Forsyth K.
      • Schimmel P.
      Double mimicry evades tRNA synthetase editing by toxic vegetable-sourced non-proteinogenic amino acid.
      ). However, owing to its toxicity, free A2C is generally avoided and instead the azetidinone moiety is preferred in drug development. Synthetic A2C derivatives are effective against bacterial and fungal pathogens (
      • Keri R.S.
      • Hosamani K.M.
      • Reddy H.S.
      • Shingalapur R.V.
      Synthesis, in-vitro antimicrobial and cytotoxic studies of novel azetidinone derivatives.
      ). Of interest, azetidinone incorporated into semisynthetic penicillins result in low cytotoxicity and improved efficacy against Staphylococcus sp. (
      • De Rosa M.
      • Vigliotta G.
      • Palma G.
      • Saturnino C.
      • Soriente A.
      Novel Penicillin-type Analogues Bearing a variable substituted 2-azetidinone ring at position 6: Synthesis and biological evaluation.
      ).

      Alkaloids

      Alkaloids are widely distributed in crop species and in medicinal plants employed over several millennia (
      • Amirkia V.
      • Heinrich M.
      Alkaloids as drug leads – a predictive structural and biodiversity-based analysis.
      ). Alkaloids are undeniably the best understood plant secondary metabolites and include atropine, caffeine, codeine, morphine, quinine, strychnine, theobromine, and xanthine. The alkaloid class covers many defense compounds and comprise a paraphyletic chemical group with regards to their biosynthesis where small molecules with one or more basic nitrogen atoms are considered alkaloids (
      • Manske R.H.F.
      • Holmes H.L.
      The Alkaloids: Chemistry and Physiology.
      ). These include compounds incorporating nitrogen from amino acids into heterocyclic rings (true alkaloids) or outside of the heterocyclic ring (protoalkaloids) (
      • Eagleson M.
      Concise Encyclopedia Chemistry.
      ). Over 27,000 alkaloids are currently listed in the Dictionary of Natural Products (
      • Hocking G.
      A Dictionary of Natural Products.
      ), and the number is continuously growing. The true alkaloids are derived primarily from the aromatic amino acids, namely, phenylalanine, tyrosine, and tryptophan, whereas NPAAs can also contribute to their biosynthesis. True alkaloids are classified based on the heterocyclic structure (
      • Dey P.
      • Kundu A.
      • Kumar A.
      • Gupta M.
      • Lee B.
      • Bhakta T.
      • Dash S.
      • Kim H.
      Analysis of alkaloids (indole alkaloids, isoquinoline alkaloids, tropane alkaloids).
      ) and more than 2500 sub-ring skeleton types have been detected in the KNApSAcK Core Database of 12,000 alkaloids (
      • Eguchi R.
      • Ono N.
      • Horai H.
      • Altuf-Ul-Amin M.
      • Hirai A.
      • Kawahara J.
      • Kasahara S.
      • Endo T.
      • Kanaya S.
      Classification of alkaloid compounds based on subring skeleton (SRS) profiling: On finding relationship of compounds with metabolic pathways.
      ). The enormous potential of alkaloids as drug leads is far from exhausted and a variety of pharmacological effects continues to be reported and reviewed (
      • Khadem S.
      • Marles R.J.
      Chromone and flavonoid alkaloids: Occurrence and bioactivity.
      ,
      • Cragg G.M.
      • Grothaus P.G.
      • Newman D.J.
      New horizons for old drugs and drug leads.
      ,
      • Ng Y.P.
      • Or T.C.
      • Ip N.Y.
      Plant alkaloids as drug leads for Alzheimer's disease.
      ,
      • Ain Q.U.
      • Khan H.
      • Mubarak M.S.
      • Pervaiz A.
      Plant alkaloids as Antiplatelet agent: Drugs of the future in the light of recent developments.
      ,
      • Hamid H.A.
      • Ramli A.N.
      • Yusoff M.M.
      Indole alkaloids from plants as potential leads for Antidepressant drugs: A mini review.
      ,
      • Davison E.K.
      • Brimble M.A.
      Natural product derived privileged scaffolds in drug discovery.
      ). Owing to the extensive diversity and immense number of alkaloids, we will only discuss selected examples of antibacterial, antifungal, antiviral, and antiparasitic molecules, with an emphasis on those reported in the last 10 years. A brief summary of the alkaloids with the most promising in vivo studies is shown in Table 2.
      Table 2A summary of the in vivo antimicrobial activity of promising alkaloids and organosulfur compounds
      Compound (class, plant source)Mechanism of actionTarget pathogen/infection (reference)Relevance
      α-Chaconine (steroidal glycoalkaloid, Solanaceae)Suppresses 70% of the parasites over 4 daysPlasmodium yoelli (
      • Chen Y.
      • Li S.
      • Sun F.
      • Han H.
      • Zhang X.
      • Fan Y.
      • Tai G.
      • Zhou Y.
      In vivo antimalarial activities of glycoalkaloids isolated from Solanaceae plants.
      )
      Pervasive drug resistance of malarial parasites
      Lycorine (phenylethylamine alkaloid, wild daffodil)Inhibits RNA-dependent RNA polymerase, reduces viral loadZika virus (
      • Chen H.
      • Lao Z.
      • Xu J.
      • Li Z.
      • Long H.
      • Li D.
      • Lin L.
      • Liu X.
      • Yu L.
      • Liu W.
      • Li G.
      • Wu J.
      Antiviral activity of lycorine against Zika virus in vivo and in vitro.
      )
      Approved vaccines/specific antivirals not available
      l-Ephedrine, d-pseudoephedrine (phenylethylamine alkaloid, Ephedra spp.)Mitigate lung injury, decrease viral load and serum interleukin IL-1β, reduce levels of inflammatory factors, increase serum interleukin 10 and interferon γInfluenza A virus (
      • Wei W.
      • Du H.
      • Shao C.
      • Zhou H.
      • Lu Y.
      • Yu L.
      • Wan H.
      • He Y.
      Screening of antiviral components of Ma Huang Tang and investigation on the Ephedra alkaloids efficacy on influenza virus type A.
      )
      Improves host immune defenses post infection
      Berberine (isoquinoline alkaloid, Berberidaceae)Globally reduces viral activation of major mitogen-activated protein kinase pathways, reduces viral titer and inflammatory symptomsChikungunya virus (
      • Varghese F.S.
      • Thaa B.
      • Amrun S.N.
      • Simarmata D.
      • Rausalu K.
      • Nyman T.A.
      • Merits A.
      • McInerney G.M.
      • Ng L.F.P.
      • Ahola T.
      The antiviral alkaloid berberine reduces chikungunya virus-induced mitogen-activated protein kinase signaling.
      )
      Attacks multiple targets and suppresses host inflammation
      MFM501 (synthetic derivative of pyrrolidine alkaloid from Codonopsis clematidea)Bacteriostatic against over 40 clinical strains, targets the bacterial membraneMethicillin-resistant S. aureus (
      • Johari S.A.
      • Mohtar M.
      • Mohammad S.A.
      • Sahdan R.
      • Shaameri Z.
      • Hamzah A.S.
      • Mohammat M.F.
      In vitro inhibitory and cytotoxic activity of MFM 501, a novel codonopsinine derivative, against Methicillin-Resistant Staphylococcus aureus clinical isolates.
      ,
      • Johari S.A.
      • Mohtar M.
      • Syed Mohamad S.A.
      • Mohammat M.F.
      • Sahdan R.
      • Mohamed A.
      • Mohamad Ridhwan M.J.
      In vitro evaluations and in vivo toxicity and efficacy studies of MFM501 against MRSA.
      )
      Clinical strains suppressed with no toxicity
      Voacamine (indole alkaloid, Tabernaemontana coronaria)Kills parasites by poisoning topoisomerase 1B; does not inhibit human topoisomerases I and IIL. donovani, L. amazonensis, T. cruzi (
      • Chowdhury S.R.
      • Kumar A.
      • Godinho J.L.P.
      • De Macedo Silva S.T.
      • Zuma A.A.
      • Saha S.
      • Kumari N.
      • Rodrigues J.C.F.
      • Sundar S.
      • Dujardin J.C.
      • Roy S.
      • De Souza W.
      • Mukhopadhyay S.
      • Majumder H.K.
      Voacamine alters Leishmania ultrastructure and kills parasite by poisoning unusual bi-subunit topoisomerase IB.
      )
      First molecule active against L. donovani strains resistant to sodium antimony gluconate, amphotericin B, and miltefosine
      Allicin (organosulfur, garlic)S-allylmercaptyl addition to bacterial cysteine sulfides, depletion of glutathione pools, induction of heat stress response; inhibits diesterases and oxidoreductases, disrupts plasma and endomembranes, promotes apoptosis and cell cycle arrest in parasites (reduces load, kills trophozoites)Lung pathogenic bacteria, Giardia duodenalis (
      • Reiter J.
      • Levina N.
      • van der Linden M.
      • Gruhlke M.
      • Martin C.
      • Slusarenko A.J.
      Diallylthiosulfinate (allicin), a Volatile antimicrobial from garlic (Allium sativum), kills human lung pathogenic bacteria, including MDR strains, as a vapor.
      ,
      • Argüello-García R.
      • de la Vega-Arnaud M.
      • Loredo-Rodríguez I.J.
      • Mejía-Corona A.M.
      • Melgarejo-Trejo E.
      • Espinoza-Contreras E.A.
      • Fonseca-Liñán R.
      • González-Robles A.
      • Pérez-Hernández N.
      • Ortega-Pierres M.G.
      Activity of Thioallyl compounds from garlic against Giardia duodenalis trophozoites and in experimental giardiasis.
      )
      Only inhalable antibiotic to clear lung infection; resistance to anti-giardial metronidazole rising, poor vaccine availability

      Pseudoalkaloids

      In these compounds, the carbon skeletons are not derived from amino acids and the nitrogen is usually incorporated by a transamination reaction. Pseudoalkaloids include the steroidal alkaloids of the Solanaceae family and glycoalkaloids. The identification of biosynthetic genes for steroidal alkaloids (
      • Szeliga M.
      • Ciura J.
      • Grzesik M.
      • Tyrka M.
      Identification of candidate genes involved in steroidal alkaloids biosynthesis in organ-specific transcriptomes of Veratrum nigrum L.
      ,
      • Cárdenas P.D.
      • Sonawane P.D.
      • Heinig U.
      • Bocobza S.E.
      • Burdman S.
      • Aharoni A.
      The bitter side of the nightshades: Genomics drives discovery in Solanaceae steroidal alkaloid metabolism.
      ) and the optimization of yeast platforms (
      • Xu S.
      • Li Y.
      Yeast as a promising heterologous host for steroid bioproduction.
      ) allow for the customization and biotechnological production of these molecules.

      Tomato alkaloids

      The tomato plant, S. lycopersicum L. produces the cholesterol-derived steroidal alkaloids tomatine and tomatidine. A summary of their biosynthesis from the precursor dehydrotomatidine via enzymatic dehydrogenation, isomerization, and successive reductions is shown in Figure 2, which is based on (
      • Akiyama R.
      • Lee H.J.
      • Nakayasu M.
      • Osakabe K.
      • Osakabe Y.
      • Umemoto N.
      • Saito K.
      • Muranaka T.
      • Sugimoto Y.
      • Mizutani M.
      Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes.
      ). Tomatidine exerts a selective and potent inhibitory effect against small-colony variants of S. aureus that cause opportunistic infections in patients with cystic fibrosis (
      • Mitchell G.
      • Gattuso M.
      • Grondin G.
      • Marsault É.
      • Bouarab K.
      • Malouin F.
      Tomatidine inhibits replication of Staphylococcus aureus small-colony variants in cystic fibrosis airway epithelial cells.
      ). Mutant and pharmacological analysis identified electron transport dysfunction as the major mechanism for the effect of tomatidine, which holds promise as a novel antibiotic lead against persistent forms of chronic S. aureus infections. Tomatidine also has potent fungistatic activity against Candida spp. with low toxicity to human cells (
      • Dorsaz S.
      • Snäkä T.
      • Favre-Godal Q.
      • Maudens P.
      • Boulens N.
      • Furrer P.
      • Ebrahimi S.N.
      • Hamburger M.
      • Allémann E.
      • Gindro K.
      • Queiroz E.F.
      • Riezman H.
      • Wolfender J.L.
      • Sanglard D.
      Identification and mode of action of a plant natural product targeting human fungal pathogens.
      ). Transcriptional and biochemical analysis led to the finding that tomatidine inhibits sterol methyltransferase and reductases. It is remarkable that tomatidine also shows antiviral activity in vitro against the chikungunya virus (CHIKV), for which vaccines and antiviral compounds are not currently available (
      • Troost B.
      • Mulder L.M.
      • Diosa-Toro M.
      • van de Pol D.
      • Rodenhuis-Zybert I.A.
      • Smit J.M.
      Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro.
      ). Tomatidine inhibits viral particle production after the entry of the virus into mammalian cells, and its activity persisted for 24 h after infection, suggesting that it blocks multiple rounds of viral replication.
      Figure thumbnail gr2
      Figure 2The biosynthetic pathway of the tomato alkaloids based on Akiyama et al. (
      • Akiyama R.
      • Lee H.J.
      • Nakayasu M.
      • Osakabe K.
      • Osakabe Y.
      • Umemoto N.
      • Saito K.
      • Muranaka T.
      • Sugimoto Y.
      • Mizutani M.
      Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes.
      ). The nitrogen incorporation occurs in the earlier phase of the biosynthesis from cholesterol (
      • Ohyama K.
      • Okawa A.
      • Fujimoto Y.
      Biosynthesis of steroidal alkaloids in Solanaceae plants: Incorporation of 3β-hydroxycholest-5-en-26-al into tomatine with tomato seedlings.
      ). The genes names in tomato are shown as yellow entries, while the blue entries are the enzyme activities. 3βHSD, 3β-hydroxysteroid dehydrogenase; 3KSI, 3-ketosteroid isomerase; S5αR, steroid 5α-reductase; 3KSR, 3-ketosteroid reductase.

      Other Solanaceae alkaloids

      The surfactant-like saponins are widely distributed in over 100 plant families and consist of terpenoid or steroidal glycoalkaloid compounds (
      • Kregiel D.
      • Berlowska J.
      • Witonska I.
      • Antolak H.
      • Proestos C.
      • Babic M.
      • Babic L.
      • Zhang B.
      Saponin-Based, Biological-Active Surfactants from Plants.
      ,
      • Moses T.
      • Papadopoulou K.K.
      • Osbourn A.
      Metabolic and functional diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives.
      ). Solanaceous plants produce steroidal alkaloids possessing broad spectrum activity against multiple groups of pathogens. The commonly occurring α-chaconine and α-solanine show strong antifungal properties (
      • Fewell A.M.
      • Roddick J.G.
      Interactive antifungal activity of the glycoalkaloids α-solanine and α-chaconine.
      ). The glycoalkaloids chaconine, solanine, solamargine, and tomatine were tested for antimalarial activity against Plasmodium yoelli in murine models (
      • Chen Y.
      • Li S.
      • Sun F.
      • Han H.
      • Zhang X.
      • Fan Y.
      • Tai G.
      • Zhou Y.
      In vivo antimalarial activities of glycoalkaloids isolated from Solanaceae plants.
      ), with the best antimalarial activity shown by chaconine. The replacement of the sugar moiety reduced the activity of the glycoalkaloids, suggesting that carbohydrate interactions are required for their antimalarial properties (
      • Chen Y.
      • Li S.
      • Sun F.
      • Han H.
      • Zhang X.
      • Fan Y.
      • Tai G.
      • Zhou Y.
      In vivo antimalarial activities of glycoalkaloids isolated from Solanaceae plants.
      ). Furthermore, sulfation of the 6-OH group led to loss of activity, demonstrating that this group is also critical for the pharmacological effects (
      • Chen Y.
      • Li S.
      • Sun F.
      • Han H.
      • Zhang X.
      • Fan Y.
      • Tai G.
      • Zhou Y.
      In vivo antimalarial activities of glycoalkaloids isolated from Solanaceae plants.
      ). Oral doses of most Solanaceae alkaloids of 3 to 5 mg/kg body weight in humans are toxic. However, mice can tolerate injections of chaconine at 7.5 mg/kg body weight with an ED50 (effective dose to reach 50% response in 50% of the subjects) of about 4.5 mg/kg body weight and a therapeutic index of 9 against P. yoelli infections (
      • Chen Y.
      • Li S.
      • Sun F.
      • Han H.
      • Zhang X.
      • Fan Y.
      • Tai G.
      • Zhou Y.
      In vivo antimalarial activities of glycoalkaloids isolated from Solanaceae plants.
      ). In the light of pervasive resistance to antimalarials, the Solanaceae alkaloids hold promise for further development.

      Protoalkaloids

      Protoalkaloids contain the amino acid–derived nitrogen outside of the heterocyclic ring. The two major families in this category are the terpenoid-containing indole alkaloids and the phenylethylamine alkaloids.

      Terpenoid indole alkaloids

      These are commonly found in plants of the dogbane (Apocynaceae) family, which includes Ervatamia chinensis, Voacanga africana, and the blackboard tree (A. scholaris). Indole alkaloids of E. chinensis possess antibacterial and antifungal activities (
      • Yu H.F.
      • Qin X.J.
      • Ding C.F.
      • Wei X.
      • Yang J.
      • Luo J.R.
      • Liu L.
      • Khan A.
      • Zhang L.C.
      • Xia C.F.
      • Luo X.D.
      Nepenthe-like indole alkaloids with antimicrobial activity from Ervatamia chinensis.
      ). The bioactive compounds erchinine A and B contain a unique 1,4-diazepine structure joined to an oxazolidine and showed activities against the fungus T. rubrum comparable with the standard antifungal drug griseofulvin, whereas the inhibitory effect on the bacterium B. subtilis was comparable with that of the antibiotic cefotaxime (
      • Yu H.F.
      • Qin X.J.
      • Ding C.F.
      • Wei X.
      • Yang J.
      • Luo J.R.
      • Liu L.
      • Khan A.
      • Zhang L.C.
      • Xia C.F.
      • Luo X.D.
      Nepenthe-like indole alkaloids with antimicrobial activity from Ervatamia chinensis.
      ). Although T. rubrum is generally non-life threatening, chronic T. rubrum infections facilitate secondary fungal infections, which can become lethal when systemic (
      • Warrilow A.G.S.
      • Parker J.E.
      • Price C.L.
      • Garvey E.P.
      • Hoekstra W.J.
      • Schotzinger R.J.
      • Wiederhold N.P.
      • Nes W.D.
      • Kelly D.E.
      • Kelly S.L.
      The Tetrazole VT-1161 is a potent inhibitor of Trichophyton rubrum through its inhibition of T. Rubrum CYP51.
      ,
      • Rouzaud C.
      • Hay R.
      • Chosidow O.
      • Dupin N.
      • Puel A.
      • Lortholary O.
      • Lanternier F.
      Severe Dermatophytosis and Acquired or innate immunodeficiency: A review.
      ). Although the mechanisms of action are not yet understood, erchinine A and B are promising for the development of novel antifungal leads.
      Aspidosperma olivaceum is a Brazilian medicinal plant, which contains several antimalarial compounds, with aspidoscarpine displaying promising activity and selectivity against the bloodstream forms of chloroquine-resistant Plasmodium falciparum and T. brucei (
      • Chierrito T.P.
      • Aguiar A.C.
      • de Andrade I.M.
      • Ceravolo I.P.
      • Gonçalves R.A.
      • de Oliveira A.J.
      • Krettli A.U.
      Anti-malarial activity of indole alkaloids isolated from Aspidosperma olivaceum.
      ). Buxus sempervirens extracts are used as an antimalarial, and its pharmacological effect is best explained by the presence of the cycloartane alkaloid O-tigloylcyclovirobuxeine-B, which shows selectivity against P. falciparum at low concentrations (
      • Althaus J.B.
      • Jerz G.
      • Winterhalter P.
      • Kaiser M.
      • Brun R.
      • Schmidt T.J.
      Antiprotozoal activity of Buxus sempervirens and activity-guided isolation of O-tigloylcyclovirobuxeine-B as the main constituent active against Plasmodium falciparum.
      ). Of importance, it was shown that cytotoxic and antimalarial/antitrypanosomal activities are due to other compounds in the extracts, and these compounds could be readily separated (
      • Althaus J.B.
      • Jerz G.
      • Winterhalter P.
      • Kaiser M.
      • Brun R.
      • Schmidt T.J.
      Antiprotozoal activity of Buxus sempervirens and activity-guided isolation of O-tigloylcyclovirobuxeine-B as the main constituent active against Plasmodium falciparum.
      ). The antibacterial and antiparasitic mechanism of this compound class is unclear, but earlier work suggests that they may inhibit DNA topoisomerase or intercalate DNA (
      • Wright C.W.
      • Bray D.H.
      • O'Neill M.J.
      • Warhurst D.C.
      • Phillipson J.D.
      • Quetin-Leclercq J.
      • Angenot L.
      Antiamoebic and antiplasmodial activities of alkaloids isolated from Strychnos usambarensis.
      ,
      • Bonjean K.
      • De Pauw-Gillet M.C.
      • Defresne M.P.
      • Colson P.
      • Houssier C.
      • Dassonneville L.
      • Bailly C.
      • Greimers R.
      • Wright C.
      • Quetin-Leclercq J.
      • Tits M.
      • Angenot L.
      The DNA intercalating alkaloid cryptolepine interferes with topoisomerase II and inhibits primarily DNA synthesis in B16 melanoma cells.
      ).

      Phenylethylamine alkaloids

      Lycorine (Fig. 3) is a benzyl phenethylamine alkaloid that was first isolated from the wild daffodil (Narcissus pseudonarcissus). Cedrón et al. synthesized and evaluated 27 derivatives of lycorine and found that the hydroxylation/esterification of the C1 or C2 positions and the presence of the double bond between C2 and C3 positions were essential for its antimalarial activity (
      • Cedrón J.C.
      • Gutiérrez D.
      • Flores N.
      • Ravelo A.G.
      • Estévez-Braun A.
      Synthesis and antiplasmodial activity of lycorine derivatives.
      ). Lycorine also inhibits flaviviruses such as West Nile virus (WNV), dengue virus (DENV), and yellow fever virus; however, a single amino acid substitution in the WNV 2K peptide was sufficient to confer lycorine resistance (
      • Zou G.
      • Puig-Basagoiti F.
      • Zhang B.
      • Qing M.
      • Chen L.
      • Pankiewicz K.W.
      • Felczak K.
      • Yuan Z.
      • Shi P.Y.
      A single-amino acid substitution in West Nile virus 2K peptide between NS4A and NS4B confers resistance to lycorine, a flavivirus inhibitor.
      ). In mice models, lycorine also possesses antiviral activity against the Zika virus (ZIKV) and inhibits RNA-dependent RNA polymerase (
      • Chen H.
      • Lao Z.
      • Xu J.
      • Li Z.
      • Long H.
      • Li D.
      • Lin L.
      • Liu X.
      • Yu L.
      • Liu W.
      • Li G.
      • Wu J.
      Antiviral activity of lycorine against Zika virus in vivo and in vitro.
      ) and, as a consequence, decreases the viral load. This is an important development since currently no vaccine or specific antiviral treatment is approved for ZIKV.
      Figure thumbnail gr3
      Figure 3Selected alkaloids, which have been utilized in in vivo studies: lycorine, berberine, cepharanthine, codonopsinine derivatives and voacamine.
      Substituted phenylethylamines are among the bioactive substances produced by Ephedra spp. (Ephedraceae) and commonly used as bronchodilators. The major Ephedra alkaloids are l-ephedrine, d-pseudoephedrine, and l-methylephedrine, which have antiviral effects on influenza A virus (IAV) in vitro, through inhibition of viral replication and modification of the inflammatory response (
      • Wei W.
      • Du H.
      • Shao C.
      • Zhou H.
      • Lu Y.
      • Yu L.
      • Wan H.
      • He Y.
      Screening of antiviral components of Ma Huang Tang and investigation on the Ephedra alkaloids efficacy on influenza virus type A.
      ). Of more importance, studies in mice showed that, after infection, l-ephedrine and d-pseudoephedrine mitigated lung injury, decreased the viral load and serum interleukin 1β, reduced transcription and translation of several inflammatory factors, and also increased the level of serum interleukin 10 and interferon γ (
      • Wei W.
      • Du H.
      • Shao C.
      • Zhou H.
      • Lu Y.
      • Yu L.
      • Wan H.
      • He Y.
      Screening of antiviral components of Ma Huang Tang and investigation on the Ephedra alkaloids efficacy on influenza virus type A.
      ) expression. Apart from their in vitro activity, the ability of Ephedra alkaloids to ameliorate host inflammation and induce antiviral host defenses against IAV make them promising candidates for clinical application.

      True alkaloids

      These contain one or more basic nitrogen elements and carbon skeletons derived from preotegenic and nonproteogenic amino acids. Selected classes are discussed here.

      Cyclopeptide alkaloids

      These are compounds with a 13-, 14-, or 15-membered macrocyclic ring system with 4 to 5 moieties comprising an amino acid, a β-hydroxy-amino acid, a hydroxystyrylamine moiety, and further substituents on the rings (
      • Gournelis D.C.
      • Laskaris G.G.
      • Verpoorte R.
      Cyclopeptide alkaloids.
      ,
      • Joullié M.M.
      • Richard D.J.
      Cyclopeptide alkaloids: Chemistry and biology.
      ). They are most widely distributed in the Acanthaceae, Malvaceae, Phyllanthaceae, Rhamnaceae, and Rubiaceae families; their structural diversity, pharmacological activities, syntheses, and antimalarial activity have been recently reviewed (
      • Tuenter E.
      • Exarchou V.
      • Apers S.
      • Pieters L.
      Cyclopeptide alkaloids.
      ,
      • Tuenter E.
      • Segers K.
      • Kang K.B.
      • Viaene J.
      • Sung S.H.
      • Cos P.
      • Maes L.
      • Heyden Y.V.
      • Pieters L.
      Antiplasmodial activity, cytotoxicity and structure-activity relationship study of cyclopeptide alkaloids.
      ). Fourteen-membered cyclopeptide alkaloids from the Brazilian medicinal plant Discaria americana (Rhamnaceae) showed antibacterial activity against E. coli, Enterobacter aerogenes, Enterobacter faecium, and S. enterica (
      • Dahmer J.
      • do Carmo G.
      • Mostardeiro M.A.
      • Neto A.T.
      • da Silva U.F.
      • Dalcol I.I.
      • Morel A.F.
      Antibacterial activity of Discaria americana Gillies ex Hook (Rhamnaceae).
      ). Mauritine-M and nummularine-H showed satisfying activity against Mycobacterium tuberculosis; the latter had an effect comparable with that of the frontline antibiotic isoniazid and was also able to target MDR strains (
      • Panseeta P.
      • Lomchoey K.
      • Prabpai S.
      • Kongsaeree P.
      • Suksamrarn A.
      • Ruchirawat S.
      • Suksamrarn S.
      Antiplasmodial and antimycobacterial cyclopeptide alkaloids from the root of Ziziphus mauritiana.
      ).
      Hymenocardia acida produces the antimalarial hymenocardine and other cyclopeptide alkaloids endowed with moderate activity, good selectivity, and low human cytotoxicity, and these could be employed as lead compounds for further optimization (
      • Tuenter E.
      • Exarchou V.
      • Ahmad R.
      • Baldé A.
      • Cos P.
      • Maes L.
      • Apers S.
      • Pieters L.
      Antiplasmodial activity of cyclopeptide alkaloids from Hymenocardia acida and Ziziphus oxyphylla.
      ). A 2017 study of several cyclopeptide alkaloids revealed that their antimalarial activity is increased if their macrocycle is 13-membered and methoxylated at position 2 of the styrylamine. The effect of modification of the β-hydroxy proline and aliphatic amino acids in the macrocycle remain unclear (
      • Tuenter E.
      • Segers K.
      • Kang K.B.
      • Viaene J.
      • Sung S.H.
      • Cos P.
      • Maes L.
      • Heyden Y.V.
      • Pieters L.
      Antiplasmodial activity, cytotoxicity and structure-activity relationship study of cyclopeptide alkaloids.
      ).

      Isoquinoline alkaloids

      These are found in several plant families such as Berberidaceae, Fumariaceae, Lauraceae, Menispermaceae, Papaveraceae, and Ranunculaceae and often possess antibacterial activity. Recently, enantioselective synthetic methods were developed for the reduced isoquinoline alkaloids, norglaucine, nordicentrine, and dicentrine, which showed promising activity against the parasites Leishmania infantum and Trypanosoma cruzi (
      • Pieper P.
      • McHugh E.
      • Amaral M.
      • Tempone A.G.
      • Anderson E.A.
      Enantioselective synthesis and anti-parasitic properties of aporphine natural products.
      ). The anti-T. cruzi alkaloid dicentrinone from Ocotea puberula (Lauraceae) causes disruption of parasite cell membranes via multiple mechanisms (
      • Barbosa H.
      • da Silva R.L.C.G.
      • Costa-Silva T.A.
      • Tempone A.G.
      • Antar G.M.
      • Lago J.H.G.
      • Caseli L.
      Interaction of dicentrinone, an antitrypanosomal aporphine alkaloid isolated from Ocotea puberula (Lauraceae), in cell membrane models at the air-water interface.
      ). From over 140 alkaloids tested, the most effective antimalarial was jozimine A2 from Ancistrocladus spp., which inhibited P. falciparum NF54 in the low-nanomolar range. Jozimine A2 was also nontoxic to mammalian cells and highly selective for P. falciparum as opposed to other parasites (
      • Bringmann G.
      • Zhang G.
      • Büttner T.
      • Bauckmann G.
      • Kupfer T.
      • Braunschweig H.
      • Brun R.
      • Mudogo V.
      Jozimine A2: The first dimeric Dioncophyllaceae-type naphthylisoquinoline alkaloid, with three chiral axes and high antiplasmodial activity.
      ), making it an excellent lead molecule for further antimalarial research.
      Berberine (Fig. 3) is a well-known benzylisoquinone alkaloid from the family Berberidaceae and inhibited the CHIKV in various cell lines (
      • Varghese F.S.
      • Thaa B.
      • Amrun S.N.
      • Simarmata D.
      • Rausalu K.
      • Nyman T.A.
      • Merits A.
      • McInerney G.M.
      • Ng L.F.P.
      • Ahola T.
      The antiviral alkaloid berberine reduces chikungunya virus-induced mitogen-activated protein kinase signaling.
      ). Furthermore, berberine is effective against several CHIKV strains without any direct effect on viral replication and significantly decreases the viral activation of the major mitogen-activated protein kinase (MAPK) signaling pathways. However, unlike specific kinase inhibitors, berberine decreased the viral activation of all major MAPK pathways, resulting in a marked reduction of the viral titer. Finally, in vivo mice models treated with berberine showed strong efficacy with an appreciable reduction of the Chikungunya-associated inflammatory symptoms (
      • Varghese F.S.
      • Thaa B.
      • Amrun S.N.
      • Simarmata D.
      • Rausalu K.
      • Nyman T.A.
      • Merits A.
      • McInerney G.M.
      • Ng L.F.P.
      • Ahola T.
      The antiviral alkaloid berberine reduces chikungunya virus-induced mitogen-activated protein kinase signaling.
      ).
      Cepharanthine (Fig. 3) is a bisbenzylisoquinoline alkaloid from the Asian medicinal plant Stephania cepharantha (Menispermaceae) and approved for clinical use in Japan. It has an established safety record and is employed for its antiparasitic and antiviral properties, as well as several health benefits (
      • Rogosnitzky M.
      • Danks R.
      Therapeutic potential of the biscoclaurine alkaloid, cepharanthine, for a range of clinical conditions.
      ). Several mechanisms explain its antimicrobial activities including interference with efflux pumps, membrane rigidification, modulation of the AMP-activated protein kinase, and impacting the nuclear factor kappa-light-chain (NF-κB) signaling pathways (
      • Bailly C.
      Cepharanthine: An update of its mode of action, pharmacological properties and medical applications.
      ). It suppresses several processes critical for both viral replication and the host inflammatory response, such as activation of nuclear factor NF-κB, lipid peroxidation, cyclooxygenase expression, and nitric oxide (NO) and cytokine production (
      • Rogosnitzky M.
      • Okediji P.
      • Koman I.
      Cepharanthine: A review of the antiviral potential of a Japanese-approved alopecia drug in COVID-19.
      ). Among the over 2400 clinically approved drugs screened in a repurposing effort for the current COVID-19 pandemic, cepharanthine was the most potent and capable of inhibiting both the entry and replication of SARS-CoV-2 and similar viruses providing solid rationale for its use in antiviral development (
      • Rogosnitzky M.
      • Okediji P.
      • Koman I.
      Cepharanthine: A review of the antiviral potential of a Japanese-approved alopecia drug in COVID-19.
      ). However, it has so far not been economically synthesized (
      • Bailly C.
      Cepharanthine: An update of its mode of action, pharmacological properties and medical applications.
      ).

      Pyrrolidine alkaloids

      Plants of the Amaryllidaceae family commonly produce pyrrolidines. The Asian bellflower (Codonopsis clematidea; Campanulaceae) contains unusual aromatic substituted pyrrolidines with antibiotic activities including codonopsinine (
      • Matkhalikova S.F.
      • Malikov V.M.
      • Yunusov S.Y.
      The structure of codonopsinine.
      ,
      • Zaurov D.
      • Belolipov I.
      • Kurmukov A.
      The medicinal plants of Uzbekistan and Kyrgyzstan.