Potential of agricultural fungicides for antifungal drug discovery

Abstract

While it is true that only a small fraction of fungal species are responsible for human mycoses, the increasing prevalence of fungal diseases has highlighted an urgent need to develop new antifungal drugs, especially for systemic administration. This contribution focuses on the similarities between agricultural fungicides and drugs. Inorganic, organometallic and organic compounds can be found amongst agricultural fungicides. Furthermore, fungicides are designed and developed in a similar fashion to drugs based on similar rules and guidelines, with fungicides also having to meet similar criteria of lead-likeness and/or drug-likeness. Modern approved specific-target fungicides are well-characterized entities with a proposed structure–activity relationships hypothesis and a defined mode of action. Extensive toxicological evaluation, including mammalian toxicology assays, is performed during the whole discovery and development process. Thus modern agrochemical research (design of modern agrochemicals) comes close to drug design, discovery and development. Therefore, modern specific-target fungicides represent excellent lead-like structures/models for novel drug design and development.

Keywords:

• agricultural fungicides
• antifungal drugs
• drug design and discovery
• drug-likeness
• lead-likeness
• mycoses

1. Introduction

Fungi have existed on Earth for hundreds of millions of years and have gone along with humans throughout their history. There are approximately 2 million different species of fungi on Earth that live outdoors in soil and on plants and trees, animals, mammals as well as on many indoor surfaces and on human skin.[1,2] Higher fungi have a long history of use in national cuisines and folk medicine, and their study has become a matter of great significance in recent decades. Investigations on their secondary metabolites have been mostly aimed at isolating bioactive compounds as potential lead structures for the development of new drugs, products for crop protection and even cosmetics, for example, Ganoderma lucidum or Cordyceps sp. The investigation of microfungi for bioactive metabolites was initiated by the discovery of penicillin G from Penicillium notatum by A. Fleming in 1928. Since then, fungal microorganisms became a hunting ground for novel drug leads. In the following years, several other important drugs and lead bioactive agents from fungi were discovered, such as cephalosporins, cyclosporine, echinocandins, emodepside, fusidic acid, griseofulvin, lovastatin, mevastatin, nodulisporic acid, pneumocandins, scyphostatin, strobilurin and various anticancer agents. Many of these compounds can be produced in large quantities and at a reasonable cost by fermentation employing wild-type or genetically altered fungi. These achievements stimulated pharmaceutical companies to sample and screen large collections of fungal strains and analyze their secondary metabolites. Microfungi for drug discovery were mainly isolated from soil samples; later, attention was given to other, alternative sources including marine microorganisms and endophytic fungi associated with plants. Various fungi are also used for biotransformation of steroids that have been extensively used in medicine.[3–5] Currently, microorganisms are used for synthesis of nanoparticles within green nanotechnology; biosynthesis of gold, silver, gold-silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, magnetite and uraninite nanoparticles by Aspergillus sp., Colletotrichum sp., Fusarium sp., Neurospora sp., Penicillium sp., Phoma sp., Trichoderma sp., Verticillium sp., yeasts and other fungal species has been reported.[6,7]

Fungi are ubiquitous in nature and vital for recycling of nutrients contained in organic matter. The vast majority of the known fungal species are strict saprophytes,[8] and based on the above-mentioned facts they are very useful. But some of them can attack human, animals and plants; it is estimated that 270,000 fungal species are associated with plants, and 325 are known to infect humans. It can be stated that virtually every living species has its own parasitic fungal pathogens to cope with.[1,2,9,10] The most significant fungal species that cause human infections, allergies and toxicosis include Absidia, Alternaria, Aspergillus, Blastomyces, Candida, Cladosporium, Coccidioides, Colletotrichum, Cryptococcus, Curvularia, Exserohilum, Fusarium, Histoplasma, Microsporum, Mucor, Pneumocystis, Rhizomucor, Rhizopus, Sporothrix and Trichophyton. It is evident that fungal diseases are caused by fungi that are common in the environment.[1,2,8,11] These fungi can be categorized into two groups in regard to infection: (i) saprophytic fungi that can be opportunistic pathogens that enter via wounds or due to a weakened state of the host and (ii) true pathogens that may depend on human tissues for nutrients but can also survive outside of the hosts. Human fungal infections range from superficial nail and skin infections to invasive, systemic infections that are really harmful to health and life that are caused by genera Aspergillus, Candida, Cryptococcus, Fusarium and Pneumocystis. Human fungal infections generally receive less attention than bacterial and viral diseases,[12] since the incidence of systemic fungal infections is considerably lower than that of superficial infections; however, mortality rates from invasive fungal infections are very high, often exceeding 50%, despite the use of antifungal drugs.[1,8,11–13]

Early diagnosis of disease and identification of the fungal pathogen remain crucial for the treatment of invasive fungal infections, because the efficacy of currently used drugs is limited by issues with administration route, narrow treatment window, activity spectrum, bioavailability, toxicity, drug resistance and cost. The increase in the number of fungal infections and the occurrence of new fungal opportunistic species is caused by general immunosuppression (primarily by tumor treatment, administration of immunosuppressive agents, wide-spectrum antibacterial chemotherapeutics and corticoids), a significant increase in the number of HIV-positive and, e.g., diabetic patients.[12–15]

2. Current situation of antifungal drugs

Antifungals (antifungal agents) are drugs that destroy or prevent the growth of fungi (yeasts, molds). They can be divided into two main classes: (i) nonspecific antifungals, and (ii) targeted site-specific antifungals. Nonspecific antifungals can be considered as disinfectants-antiseptics especially for superficial/local treatment of skin or mucosa. They can be divided into (i) aldehydes (e.g., polynoxylin); (ii) acids (e.g., benzoic acid, salicylic acid, 5-bromo-salicylic acid, undecylic acid); (iii) phenols/halogenated phenols (e.g., chlorocresol, 2-chloro-4-nitrophenol, chlorophene, chlorophetanol, chloroxylenol, haloprogin, hexachlorophene, parabens, tetrabromo-o-cresol); (iv) quinolinols (e.g., chloroxine); (v) amides/amidines (e.g., dimazole, ticlatone); (vi) quaternary ammonium/phosphonium salts (e.g., dequalinium dichloride, dodecyltriphenylphosphonium bromide); and (vii) dyes (e.g., methylrosanilinium chloride). Clinically used site-specific antifungal drugs classified according to the mode of action and their chemical structure are listed in Table 1.[16] Other potential antifungal agents are under basic research, for example, (benz)azoles, benz(thia/oxa)zoles, pyrroles, (mono/di-aza)naphthalenes, naphthoquinones, morpholines, phenothiazines, etc.[16–18]

Table 1. Approved human antifungal drugs.[16,19,20]

Classification/mode of action			General structure	Examples
agents affecting ergosterol	synthesis inhibitors	imidazoles (14-α-demethylase inhibitors)		clotrimazole, isoconazole, ketoconazole, luliconazole, miconazole, oxiconazole, tioconazole
		triazoles (14-α-demethylase inhibitors)		fluconazole, isavuconazole, itraconazole, posaconazole, terconazole, voriconazole
		thiocarbamates (squalene 2,3-epoxidase inhibitors)		tolnaftate
		naphthylmethylamines (squalene 2,3-epoxidase inhibitors)		naftifine, terbinafine, butenafine
		phenylpropylmorpholines (Δ^14 reductase and Δ^7 – Δ^8 isomerase inhibitors)		amorolfine
	ergosterol binding polyenes	macrolide antibiotics with glycosidic bounded mycosamine (bond to ergosterol in cell membrane, transmembrane channels creation, change in membrane permeability)		amphotericin B, nystatin, natamycin
agents interacting with cell wall	glucan synthesis inhibitors	echinocandins, pneumocandins (β-(1,3)-glucan synthase inhibitors)	cyclic lipopeptide antibiotics	caspofungin, anidulafungin, micafungin
transport processes inhibitors	proton pump inhibitors	N-hydroxypyridones (multiple mode of action; also disrupt membrane and intracellular transport systems)		ciclopirox
inhibitors at nuclear level	nucleic acid synthesis inhibitors	pyrimidines/thymidylate (synthase inhibitors – competitive synthesis inhibitor of DNA and RNA)		flucytosine
	protein synthesis inhibitors	benzoxaboroles (cytoplasmic leucyl-aminoacyl transfer RNA synthetase inhibitors)		tavaborole
	microtubules synthesis inhibitors	benzofurans (inhibitor of mitosis (bind to α- and β-tubulin) and nuclear acid synthesis)		griseofulvin

Nevertheless, in spite of this list, the fact remains that majority of the drugs are for topical antifungal medication for the treatment of mycoses of nails, skin and mucosa. Only some of them were approved for the treatment of systemic fungal infections (ATC J02A). These include azoles, especially triazoles (fluconazole, isavuconazole, itraconazole, posaconazole, voriconazole), naphthylmethylamines (terbinafine), pyrimidine (flucytosine), benzofurans (griseofulvin), echinocandins (caspofungin, anidulafungin, micafungin) and different (nano)formulations of polyene amphotericin B.[19,20] Development of resistance/cross-resistance to commonly used drugs and multidrug-resistance of fungal pathogens (Candida sp., Aspergillus sp. and Cryptococcus sp.) constitutes a serious problem, and it is evident that new systemic (preferably orally administered) antifungal drugs are urgently needed.[15,21,22]

3. Agricultural fungicides

Fungi cause crop losses worldwide. Fungal diseases have a significant economic impact on plant yield and quality, thus managing such diseases is an essential component of production for most crops. A fungicide is a specific type of pesticide that controls fungal disease by specifically inhibiting or killing the fungus that causes the disease. Not all diseases caused by fungi can be adequately controlled by fungicides. These include the vascular wilt fungal diseases Fusarium and Verticillium. Fungicides are used to control a disease during the establishment and development of a crop, to increase productivity of a crop and to reduce blemishes and to improve the storage life and the quality of harvested plants. Therefore, fungicides are extensively used in agriculture to control soil borne, seed borne or air borne fungal pathogens.[23,24]

Commercial agricultural fungicides are classified according to their target sites by the international Fungicide Resistance Action Committee (FRAC).[25] Based on this FRAC classification, fungicides that can be potentially used as model compounds in drug design were selected and are mentioned in Table 2. It is important to note that in Table 2 the following is not listed: inorganic, metallo-organic and human hazardous fungicides, compounds with the same mode of action as used antifungal drugs (inhibitors of 14-α-demethylase, squalene 2,3-epoxidase, Δ¹⁴ reductase and Δ⁷–Δ⁸ isomerase), fungicides with already known resistance/cross-resistance and fungicides that are used as drugs in other indications (e.g., show significant antibacterial or antiproliferative properties).[26] Due to the above-mentioned properties, many noteworthy fungicide classes, which can be considered as very important agricultural fungicides, are not included in Table 2, e.g., RNA polymerase I inhibitors (code A1), compounds affecting mitosis (codes B1, B2), succinate dehydrogenase inhibitors (code C2), strobilurins (cytochrome bc1 (ubiquinol oxidase) inhibitors, code C3), amino acids and protein synthesis inhibitors (codes D1-5). From Tables 1 and 2 it is evident that agricultural fungicides show much higher diversity in their chemical structures and modes of action than human antifungals. Although mostly specific-target fungicides are used, multisite fungicides, e.g., copper and sulfur formulations, dithiocarbamates, phthalimides, anthraquinones and chloronitriles, are needed to control many plant pathogens.[25] Nevertheless, this trend in development of multisite antifungal drugs was also recently described. [27,28]

Table 2. Selected agricultural fungicides (including mode of action and FRAC target site code) potentially interesting for drug design.[25,26]

Classification/mode of action (code)			General structure	Examples
nucleic acids synthesis inhibitors	adenosine-deaminase inhibitors (A2)	2-amino-4-hydroxy-pyrimidines		bupirimate, ethirimol
	DNA/RNA synthesis inhibitors (A3)	isoxazoles, isothiazolones		hymexazole, octhilinone
affecting mitosis and cell division	β-tubulin assembly in mitosis (B3)	(hetero)arylcarboxamides		zoxamide, ethaboxam
	cell division (B4)	phenylureas		pencycuron
	delocalization of spectrin-like proteins (B5)	benzamides		fluopicolide
respiration inhibitors	NADH oxidoreductase inhibitors (C1)	pyrimidinamines		diflumetorim
		pyrazole-5-carboxamides		tolfenpyrad
	cytochrome bc1 (ubiquinone reductase) inhibitors (C4)	azole-sulfonamides (cyano-imidazole, sulfamoyl-triazole)		cyazofamid, amisulbrom
	cytochrome bc1 (unknown binding site) (U16)	4-quinolyl-acetate		tebufloquin
	uncouplers of oxidative phorylation (C5)	dinitrophenyl crotonates, 2,6-dinitro-anilines		dinocap, fluazinam
	ATP production inhibitors (C7)	thiophene-3-carboxamides		silthiofam
affecting signal transduction	signal transduction (E1)	azanaphthalenes (aryloxyquinolines, quinazolinones)		quinoxyfen, proquinazid
	osmotic signal transduction MAPK/HK inhibitors (os-2, HOG1) (E2)	phenylpyrroles		fenpiclonil, fludioxonil
affecting lipid synthesis and membrane integrity	cell membrane permeability, fatty acids (F4)	(thio)carbamates		propamocarb, prothiocarb
ergosterol biosynthesis inhibitors	3-keto reductase, C4-demethylation (G3)	hydroxyanilides, amino-pyrazolinone		fenhexamid, fenpyrazamine
cell wall biosynthesis inhibitors	trehalase- and inositol-synthesis inhibitors (H3)	glucopyranosyl antibiotics		validamycin
	cellulose synthase inhibitors (H5)	amides of cinnamic and mandelic acids		dimethomorph, mandipropamid
		valinamide carbamates		benthiavalicarb, valifenalate
inhibitors of melanin biosynthesis in cell wall	reductase inhibitors (I1)	isobenzofuranones		fthalide
		pyrroloquinolinones		pyroquilon
		triazolobenzothiazoles		tricyclazole
	dehydratase inhibitors (I2)	carboxamides		carpropamid, diclocymet, fenoxanil
	polyketide synthase inhibitors (I3)	trifluoroethyl carbamates		tolprocarb
actin disruptors	unknown binding site (U8)	(hetero)aryl- trimethoxyphenyl-ketones		metrafenone, pyriofenone
oxysterol binding protein inhibitors	unknown binding site (U15)	piperidinylthiazole- isoxazolines		oxathiapiprolin

4. Similarity of drugs and agrochemicals

The most valuable drug or agrochemical R&D approach is to design a new structure with a new mechanism of action. However, this process is very expensive, lengthy and uncertain as to the outcome. When a new molecule is designed, knowledge of related branches or retesting of previously described compounds for new effects can be used. An effective approach for obtaining new active agents is structure modification of existing drugs. These “me too” drugs are developed based on the knowledge of structure–activity relationships but rarely have a new mode of action.

The increase in the number of fungal infections, their more difficult treatment and the emergence of resistant fungal strains make the discovery of new molecular scaffolds a priority, and the current situation even necessitates re-engineering and repositioning of some old drug families to achieve effective control. Based on the mentioned data and the structures from Tables 1 and 2, it can be stated that agricultural fungicides constitute an excellent source of lead structures. Moreover, in the past, most pharmaceutical companies had pesticide divisions, because drugs and pesticides are designed to target particular biological functions, and in some cases, these functions overlap in their molecular target sites or target similar processes or molecules. All compounds generated by either division of the company were evaluated for pesticide and pharmaceutical uses, and this arrangement allowed knowledge to flow freely between the two divisions. Thus, formerly, some lead pesticides became pharmaceuticals and vice versa. However, little information of this type was published and must usually be deduced from patent literature. One of the exceptions is fluconazole, a fungicide product discovered by the pharmaceutical sector that is now used as a pharmaceutical but also was patented as a chemical with applications in crop production.[29–34]

Bioavailability is highly important for any bioactive agent. The Lipinski’s Rule of Five [35] can be considered as the reference for defining physicochemical and structural parameters for oral drug bioavailability. Molecules that meet these criteria can be considered as drug-like. The Rule of Three published by Carr et al. [36] establishes parameters for individual fragments (fragment-like properties) in Fragment-Based Drug Design; that is, based on this rule rather lead-likeness of new structures than drug-likeness can be defined. Other simplified rule-based drug-likeness and/or lead-likeness definitions were summarized by, for example, Abad-Zapatero or Walters.[37,38] Rules based on the Lipinski’s approach aimed at improvement of oral bioavailability and redistribution of agrochemicals were adopted quickly, see Table 3, where the most significant rules are summarized. The pioneer work was published by Briggs as the Rule of Three for agrochemical in vivo high-throughput screening,[39] but the notable paper was published by Tice.[40] Clarke [41] added other molecular properties known to influence absorption and distribution of agrochemicals and published it as the Rule of Two for lead progression. Hao et al. [42] published recently a new comparative study on the constitutive properties of newly marketed pesticides. He defined simple and easy to implement rules for pesticide-likeness, by including molecular weight (MW), lipophilicity (expressed as log P), number of H-bond acceptors (HBA) and donors (HBD), number of rotatable bonds (RB) and number of aromatic bounds. Comprehensive investigations and matching of the discussed parameters were performed by Lindell et al. [43] and Avram et al. [44] There are some small differences in the optimal properties for agrochemicals and drugs. Agrochemicals show a slightly lower tolerance for H-bond donors than drugs. This is caused by the requirement to agrochemicals to resist to metabolic attack by the pest species.[30,40,41] Nevertheless, it can be stated that the conclusions reached in all these studies were very similar to each other and to the Lipinski’s work.

Table 3. Bioavailability guidelines – summary of relevant rules concerning physicochemical property limits of drugs and agrochemicals.

Parameter	Drugs		Agrochemicals
	Lipinski [35]	Carr [36]	Briggs [39]	Tice [40]	Clarke [41]	Hao [42]
MW	≤500	≤300	300 ± 100	≤500	200–400	≤435
log P	≤5	≤3	3 ± 3	≤5	≤4	≤6
HBD	≤5	≤3	≤3	≤3	≤2	≤2
HBA	≤10	≤3	–	≤12	–	≤6
RB	–	≤3	–	≤12	–	≤9

HBA: number of H-bond acceptors; HBD: number of H-bond donors; log P: lipophilicity; MW: molecular weight; RB: number of rotatable bonds.

Leads are usually smaller and less lipophilic than launched active ingredients; MW and lipophilicity generally increase during the lead-to-drug optimization process. According to Myung and Klittich, most marketed fungicides possess physicochemical parameters within the lead-like range for drugs.[34] Also modern fungicides have been targeted to specific active sites (often have nanomolar (or better) affinity for their target site); they are subjected to lead optimization and thus fulfill other requirements of lead chemistry such as tractability in structure–activity relationships and lack of reactivity or promiscuous binding.[45] The similarity of drugs and pesticides was supported by comparison of the frequency of occurrence of structural fragments in the two types of compounds [30 and refs. thereon].

Toxicity assays are crucial in development of each drug. Various mammalian-based toxicity assays are key issues for modern pesticide research. Toxicity indicators are evaluated at various stages of discovery and development, from eliminating undesirable modes of action early in development to the detailed good laboratory practice studies before registration of a new product. Acute and subchronic tests for oral toxicity, dermal toxicity, irritation and sensitization, inhalation toxicity, eye irritation and neurotoxicity are also standardly conducted within pesticide research. Chronic toxicity and mutagenicity tests are required to determine effects of prolonged and repeated exposure, including potential for carcinogenicity, teratogenicity and reproduction effects. As the use pattern for agricultural fungicides is designed to minimize human exposure and thus is very different from intentional dosing to humans to control a fungal infection, there is no guarantee that fungicides with excellent safety in agricultural applications will avoid toxicity issues in humans when administered at much higher doses; nevertheless, the complexity of toxicity information available for registered fungicides should provide a well-informed starting point for new potential drug leads [30,34 and refs. thereon].

5. Conclusion

Infections represent an increasing worldwide threat. This increase in the number of new infections is caused by general immunosuppression of world population and development of resistance of strains to commonly used drugs. Also development of cross-resistant or multidrug-resistant strains constitutes a great problem. Development in the field of new potential antifungal drugs is not so common/extensive as, e.g., design and discovery of new potential antibacterial drugs. The majority of approved antifungal drugs are for the treatment of topical mycoses. Only 12 drugs have been approved for the treatment of systemic fungal infections, and they feature serious undesirable/toxic effects. The “me too” drugs cannot be a solution in this critical situation. As a source of inspiration for design of qualitatively new antifungal drugs, some of classes (structures) of new modern agricultural fungicides can be used, since many of them meet the criteria of lead-likeness and/or drug-likeness.

6. Expert opinion

Although macro and micro fungi are beneficial to human, a small fraction of the estimated 2 million fungal species is responsible for devastating diseases affecting crops, food security and human health. The increasing emergence of fungal diseases is furthermore promoted by human activity, primarily through global trade, which lacks sufficient biosecurity measures, and may be exacerbated by the impact of climate change. The massive use of fungicides to protect crops may contribute to resistance against drugs used to treat people with life-threatening infections. Fungal pathogens are characterized by a remarkable genetic flexibility that facilitates rapid evolution and adaptation to the host or environment. Among agricultural fungicides inorganic, organometallic or organic compounds can be found. Also, encapsulated fungicides in nanoformulations primarily designed to decrease dose-dependent toxicity for non-target organisms and environmental burden by means of their enhanced bioavailability (targeted site-specific delivery and controlled release) and protection against degradation especially are developed. Thus, modern agrochemical research as well as design of modern agrochemicals is close to drug design, discovery and development. Modern approved specific-target fungicides (sometimes multisite fungicides) are well-characterized entities (also in relation to stereochemistry) with proposed SAR hypotheses related to defined modes of action. Extensive toxicology evaluation, including mammalian toxicology assays, is routinely performed during the whole discovery and development process. Therefore, in my opinion, utilizing modern specific-target agricultural fungicides can accelerate the process of identification of new modes of action and leads/lead-like structures for a pharmaceutical pipeline that will control human fungal pathogens.

Declaration of interest

J Jampilek is supported by the grant projects of the Internal Grant Agency of the Veterinary and Pharmaceutical University Brno 37/2014/FaF, 52/2014/FaF and 304/2015/FaF. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.