Many superficial mycoses have been treated with a range of topical medicines that have had moderate efficacy in reducing common infections over the last century. The fact that there are limited antifungal medications available to treat systemic fungal infections increases the impact of fungus on human health (Perfect, 2017). There are only four primary classes of available antifungal drugs, which include- polyenes, flucytosine, azoles, and echinocandins (Carmona and Limper, 2017). Many opportunistic fungi such as Candida auris and Lomentospora prolificans are innately resistant to many antimycotics (Arastehfar et al., 2020). Some species, like C. glabrata, have strong intrinsic resistance to antifungals like azoles, while other species such as C. albicans, can acquire resistance to antifungals after being exposed to them, mainly during preventive treatments (Arastehfar et al., 2020). Despite the limited number of antimicrobials available, microorganisms have a remarkable capacity to develop resistance to them, posing a serious threat to public health. Fungal pathogens, which can only be treated with a limited number of antifungal medications and produce catastrophic invasive infections, are of particular concern. The rates of resistance for fungal infections vary by geography, species, and accessible treatments; nonetheless, the spread of drug-resistant fungus species is global. In general, fungal pathogens’ innate and acquired drug resistance strategies involve lowering the effective drug concentration, changing the drug target, or rerouting antifungal toxicity through metabolic change (Sanglard, 2016).

Some of the challenges that hinder antifungal drug development can be summarized as follows:

Cockroaches have been found to carry members of several related bacterial families, most notably the Enterobacteriaceae, Staphylococcaceae, and Mycobacteriaceae species. The most often reported genera from the cockroach gut were Streptomyces, Bacillus, Enterococcus, and Pseudomonas (Guzman and Vilcinskas, 2020). Feces of the cockroach Cryptocercus punctulatus demonstrated antifungal action linked to compounds made by gut microorganisms (Rosengaus et al., 2013). A species of actinobacteria, Streptomyces globisporus WA5-2-37 that produces Actinomycin X₂ and Collismycin, was just recently discovered in the intestine of Periplaneta americana (Chen et al., 2020). Bacillus atrophaeus, Bacillus subtilis, and Pseudomonas reactans are a few bacterial strains that have been successfully isolated from the gut of Blatella germanica and have the ability to limit the growth of entomopathogenic fungus Beauveria bassiana (Huang et al., 2013).

Vertebrate skin microbiomes act as a defense against infections and can influence disease risk. Higher bacterial species richness, certain microbial community assemblages, and the presence of microorganisms that produce compounds that impede pathogen growth have all been linked to lower infection risk in vertebrates. Many amphibians’ population declines and extinctions have been attributed to the fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes chytridiomycosis (Berger et al., 2016). Since these circumstances match with the pathogen’s ideal development range, chytridiomycosis has primarily afflicted tropical amphibian populations at high elevations and during cooler seasons (Sapsford et al., 2013). Amphibians have a variety of defence mechanisms, including the presence of skin bacteria that create antifungal metabolites. For example, the bacteria Janthinobacterium lividum, which lives in the salamander Plethodon cinereus, produces anti-Bd secondary metabolites such as violacein and indole-3-carboxaldehyde (Brucker et al., 2008).

Communities of symbiotic microorganisms are found in insect digestive systems, and these communities are the focus of research on microbial diversity and new bioactive microbial compounds (Breznak, 2003). Fungus-farming termites belonging to the subfamily Macrotermitinae, cultivate basidiomycetes (genus Termitomyces) as their main source of food (Lever et al., 2015) and these basidiomycetes are constantly susceptible to parasitism by other fungi. The termites probably use a variety of tactics to manage such antagonists, with the production of antifungals being one among them (Um et al., 2013). The gut flora of the Macrotermitinae has so far been found to contain seven different bacterial phyla (Otani et al., 2014). Bacillus strains are the most prevalent and can produce antifungals among them. An initial analysis of an extract of the Bacillus strain cultures using liquid chromatography and mass spectrometry identified a significant secondary metabolite: Bacillaene, a polyene polyketide that is present in all strains and which inhibits the growth of Pseudoxylaria, Trichoderma, and Fusarium (Um et al., 2013). From the nests of the French Guiana termite Nasutitermes corniger, the fungus Neonectria discophora SNB-CN63 was isolated (Sorres et al., 2018) that produced Ilicicolinic acid C and D. These are non-macrocyclic, non-polycyclic polyketide that showed antifungal activities against Trichophyton rubrum and Candida albicans (Nirma et al., 2015). There is proof that the hindgut microbiota of the termite Zootermopsis angusticollis produces many functionally active b-1,3-glucanases with a probable involvement in fungal pathogen protection (Rosengaus et al., 2014). Termites also support the Streptomyces species, which makes the antifungal Natalamycin (Kim et al., 2014). Other antibiotics known as microtermolides A and B are produced by the Streptomyces strain connected to the fungus-farming termites (Carr et al., 2012).

The solitary wasps Sceliphron caementarium and Chalybion californicum are associated with Streptomyces spp. that produce Streptochlorin, a range of Piericidin analogs, and other chemicals that protect their larvae against bacteria and fungi (Poulsen et al., 2011). Sceliphrolactam, an antifungal substance, was discovered from Streptomyces that was associated to the mud dauber wasp Sceliphron caementarium (Oh et al., 2011). The substance is a polyene macrocyclic lactam with antifungal action against Candida albicans that is resistant to amphotericin B (Oh et al., 2011). Kumar et al. (2012, 2014) also reported novel species of Streptomyces from solitary wasp producing antimicrobial agents showing promising activity against drug resistant bacterial and fungal pathogens.

The leaf cutter ants have many defense mechanisms against the risk of pathogenic infection of their garden fungus, including a tripartite mutualistic interaction in which they host bacteria that produce antibiotics on their bodies (Barke et al., 2010). Numerous of these bacteria have coevolved with their hosts, creating antifungals to inhibit parasitic fungi (Escovopsis spp. and allied taxa), and in exchange, the ants feed the bacteria through special exocrine glands inside elaborate cuticular crypts, which also provide the bacteria with their preferred microclimate (Currie et al., 2006). L. gongylophorus, a mutualist fungus, is the only source of nourishment for Acromyrmex, which also harbors Pseudonocardia bacteria in its metapleural glands (Heine et al., 2018). Another fungus, Escovopsis, parasitizes the L. gongylophorus cultivar (Yek et al., 2012). To suppress Escovopsis, the Pseudonocardia produce various variations of the broad-spectrum polyene antifungal nystatin P1 (Holmes et al., 2016). Additionally, it has recently been discovered that Pseudonocardia associated with the attines Apterostigma dentigerum and Trachymyrmex cornetzi create novel cyclic depsipeptide substances known as gerumycins A-C (Holmes et al., 2016).

Cave habitats encourage the development of antimicrobials due to their unique properties, such as darkness, high humidity, consistent low temperature, and lack of nutrition which influence the microflora that thrives there (Ghosh et al., 2017). Such extreme environments have caught the attention of researchers due to the presence of undiscovered microbial communities in these caves. Due to a scarcity of nutrients in cave habitats, microorganisms become more competitive and adopt survival tactics such as secreting metabolites (antibiotics and enzymes; Ghosh et al., 2017). Various caves have been studied for potential bioactive metabolites all over the world. The Streptomyces genus has been found in a variety of caves in various countries including Thailand (Nakaew et al., 2009a), Turkey (Yücel and Yamaç, 2010), Pakistan (Zada et al., 2016), India (Yogita et al., 2012), China (Jiang et al., 2015), Russia (Axenov-Gibanov et al., 2016; Voytsekhovskaya et al., 2018), Canada (Riquelme et al., 2017)^, and Poland (Herold et al., 2005). Other Actinobacteria genera have also been found in cave habitats including Arthrobacter, Nocardia, Saccharothrix, Lentzea, Micrococcus, Nonomuraea, Spirillospora, Micromonospora, Microbacterium, Cryobacterium, and Lysinibacter (Lee et al., 2000; Ningthoujam et al., 2009; Nakaew et al., 2009b). But only a few bioactive substances have been documented so far and the chemical characteristics of the majority of the reported metabolites released by particular bacteria are unknown. The identified bioactive substances include undecylprodigiosin (Stankovic et al., 2012), xiakemycin and huanglongmycin (Jiang et al., 2015; Jiang L. et al., 2018), chaxalactin B (Castro et al., 2018), diazepinomicin (Gosse et al., 2019) from Streptomyces sp., a mixture of antibiotics of Streptomyces sp., Bacillus sp. and Bacillaceae (Axenov-Gibanov et al., 2016), a mixture of polyene and non-polyene metabolites of Streptomyces sp. and Penicillium sp. (Belyagoubi et al., 2018), lanthipeptides, polymyxin B, paenicidin B, fusaricidin, tridecaptin, and colistin A of Paenibacillus (Baindara et al., 2020; Lebedeva et al., 2021), lipids of Toxopsis calypsus and Phormidium melanochroun (Lamprinou et al., 2015).

Exocrine glands in arthropods secrete a variety of secondary compounds. The outer cuticle of the insect serves as the first line of defence against fungal infection. The first spider AMPs, known as lycotoxins-I and II from Lycosa carolinensis, possessed both antibacterial and antifungal properties that were able to prevent the growth of bacteria and the Candida glabrata yeast (Yan and Adams, 1998). Venom can have neurotoxic effects, but it can also have analgesic, anticancer, antiarrhythmic, and most importantly, antibacterial properties. Spider venom contains salts, acylpolyamines, peptides, enzymes, amino acids, nucleotides, and low molecular mass molecules (Vassilevski et al., 2009). The venom of Ornithoctonus hainana also contained an antibacterial substance called Oh-defensin that was active against both Gram-positive and Gram-negative bacteria as well as fungi (Zhao et al., 2014). When extracted from the hemolymph of scorpions of the species Androctonus australis, the peptide androctonin was found to be effective against both bacteria and fungus (Ehret-Sabatier et al., 1996). Melectin and halictines are two antimicrobial substances that can be found in the venom of Hymenopterans (Slaninová et al., 2011). Ponericins from the venom of the ponerine ant Pachicondyla gueldi has been observed to be effective against bacteria and yeasts (Orivel et al., 2001). Unknown toxins in the venom of the paper wasp Polistes flavus are also effective against Candida and Aspergillus niger (Prajapati and Upadhyay, 2016). The antimicrobial properties of the venom of wasps and bees have also been demonstrated in some studies. For instance, the crude venom of the wasp Vespa orientalis effectively exhibited antifungal efficacy against the fungal strain Candida albicans (Farag and Swaby, 2018). The venom of Pseudopolybia vespiceps wasps contains another mastoparan peptide that exhibits substantial antifungal properties against Candida albicans and Candida neoformans (Silva et al., 2017). Other recently discovered peptides with certain antifungal activities obtained from natural sources, i.e., animal secretions or microbes, have been summarized in Table 2.

Figure 3 depicts the old as well as emerging fungal targets along with their respective antifungal agents that are either currently available for use or undergoing clinical trials. Some of the fungal targets that have recently gained attention of researchers to develop novel antifungals are described below.

An interesting potential target for developing novel antifungal drugs is the covalent attachment of proteins to glycosylphosphatidylinositol (GPI), which anchors them to the plasma membrane (Yadav and Khan, 2018). The Eisai Company in Japan developed the novel antifungal drug E1210 (fosmanogepix or APX001), which inhibits an initial stage of the GPI-dependent anchoring of mannosylated cell wall proteins linked to the polysaccharide wall component; the cell wall becomes weaker as a result of this interruption, which stops the growth of fungi. The fourth enzyme in the GPI-anchoring pathway, Gwp1p, which is in charge of inositol acylation, is the target of E1210 (the mammalian homolog is not inhibited; Watanabe et al., 2012). In vitro tests showed that E1210 was highly effective against the majority of fungi, including strains that were resistant to triazoles and polyenes as well as yeasts (Candida species other than Candida krusei) and molds (Aspergillus species, Fusarium species, and black moulds; Miyazaki et al., 2011; Pfaller et al., 2011). In a recent study, fosmanogepix was also found to be effective against Fusariosis in immunocompromised mice (Alkhazraji et al., 2020). A second GPI inhibitor named gepinacin (Phenoxyacetanilide), which is a monocarboxylic amide with antifungal activity against various yeasts and molds was found (Ivanov et al., 2022). An essential acyltransferase needed for the manufacture of fungal GPI anchors, Gwt1, is selectively inhibited by gepinacin (McLellan et al., 2012). The GPI anchor synthetic pathway enzyme Mcd4 is inhibited by M720, a phosphoethanolamine transferase-I inhibitor with in vivo activity against Candida albicans (Mann et al., 2015). The SPT14 gene (YPL175W), which encodes the fungal UDP-glycosyltransferase catalytic subunit known as Spt14, is crucial for the production of GPI. A natural substance called jawsamycin (FR-900848) contains oligomeric cyclopropyl that targets the catalytic component Spt14 (Fu et al., 2020). Jawsamycin targets the fungal Spt14 but not the human PIG-A, although the human homolog PIG-A and the S. cerevisiae Spt14 share 40% similarity (Fu et al., 2020). Jawsamycin demonstrated antifungal action with broad-spectrum and potency against Mucorales fungi, such as Rhizopus oryzae and Absidia corymbifera, as well as Fusarium species, Scedosporium species, and Mucor circinelloides (Fu et al., 2020).

Enolase catalyzes the conversion of 2-phosphoglycerate (2-PG) and phosphoenolpyruvate in the penultimate phase of glycolysis (Ji et al., 2016). A genetic knockout of enolase in C. albicans has been demonstrated to decrease germination tube and hyphal development, leading to decreased virulence and growth rate, which is consistent with its participation in core metabolic activities (Ji et al., 2016). It’s interesting to note that while this enzyme performs its glycolytic functions in the cytoplasm, many bacterial and fungal species also express it on their cell surfaces. This has been seen in Aspergillus flavus, A. terrus, A. nidulans, Candida glabrata, Saccharomyces cerevisiae (Funk et al., 2016). The potential of A. fumigatus surface-expressed enolase to interact with human immunological regulators, such as factor H, factor-H-like protein 1 (FHL-1), C4b-binding protein (C4BP), and plasminogen, was demonstrated in a thorough investigation (Dasari et al., 2019). When human A549 epithelial cells or an epithelial monolayer were exposed to swollen A. fumigatus conidia covered with human plasminogen, cell retraction, as well as cellular metabolic activity, was observed to be lowered significantly (Dasari et al., 2019). The structure-guided design of a peptidomimetic or small molecule inhibitor that precisely targets the enolase plasminogen docking site is a potential route for novel therapies (Dasari et al., 2019). A thorough understanding of the interactions between the two proteins is necessary for this method. Although full-length human Type II plasminogen (4DUR) and fungal enolase from Saccharomyces cerevisiae (3ENL) have high-resolution crystal structures, the interface for their interactions has not yet been established (Law et al., 2012).

A vital mechanism that promotes fungal virulence and survivability is mannitol production. Mannitol is acyclic, six-carbon sterol alcohol that is produced by the major biosynthetic enzymes mannitol-2-dehydrogenase (M2DH) and mannitol-1-phosphate 5-dehydrogenase (M1P5DH). Mannitol is a molecule that is found in almost all the structures of fungi, including mycelia, fruiting bodies, and conidia. It functions as a molecule that stores carbohydrates, an osmolyte, and a source of reducing power (Meena et al., 2015). Mannitol synthesis is boosted during pathogenesis to take advantage of its capacity to squelch reactive oxygen species (ROS) and evade host defenses (Wyatt et al., 2014; Meena et al., 2015). Interest in the underlying mechanism of mannitol’s manufacture and secretion during fungal infection has increased because of the special and varied features of mannitol, particularly concerning the human disease. The fact that humans do not manufacture mannitol and hence lack corresponding enzymes adds to the appeal of targeting mannitol production pathways in fungus. The discovery and development of inhibitory drugs that selectively target these fungal enzymes are made much easier by the lack of a human equivalent.

Due to its involvement in vital activities like DNA and RNA synthesis, energy metabolism, and signal transmission, disruption of the purine and pyrimidine biosynthesis pathway using small molecules has been investigated as a potential strategy to produce antimicrobials (Trapero et al., 2018). It is crucial in the drug design process to achieve species-selective targeting of the biosynthesis enzymes since many of these are shared by humans, fungi, and bacteria (Chitty and Fraser, 2017). Thus, the creation of high potency and selectivity inhibitors will benefit greatly from a thorough understanding of the structural and kinetic properties of the fungal and human homologs of these enzymes. A novel substance known as ECC1385 that was discovered in a library of synthetic compounds has been demonstrated to inhibit C. neoformans and C. albicans GMP synthase activity (Rodriguez-Suarez et al., 2007). It exhibited whole cell action against a wide range of fungi and bacteria in vitro, including various species of Candida, A. fumigatus, C. neoformans and Staphylococcus aureus (Rodriguez-Suarez et al., 2007). Antiretroviral medicine azidothymidine (AZT; brand name RETROVIR) is being used to treat HIV demonstrated antifungal efficacy in vivo and in vitro for A. flavus (Wang X. et al., 2019). Since AZT has FDA approval, there is potential to employ the AZT scaffold to enhance its antimycotic capabilities or to repurpose it as an antifungal medication. F901318 (olorofim), which was found using an Aspergillus nidulans library screen, inhibits the enzyme dihydroorotate dehydrogenase (DHODH), which is responsible for catalyzing the fourth enzymatic stage of pyrimidine biosynthesis (Oliver et al., 2016). Despite being ineffective against Mucorales and Candida species, F901318 is quite effective in vitro against triazole-resistant Aspergillus, Scedosporium, Madurella, and Fusarium species (Law, 2016). Mammalian cells also have DHODH, however, F901318 is unable to inhibit the human version of the enzyme. In addition, olorofim exhibits effectiveness both in vitro and in vivo against A. fumigatus isolates that are resistant to other antifungal medications (Du Pré et al., 2018). The FDA designated olorofim as a “breakthrough drug therapy” in November 2019. It was subsequently given the orphan drug designation for the treatment of invasive aspergillosis, Lomentospora/Scedosporium infection (March 2020), and coccidiosis (June 2020), and just recently became an approved medication for the treatment of infectious diseases (June 2020; Wiederhold, 2020).

In eukaryotes, Hsp90 is a crucial molecular chaperone that controls the proper transport, maturation, and destruction of client proteins. In addition, Hsp90 controls the growth of mycelium, which is essential for virulence. Drug-resistant fungi are becoming an increasingly serious problem, and Hsp90 may offer a fresh way to address this growing danger (Arastehfar et al., 2020). Hsp90 impacts the survival, pathogenicity, and drug resistance of many diseases. The nucleotide-binding domains (NBD) of Hsp90 are conformationally flexible, according to research by Whitesell L. and colleagues in which they created CMLD013075, which had a more than 25-fold binding selectivity for fungal Hsp90 even though Hsp90 is extremely conserved (Huang et al., 2019; Whitesell et al., 2019). In contrast to previous Hsp90 inhibitors, CMLD013075 demonstrated little toxicity to mammalian cell lines (Whitesell et al., 2019). When used against resistant clinical isolates of Candida albicans, CMLD013075 was able to reduce the development of Candida albicans, and also improved the antifungal effects of azole antifungals. As a highly selective fungal Hsp90 inhibitor, CMLD013075 is a successful illustration of how IFIs can be treated by preventing fungal Hsp90 from doing its job. Histone deacetylase 2 (Hos2) controls the deacetylation of Hsp90, which is necessary for the interaction of Hsp90 with specific co-chaperones and the stability of several Hsp90 client proteins (van Daele et al., 2019). MGCD290 is a fungus Hos2 inhibitor created by MethylGene, Inc. of Montreal, Canada which not only prevents the deacetylation of histone proteins but also that of non-histone proteins like Hsp90 (Pfaller et al., 2015). Although MGCD290 only exhibits mild anti-Candida activity it exhibits good synergistic antifungal activity with azoles against a variety of Candida species strains that are resistant to several drugs in an in vitro setting (Pfaller et al., 2015). To increase the antifungal effectiveness of antifungal medications against drug-resistant pathogenic fungi, MGCD290 can be used as an adjuvant. It is necessary to confirm the antifungal efficacy of MGCD290 utilizing additional animal models of IFIs and, ultimately, in carefully planned clinical studies.

Chitin is produced by a family of membrane-localized chitin synthase enzymes and is necessary for cell survival (Chs1 to 3, and Chs8 in C. albicans). Due to their structural similarity, polyoxins and nikkomycins are powerful inhibitors of the Chs enzyme that compete with the Chs substrate UDP-GlcNAc for Chs binding, although they only have a little impact on whole cells (Shubitz et al., 2014; Ibe and Munro, 2021). Streptomyces tendae and Streptomyces ansochromogenes both generate the structurally related antifungal drugs nikkomycin X and nikkomycin Z, which have activity against a range of human fungal infections. The respiratory infections caused by Coccidioides species can be treated with nikkomycin Z (Shubitz et al., 2014). Recent research has led to the development of two new nikkomycin analogs, nikkomycin Px and Pz, which share the same antifungal properties as nikkomycin X and Z but exhibit significantly greater pH and temperature stability (Feng et al., 2014). Despite having specialized roles, the enzymes involved in the manufacture of chitin might become redundant in certain circumstances. The creation of effective chitin synthase inhibitors has also been hampered by minute variations in protein structure across members of the Chs family.

The lack of access to specific diagnostic tests and the restricted availability of antifungal drugs are the major challenges faced by clinicians to manage drug-resistant pathogens in present times. Also, the vast diversity of the fungal kingdom ensures an endless supply of new pathogens as well as new variants of known pathogens that are capable of adapting and evolving resistance themselves when exposed to antifungal agents. The key issue is that mostly in-vitro experiments have been used to investigate the activity of newly discovered antifungals. As the novel antifungal research moves forward, more studies using in-vivo infection models and subsequent clinical studies are required. Nevertheless, several naturally available agents all around us show promise in fending against the growing threat posed by fungi. There has been significant progress in identifying possible targets for new agents that take advantage of intrinsic dissimilarities between humans and fungi. Even while many of these targets are still hypothetical, some compounds have just recently started their early stages of clinical research. By increasing funding for research, exploring unexplored as well as underexplored sources of natural antimicrobials, taking into account novel alternative targets, repurposing already-existing non-antifungal medications, fortifying ties between academia and industry, and offering financial incentives for the development of new therapeutic options to treat these uncommon but deadly infections, we must address this unmet need.