Bioactive and unusual steroids from Penicillium fungi

Abstract

Penicillium fungi are represented by various species and can be found worldwide and thrive in a range of environments, such as in the soil, air, and indoors, and in marine environments, as well as food products. Chemical investigation of species of this genus has led to the discovery of compounds from several structural classes with varied bioactivities. As an example, this genus has been a source of bioactive and structurally unusual steroids. The scope of this short review is to cover specialized metabolites of the steroid class and the cytotoxic, antimicrobial, anti-inflammatory as well as phytotoxic activities of these compounds. Other steroids that possess unusual structures, with significant bioactivity yet to determined, will also be discussed to further demonstrate the structural diversity of this compound class from Penicillium fungi, and hopefully inspire the further exploration of such compounds to uncover their activity.

Graphical Abstract

Penicillium spp. are producers of steroids with interesting structures and biological activity.

1. Introduction

Penicillium spp. are filamentous and usually monomorphic fungi, which occur in a very well-known and commonly found genus. It features two subgenera and occurs in the family Aspergillaceae of which 26 sections are affiliated with Penicillium, and with over 400 species having been reported to date (Shah et al., 2022). Morphological features of these fungi are a brush-like appearance due to the presence of separated hyphae, 2 to 5 μm in diameter, which appear as branched and unbranched conidiophores with secondary branches (Langlois et al. 2014). Species of these fungi also act as decomposers (saprobic) and are responsible for the production of a wide range of mycotoxins (Visagie et al., 2014).

The genus began to have medicinal significance over ninety years ago when Alexander Fleming discovered that a fungal strain, later identified as Penicillium notatum Westling, had contaminated a bacterial Staphylococcus colony and was able to inhibit its growth (Fleming, 1929). This serendipitous discovery led to the development of the very important antibiotic penicillin (1, Fig. 1), which is associated with the “antibiotic era” and had a tremendous impact on antimicrobial discovery (Aminov, 2010; Ribeiro da Cunha, et al. 2019).

Figure 1.

Examples of medicinally important compounds isolated from Penicillium spp. (penicillin, pravastatin and compactin; 1–3, respectively).

Today, the genus Penicillium has been investigated widely for applications in agriculture, biotechnology, biology, and drug discovery. Compounds successfully isolated from Penicillium spp. include alkaloids, polyketides, terpenoids, lactones, and steroids (Shah et al., 2022; Xue et al., 2014; Sun et al., 2006; Gao et al., 2011). These compounds have bioactivities such as cytotoxic, antioxidant, and antibacterial effects. For instance, the antithrombotic, anti-inflammatory, and antifungal medicinally important compounds pravastatin (2) and compactin (3) have been reported from Penicillium species (Shah et al., 2022).

Although many compounds of the genus Penicillium have shown potential in medicinal applications, only those that are steroids with bioactivities or displaying unusual structures will be discussed herein.

2. Methodology

A comprehensive survey of the bioactive and unusual steroids from Penicillium fungi was conducted by using the CAS SciFinderⁿ, Reaxys, Google Scholar, and PubMed databases. To obtain the articles reporting bioactive or unusual steroids the following search terms were used, “cytotoxic sterols – Penicillium”, cytotoxic steroid – Penicillium”, “ergostane Penicillium”, “antiproliferative sterol – Penicillium”, “cytotoxic steroidal saponins – Penicillium”, “ergosteroids from Penicillium”, “antibacterial steroids”, “Penicillium steroids”, “antifungal Penicillium steroids”, “antibacterial Penicillium steroids”, “antimicrobial steroids Penicillium”, “neuroprotective Penicillium steroids”, and “anti-inflammatory sterols – Penicillium”. A total of 19 articles were obtained which specifically discussed the bioactive and or unusual steroids. The studies reviewed covered the period 2003 to December 2022 to demonstrate more recent investigations on steroids in Penicillium fungi. During the review of the literature, several compounds with features not typically seen in steroids were observed, and from these a section of the review was dedicated to highlight a selection of these unusual steroids.

3. Steroids

Steroids are aliphatic compounds that are tetracyclic isoprenoid derivatives of triterpenoids, but do not possess methyl groups at either C-4 or C-14. Steroids occur in sub-types based on the hydrocarbon framework of the parent skeleton, namely, gonane, estrane, androstane, pregnane, cholane, cholestane, ergostane, campestane, and stigmastane (4–12, Fig. 2) (Dewick, 2009).

Figure 2.

The gonane, estrane, androstane, pregnane, cholane, cholestane, ergostane, campestane, and stigmastane (4–12) steroid parent molecules.

The core structure of all steroids resembles cholesterol (13), but they can be differentiated as belonging to other classes such as sterols, corticosteroids, cardenolides, sex hormones of mammals, and steroidal saponins. An example of each is depicted in figure 3 for compounds 13–17, respectively. Since the core structure of these compounds is like cholesterol (Dewick, 2009), its structure will be used as a frame of reference to discuss their biosynthesis and structural features.

Figure. 3.

Structures of a sterol, a corticosteroid, a cardenolide, a mammalian sex hormone, and a steroidal saponin (13 – 17).

The initial stages of sterol biosynthesis occur similarly to those of triterpenoids, involving the reaction of two farnesyl pyrophosphate (C₁₅) units to form the long-chain aliphatic intermediate, squalene (C₃₀). The cyclization of this intermediate eventually leads to squalene oxide, and another cationic intermediate that produces lanosterol, the first cyclic sterol/steroid occurring in fungi (Dewick, 2009). Following the preceding reactions, the C₂₈ sterols typical of fungi are then formed after a C-24 methylation, C-4 and C-14 demethylations, and double bond transformations, as can be seen in sterols such as cholesterol (13) (Weete et al., 2010).

In the present report, the process of sterol production in fungi was further elaborated using the biosynthesis of ergosterol, the most well-known fungi-associated sterol. According to Hu et al., the biosynthesis of ergosterol involved the use of 30 enzymes (Erg proteins). The biosynthesis can be divided into three modules: the mevalonate, the farnesyl pyrophosphate, and the ergosterol biosynthesis (Hu et al., 2017). The mevalonate biosynthesis begins with two acetyl-CoA molecules condensing to form acetoacetyl-CoA via acetyl-CoA acetyltransferase (Erg10). Another condensation of a third acetyl-CoA molecule with acetoacetyl-CoA using hydroxymethylglutaryl-CoA synthase (Erg13) yields 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). This latter intermediate was then reduced by the HMG-CoA reductases (HMGR, known as Hmg1 and Hmg2) to produce mevalonic acid (Hayakawa et al., 2017). During the second module, mevalonate underwent successive phosphorylation processes using mevalonate kinase (Erg12) and phosphomevalonate kinase (Erg8) to give mevalonate-5-pyrophosphate. The formation of farnesyl pyrophosphate (FPP) also occured in this module, as follows. Isopentenyl pyrophosphate (IPP) was first synthesized via mevalonate pyrophosphate decarboxylase (Erg19) in a decarboxylation step. IPP was then isomerized to form dimethylallyl pyrophosphate (DPP) by the action of IPP isomerase (Idi1). Furthermore, condensation of DPP with two molecules of IPP yielded geranyl pyrophosphate and ultimately FPP. This process was catalyzed by geranyl/FPP synthase (Erg20). In the third module, squalene (the precursor molecule of all steroids) was formed after two FPP molecules are catalyzed via squalene synthase (Erg9). This squalene was then epoxidized to form squalene oxide via squalene epoxidase (Erg1). The latter was converted to lanosterol via lanosterol synthase (Erg7). Several other steps then take place to ultimately yield ergosterol. For instance, conversion of lanosterol to zymosterol occurred in complex processes involving demethylations, reduction, and desaturation reactions, carried out by lanosterol C-14 demethylase (Erg11) and (Erg24-Erg27), respectively. Zymosterol was then converted to fecosterol, which was further converted to episterol, via the enzymes C-24 methyltransferase (Erg6), and the sterol C-8 isomerase (Erg 2), respectively. Finally, denaturation and reduction of episterol by Erg3, Erg5, and Erg4 yielded ergosterol (Klug and Daum 2014). A summary of the biosynthetic pathway is displayed in figure 4.

Figure. 4.

The three-module biosynthetic pathway of ergosterol from acetyl-CoA in fungi and the enzymes involved. Adopted from Hu et al. (2017).

The arrangement of the rings of sterols represents a characteristic feature of these molecules that contributes both to their configuration and the resultant bioactivities that are found. The striking differences in the biological activities of steroids are evident as a result of the functional groups attached to the nucleus as well as the general stereochemistry of the steroidal nucleus caused by the ring fusions. These ring fusions typically appear with the A/B ring having a trans or cis conformation, with unsaturation at Δ⁴ and Δ⁵, or in some cases ring A is aromatized (as seen in the estrogens, such as estradiol, 16). The B/C, as well as the C/D ring fusions of natural steroids are often trans, but exceptions do exist, as for example in the C/D rings of some cardiac glycosides (Dewick, 2009).

Taking the structure of cholesterol (13) as an example, the features of its overall structure can be used to represent other steroids of similar structure. In domain A, the C-3 OH facilitates hydrogen bonding through its polarity and tilt (figure 5, in pink brackets). Based on the location and number of double bonds (if present) in a sterol, the overall shape of the molecule and how the 17(20)-bond will be oriented can be affected. The C-20, R configuration of a sterol results in the side chain being arranged “right-handed”. In domain D (Fig. 5 in red brackets) of the molecule, the length and conformation of the side chain is crucial for intermolecular interactions. Consequently, the above molecular properties allow the Δ⁵ -sterol, cholesterol, to be oriented as a flat and elongated molecule in membranes. Fig. 5 displays a schematic drawing of cholesterol with its domains (Nes, 2011).

Figure 5.

Domains of cholesterol (13) that result from its stereochemistry; adopted from Nes (2011).

4. Role of Sterols in Fungi

Sterols play a crucial role in the growth of fungi (Weete et al., 2010). Ergosterol (18), the most widely known sterol associated with fungi, is required for membrane permeability and fluidity (Kodedová and Sychrová, 2015). Other functions of sterols in fungi include involvement in the formation of microdomain structures as seen in Saccharomyces cerevisiae (Desm.) Meyen (Malínský et al., 2010), Schizosaccharomyces pombe Lindner, Candida albicans (C.P. Robin) Berkhout, and Cryptococcus neoformans (San Felice) Vuill., which possess membrane proteins that are used in the homeostases of Na⁺, K⁺, and pH, in the transport of nutrients into fungal cells, mating, and drug efflux (Mollinedo 2012), and that are used as multi-drug-resistant pumps in pathogenic fungi (Spira et al. 2012).

For over 125 years, the major C₂₈ sterol found in fungi was thought to be ergosterol (18). More recent molecular phylogeny studies, however, show that the major sterols can vary across the fungal kingdom. The four other predominant sterols found are cholesterol (13), 24-methyl cholesterol (19), 24-ethyl cholesterol (20), and brassicasterol (21, figure 6). In addition to these, intermediates of 24-methyl cholesterol are also found in high amounts in some fungi (Weete et al., 2010).

Figure 6.

Structures of the major sterols, cholesterol, ergosterol, 24-methyl cholesterol, 24-ethyl-cholesterol, and brassicasterol (13, 18 – 21) occurring in fungi.

5. General Bioactivities of Steroids from Fungi

The activities of steroids expand beyond those evident in the metabolism of fungi and investigation of these compounds reveals a treasure trove of bioactive secondary products. Steroids of fungal origin have been shown to have potential value for inhibition of neurotoxicity and neuronal cell death, which may result in their applicability in the prevention of the onset of dementia (Ano et al., 2015). Fungal steroids have also shown potential value for preventing chronic inflammatory and autoimmune diseases (Ano et al. 2017). Promise for their use against type 2 diabetes has also been proposed by being able to reduce the effects of hyperlipidemia (Kuo et al., 2015). Their potential for applicability in the prevention of cancer metastases was indicated using an experimental model of lung metastasis (Shin et al., 2021). Steroids from fungi have also been shown to possess cytotoxicity against a host of cancer cell lines. Included were the Hep G2 human liver cancer cell line, the Huh7/S cell line from male hepatoma tissue, the A549 lung carcinoma epithelial cell line, and the MCF-7 human breast cancer cell line, among others (Zhabinskii et al., 2022).

6. Penicillium Species Are Producers of Bioactive and Structurally Diverse Steroids

Since many fungal species display biological activities, this review will explore Penicillium species as a source of bioactive compounds, specifically steroids. In addition, steroids from the genus that were not identified as significantly bioactive in the respective study or studies mentioned but possess unusual steroid structural features are of interest. These include a compound with a moiety previously observed only from a plant, and another with a typical ring fusion that is uncommon for natural steroids at the time of publication. The aim is to demonstrate that Penicillium fungi can produce new and bioactive steroids, and to pique interest in the exploration of their activity in other bioassays for those with fascinating structures, but for which no significant activity has yet been reported in the studies cited.

7. Cytotoxic Steroids from Penicillium Species

Cancer is currently a major global health care concern, and, in 2019, it was predicted that cancer cases would rise by 50%, thus resulting in 21 million new cases in the next two decades worldwide. While there exists many therapeutic options for the treatment of cancer such as surgery and chemotherapy using natural and/or synthetically derived drugs, these treatment options have often proven unsuccessful in providing the desired result of cancer cessation and tumor relapse, and the arrest of metastasis taking place. As such, there is a need to discover compounds with lower side effects, better medicinal properties, and decreased chances for causing disease resistance (Sharifi-Rad et al., 2019). Natural products are a promising source of new anticancer agents, not only for the promise of compounds representative of new chemical classes, but also for determining new cellular mechanisms of activity (Khlaifa et al., 2019). Furthermore, they are the best source for obtaining novel agents/active templates that can aid in the development of valuable agents against several human diseases (Newman and Cragg, 2020).

Penicillium fungi represent one potential natural source of new therapeutic agents for cancer (Uzma et al., 2018). Published findings on examples of steroids from this genus with demonstrated cytotoxic activity will be discussed below.

Penicillitone (22), a sterol with a rare tetracyclic backbone, was isolated from the fungus Penicillium purpurogenum. This compound possessed good potency toward several cancer cell lines with IC₅₀ values of 5.57 ± 0.19 μM (A549 human lung carcinoma/alveolar cells), 4.44 ± 0.24 μM (HepG2 human hepatoma cells), and 5.98 ± 0.22 μM (MCF-7 human breast cancer cells). For this study, adriamycin was used as a positive control with respective IC₅₀ values of 0.35 ± 0.07, 0.37 ± 0.06, and 0.17 ± 0.028 μM against the three cell lines listed above (Xue et al., 2014). The steroids 8(14),22E-diene-3β,5α,6β,7α-tetraol (23), ergosta-8(9),22E-diene-3β,5α,6β,7α-tetraol (24), 5α,8α-epidioxy-24(S)-methylcholesta-6,22E-diene-3β-ol (25), 5α,8α-epidioxy-24(S)-methylcholesta-6,9(11),22-triene-3β-ol (26), and 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one (27) were obtained from a Penicillium sp. isolated from a sea moss from a region in the South Pole. The IC₅₀ values observed for each were 10.4, 15.6, 20.7, 16.8, 21.3 μg/mL, respectively, when tested against Hep G2 human liver cancer cells. Among the compounds evaluated, 23 exhibited the most potent activity (Sun et al., 2006). Penicisteroid A (28, Fig. 7), a polyoxygenated sterol, was isolated from the endophytic marine-derived fungus Penicillium chrysogenum Thom QEN-24S. It displayed weak cytotoxic activities against HeLa (cervical cancer cells), SW1990 (pancreatic cancer cells), and NCI-H460 (hypotriploid human cells), with IC₅₀ values of 15, 31, and 40 μg/mL, respectively. It was reported that the presence of a hydroxy group at the C-6 position in the B ring of penicisteroid A is necessary for activity, as an analogue, penicisteroid B, which lacks this feature, displayed no discernible cytotoxicity (Gao et al., 2011).

Figure 7.

Cytotoxic steroids penicillitone, 8(14),22E-diene-3β,5α,6β,7α-tetraol, ergosta-8(9),22E-diene-3β,5α,6β,7α-tetraol, 5α,8α-epidioxy-24(S)-methylcholesta-6,22E-diene-3β-ol, 5α,8α-epidioxy-24(S)-methylcholesta-6,9(11),22-triene-3β-ol, and 3β,5α,9α-trihydroxyergosta-7,22-diene-6-one, and epidioxyergosterol (22 – 29) obtained from Penicillium spp.

In another study of a P. chrysogenum strain S003, obtained from a deep-sea sediment sample from the Red Sea, the well-known fungal sterol ergosterol (18) and another compound identified as epidioxyergosterol (29, Fig. 7) were isolated and were found to display cytotoxicity against the A549, DU-145 (human prostate carcinoma cells), MCF-7, and HepG2 cancer cell lines, with IC₅₀ values of 21.3, 19.3, 1.5, 6.1, and 17.0, 13.6, 2.9, 3.1 µM, respectively (Alshehri et al., 2020).

Penicillium citrinum Thom strain SCSIO 41017 was isolated from a Callyspongia sp. sponge that was collected from Guangdong Province in China and, following culture, extraction, and isolation procedures, a steroid, 16α-methylpregna-17α-hydroxy-(9,11)-epoxy-4-ene-3,18-dione-20-acetate (30), which was initially reported only as a synthesized compound, was obtained. This compound possessed moderate activity with IC₅₀ values of 13.5–18.0 µM against the SF-268 (glioblastoma cells), MCF-7, HepG2, and A549 cancer cell lines (Salendra et al. 2021). Another marine-derived fungus, Penicillium stoloniferum Thom QY2-10, associated with a sea squirt, was found to produce 5α,8α-epidioxy-23-methyl-(22E,24R)-ergosta-6,22-dien-3β-ol (31), which was cytotoxic towards P388 (murine leukemia) cells with an IC₅₀ value of 4.07 µM (Xin et al., 2007).

Solitumergosterol A (32, Fig. 8), a steroid with a 6/6/5/6-pentacyclic backbone, was isolated from the deep-sea fungus Penicillium solitum Westling strain MCCC 3A00215. This compound exhibited weak antiproliferative activity against MB231 (triple-negative breast) tumor cells. Solitumergosterol A has been reported to have a proposed biosynthetic pathway that utilizes a 14-dihydroergosterol (DHE) unit during Diels–Alder cycloaddition with maleic acid or maleimide, followed by decarboxylation to produce the aromatic moiety fused to rings B, C, and D (He et al., 2021).

Figure 8.

Cytotoxic steroids 16α-methylpregna-17α-hydroxy-(9,11)-epoxy-4-ene-3,18-dione-20-acetate, 5α,8α-epidioxy-23-methyl-(22E,24R)-ergosta-6,22-dien-3β-ol, solitumergosterol A, penicisteroids E, G, H, C, A, auransterol, antineocyclocitrinol A, 23-O-methylantineocyclocitrinol, neocyclocitrinol A, 24-epi-cyclocitrinol, and 20-O-methyl-24-epi-cyclocitrinol (30 – 42) from Penicillium spp.

The ergostane derivatives of penicisteroids E, G, H, C and A (33-36, and 28, respectively) were isolated from the deep-sea fungus Penicillium granulatum Bainier, strain MCCC 3A00475. Compounds 33-36, and 28 exhibited antiproliferative activities against 12 different cancer lines demonstrating overall IC₅₀ values of around 5 µM. An assessment of the mechanism of action of penicisteroids 33 and 28 revealed that both compounds caused a significant decrease in the transcriptional activation of RXRα induced by 9-cis-retinoic acid, and, in turn have the potential for affecting RXRα-mediated growth inhibition and inducing apoptosis in cancer cells (Xie et al., 2019). Recent work performed at the authors’ institution has resulted in the isolation of the cytotoxic sterol, auransterol, from the lichen-associated fungus Penicillium aurantiacobrunneum Houbraken, Frisvad & Samson, which was obtained from the U. S. endemic lichen Niebla homalea (Ach.) Rundel & Bowler. Compound 37 showed an IC₅₀ value of 9.8 μM against the proliferation of HT-29 colorectal adenocarcinoma cell line, with the control used being paclitaxel with an IC₅₀ of 0.001 μM (Tan et al., 2019). A study by Xia et al. of the fungus P. purpurogenum AD-1-2 yielded C₂₅ steroids which, although weak, demonstrated varying inhibitory effects across several cancer cell lines. These compounds were antineocyclocitrinol A, 23-O-methylantineocyclocitrinol, neocyclocitrinol A, 24-epi-cyclocitrinol, and 20-O-methyl-24-epi-cyclocitrinol (38–42). The cytotoxicity of these compounds was reported as the inhibition rate (IR%) at 100 µg/mL, as follows: 38, 34.7% (HL-60, human leukemia cells); 39, 33.8% (HL-60); 40, 34.7% (HL-60); 41, 31.4% (K562, myelogenous leukemia cells); and 42 (Fig. 8), 37.3% (K562); 40.4% (HL-60), 63.5% (HeLa). The latter compound exhibited comparable results to the positive control docetaxel (tested at 100 µg/mL) and even surpassed the IR% values observed, 42.90% (K562), 46.90% (HL-60), and 41.70% (HeLa), determined in some cell lines. All these compounds were only produced by P. purpurogenum AD-1-2 after mutagenesis using diethyl sulphate of the strain G59 which prompted the production of these silent secondary products (Xia et al. 2014).

8. Antimicrobial Steroids from Penicillium Species

Penicillium fungi are a rich source of compounds with antimicrobial activity, and it was from this genus that the dawn of the golden age of natural product antibiotic drug discovery emerged (Hutchings et al., 2019). The need still exists, and urgently so, for new antimicrobial compounds to be elucidated that can combat multi-resistant fungal strains and do not cause toxicity to mammalian cells (Singh et al., 2015). As in the case of fungal resistance, also bacterial resistance a cause for concern. There is a great need for new antibacterial compounds that can combat multi-drug-resistant bacteria, especially those with new modes of action as well as having an ability to combat biofilm formation, which is a successful means whereby bacteria can evade elimination and also enables an increase in virulence (Miethke et al., 2021; Attia et al., 2022). Penicillium fungi still represent a potential source of antimicrobial compounds, even many years after the discovery of penicillin. As such, steroids from this genus with antifungal and antibacterial activity will be discussed.

Penicisteroid A (28), isolated from P. chrysogenum QEN-24S, exhibited potent inhibition of the fungus Aspergillus niger Tiegh. and moderate activity against Alternaria brassicae (Berk.) Sacc., with an inhibition zone of 18 mm in diameter at a 20 µg assay concentration and 8 mm at a 20 µg assay concentration, respectively. The study also yielded another penicisteroid derivative, anicequol (43), which demonstrated moderate activity against the plant fungal pathogen Alternaria brassicae, with an inhibition zone of 6 mm. The positive control, amphotericin B, gave inhibition zones of 24 and 16 mm against A. niger and A. brassicae, respectively at a 20 µg assay concentration. The hydroxy group at C-6 may be important for activity, as another derivative lacking this feature was not active against A. niger. However, the activity seen against A. brassicae, which is responsible for Alternaria leaf blight in oilseed brassicas, indicates that one or more substitutions of hydroxy groups in the B ring may be required for antifungal activity (Gao et al., 2011). A study by Singh et al. yielded the promising antifungal compound wortmannin (44), isolated from Penicillium radicum A.D. Hocking & Whitelaw, after assessing its antifungal activity against three fungal targets, using molecular docking. The bioactivity of 44 was first established using six strains of Candida spp., and Cryptococcus terreus Di Menna, strain ATCC 11799, Alternaria alternata (Fr.) Keissl., strain ATCC 6663, Trichophyton rubrum (Castell.) Sabour., strain ATCC 296, and Rhizoctonia oryzae (synonym for Rhizopus arrhizus A. Fisch.), strain ATCC 52545, five strains of Aspergillus spp., and Aureobasidium pullulans (De Bary) G. Arnaud ex Cif., Ribaldi & Corte, strain DSM 2404, Fusarium moniliforme J. Sheld., strain ATCC 14164, and Saccharomyces cerevisiae ATCC 2365. The best activity was observed against Candida albicans ATCC 24433 with a MIC value of 0.39 ± 0.05 μg/mL. The controls used were amphotericin B, with a MIC value of 0.39 ± 0.02 μg/mL and fluconazole, with a MIC value of 6.25 ± 0.44 μg/mL. In silico analysis was then performed on the binding of 44 to the proteins mevalonate-5-diphosphate decarboxylase (1FI4), exocyst complex component SEC3 (3A58), and Kre2p/Mnt1p, a Golgi alpha1,2-mannosyltransferase (1S4N). These proteins are used for steroid/isoprenoid biosynthesis, for Rho- and phosphoinositide-dependent localization, and play a role in yeast cell wall glycoprotein biosynthesis. The results revealed binding with lower docking energies for 44 in all three targets and the interaction with differing active amino acid residues than those observed for the known positive controls voriconazole, pyridobenzimidazole, and nikkomycin. This molecular docking study, therefore, has suggested a better antifungal efficacy for 44 compared to these known compounds and indicated that wortmannin is a promising lead compound that also can be derivatized and chemically improved (Singh et al. 2015). However, before additional work is carried out, it would seem prudent to assess wortmannin’s activity in comparison with voriconazole, pyridobenzimidazole, and nikkomycin in antifungal assays.

As for fungal infections, those from bacteria also pose cause for concern and there is a great need for the discovery of new antibiotics that can reduce the growth of multidrug-resistant bacteria. A study of Penicillium janthinellum Biourge, a marine-derived fungus, afforded the isolation of penijanthoids A and B (45 and 46). These steroidal epimers were tested against bacterial species belonging to the genus Vibrio, Gram-negative halophilic bacteria, that can lead to vibriosis in crustacean species, but only exhibited weak activities with MICs ranging between 25 to 50 μM (Guo et al., 2019). To combat biofilm-driven resistance of the Gram-negative bacterium, Pseudomonas aeruginosa, the endophytic fungus Penicillium chrysogenum AJEF2 was investigated for metabolites that could inhibit LasR protein-specific quorum sensing. Docking studies revealed that the steroid citreoanthrasteroid A (47) can exhibit favorable binding via interactions by π-stacking with the amino acid residues Phe101 and Trp88 and hydrogen bonding to a key amino acid, Trp60 (Attia et al., 2022). Another endophytic Penicillium sp., isolated from the tuber Pinellia ternata Thunb., yielded the steroidal compound helvolic acid (48, Fig. 9), which was active against the Gram-positive bacterium Staphylococcus aureus as well as P. aeruginosa, with MIC values of 5.8 and 4.6 μg/mL, respectively (Yang et al., 2017).

Figure 9.

Antimicrobial steroids anicequol, wortmannin, penijanthoids A and B, citreoanthrasteroid A, helvolic acid, isocyclocitrinol A, and 22-O-acetylisocyclocitrinol A (43 – 50) from Penicillium spp.

The steroids, isocyclocitrinol A (49) and 22-O-acetylisocyclocitrinol A (50) were isolated from a sponge-associated fungus, Penicillium citrinum. These compounds possess a bicyclo [4.4.1] A/B ring system. The ring system of both compounds was proposed to have resulted from a 1,2 migration of the C-5--C-10 bond to give rise to a C-5−C-18 bond (Amagata et al., 2003). Compounds 49 and 50 (Fig. 9) exhibited weak antibacterial activity against Staphylococcus epidermidis and Enterococcus durans with MIC values of 100 μg/mL, for each compound and bacterial strain. Thus, these compounds discussed demonstrate the potential for production of steroids from Penicillium spp. with antimicrobial activity (Amagata et al., 2003).

9. Steroids from Penicillium species with miscellaneous bioactivities

Penicillitone (22), from P. purpurogenum SC0070, in addition to exhibiting cancer cell line cytotoxicity, also showed an ability to diminish the secretion of the pro-inflammatory cytokines TNF-α and IL-6 of LPS-stimulated RAW 264.7 macrophages by 70.7% and 96.6%, respectively (both at a 5 μM treatment). This observation is especially promising as this compound exhibited comparable activities to the known steroid and anti-inflammatory drug dexamethasone, which inhibited the above-mentioned pro-inflammatory cytokines at 87.3% and 96.7%, respectively (both with a 100 μM treatment) (Xue et al., 2014).

A study of Penicillium citrinum strain HGY1-5 from the volcanic crater ash of the extinct Huguangyan volcano in China, resulted the isolation and elucidation of three C₂₅ steroid isomers. These compounds possess an unusual of bicyclo[4.4.1]A/B ring system and were identified as neocyclocitrinol B (51), neocyclocitrinol C (52), and threo-23-O-methylneocyclocitrinol (53), respectively. When tested for their ability to affect cAMP generation, the results showed that all three compounds caused the production of cAMP in GPR12-transfected CHO cells at 10 μM. However, it is worthy of note that these three compounds were later determined to be extraction artifacts. Since they showed anti-inflammatory effects and share a structural similarity to known compounds from their fungal source, they are discussed herein. The data obtained from this study indicate that these steroids may hold promise for application in the treatment against damaged neurons caused by spinal cord injuries and strokes as cAMP plays a role in neurite outgrowth and axonal regeneration (Du et al., 2008).

To study the phytotoxic effect of metabolites from Penicillium brasilianum Bat., the steroid ergosterol peroxide (54, Fig. 10) was isolated and tested against Raphanus sativus L. (Domin) (synonym of Raphanus raphanistrum subsp. sativus) roots and was able to inhibit growth at 200 μM with a root inhibition value (RI) − 0.28, which was similar to glyphosate (RI − 0.39) at the same concentration (Tang et al., 2015).

Figure 10.

Anti-inflammatory steroids neocyclocitrinol B, neocyclocitrinol C, threo-23-O-methylneocyclocitrinol, and ergosterol peroxide (51 – 54) from Penicillium spp.

10. Steroids from Penicillium spp. with unusual structural features

As described earlier, Penicillium spp. produce structurally diverse steroids. While the following compounds were not found to be significantly bioactive in the context of their respective studies, they represent unusual sterol derivatives. These may prove useful in studying other disease applications.

Sterolic acid (55) is a sterol with an intriguing structure and was isolated from a deep-sea sediment Penicillium sp. fungus. Sterolic acid contains a diepoxy moiety in its A ring and possesses a oxabicyclo[2.2.2] octane moiety, which up to the time of its isolation, was found only in a plant metabolite. Also, of interest is the presence of a carboxylic acid group at the C-27 position in sterol acid (55) (Li et al., 2012).

Penicillium citrinum strain HGY1-5, collected from the crater ash of the extinct volcano Huguangyan in Guangdong Province of China yielded an extract from which eighteen C₂₅ steroids with bicyclo[4.4.1]A/B rings were characterized, of which seven were identified as extraction artifacts. Compounds that have been identified as being biosynthesized by P. citrinum HGY1-5 were 24-oxocyclocitrinol (56), 12R-hydroxycyclocitrinol (57), isocyclocitrinol B (58), and precyclocitrinol B (59, Fig. 11) (Du et al., 2008).

Figure 11.

Other steroids sterolic acid, 24-oxocyclocitrinol, 12R-hydroxycyclocitrinol, isocyclocitrinol B, precyclocitrinol B, 24-O-methyl-24-epi-cyclocitrinol, and 24-O-methylcyclocitrinol, and penicillisterol (55 – 62) from Penicillium spp.

Investigation of compounds produced by Penicillium ubiquetum Houbraken, Frisvad & Samson, strain MMS330, which was associated with the blue mussel Mytilus edulis, resulted in the isolation of two C₂₅ steroids, 24-O-methyl-24-epi-cyclocitrinol (60) and 24-O-methylcyclocitrinol (61, Fig. 11). These compounds are epimers, which is not surprising, as other similar steroids have often been isolated as epimers or diastereomers (specifically, at the C-24 or C-23, and C-24 position, respectively (Hoang et al., 2019). C₂₅ steroids are precursors of ergosterol (18), and though considered rare seem to be more widely distributed in fungi than originally thought (Du et al., 2008). According to Wang et al. and Wu et al., the biosynthesis C₂₅ steroids from ergosterol begins with enzymatic activation at the C-19 position of 18 to produce 63 (displayed in figure 12). Following this, the C-5−C-6 alkene of 63 acts as a nucleophile in a C-5−C-19 bond reaction which results in cyclopropane formation and a C-6 oxidation to yield 64. Following this step, a deprotonation at C-1, aided by the carbonyl at C-6, then causes a cyclopropane fragmentation. The side chain of the steroid intermediate 64 also undergoes side chain oxidations. This ultimately gives cyclocitrinol (65), which is reported as the first C₂₅ steroid isolated and was produced by Penicillium citrinum (Wang et al., 2019, Wu et al., 2021). Thus, the preceding unusual C₂₅ steroids are proposed to be biosynthesized in a similar fashion and obtained by further elaboration or other derivatization during biosynthesis.

Figure. 12.

Proposed biosynthetic scheme from ergosterol (18) to cyclocitrinol (65), a C₂₅ steroid is shown. Adopted from Wang et al. (2019).

Last, P. purpurogenum strain SC0070 produced an unusual compound known as penicillisterol (62, Fig. 11), which possesses an unusual cis-ring fusion of both A/B and C/D rings, which is not typically observed in naturally occurring sterols (Xue et al., 2014).

11. Conclusion

The current short review describes steroids that have been isolated recently from Penicillium fungi. It covers a sampling of those steroids that have been studied more recently and reviews literature articles from 2003 to 2022. Thus, from the survey conducted, a total of 41 steroids have been included. Several of these steroids show activities using assays related to more than one potential disease application. The steroids obtained were isolated from terrestrial as well as marine habitats and those with cytotoxic, antifungal, antibacterial, anti-inflammatory activity, and phytotoxic activity were reported.

The cytotoxicity observed from reported studies was especially potent for penicillitone (22), ergosterol (18), epidioxyergosterol (29), 5α,8α-epidioxy-23-methyl-(22E,24R)-ergosta-6, 22-dien-3β-ol (31), and auransterol (37) (Table 1), with ergosterol showing the highest IC₅₀ value of 1.5 μM against the DU-145 prostate cancer cell line. In turn, the highest antimicrobial activity observed from the literature was observed for penicisteroid A (28) and wortmannin (44), (Table 2), with 28 having a zone of inhibition of 18 mm against Aspergillus niger and 44 with activity observed at 0.39 ± 0.05 μg/mL against Candida albicans strain ATCC 24433. Penicillitone (22) showed an anti-inflammatory effect on pro-inflammatory cytokine IL-6 of 96.6% when treated using a 5 μM concentration level, which was comparable to the result seen by treatment using dexamethasone.

Table 1.

Cytotoxic activities of compounds with IC₅₀ values below 10 μM

Compound	Source	Cancer cell line	Activity (IC50, μM)	Reference
Penicillitone (22)	Penicillium purpurogenum	A549	5.57 ± 0.19	Xue et al., 2014
Ergosterol (18)	Penicillium chrysogenum S003	DU-145	1.5	Alshehri et al., 2020
		HepG2	2.9
Epidioxyergosterol (29)	Penicillium chrysogenum S003	DU-145	6.1
		HepG2	3.07
5α,8α-Epidioxy-23-methyl-(22E,24R)-ergosta-6, 22-dien-3β-ol (31)	Penicillium stoloniferum QY2-10	P388	4.07	Xin et al., 2007
Auransterol (37)	Penicillium aurantiacobrunneum	HT-29	9.8	Tan et al., 2019

Table 2.

Antifungal compounds from Penicillium spp. with activities observed above 10 mm zone of inhibition or MIC below 0.5 μg/mL

Compound	Source	Pathogenic fungi	Assay Measurement Method	Activity	Reference
Penicisteroid A (28)	Penicillium chrysogenum QEN-24S	Aspergillus niger	Zone of inhibition	18 mm	Gao et al., 2011
Wortmannin (44)	Penicillium radicum	Candida albicans ATCC 24433	MIC measurement	0.39 ± 0.05 μg/mL	Singh et al. 2015

The studies to date not only demonstrate the propensity of Penicillium spp. to produce bioactive steroidal compounds but also those with rare structural features. This was especially seen in the C₂₅ steroids with bicyclo[4.4.1]A/B rings. Though several of these compounds were not significantly active in the context of the study explored, some were found to possess activity, such as 38 – 42, which were produced following mutagenesis to activate silent metabolites. Other C₂₅ extraction artifacts were also active (51 – 53). Hence this revealed that mutagenesis and chemical modification may provide an additional avenue for generating steroids with interesting structures. Additionally, because steroids from the list of those identified possess activity in assays applicable to various diseases, it is recommended that C₂₅ steroids and other steroids with unusual structures may be assessed in other assays outside of those already studied in order to unearth new activity.

The present communication indicates promise in studying this group of compounds from the penicillin-producing genus Penicillium and their potential therapeutic application. Notably, the steroids 33 and 28 were reported to bind to RXRα and induce apoptosis following this binding. This receptor plays an important role in the treatment of cancer, metabolic, and neurodegenerative diseases and is a promising target of study among the nuclear receptor superfamily (Xie et al. 2019). Furthermore, natural products with bridgehead double bonds have been shown to possess biological activity, among which the best-known example has been paclitaxel, used to treat several cancers (Wu et al., 2021). Interestingly, some C₂₅ steroids reported here possess a bridgehead double bond in the bicyclo[4.4.1]undec- 7,10-diene A/B ring system. Thus, these latter examples and the reported active steroids show that Penicillium fungi represent a potential source of new and structurally important steroids that could possess biological activities such as, cancer cell cytotoxicity, antimicrobial and anti-inflammatory activities, among others.

Research Highlights.

• Penicillium spp. are rich sources of steroids

• Penicillium spp. have yielded unusual steroids

• Steroids from Penicillium spp. display antimicrobial and cytotoxic activities

• The biological role of many unique steroids from Penicillium spp. has not been identified

Biographies

Ms. CHARMAINE A. LINDSAY – BRIEF C.V.

Ms. Charmaine A. Lindsay is a Ph.D. candidate in Dr. Harinantenaina Rakotondraibe’s research group. She originates from Jamaica and graduated (BSc, Biology) from Claflin University (South Carolina) with chemistry and applied computing minors in 2019. She joined the Division of Medicinal Chemistry and Pharmacognosy of The Ohio State University in 2019 and is working on discovering bioactive compounds from lichen mycobionts.

Dr. HARINANTENAINA L. RAKOTONDRAIBE – BRIEF C.V.

Dr. Harinantenaina L. Rakotondraibe, is an Associate Professor at the College of Pharmacy, The Ohio State University (OSU). He got his Ph.D. degree from the Institute of Pharmaceutical Sciences, School of Medicine, Hiroshima University (Japan) in 2003. From 2003–2007, he was a postdoctoral fellow at Tokushima Bunri University (partly supported by the Japan Society for the Promotion of Science) and then served on the faculty of the Graduate School of the Biomedical Sciences of Hiroshima University. Before joining the College of Pharmacy of The Ohio State University in 2013 as faculty, he was a Research Scientist at the Department of Chemistry of the Virginia Polytechnic Institute and State University and worked on bioactive compounds from natural products of Madagascar for four years. He is an Editor of the Journal of Natural Medicines and Molecules.

PROF. A. DOUGLAS KINGHORN – BRIEF C.V.

Prof. A. Douglas Kinghorn is Jack L. Beal Chair and Distinguished University Professor, at the College of Pharmacy, The Ohio State University (OSU). From 1977–2004, he served on the faculty of the College of Pharmacy of the University of Illinois at Chicago, where he was promoted to Full Professor in 1986. He has Ph.D. (1975) and D.Sc. (1990) degrees from The School of Pharmacy, University of London (now the School of Pharmacy, University College London). He received the 2010 Norman R. Farnsworth Research Achievement Award of the American Society of Phamacognosy and the 2020 Egon Stahl Award in Gold from GA (Society for Medicinal Plant and Natural Product Research), both for lifetime contributions to natural products research. He is the former Editor-in-Chief (1994–2019) and current Emeritus Editor (2020–2023) of the Journal of Natural Products.

Data availability

Data will be made available on request.