Epigenetic Strategies to Discover Novel Fungal Secondary Metabolites

Natural product search is an enduring revitalization upon the exploration of a huge already exotic potential for Secondary Metabolite (SM) production obscure in microbial genomes. Filamentous fungi genomes have an immense number of “orphan” SM gene clusters. Current evaluation indicates that only 5% of extant fungal species have been explored, thus the apparent for the disclosure of novel metabolites in fungi is extensive. In this situation, fungi burgeoning in severe environments are of special interest since they are distinguished producers of astonishing chemical structures. Genome mining strategies, more specifically epigenetic strategies are playing an important role in natural product discovery. This review has been organized and written to focus on available epigenetic approaches, targeting on DNA methyltransferase and histone deacetylase inhibitors along with reported novel secondary metabolites. To the best of our knowledge, this review article is the first attempt to incorporate the facts regarding DNA methyltransferase inhibitors and histone deacetylase inhibitors along with reported novel secondary metabolites with their recorded bioactivities.

In the repetitive research by pharmacists for new products, natural selection is superior to combinatorial chemistry for discovering novel substances that have the potential to be developed into this era [1]. Search for natural products is undergoing rebirth upon the discovery of a huge previously unknown potential for Secondary Metabolite (SM) production hidden in microbial genomes. Many fungi can effectively produce many natural products, more specifically bioactive natural products. Fungi are biosynthetically nature gifted organisms efficient of producing an extensive range of chemically diverse and biologically fascinating small molecules. The majority of scientific insight in fungal natural products has focused on their pharmaceutical applications, roles as mycotoxins, and diverse ecological functions. Unfortunately, characteristic fungal fermentation approaches such as an axenic shake or static culture strategies on artificially defined media are a poor replacement for mirroring an organism’s native habitat. The significance of these procedures is that only a subgroup of the biosynthetic pathways which encrypt for secondary metabolite production in fungi are ever expressed, thus restraining prospects for understanding the comprehensive drug discovery potential of these organisms [2].

It has been revealed that there is a biological relationship between the methylation of DNA and histone, which both are interrelated with diverse chemical reactions. This connection has a dynamic function in gene silencing from fungi to mammals [3-7]. The histone modification markers, such as H3K4me3, can work as indirect regulators to affect DNA methylation [8-10]. The primary methyltransferase of histone arginine methylation (H3R8me) is PRMT5, and its decreased levels can reduce the binding between DNMT3A and chromatin, decrease DNA methylation, and afterward facilitate genetic transcription [11]. Current data recommend that DNA and histone methylation commonly control fungal development and biosynthesis of toxic secondary metabolites [12]. Histone methylation is willingly reversible and usually precedes DNA methylation in N. crassa, whereas DNA methylation is comparatively stable and conduces to form a stable heterochromatic state [4,7].

DNA methylation is an epigenetic mechanism comprising the transfer of a methyl group onto the cytosine C5 position to form 5-methylcytosine. DNA methylation controls gene expression by engaging proteins involved in gene repression or by hindering the binding of transcription factor(s) to DNA. In the course of development, the DNA methylation pattern in the genome changes as a result of a vigorous process comprising both de novo DNA methylation and demethylation. As a result, discriminated cells develop a steady and exceptional DNA methylation pattern that controls tissue-specific gene transcription [13].

Among fungi, DNA methylation consists of imperceptible levels i.e. ≤ 0.1% of cytosine residues [14] to low but detectable levels i.e. 0.2-4.3% of cytosine residues [15-17] to markedly high level i.e. 10-30% of cytosine residues [18]. Furthermore, the methylated locates are usually clustered away from principally unmethylated regions. Though the significance of DNA methylation in fungi is still uncertain, Ascobolus immersus and Neurospora crassa are two documented examples of DNA methylation that plays roles in genome protection. Current studies on the genome-wide methylation study specified that DNA methylation ensues in and around genes, and fungal epigenetic entities subsidized to fungal growth as well as genome protection [19,20]. Furthermore, while some functions of DNA methylation have been identified, its regulation is not well understood (Figure 1).

Figure 1: Mechanism of DNA methylation by the addition of methyl group on 5ʹ of cytosine.

Histone acetylation and deacetylation are crucial processes of gene regulation. These reactions are naturally accelerated by an enzyme including "Histone Acetyltransferase" (HAT) or "Histone Deacetylase" (HDAC). The process of acetylation in terms of gene regulation is the transfer of an acetyl functional group from a molecule i.e. from Acetyl-Coenzyme A to histone. Whereas deacetylation is the reverse process of acetylation in which an acetyl group is removed from a histone molecule.

Acetylated histones which are octameric proteins organize chromatin into nucleosomes, basic structural units of the chromosomes and finally upper order structures characterize a type of epigenetic marker within chromatin. Acetylation reaction eliminates the positive charge on the histones, thus reducing the interaction of the N-terminal of histones with the negatively charged phosphate groups of DNA. As a result, the compressed chromatin is converted into a more relaxed structure that is concomitant with greater levels of gene transcription. This recreation can be inverted by HDAC activity. Relaxed, transcriptionally active DNA is stated as euchromatin. More compressed DNA is termed Compression can be brought about by processes including deacetylation and methylation [21] (Figure 2).

Figure 2: General mechanism of histone acetylation and deacetylation.

So far it can be said that both processes of DNA methylation and histone deacetylation among fungi are not biosynthetically essential. These processes can alter the transcription of many essential genes which may require either for defense mechanism [22] (Figure 3). Transcription of such genes usually not happened until such fungi have not been exposed to special modifiers. For that purpose, naturally or synthetically some modulators are available which were studied with different aspects including cancer treatment among eukaryotes [23]. These kinds of modulators are well known as epigenetic modulators. In the research of novel bioactive natural products by epigenetic strategies, these epigenetic modulators are serving as inducers for the induction of silent biosynthetic gene clusters in fungi [24,25]. In literature, several examples are available for the use of such epigenetic modifiers to be used for the induction of antimicrobial metabolites [26], toxicities induction [27], anticancer candidates [28], and others [29].

Figure 3: DNA demethylation and histone deacetylase inhibition mechanism.

Practicing epigenetic strategies with some examples

The term ‘epigenetic’ was invented by Waddington CH [30] and from that time; the definition of ‘epigenetic’ has developed. Epigenetics is the study of molecular procedures that affect the sequence of information between an adaptable gene expression patterns and a constant DNA sequence. The well-mannered definition of epigenetic is stated as the range of biochemical features that serve to modify the transcription of a gene or genes, nevertheless, do not straight forward change the conformation of DNA. Additionally, the epigenome functions as a biological filter that is accountable for controlling the availability of cells to ‘inbound’ interspecies and intraspecies signaling events. Likewise, the epigenome also can act as an ‘outbound’ filter that can chunk the DNA transcription and, in that way, successfully results signal generation [2,31].

Fungal genome manipulation via chemical epigenetics

The scientific researcher community's abilities to an emerging collection of small-molecule tools has permitted the expansion of chemical epigenetic techniques [32] that are directed toward searching how epigenome features control biological processes. This includes the biosynthetic mechanisms regulating potential for secondary metabolite production [33]. A considerable body of information has accrued relating to the effects of epigenetic modulators on fungi. Maximum examples have engrossed on the use of DNMT inhibitors such as 5-azacytidine (1) and 5-aza-2′deoxycytidine (decitabine) (2), which have validated the capability to reduce the DNA methylation- interceded silencing of a phleomycin-resistance gene in Phanerochaete Chrysosporium [33] and hygromycin-resistance genes in N. crassa [34] and Schizophyllum commune [35]. Some of the data related to the role of 5-azacytidine (1) revealed the capability of this in induction of heritable epigenetic modifications in fungi with respect to the acquisition of new and mitotically stable phenotypic characteristics [36]. However, a mutation-inducing effect for 5-azacytidine as a result of its DNA integration cannot be completely ignored in some situations [34,35]. Another unusual DNMTi is RG-108 (3), reported in the article of Asai, et al. [37] with the isolation of novel secondary metabolites [38]. Other less common DNMTi are mathyladenosine (4), sinefungin (5), and S-adenosylhomocystein (6) [36] (Figure 4).

Figure 4: Chemical structures of DNMTi and HDACi.

Chemical-epigenetic approaches and mutant studies were positively employed for the de novo or improved production of structurally diverse fungal natural products (e.g., mycotoxins, anthraquinones, nygerones, cladochromes, and lunalides) [36]. Some reported epigenetic modifiers apart of DNMTi are HDAC inhibitors (HDACi). Several research articles have described the differential effects of HDAC inhibitors on fungi. A chemical genetics approach retaining hydroxamic-acid-containing compounds such as trichostatin A (7) has also been used to reveal HDAC functions and universal transcriptional control mechanisms in Saccharomyces cerevisiae [39]. It is notable that Cochliobolus caronum, a fungal pathogen, which is responsible for the production of HC-toxin which is a potent HDAC inhibitor [40], synthesize a structurally modified HDAC that is unaffected to both trichostatin A (7). It is supposed that this exclusively adapted HDAC serves to defend C. caronum from the autotoxic effects of HC-toxin throughout the fungus's invasion and chemical attack upon its maize host [41]. A synthetic derivative of trichostatin A (7), suberoylanilide hydroxamic acid (SAHA) (8) also known as vorinostat, is another noteworthy examples of potent HDACi that have been effectively used as chemical epigenetic probes in a variety of eukaryotic systems, including filamentous fungi [10].

Other examples of amending media with HDACi are nicotinamide (9), suberoyl bishydroxamic acid (SBHA) (10), and sodium butyrate (11) in several studies directed not only to the improved production of compounds by fungi besides to the biosynthesis of novel compounds that were not produced without epigenetic modification [42,43]. Apart of that, some other unusual HDACi which uncommonly used are valproic acid (12), trapoxin (13), apicidin (14), and HC-toxin (15) [36] (Figure 4).

DNA Methyltransferase Inhibitors (DNMTi)

5ʹ Azacytidine: A chemical analog of cytidine, azacitidine and its deoxy derivative, decitabine which is also known as 5-aza-2′-deoxycytidine are used in the treatment of the myelodysplastic syndrome. Czechoslovakia firstly synthesized both of these drugs which were used as potential chemotherapeutic agents for cancer [76]. Perturbation of 5-azacytidine to Cladosporium cladosporioides can stimulate the production of numerous oxylipins including a glycerol conjugate (16), (9Z, 12Z)-11-hydroxyoctadeca-9,12-dienoic acid (17), and its methyl ester (18). Treatment of a Diatrype species with 5-azacytidine elicited the formation of lunalides A (19) and B (20) [2]. Yang and co-workers also reported the production of aflatoxins by Aspergillus flavus via 5azacytidine treatment [12].

Chemical epigenetic manipulation of Penicillium citreonigrum directed to profound changes in the secondary metabolic profile of its guttate. Fungi treated with 50 µM 5-azacytidine results in the 2 new metabolites production, meroterpenes atlantinones A (21) and B (22). Both metabolites from the P. citreonigrum guttate were tested for antimicrobial activity in a disk diffusion assay but found to be inactive [44]. Aspergillus sp. XS-20090066 was treated with a DNA methyltransferase inhibitor, 5-azacytidine, resulted the production of six bisabolane-type sesquiterpenoids, including (R)-(–)-hydroxy sydonic acid (31) [48], (S)-(+)-sydonic acid (32) [48], (S)-(–)-5-(hydroxymethyl)-2-(2_,6_,6_-trimethyltetrahydro-2H-pyran-2-yl)phenol (33) [51,52], (7S,11S)-(+)-12-hydroxy sydonic acid (34) [47], (S)-(+)-11-dehydro sydonic acid (35) [50], and (S)-(–)-sydowic acid (36) [51]. It has been supposed that 5-azacytidine may defeat DNA methyltransferase and consequently activate genes that express the bisabolane-type sesquiterpenoids. All compounds were tested for their antibacterial activities against various pathogenic bacteria strains. Compounds 31-36 showed broad spectrum activities against tested bacteria, while the others had weak or no antibacterial activities. In particular, (31) showed pronounced antibacterial activity against S. aureus with a MIC value of 3.13 µM, which was close to the positive control ciprofloxacin (MIC = 2.5 µM). N. crassa was treated with 5-azacytidine and result in the production of new carotenoids while its bioactivity was not tested [45].

Incorporation of 5-azacytidine in endophytic fungus Pestalotiopsis crassiuscula culture can change the metabolic profile with biosynthesizing a novel coumarin (80) that was confirmed by NMR spectra. Compound 80 was reported to not possess antifungal activity [77].

Two new aromatic polyketide glycoside indigotide A (82) and indigotide B (83), were isolated from the culture broth of the entomopathogenic fungus, Cordyceps tenuipes culture broth with the treatment of 5-azacytidine with no tested bioactivity [69] (Figure 5).

Figure 5: Chemical structures of novel secondary metabolites via 5-azacytidine stimulation.

Histone Deacetylase Inhibitors (HDACi)

Suberanilohydroxamic Acid (SAHA): Suberanilohydroxamic Acid (SAHA) also known as vorinostat with trade name Zolinza is a member of a larger class of compounds that inhibit Histone Deacetylases (HDAC). Histone deacetylase inhibitors have a broad spectrum of epigenetic activities. The compound was introduced by Ronald Breslow, the Columbia University chemist and Paul Marks, A Memorial Sloan-Kettering researcher [78,79]. The first Histone Deacetylase Inhibitor (HDACi) was vorinostat that was approved by the U.S. Food and Drug Administration (FDA) for the treatment of Cutaneous T-cell lymphoma (CTCL) on October 6, 2006 [78].

SAHA as an epigenetic modifying agent used by many researchers to stimulate the production of many secondary metabolites. Fungi, C. cladosporioides, treated SAHA resulted in the production of a complex series of perylenequinones including cladochromes A-D, F, and G (23-28) and calphostin B (29). The Cichewicz group also isolated nygerone A (30) from Aspergillus niger when culturing with suberoylanilide hydroxamic acid [79]. Metabolic profiles of P. mallochii CCH01 were reported to be changed by SAHA treatment. Two new natural sclerotioramine derivatives, isochromophilone XIV (37) and isochromophilone XV (38) were purified with no reported bioactivity [53]. Two new compounds, (+)- and (-)-brevianamide X ((+)- and (-)- 39 and 40)), in addition to a new naturally occurring one, 3-[6-(2-methylpropyl)-2-oxo1H-pyrazin-3-yl] propanamide (41) were purified from the chemical-epigenetic cultures of Aspergillus versicolor OUCMDZ-2738 with 10 µM vorinostat (SAHA) [54]. Similarly, another research reported about SAHA significance to improve the diversity of secondary metabolites of Aspergillus versicolor by the production of a new biphenyl derivative, named versiperol A (42) [10]. Two novel polyketides from a fermentation broth of A. cruciatus named primarolides A (43) and B (44) and were purified when treated with SAHA in combination with sodium chloride (NaCl). Bioactivity of 39, 40, 41, 42, 43, and 44 were not reported [80].

SAHA has been used to suppress HDAC in Chaetomium indicum, giving rise to six novel prenylated aromatic polyketides, chaetophenols A-F (45-50). Among these, compounds 48, 49, and 50 contained extraordinary polycyclic skeletons [42]. More recently, chemical analysis of the culture broth of the plant endophyte Penicillium sp. HS-11 in the modified Martin’s medium improved with SAHA, led to the isolation and identification of one novel chemical structure, 4epipenicillone B (51) and one previously undescribed polyketide with a rarely occurring tricycle [5.3.1.03,8] undecane skeleton (R)-(+)-chrysogine (52). Acquisition of 4-epipenicillone B (51) enriched the chemical diversities of fungal natural products possessing a tricyclo [5.3.1.03,8] undecane skeleton. The cytotoxic activity of 52 was also evaluated [57]. Vervoort, et al. in 2010 reported that culturing marine sediment-derived fungus Microascus sp. in the presence of SAHA can lead to the biosynthesis of EGM-556, a new cyclodepsipeptide (70) of hybrid biosynthetic origin [65]. Although bioactivity of that metabolite has not performed it was reported that the 16 atom peptolide center of 70 is rare; the solitary additional examples are the antimicrobial unnarmicins from a marine-derived Photobacterium sp. MBIC06485 [81] and the histone deacetylase inhibitory/ antitumor active FK228 (FR901228, 4) from Chromobacterium violaceum No. 968 [82].

Similarly, incorporation of SAHA to a culture broth of the endophytic fungus Phoma sp. nov. LG0217 isolated from Parkinsonia microphylla altered its metabolite profile and give rise to the production of (10ʹS)-verruculide B (71), vermistatin (72) and dihydrovermistatin (73). Compound 71 repressed the activity of Protein Tyrosine Phosphatases (PTPs) 1B (PTP1B), Src homology 2-containing PTP 1 (SHP1) and T-cell PTP (TCPTP) with IC₅₀ values of 13.7 ± 3.4, 8.8 ± 0.6, and 16.6 ± 3.8 μM, respectively [94]. The addition of SAHA to a culture of the filamentous fungus Beauveria felina was reported to significantly changed its secondary metabolite profile and results in the isolation of three new compounds including cyclodepsipeptides, desmethylisaridin E (107), desmethylisaridin C2 (108), and isaridin F (109). The anti-inflammatory activity of these compounds was evaluated by assessing their effect on superoxide anion production and elastase release by FMLP-induced human neutrophils. Among all three compounds, desmethylisaridin E (107) repressed superoxide anion production and desmethylisaridin C2 (108) repressed elastase release, with IC₅₀ values of 10.00 ± 0.80 and 10.01 ± 0.46 μM, respectively [73] (Figure 6).

Figure 6: Chemical structures of novel secondary metabolites by SAHA stimulation.

Trichostatin A

An organic compound, trichostatin A is an antifungal antibiotic that selectively inhibits the class I and II histone deacetylase families of enzymes, but not class III HDACs among mammals. Researchers are using trichostatin A as an epigenetic modifying agent to selectively effect on HDAC machinery of fungus to stimulate its secondary metabolite production. Four new meroterpenoids named as (4S)-4-decarboxylflavipesolide C (55), 1-(2,2- dimethylchroman-6-yl)3-(4-hydroxyphenyl)propan-2-one (56), (R,E)-3-(2,2-dimethyl chroman-6-yl)-4-hydroxy-5-((2(2-hydroxypropan-2-yl)-2,3-dihydrobenzofuran-5 yl)methylene) furan- 2(5H)-one (57), methyl (R)-2-(2-(2-hydroxypropan-2-yl)-2,3-dihydrobenzofuran-5-yl) acetate (58), were identified from a 10 µM trichostatin A treated strain of Aspergillus terreus OUCMDZ-2739. Compound 57 showed potent α-glucosidase inhibitory activity in comparison with others [59]. Treatment of A. alternate and P. expansum with trichostatin A results the synthesis of new unidentified compounds with untested bioactivity [46] (Figure 7).

Figure 7: Chemical structures of secondary metabolites stimulated by others HDACi.

Nicotinamide

Nicotinamide, also known as niacinamide, serves as a component of the coenzyme Nicotinamide Adenine Dinucleotide (NAD). Nicotinamide acts as a radio and chemosensitizing agent via increasing tumor blood flow as a result to reduce tumor hypoxia. Nicotinamide also inhibits poly (ADP-ribose) polymerases, enzymes involved in the rejoining of DNA strand breaks induced by radiation or chemotherapy.

Nicotinamide is a histone deacetylase inhibitor that also serves as epigenetic modulators to stimulate the secondary metabolites production among fungi. Epigenetic perturbation of the endophytic fungus led to enhanced production of two new decalin-containing compounds, eupenicinicols C and D (53) and (54). Compound 53 was active against Staphylococcus aureus with an MIC of 0.1 μg/mL and confirmed obvious cytotoxicity against the human acute monocytic leukemia cell line (THP-1) and compound 54 was active against Escherichia coli with a MIC of 5.0 μg/mL [36]. Nicotinamide can also induce the production of chaetophenols G (67) and cancrolides A (68) and B (69) when treated with the culture Chaetomium cancroideum [64]. Another species of the same genus i.e. Chaetomium mollipilium can produce five new C13polyketides, mollipilin A-E (89-93) when cultivated with nicotinamide. Mollipilin A (89) and B (90) showed moderate inhibitory activity on cell growth with GI50 values of 1.8 and 3.7 μM, respectively [71].

Asai, et al. [72] reported the addition of nicotinamide, to the culture medium of the endophytic G. chlorocephala, can significantly stimulate its benzophenone production. A set of new benzophenones, cephalanones A-F (101-106) were isolated with no tested bioactivities (Figure 7).

Suberoyl Bis-Hydroxamic Acid (SBHA)

Suberoyl Bis-Hydroxamic Acid (SBHA) is a Histone Deacetylase (HDAC) inhibitor that hinders the activity of HDAC1 and HDAC3. SBHA suppress the proliferation and brings apoptosis in several cancer cell lines. SBHA has been shown to trigger Notch signaling in Medullary Thyroid Carcinoma (MTC) cells.

SBHA can significantly effect on the secondary metabolism of an entomopathogenic fungus, Torrubiella luteorostrata by the production of three new prenylated tryptophan analogs, luteorides A–C (59-61) with no record of bioactivity [60]. Similarly, another new compound, named 13-angeloyloxy-diplosporin (62) was isolated from the endophytic Phomopsis sp. 0391 cultivated in the presence of SBHA. 62 was tested for lipase inhibitory activity but not found to be active [61].

By the treatment of SBHA to the culture of F. oxysporumiz can produce two new fusaric acid derivatives 5-butyl-6-oxo-1,6- dihydropyridine-2-carboxylic acid (65) and 5-(but-9-enyl)-6oxo-1,6-dihydropyridine-2-carboxylic acid (66). Antibacterial activities were tested but unfortunately, none of the compounds was reported to be bioactive [62]. The accumulation of the epigenetic modifier, specifically, SBHA to the culture medium of Alternaria sp. intensely altered the metabolic profile. Six new compounds named alternariol (74), alternariol-5-O-methyl ether (75), 3′-hydroxyalternariol-5-O-methyl ether (76), altenusin (77), tenuazonic acid (78), and altertoxin II (79) [67].

Screening of the entomopathogenic fungi Isaria tenuipes that were cultured in the presence of SBHA showed significant changes in the production of secondary metabolites. This approach led to the isolation of tenuipyrone (81), a novel skeletal polyketide with no tested bioactivity [68]. The secondary metabolite production of an entomopathogenic fungus Cordyceps annullata, was improved by the accumulation of SBHA to the culture medium. Four new 2,3-dihydrobenzofurans, annullatins A-D (84-87), and a new aromatic polyketide, annullatin E (88) were purified.

Dihydrobenzofurans serve as cannabinoid receptor ligands [37] (Figure 7).

Sodium butyrate

Sodium butyrate is the sodium salt of butyric acid. It has numerous effects on cultured mammalian cells comprising inhibition of proliferation, induction of differentiation and induction or repression of gene expression [43]. Sodium butyrate can be used in a lab to bring about any of these effects. Precisely, butyrate treatment of cells results in histone hyperacetylation, and butyrate itself hinders class I Histone Deacetylase (HDAC) activity [83], specifically HDAC1, HDAC2, HDAC3, and butyrate can be used in defining histone deacetylate in chromatin structure and function [84].

Sodium butyrate is also a histone deacetylase inhibitor, effective to stimulate the metabolite production of fungi by reverse the effect of histone acetylase. Treatment of sodium butyrate to an endophytic fungus Phomopsis species led to the isolation of two new 14-membered resorcylic acid lactones described with bromine substitution, 5-bromozeaenol (63) and 3, 5-dibromozeaenol (64). Both compounds were found to be inactive when tested for cytotoxicity, antifouling and zebrafish teratogenicity [62].

Epigenetic modifier sodium butyrate was incorporated into the culture medium Leucostoma persoonii and induced the new secondary metabolite Cytosporone R (111) with no reported activity [75] (Figure 7).

DNMTi and HDACi Combined Treatment to Stimulate the Secondary Metabolite Production

The combined treatment of DNMTi and HDACi has studied to activate those secondary metabolites which could produce neither usual laboratory condition nor single epigenetic modifier treatment. Asai and co-workers have applied that approach by using RG-108 and SBHA as DNMTi and HDACi, respectively. Both modifiers were added to the culture medium of G. formosana and found significant enhancement of the secondary metabolite production. Two types of natural products were isolated include highly oxidized ergosterols and isariotin analogs. Highly oxidized ergosterols, include formosterols A (94) and B (95), while five new isariotin analogs include 120O-acetylisariotin A (96), 1-epi-isariotin A (97), and isariotins K-M (98-100). None of these compounds were tested for any bioactivities [72]. Likewise, incorporation of 5-azacytidine or SAHA led to the induction of isosulochrin (110) in Chaetomium sp with no reported bioactivities [74] (Figure 8).

Figure 8: Chemical structures of secondary metabolites produced by the combined exposure of Epigenetic modifiers.

Valproic acid (valproate), an anticonvulsant and a mood stabilizer, is a potent Histone Deacetylase Inhibitor (HDACi). The addition of valproic acid in the culture medium can improved the metabolic profile of A. fumigatus (GA-L7) by the enrichment of fumiquinazoline C (112). This compound was produced in trace amounts under normal laboratory conditions. Fumiquinazolines are peptidyl alkaloids that are reported to possess substantial antitumor [85], antifungal [86] and antibacterial properties [87].

It was reported that endophytic fungal isolate, diaporthe sp. PF20. When exposing to epigenetic treatment along with previously characterized piperine producing Colletotrichum sp. and Mycosphaerella sp. from the Piper nigrum L. plant can overproduce piperine (113) by the use of SAHA. In this report, the epigenetic modulator (SAHA) mediated enrichment of phytochemical biosynthetic potential of endophytic fungi [47]. A. nidulans can overexpress the genes for fellutamides A-D (114-117), proteasome inhibitors when treated with SAHA, an HDACi [88]. Fungi, Botryosphaeria rhodina when cultured in the presence of 5-azacytidine can enhance the production of Camptothecin (CPT) (118) as compared to wild type. CPT was reported to have anticancer activity [89].

The histone deacetylase inhibitor SAHA was also reported to significantly improve the alkaloid productivity of the strain Claviceps purpurea Cp-1. Principally, the titers of total ergot alkaloids, ergometrine (119) were progressively improved with the increasing concentration of SAHA in the fermentation medium, and the maximum production of ergot alkaloids could be attained at the concentration of 500 µM SAHA. Particularly, the titers of ergometrine and total ergot alkaloids were as high as 95.4 mg/L and 179.7 mg/L, respectively, which were twice those of the control [90].

The histone deacetylase inhibitor sodium butyrate can lead to the enhancement of know bioactive secondary metabolites in Leucostoma persoonii including cytosporones B (120), C (121) and E (122). Cytosporone E (122) was reported to be the most bioactive, displaying an IC90 of 13 μM toward Plasmodium falciparum, with A549 cytotoxicity IC90 of 437 μM, demonstrating a 90% inhibition therapeutic index (TI90 = IC90 A459/IC90 P. falciparum) (Table 2). Including, cytosporone E (122) was active against MRSA with a Minimal Inhibitory Concentration (MIC) of 72 μM [75]. It was reported by Xiao, et al. in 2013 that SAHA exhibited a positive impact on (+) terrein (123) production in Aspergillus terreus strain PF26 which was resulting from endorsing the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein (123). (+)-terrein (123) has many reported bioactivities [91] (Figure 9).

Figure 9: Chemical structures of secondary metabolites enhanced exposure of DNMTi and HDACi.

Up to date above 500 compounds have been tested as inhibitors of DNMTs. Their structure and coverage in chemical space have been studied using chemoinformatic methods [101]. The DNMT inhibitors targets have been compared with inhibitors of other epigenetic targets [102]. Additionally, the Structure-Activity Relationships (SAR) of DNMT inhibitor using the idea of the activity landscape have been acknowledged [103] (Table 3).

Several different strategies including virtual, organic synthesis, and high throughput screening were used to isolate DNA methyltransferase inhibitors [131]. For principal optimization, organic synthesis has been work in several instances [132,133]. Food chemicals and natural products have been serving as major sources of active compounds. Zwergel, et al. [104] have extensively reviewed the natural products as known DNMT inhibitors or demethylating agents. The natural products which were isolated belong to the class of flavonoids, polyphenols, anthraquinones, and others. We have collected some specific data from that review and accumulated here. Based on such DNMTi sources/origin and type/class, those DNMTi include flavonoids, genistein (124) from soybean Genista tinctoria, quercetin (125) from fruits, vegetables and beverages [104-108], luteolin (130) from Terminalia chebula [104,114,115], silibinin (134) from Silybum marianum, and kazinol Q (135) from Broussonetia kazinoki [104,119-122]. Including quinones, nanaomycin A (136) from Streptomyces, laccaic acid (137) from Kerria lacca, hypericin (138) from Hypericum [104,124-128]. Among polyphenols are (-)-epigallocatechin-3-gallate (EGCG) (126) from Camellia Sinensis (green tea) [104,109], curcumin (129) from Curcuma longa [21,133], caffeic acid (132), and chlorogenic acid (133) from Coffea Arabica [104,118]. Some other reported DNMTi were carbazole alkaloid, mahanine (128) from Micromelum minutum and Murraya koenigii, terpenoid, boswellic acid (131) from Boswellia serrata, nonmetal, selenium (127) is an essential trace element which also reported to act like DNMTi [104,110-111,116], and bright red carotene, lycopene (139) from lycopersicum [104,110-112,116,129]. The bioactive profile, mechanisms, and techniques of such natural products well described in the review of Zwergel, et al. [104] with their IC₅₀ values (Figure 10).

Another study conducted by Wei, et al. in 2018 in which they examine the crude extract of C. arbuscula with deleted hdaA (ΔhdaA) strain resulted in the separation of twelve new diterpenoids including three cassanes A-C (140-142), onecleistanthane (143), six pimaranes A-F (144-149), and two isopimaranes A-B (150-151). Compounds 141 and 142 has reported to showed strong inhibitory effects on the expression of MMP1 and MMP2 (matrix metallo proteinases family) in human breast cancer (MCF-7) cells [130] (Figure 10).

Figure 10: Chemical structures of r1epresentative epigenetic modifiers from natural sources.

More research on the effect of epigenetic modulators on fungi could be recommended to discover newly structured compounds in future studies. The rate of emerging diseases and infections is increasing day by day, and the efficacy and selectivity of available drugs are decreasing. However, many efforts still need to be devoted to addressing these strategies towards the infectious era. In this review, 15 DNMTi and HDACi are reported; 6 commonly used inhibitors are discussed in detail. A total of 96 new compounds with reported bioactivities from different research articles are mentioned, isolated from those 6 inhibitors, either in combination or a single effect. Epigenetic techniques have several noteworthy benefits related to presently available molecular or culture-dependent techniques. First and leading, it provides a needed tool for rapidly retrieving possible pools of cryptic fungal natural products in their natural hosts. Second, this approach can be readily implemented in most laboratories deprived of widespread retooling, giving it a varied scope of consumption. Third, this technique will suggestively reduce the cost and exertion of obtaining the products of silent secondary metabolic pathways since fungi do not need to be pre-screened using a multitude of culture conditions.

All the authors declared no potential conflict of interest.

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