Key words
fungi - Mexico - bioprospecting - antidiabetic - agrochemical - anticancer
Introduction
Fungi represent a prized source of natural product bioactive compounds with an enormous
and relevant impact in human medicine and crop protection. Some important drugs and
products for agriculture have been derived from or inspired by fungal natural products;
some important examples include β-lactam antibiotics, griseofulvin, cyclosporine, statins, echinocandins, PF1022A,
nodulisporic acid, scyphostatin, and strobilurins. According to new evidence, the
number of fungal species might be between 2.2 and 3.8 million, with 120 000 currently
accepted [1]. Since the estimations of fungal biodiversity exceed, by far, the number of already
identified species, chances of finding unidentified fungal species and novel bioactive
products are still high [2]. Furthermore, the advances in genomic analysis techniques have permitted the discovery
of innumerable putative gene clusters for potentially bioactive compounds.
The knowledge of fungal biodiversity in Mexico is still poor. A recent analysis proposed
that there are 200 000 fungal species, but only 5% of these have been studied [3]. Consequently, the potential of fungal biodiversity from Mexico as a source of bioactive
compounds remains largely unexplored. Thus, the aim of this paper is to provide an
overview on the status of fungal bioprospecting in Mexico in the last two decades,
considering only the work pursued in the authors laboratories at the National Autonomous
University of Mexico. Twenty fungi have been investigated from the biological and
chemical point of view, leading to the isolation of over 100 bioactive secondary metabolites
of medicinal and/or agrochemical relevance. This information has been reported in
over 50 peer-reviewed publications according to Scopus and SciFinder databases. In
the medicinal area, the studies were focused on the discovery of new leads for the
development of antitumoral, Ca2+-calmodulin (CaM) antagonists, and antidiabetic drugs with α-glucosidase inhibitory activity. In the field of agrochemicals, the studies emphasized
the discovery of potential herbicidal, antifungal, and anti-oomycete agents.
The fungi discussed in this review were isolated from diverse substrates, mainly from
plants (endophytes), dung, soil, insects, and marine environments belonging to different
ecosystems in Mexico. Bioprospecting of endophytes from wild medicinal plants is a
successful strategy for the discovery of novel and known bioactive compounds [4]. Accordingly, we analyzed endophytes from selected medicinal plants, including Hintonia latiflora (Sessé & Moc. ex DC.) Bullock (Rubiaceae), Bursera simaruba (L.) Sarg. (Burseraceae), Callicarpa acuminata Kunth. (Verbenaceae), Haematoxylon brasiletto H. Karst. (Fabaceae), Sapium macrocarpum Muell. Arg. (Euphorbiaceae), and Gliricidia sepium (Jacq.) Kunth ex Walp. (Fabaceae). The endophytic fungi reviewed includes strain
MEXU 27095 (Chaetomiaceae), Preussia minimoides (S. I. Ahmed & Cain) Valldos. & Guarro (Sporormiaceae), Xylaria feejeensis (Berk.) Fr. (Xylariaceae), Edenia gomezpompae M. C. González, Anaya, Glenn, Saucedo & Hanlin (Pleosporaceae), Muscodor yucatanensis M. C. González, Anaya, Glenn & Hanlin (Xylariaceae), Acremonium camptosporum W. Gams (Clavicipitaceae), X. feejeensis SM3e-1b, and Hypoxylon anthochroum Berk. & Broome strains Blaci and GS4d2II (Xylariaceae).
Coprophilous fungi are a large group of saprotrophic fungi, which represent an important
reservoir of useful novel bioactive secondary metabolites. The number of dung fungi
undergoing investigation is continuously increasing with new species and genera constantly
described [5], [6]. On these grounds, a few coprophilous fungi isolated from bat dung in different
regions of Mexico have been investigated in our laboratories. Some of these fungi
include Penicillium spathulatum B35 Frisvad & Samson (Trichocomaceae), Penicillium sp. G1-a14, Guanomyces polythrix M. C. González, Hanlin & Ulloa (Chaetomiaceae), and Malbranchea aurantiaca Sigler and Carmich (Myxotrichaceae).
Marine fungi (sensu stricto) or those isolated from a marine substratum are a rich source of new natural products
with interesting chemical scaffolds and potent biological activities [7], therefore Aspergillus stromatoides Raper & Fennell (Trichocomaceae), Aspergillus sp. MEXU 27854, and Emericella sp. MEXU 25379 (Trichocomaceae) were analyzed. Among the entomophatogenic fungi,
we have only investigated Isaria fumosorosea Wize (Clavicipitaceae), a fungus currently used for the biological control of whiteflies
(Bemisia tabaci Gennadius). Phytopathogenic fungi are known to be involved in several plant diseases
that damage agrarian and forest crops, besides having general adverse effects on wild
plant species. These damages are produced by toxins which, in many cases, have been
used to obtain products for plant protection. Based on this consideration, we investigated
Phoma herbarum Westend. (Pleosporales). Finally, two saprophytes were investigated, Malbranchea flavorosea Sigler & J. W. Carmich. (Myxotrichaceae), isolated from soil, and Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samso (Ophiocordycipitaceae).
Alpha-Glucosidase Inhibitors
Alpha-Glucosidase Inhibitors
Type II diabetes mellitus is a disease with skyrocketing prevalence. The number of
patients today, ~ 380 million, will increase to ~ 450 million in 2030. In this scenario,
extraordinary efforts have been made to identify lead compounds for the development
of new drugs for treating this disease. Metformin remains the first-line pharmacotherapy
for patients with type II diabetes mellitus, whereas the use of other well-established
agents, such as sulfonylureas, meglitinides, thiazolidinediones, and α-glucosidase inhibitors, varies in different regions [8]. In areas with a large intake of complex carbohydrates, α-glucosidase inhibitors are widely used. These agents competitively inhibit α-glucosidase enzymes in the brush border of enterocytes lining the intestinal villi.
Inhibition of intestinal α-glucosidases delays the digestion of starch and sucrose, flattens the postprandial
blood glucose excursions, and thus mimics the effects of dieting on hyperglycemia,
hyperinsulinemia, and hypertriglyceridemia [9], [10]. They minimize hypoglycemia and weight gain, increase postprandial glucagon-like
peptide-1 secretion, decrease both postprandial hyperglycemia and hyperinsulinemia,
and thereby may improve sensitivity to insulin. The α-glucosidase inhibitors class currently comprises three compounds, namely, acarbose,
miglitol, and voglibose. Gastrointestinal adverse effects and a rapid absorption rate
of some α-glucosidase inhibitors are commonly encountered and can lead to treatment withdrawal.
Based on the above considerations, we have explored a few fungi to discover new α-glucosidase inhibitors. To detect fungi extracts suitable for α-glucosidase inhibitor isolation, first an in vitro enzymatic test is carried out using a well-known spectrophotocolorimetric procedure
[11]. Those extracts with evident activity in the assay were subjected to activity-guided
fractionation. Once the active compounds are obtained and characterized, if the quantity
of the active compounds is sufficient (over 100 mg), animal in vivo studies using an oral sucrose tolerance test in normal and hyperglycemic mice were
performed. In most cases, docking studies are performed using protein models of human
N-terminal and C-terminal subunits of maltase glucoamylase (hNt-MGAM, PDB code 2QMJ; and hCt-MGAM, PDB code 3TOP; respectively), human N-terminal sucrose isomaltase (hNt-SI, PDB code 3LPP), and yeast isomaltase (y-IM, PDB code 3A4A). Recent work in this area is summarized in the following paragraphs.
Fungal endophytes from Hintonia latiflora
H. latiflora is a Mexican medicinal plant with a long history of use for treating diabetes. Owing
to its antidiabetic effect, Mexican and several European drug companies have commercialized
herbal products containing the stem bark of this species for decades (e.g., Sucontral
D, Copalchi-Bellsolá, Copalchi, Copangel, among others). The plant has been the subject
of many chemical and pharmacological studies that have established the hypoglycemic
and antihyperglycemic actions. The active principles are a few cucurbitacins and 4-phenylcoumarins
type of compounds [12]. With the increasing demand for herbal drugs and natural health products, wild H. latiflora and related species are facing accelerated loss and are now threatened with extinction
from overexploitation. Therefore, it is important to find alternative approaches to
meet the medical demand. This can be achieved using different strategies, including
the assessment of the ability of the host microorganisms to biosynthesize pharmacologically
active secondary metabolites differently, similarly, or identically to those produced
by their host medicinal plant [13]. Therefore, a few endophytes from H. latiflora were isolated and investigated.
The first endophyte analyzed was MEXU 27095. Bioassay-guided fractionation of the
active organic extract obtained from solid media culture led to the separation of
three tridepsides, which were identified as thielavins A, J, and K (1 – 3) [14] ([Fig. 1]). All three compounds inhibited bakerʼs yeast α-glucosidase (αGHY) in a concentration-dependent manner with IC50 values of 23.8, 15.8, and 22.1 µM, respectively. Their inhibitory action was higher
than that of acarbose (IC50 = 545 µM), used as a positive control. Kinetic analysis established that the three
compounds acted as noncompetitive inhibitors with K
i values of 27.8, 66.2 and 55.4 µM, respectively. Docking analysis predicted that 1 – 3 and acarbose bind to αGHY in a pocket close to the catalytic site for isomaltose. The α-glucosidase inhibitory properties of thielavin K (3) were corroborated in vivo since it induced noted antihyperglycemic action during an oral sucrose tolerance
test (3.1, 10.0, and 31.6 mg/kg) in normal and nicotinamide-streptozotocin (NA-STZ)
hyperglycemic mice. Thielavin K (3) also showed a hypoglycemic effect in vivo at the doses of 3.1 and 10 mg/kg when tested in normal and hyperglycemic mice. In
both cases the effect was attained after 5 h and maintained throughout the experiment,
and was comparable to that of glibenclamide [14]. Sakemi and coworkers previously demonstrated that compounds 1 – 3 were glucose-6-phosphatase inhibitors in vitro
[15]. Altogether, these results revealed that thielavin-type tridepsides represent a
new lead for the development of α-glucosidase inhibitors; specifically, compound 3 shows potential as an antidiabetic agent acting at different targets, namely, inhibiting
the α-glucosidases at the intestinal levels or decreasing hepatic glucose output from glyconeogenesis
and glycogenolysis. Unfortunately, compounds 1 – 3 showed poor solubility, which caused problems for further in vitro and in vivo assays.
Fig. 1 Secondary metabolites isolated from endophyte MEXU 27095.
P. minimoides was also isolated as an endophyte from H. latiflora, although it has been previously obtained from different substrates including dung,
Pinus tabulaeformis Carr., and Trametes hirsutum (Will. Fr.) S. F. Gray, a medicinal fungus collected from a dead hardwood branch
in a dry forest in Hawaii [16]. A previous chemical investigation performed by other authors resulted in the isolation
of a depsipeptide and two polyketides, sporminarins A and B [16], with antifungal properties against Aspergillus flavus Link [17]. In our exploration, extensive fractionation of an extract from the grain-based
culture of P. minimoides led to the isolation of five new polyketides, two of them with novel skeletons, minimoidiones
A (4) and B (5), corymbiferone C (6), corymbiferan lactone E (7), and 5-hydroxy-2,7-dimethoxy-8-methylnaphthoquinone (8), along with the known compounds preussochromone C (9), corymbiferone (10), brocaenol B (11), and ziganein (12) [18], [19] ([Fig. 2]). The structures of the new compounds 4 – 8 were elucidated using 1D and 2D NMR data analysis, along with density functional
theory (DFT) calculations of NMR chemical shifts. The absolute configuration of 4 was established by a single-crystal X-ray diffraction analysis and time-dependent
DFT electronic circular dichroism (TDDFT-ECD) calculations. Polyketides 4, 5, 9, and 10 significantly inhibited yeast α-glucosidase with IC50ʼs ranging from 2.9 to 155 µM. Among them, compound 5 showed the strongest effect (IC50 = 2.9 µM). In order to envisage the putative binding mode of compounds 4 and 5 with αGHY, docking analyses were carried out using the crystallized structure of αGHY. Minimoidione A (4) and the R and S enantiomers of 5 were docked into the validated α-glucosidase model. The results predicted that the R enantiomer of compound 5 binds in a site different from the catalytic domain. On the other hand, docking of
compound 4 and the S stereoisomer of 5 suggested that they bind to the catalytic site of αGHY with higher affinities. Further work is in progress to test these polyketides
in vivo.
Fig. 2 Secondary metabolites isolated from endophyte P. minimoides.
The last endophyte from H. latiflora with α-glucosidase inhibitory activity was characterized as X. feejeensis. The genus Xylaria Hill ex Schrank comprises more than 300 species, some of which have received special
attention due to their potential as sources of novel secondary metabolites. A recent
review showed that more than 180 compounds, including sesquiterpenoids, diterpenoids,
diterpene glycosides, triterpene glycosides, steroids, N-containing compounds, pyrone derivatives, and polyketides, have been isolated from
this genus [20], [21]. Specifically, from X. feejeensis, integric acid, xylaropyrone, and the nonenolide xyolide have been isolated. During
our investigations, we isolated two new compounds, pestalotin 4′-O-methyl-β-mannopyranoside (13) and 3S,4R-(+)-4-hydroxymellein (14), from an organic extract of a solid medium culture (rice). In addition, the known
compounds 3S,4S-(+)-4-hydroxymellein (15), 3S-(+)-8-methoxymellein (16), coriloxine (17), and the quinone derivatives 18 and 19 were obtained [22] ([Fig. 3]). The absolute configuration of the stereogenic centers of 13 and 14 was determined using TDDFT-ECD calculations. Compounds 14 and 15 inhibited αGHY with IC50 values of 441 and 549 µM, respectively. Their activity was comparable to that of
acarbose. Molecular docking predicted that both compounds bind to αGHY in a site different from the catalytic domain, which could imply an allosteric
type of inhibition.
Fig. 3 Secondary metabolites isolated from endophyte X. feejeensis.
Coprophilous Penicillium spathulatum
P. spathulatum was collected aseptically from bat dung obtained in the Chontalcoatlán cave in the
State of Guerrero. This fungus has a worldwide distribution and is found commonly
in soil, air, water, ice, and food products, and is associated with different plants.
HPLC analysis of the extrolites of some strains of P. spathulatum revealed that all of the isolates produced asperphenamate (20) and perinadine, some benzomalvins, breviones, cyclopenols, and related benzodiazepine
alkaloids, and the least quinolactacins [23]. The defatted extract from P. spathulatum B35 grown in rice showed inhibitory activity against yeast α-glucosidase (IC50 = 56.5 µg/mL). Extensive chromatography of this active fraction led to the isolation
of 20, the epimeric mixture (7 : 3) of quinolactacins A1 (21) and A2 (22), quinolactacin B as a racemate (23), quinolonimide (24), benzomalvin A (25), and 2-chloro-6-[2′(S)-hydroxypropyl]-1,3,8-trihydroxy-anthraquinone (26), a new chemical entity [24] ([Fig. 4]). Compound 20, the mixture of 21 and 22, racemate 23, and compound 25 inhibited yeast α-glucosidase in a concentration-dependent fashion with IC50 values of 8.3, 273.3, 57.3, and 383.2 µM, respectively. The α-glucosidase inhibitory properties of 25 were confirmed in vivo with an oral sucrose tolerance test in normal and hyperglycemic mice. Docking studies
performed with conformers 25a and 25b indicated that they bind to yeast isomaltase (3A4A) in a different location than
acarbose, whereas for human enzymes 3LPP, 2QMJ, and 3TOP, the binding occurred at
the catalytic domain [24]. Compound 25 also showed antihyperalgesic activity in NA-STZ hyperglycemic mice, reducing formalin-induced
hyperalgesic behavior during phase I at the dose of 31.6 µg/paw, and during phase
II at 10 and 31.6 µg/paw. The benzodiazepine-GABAA receptor was ruled out as the molecular target of 25 on the basis of classical agonist-antagonist experiments.
Fig. 4 Secondary metabolites isolated from coprophilous P. spathulatum.
The last fungus selected for the search of α-glucosidase inhibitors was M. flavorosea. From the active extract prepared from the grain-based culture, two new polyketides,
namely, 8-chloroxylarinol A (27) and flavoroseoside (28), along with the known compounds xylarinol A (29), xylarinol B (30), massarigenins B and C (31 and 32), and clavatol (33), were isolated [25] ([Fig. 5]). The structures of 27, 28, and 32 were corroborated by single-crystal X-ray diffraction analysis. Compounds 29, 30, and 32 significantly inhibited αGHY. Oral administration of 32 significantly reduced the postprandial peak in a non-dose-dependent manner during
an oral sucrose tolerance test using normal and NA-STZ-induced (50 – 130 mg/kg) hyperglycemic
mice. In all cases, the effect was comparable to that of the positive control acarbose,
thus revealing the antihyperglycemic potential of compound 32. Docking of 32 to hNt-MGAM and hCt-MGAM as well as to hNt-SI and y-IM predicted that it would bind to them far from the catalytic site; the interacting
forces were mainly by hydrophobic contacts and hydrogen bonding (HB). The overall
docking results were consistent with the in vitro α-glucosidase inhibitory properties of compound 32 and predicted that its inhibitory action could be exerted throughout an allosteric
interaction with the enzymes [25]. Further research is in progress to assess how it is absorbed, distributed, metabolized,
and excreted.
Fig. 5 Secondary metabolites isolated from M. flavorosea.
CaM Inhibitors
Calmodulin is the major ubiquitous Ca2+-binding protein of all eukaryotes involved in a variety of physiological and pathophysiological
roles related to smooth muscle contraction/relaxation, cell motility, cytoskeleton
architecture and function, cell proliferation, apoptosis, autophagy, metabolic homeostasis,
ion channel function, phosphorylation/dephosphorylation of proteins, plant growth,
reproductive processes, and gene expression, to mention a few. CaM controls these
processes through the modulation of over 100 different proteins including enzymes
such as calmodulin-dependent phosphodiesterase (PDE1), nitric oxide synthases (NOS),
several kinases (NADK), ion channels, phosphatases, calcium-ATPase pumps, and ion
channels [26].
Many natural products, including drugs, pesticides, and research tools, interact with
CaM at different binding sites, provoking a series of conformational changes in the
protein. This, in turn, modifies the activity of the enzymes regulated by this important
protein. For the discovery of CaM inhibitors, several methods have been used, which
were described in a recent review [26]. In our investigations, we have employed functional enzymatic assays with bovine
brain CaM-sensitive cAMP phosphodiesterase (PDE1) and/or peas CaM-dependent nicotinamide
adenine dinucleotide kinase (NADK) as reporter enzymes, SDS-PAGE, and/or fluorescent
biological sensors (FBS) built with human CaM (hCaM) developed in recent years [27], [28], [29], [30], [31], [32], [33]. In some cases, in silico analyses were used to explore the potential binding site of the inhibitors. According
to literature data, the Fungi Kingdom has yielded many important CaM inhibitors. Perhaps
the ophiobolins isolated from several species of the genus Bipolaris has been the most thoroughly investigated [34]. In the course of our investigations, we have also discovered new CaM inhibitors
from selected fungi from Mexico, which are summarized in the following sections.
Plant pathogen Phoma herbarum
The genus Phoma Saccardo consists of over 2000 species found all over the world. Numerous species
of this genus are plant pathogens causing root infections and different spot/blotch
diseases in several economically important crops [35]. Among the most relevant pathogens of an important variety of corn (Zea mays L.) in Mexico (Cacahuacintle corn) is P. herbarum. This species is distributed worldwide and found in a variety of substrates, including
herbaceous and woody plants, soil, and water, and has also been reported as a pathogen
of wild oats and dandelions [36], [37]. Chemical studies of organic extracts prepared from the culture media of P. herbarum TOX-01020 led to the isolation of three new nonenolides, namely, herbarumins I – III
(34 – 36) ([Fig. 6]). The structure elucidation of the herbarumins was accomplished by chemical, spectroscopic,
and computational methods [38], [39]. These compounds possess a unique 10-membered macrolide core, which, according to
a recent review, has inspired many synthetic works all around the world [40].
Fig. 6 Secondary metabolites isolated from plant pathogen P. herbarum.
The interaction of the nonenolides with CaM was assessed using an SDS-PAGE and the
functional enzymatic assay with PDE1. In all cases, CaM treated with the lactones
had lower electrophoretic mobility than untreated CaM, and the effect was comparable
to that of chlorpromazine (CPZ), used as a positive control. The enzyme inhibition
studies were consistent with these results. As in the case of ophiobolin A, the CaM
inhibitor properties of herbarumins I – III (34 – 36) correlated well with a phytotoxic action because when tested against seedlings of
Amaranthus hypochondriacus L. using the petri dish bioassay, a significant phytogrowth inhibitory activity was
observed. This action was similar or higher than that of 2,2-dichlorophenoxyacetic
acid (2,4-D) used as a positive control. Altogether, our results could be related
with the observations of Schnick and Boland [41], who demonstrated that a combination of 2,4-D and P. herbarum produced enhanced control of dandelion. More recently, a few investigations on this
fungus isolated from different substrates allowed for the isolation of several new
bioactive compounds. Thus, a marine isolate of P. herbarum produced the polyketides arthropsadiol C and massarilactone H, which possessed moderate
neuraminidase inhibitory activity [42]. From a P. herbarum endophyte of Aegle marmelos (L.) Correa, a few new antibacterial naphthalenes were isolated [43], whereas from P. herbarum PSU-H256 from Hevea brasiliensis (Willd. ex A. Juss.) Müll.Arg. some terezine, tyrosine, and hydantoin derivatives
were characterized [44], [45].
Coprophilous Guanomyces polythrix
G. polythrix was isolated from bat guano in the Cueva del Diablo (Devilʼs cave) in Tepozotlán,
Morelos, Mexico. Since the morphological and molecular characteristics of this isolate
were unique, the new genus Guanomyces M. C. González, Hanlin & Ulloa was proposed to put up the new species [46], [47]. Bioassay-guided fractionation of an organic extract from the fermentation broth
and mycelium of G. polythrix led to the isolation of several compounds, including seven new naphthopyranone derivatives
(37 – 43) and the known compounds rubrofusarin B (44), p-hydroxybenzoic acid (45) and its methyl ester (46), a xanthene carboxylic acid methyl ester (47), emodin (48), citrinin (49), and the ergostatetraen-3-one (50) [47], [48], [49] ([Fig. 7]). According to an SDS-PAGE, compounds 37 – 50 interacted with both spinach and bovine brain CaMs, but 37 – 44 and 50 inhibited the activity of PDE1 in a concentration-dependent manner. The inhibitory
activity was higher or comparable to that of CPZ (IC50 = 10.6 µM), a well-known CaM inhibitor. Natural compounds 38 and 42 – 44 were also potent inhibitors of the complex spinach-CaM-NADK, with IC50 values of 22.0, 24.3, 13.3, and 17.1 µM, respectively. Phytotoxins 43 and 44 that possess a double bond between C-2 and C-3 were the most active CaM inhibitors.
Once more, all compounds significantly inhibited the radicle growth of A. hypochondriacus and Echinochloa crus-galli (L.) P. Beauv. in a concentration-dependent manner, and were more potent than 2,4-D
(IC50 = 1.8 × 10−4 and 8.8 × 10−4 M for A. hypochondriacus and E. crus-galli, respectively). Overall, the effect of the phytotoxins of G. polythrix with both enzyme complexes suggested that they may act as CaM antagonists in vivo, inhibiting the CaM-dependent phenomena during plant growth, although they could
also interfere with other cellular metabolic processes [47], [48], [49].
Fig. 7 Secondary metabolites isolated from coprophilous G. polythrix.
Marine-derived Emericella sp. and Aspergillus stromatoides
As of now, less than 80 marine fungi have been taxonomically described from the Pacific
Coast, Gulf of Mexico Coast, and Caribbean Coast, with just a small percentage of
them subjected to chemical studies [50]. Among them, the marine fungus Emericella sp. MEXU 25379 and A. stromatoides were investigated as a source of new CaM inhibitors.
The genus Emericella Berk (Aspergillus anamorph) produces a variety of secondary metabolites with important biological properties
like antitumor, antioxidant, antimicrobial, immunostimulant, and antiallergic, among
others [51]. Emericella sp. MEXU 25379 was isolated from the surface of the soft coral Pacifigorgia rutilia collected at Marietas Islands in Nayarit, Mexico. Chemical analyses of an organic
extract of the broth culture (Czapeck) afforded five new (51 – 55) and four known (56 – 59) prenylated xanthones ([Fig. 8]). Their structures were elucidated by spectroscopic, chemical (Mosher ester analysis),
and molecular modeling methods. Their anti-CaM properties were assessed with the PDE1
assay, SDS-PAGE, and/or FBS [31], [51]. Compounds 52 and 57 (IC50 = 5.54 and 5.62 µM, respectively) inhibited the activation of PDE1 by CaM in a concentration-dependent
manner and retarded the electrophoretic mobility of CaM. In both cases, the effects
were comparable to those of CPZ. Furthermore, kinetic analyses revealed that 52 and 57 acted as competitive antagonists of CaM, interfering with the formation of the CaM-PDE1
active complex [51]. The affinity values of compounds 52 – 59 were measured using the fluorescent biosensor hCaM L39C-mBBr/V91C-mBBr (K
d values between 3.7 and 498.4 nM) [52]. Finally, all compounds docked into CaM at site I, except for compounds 54 and 59, which bound to sites IV and III, respectively. These binding sites have been identified
as the most common binding regions for most CaM ligands, and all the predicted binding
affinities were in excellent agreement with the experimental values [31], [51].
Fig. 8 Secondary metabolites isolated from marine-derived Emericella sp. MEXU 25379 and A. stromatoides.
The fungal strain A. stromatoides was isolated from the sandy soil of Sánchez Magallanes beach, State of Tabasco, Mexico
[52]. Bioassay-guided fractionation of the organic extract from the mycelium and culture
medium of A. stromatoides led to the isolation of a series of polyketides identified as emodin (48), ω-hydroxyemodin (60), citrinin (49), 47, and coniochaetone A (61) ([Fig. 8]). The latter was unambiguously established by X-ray analysis. The affinity of compounds
48 and 60 with hCaM was determined using the fluorescent biosensor hCaM M124C-mBBr (K
d = 0.33 and 0.76 µM, respectively) and was slightly better than that of CPZ (K
d = 1.25 µM). These results were in agreement with those of the theoretical inhibition
constants obtained from docking analysis. The interactions observed in the docking
results for 48 and 60 showed that they bind in the same hydrophobic pocket as CPZ and some antitumoral
drugs. In turn, our finding might account for the cytotoxic effect of these well-known
anthraquinones whose antitumoral properties have been the subject of a few patents
[52].
Fungal endophyte MEXU 26343
MEXU 26343 was isolated from selected adult and healthy leaves of H. latiflora and tested for its CaM inhibitor properties [53]. The endophytic fungus was cultured in moist rice, and its organic extract was subjected
to chemical investigation, yielding the mixture E and Z of the known polyketide vermelhotin (62) and a new salicylic aldehyde derivative, namely, 9S,11R-(+)-ascosalitoxin (63) ([Fig. 9]). The mixture E and Z of 62 was not separable, but in the presence of TFA, the generation of a pyrylium trifluoroacetate
was favored and explained the water solubility of 62. On the other hand, the structure and absolute configuration of the new compound
63 were established through extensive NMR spectroscopy and molecular modeling calculations
at the DFT B3LYP/DGDZVP level, which included the comparison between theoretical and
experimental optical rotation values. In addition, chemical transformations of 63 yielded suitable derivatives for NOESY and 1H-1H NMR coupling constant analyses, which reinforces the stereochemical assignment [53].
Fig. 9 Secondary metabolites isolated from endophyte MEXU 26343.
The potential affinity of 62 and 63 with CaM was established using the fluorescent biosensor hCaM M124C-mBBr, but only compound 62 bound to the protein with a K
d value similar to that of the classical CaM inhibitor CPZ (K
d = 0.25 µM for 62, and 0.64 µM for CPZ). Docking analysis of the four possible conformers of 62 showed that all bound to CaM at site I, as did trifluoperazine, with affinities consistent
with the results from the fluorescence quenching experiments using the hCaM M124C-mBBr biosensor [53]. These findings suggested that compound 62 might be useful as a tool for studying the role of CaM in several physiological and
pathophysiological processes.
Entomopathogen Isaria fumosorosea
The entomopathogen I. fumosorosea was isolated from the whitefly B. tabaci Gennadius. Because of its wide arthropod host range, this species has been used for
the biological control of several economically important insect pests of agricultural
crops [54]. The total extract of I. fumosorosea showed significant anti-CaM activity using the CaM biosensor hCaM M124C-AF
350. Beauverolides C (64), F (65), I (66), Ja (67), L (68), M (69), and N (70) were identified as the active constituents of the extract ([Fig. 10]). This group of lipophilic and neutral cyclotetradepsipeptides, containing linear
and branched C9- or C11-β-hydroxy acid residues, has been previously reported in the genera Beauveria and Paecilomyces
[55]. All isolated cyclotetradepsipeptides interacted with CaM with affinities ranging
from 0.078 to 3.44 µM. The most active compound, beauverolide Ja (67), was almost tenfold more active than CPZ and contains a tryptophan moiety in its
structure that could be related to its highest affinity. Docking analyses of compounds
64 – 70 into CaM revealed that, in all cases, they bind in the same pocket of CPZ and showed
mainly hydrophobic interactions to the protein [55].
Fig. 10 Secondary metabolites isolated from entomopathogen I. fumosorosea.
Saprophyte Purpureocillium lilacinum
P. lilacinum was isolated from soil collected in a cave in Juxtlahuaca, State of Guerrero, Mexico,
in 1998. This species is an emergent pathogen that causes severe human infections,
but is also used as an important biological control agent against nematodes [56]. Bioassay-guided fractionation of an extract prepared from the culture medium and
mycelium of P. lilacinum yielded two CaM inhibitors, namely, acremoxanthone C (71) and acremonidin A (72) ([Fig. 11]). Both compounds showed an important affinity (70-fold higher than CPZ) for CaM
using the fluorescent biosensor hCaM M124C-mBBr. Interestingly, this level of affinity to CaM is unusual, since most CaM antagonists
bind to the protein with much lower affinity, usually in the micromolar range [56]. Based on the observed extraordinary affinity to CaM, we completed the structural
characterization of 71 using molecular modeling calculations, which included the comparison between theoretical
and experimental specific rotation, 3
J
C,H, and 3
J
H,H values. Docking analysis predicted that compounds 71 and 72 bind to CaM at a similar site to KAR-2, which is unusual. This prediction was experimentally
supported since 71 quenched the fluorescence of hCaM T110C-mBBr, a biosensor specially designed to target nonclassical inhibitors of CaM [56].
Fig. 11 Secondary metabolites isolated from saprophyte P. lilacinum.
Coprophilous Malbranchea aurantiaca
The genus Malbranchea Saccardo comprises around 30 species distributed worldwide and mainly isolated from
decaying vegetation, soil, and animal dung (http://www.indexfungorum.org). M. aurantiaca was obtained from bat guano collected at the Juxtlahuaca cave located in Ramal del
Infierno, State of Guerrero, Mexico. The mycelial and culture broth organic extract
of this fungus yielded an eremophilane derivative (73), penicillic acid (74), and four rare indole alkaloids possessing an unusual bicyclo [2.2.2] diazaoctane
ring system, namely, malbrancheamide (75) and malbrancheamide B (76), isomalbrancheamide B (77), and premalbrancheamide (78) [57], [58], [59] ([Fig. 12]).
Fig. 12 Secondary metabolites isolated from coprophilous M. aurantiaca.
Compounds 75 – 78 have been fully characterized as CaM ligands by NMR, fluorescence, circular dichroism,
computational, and enzymatic functional methods. Of the alkaloids with an hCaM M124C-mBBr biosensor, only 75 quenched significantly (K
d = 1.1 µM). The monochlorinated derivatives (76 and 77) provoked only limited decreases in fluorescence quenching, and 78 provoked none. Thus, the presence of two chlorine atoms confers 75 to have the best affinity to hCaM. Docking analysis predicted that 75 binds in the hydrophobic pocket of CaM through HB and hydrophobic interactions with
a few methionine (Met) residues of the protein. Indeed, NMR experiments confirmed
these results, since in solution, 75 induced a diamagnetic shift of most of the Met-methyl resonances of the protein,
in particular, to those of Met residues 36, 51, 71, 72, 76, 109, 124, 144, and 145
[27].
As for other CaM inhibitors, 75 – 77 induced significant vasorelaxant activity in an endothelium intact model in rat aorta
rings, and a lesser effect in an endothelium denuded model. Compound 75 was the most active (EC50 = 2.7 µM) with a maximum effect of almost 100%, which is unusual for a natural product;
the involvement of the nitric oxide-cGMP pathway was experimentally demonstrated.
Furthermore, docking studies carried out with two crystallized eNOS domains, namely, oxygenase (PDB code 3NOS) and a CaM-binding peptide bound to
CaM (PDB code 1NIW), predicted that the endothelium-independent relaxation exerted
by 75 could be mediated by its CaM inhibitory properties throughout a positive modulation
of eNOS. In addition, in a fluorescent-based experiment using the hCaM M124C-mBBr biosensor, it was demonstrated that 75 (K
d = 0.55 µM) induced a perturbation of the Ca2+-hCaM-MLCK complex, which could also account for its vasorelaxant effect [60].
Other Activities
Marine-facultative Aspergillus sp.
The marine-facultative Aspergillus sp. MEXU 27854 was isolated from sandy soil collected in the intertidal zone located
in Caleta Bay, Acapulco, Guerrero, Mexico [61]. The intertidal regions are dynamic environments that support an immense biodiversity.
In these ecosystems, fungi play important roles in the process of decomposition and
mineralization of organic matter [62]. From the organic extract of a rice-based culture of Aspergillus sp. MEXU 27854, a series of dioxomorpholine derivatives (79 – 84) were isolated ([Fig. 13]). An extensive literature search revealed that the morpholine-2,5-dione ring system
is rare in nature and mainly biosynthesized by fungi. Ergosecalinine, the first reported
dioxomorpholine, was isolated in 1959 from Claviceps purpurea (Fr.) Tul. [63], the immunomodulator metacytofilin was obtained from the fungus Metarhizium sp. TA2759 [64], the compound lateritin, isolated from Gibberella lateritium W. C. Snyder & H. N. Hansen in 1993, was recently revised to beauvericin by extensive
NMR and MS analyses [65], the cyclodidepsipeptides isolated as a mixture from the fungus Fusarium sporotrichioides Sherb., were identified by comparison with the synthetic series, and their stereostructure
was studied by quantum chemical calculations [66], and, finally, PF1233 B was isolated from Aspergillus niveus Blochwitz in 2003 as a novel inhibitor of voltage-dependent sodium channels, and
reported again as a new natural product with P-glycoprotein (Pgp) inhibitor properties
in 2014 [67], [68], [69].
Fig. 13 Secondary metabolites isolated from marine-facultative Aspergillus sp. MEXU 27854.
The structures of 79 – 84 were established by 1D and 2D NMR and HRESIMS data analysis, and the absolute configuration
of 79 and 80 was elucidated by comparison of experimental and DFT calculated vibrational circular
dichroism spectra. Finally, the cytotoxic properties of derivatives 81, 83, and 84 were determined using a panel of human cancer cell lines with different functional
status for p53 and against a cell line (A549/doxorubicin) that overexpresses Pgp and
presents developed resistance to doxorubicin [61].
Agrochemicals
Like pharmaceuticals, many pesticides are based on fungal natural products; these
could belong to the category of biopesticides or biochemical pesticides. These agents
have become attractive in recent years because the novelty of their structures offers
the possibility of finding compounds with new modes of action, thus diminishing the
induction of resistance [70]. Among the most relevant pesticides developed from fungal natural products are the
strobilurins derivatives. The natural strobilurins were originally found in several
fungi, including Strobilurus tenacellus (Pers.) Singer, Xerula spp., and Cyphellopsis anomala (Pers.) Donk. These types of compounds inhibit the electron transfer in mitochondrial
respiration by binding to the ubiquinol site of cytochrome b. In the case of biopesticides, a few formulations with Colletotrichum gloeosporioides f.sp. (Penz.) Penz. & Sacc. (BioMal and Collego), Phoma macrostoma Montagne (Ecosense), which has been developed and applied successfully for weed control,
and Myrothecium verrucaria (Alb. & Schwein.) Ditmar (DiTera) have been made against plant parasitic nematodes.
Recently, a few reviews have been published emphasizing the urgent need for new pesticides
with safer toxicological and environmental profiles and new modes of action [70], [71]. With these considerations, we have investigated several fungi from Mexico in order
to discover new leads for the development of biochemical pesticides, in particular,
for weed control affecting different molecular targets.
Endophytes from Plants Collected in El Eden Ecological Reserve, Quintana Roo, Mexico
El Eden Ecological Reserve is a natural protected area situated in the State of Quintana
Roo, Mexico, and is part of the Mesoamerican tropical rain forest. Nowadays, many
multidisciplinary research projects take place in the region, with fungal biodiversity
monitoring and bioprospection projects being two of them [72]. In this context, E. gomezpompae was isolated from surface sterilized leaves of the medicinal plant C. acuminata
[73]. A chemical investigation of the mycelium of E. gomezpompae resulted in the isolation of eight naphthoquinone-spiroketals, namely, preussomerins
EG1 – EG4 (85 – 88), palmarumycins EG1 (89), CP2 (90), CP17 (91), and CP19 (92) as well as 50
[74], [75] ([Fig. 14]).
Fig. 14 Secondary metabolites isolated from endophyte E. gomezpompae.
Compounds 85 – 87 and the acetyl derivatives 85a and 85b showed significant antifungal and anti-oomycete activities against four economically
important phytopathogens, Phythophtora capsici Leonian, Phythophtora parasitica Dastur, Fusarium oxysporum Schltdl., and Alternaria solani (Cooke) Wint. Their IC50 values ranged between 47.8 and 313.0 µg/mL. Preussomerin EG1 (85) caused complete growth inhibition of P. parasitica, F. oxysporum, and P. capsici. In the case of P. capsici, 85 (IC50 = 5.61 × 10−5 M) was more active than the commercial fungicide Captan (IC50 = 1.53 × 10−4 M). A. solani was only affected by 86 and 87. Compounds 85 – 88 inhibited the germination of A. hypochondriacus, Solanum lycopersicum L., and E. crus-galli, being more potent than the positive control Rival. Compound 86 significantly inhibited (> 50%) root elongation of the three species tested, but
87 and 92 affected (> 50%) both A. hypochondriacus and E. crus-galli. Compound 86 inhibited respiration of all seedlings tested, but 88 and 89 of S. lycopersicum and E. crus-galli. Furthermore, oxygen consumption in intact mitochondria was significantly inhibited
by compounds 86 – 88 and 90 – 93, but the effect was lower than that of 2,4-dinitrophenol [74]. The study of compounds 85, 88, 90, and 91 in a series of photosynthesis light reactions in freshly lysed spinach thylakoids
(ATP synthesis, electron transport rate basal, phosphorylating, uncoupled and partial
reactions of the photosystems I and II, and chlorophyll a fluorescence of the photosystem II) revealed that they are Hillʼs reaction inhibitors
[76]. They also interacted at the acceptor site of the photosystem II in a similar way
as the commercial herbicide Diuron, as demonstrated by chlorophyll a fluorescence experiments. Consequently, these compounds could be considered promising
leads for developing new herbicides for utilization in modern agriculture.
Muscodor yucatanenis MEXU 25511 [77], a new fungal species, and A. camptosporum MEXU 26354 [78] were isolated from the leaves of B. simaruba, which is a frequent codominant medicinal tree of the Yucatan Peninsula; the Mayan
communities use this plant as an analgesic, antimycotic, and anti-inflammatory agent.
So far, all Muscodor species are endophytes that produce mixtures of volatile organic compounds (VOCs).
These mixtures are useful as pesticides in living environments since they are readily
converted to a gaseous phase at room temperature due to their low vapor pressure [79]. The composition and relative abundance of VOCs produced by microorganisms can be
established using headspace solid-phase microextraction.
The VOCs of M. yucatanensis were lethal to the endophytes Colletotrichum sp., Phomopsis sp., and Guignardia mangiferae, A. J. Roy, and to the phytopathogens P. capsici, P. parasitica, Rhizoctonia sp., and A. solani. Root elongation of A. hypochondriacus, S. lycopersicum, and E. crus-galli was also inhibited by the volatile mixture. M. yucatanensis’ VOCs included 38 compounds that were identified by GC-MS, mainly, alcohols, acids,
esters, ketones, naphthalene derivatives, benzene derivatives, terpenoids, and aliphatic
hydrocarbons. The organic extracts from the culture medium and mycelium of M. yucatanensis were also active against the same endophytes, phytopathogens, and plants tested.
In the case of A. hypochondriacus, the culture medium extract (IC50 = 194.3 µg/mL) was more active than the commercial herbicide Rival (IC50 = 234.42 µg/mL). According to GC-MS, the extracts and VOCs shared 12 compounds, including
benzene derivatives, phenolic compounds, cyclopentadienes, esters, lactones, alkanes,
aldehydes, and carboxylic acids [80]. Recently, Qadri and coworkers [81] isolated a new strain of this fungus (Ni30) from a leaf of Elleanthus sp. at the Reserva Natural Punta Gorda, Nicaragua. The chemical composition of the
volatile mixture of Ni30 was similar to those described by Macías-Rubalcava and coworkers
[80]. The Ni30 strain also biosynthesized the macrolactone brefeldin A.
The genus Acremonium Link contains more than 200 species, which could be saprophytic, pathogenic, or endophytic.
This genus is known for producing many interesting bioactive secondary metabolites,
like the β-lactam antibiotics cephalosporins, the tremorgenic indole-diterpene alkaloids lolitrems,
sesquiterpenoids, diterpenoids and triterpenoids with different skeletons, ophiobiolin
D, a few meroterpenoids, a variety of polyketides, and peptides [81]. Bioassay-guided fractionation of the mycelial extract from A. camptosporum MEXU 26354 led to the isolation of six heterodimeric polyketides, acremoxanthone
E (93), acremoxanthone A – C (94, 95, and 71), and acremonidins A (72) and B (96) [82] ([Fig. 15]). All compounds showed anti-oomycete activity against Pythium aphanidermatum Edson, Phytophthora cinnamomi Rands, P. capsici, and P. parasitica, reducing the diameter growth of most target oomycetes in a concentration-dependent
manner, with IC50 values ranging between 6 – 38 µM, and the activity was comparable to the commercial
fungicide Ridomil Gold 4E. However, in the case of P. cinnamomi, polyketides 93, 94, and 71 were more active than the commercial fungicide. Unusually, this fungus biosynthesizes
three different types of heterodimeric polyketides linked by a bicyclo[3.2.2]nonene:
xanthoquinodins, acremonidins, and acremoxanthones.
Fig. 15 Secondary metabolites isolated from endophyte A. camptosporum MEXU 26354.
Endophytes from Plants Collected in Sierra de Huautla Biosphere Reserve
Endophytes from Plants Collected in Sierra de Huautla Biosphere Reserve
Xylaria feejeensis SM3e-1b
The Sierra de Huautla Biosphere Reserve (REBIOSH), recognized by the MAB-UNESCO program
and included in the world net of biosphere reserves, is located in Quilamula at Tlalquitenango,
Morelos, Mexico. This area represents the largest natural protected area devoted to
the conservation of a tropical dry forest in Central Mexico, and is an important reservoir
of endemic species, including fungal endophytes (www.conanp.gob.mx).
From symptomless healthy leaves of the tree S. macrocarpum, the strain X. feejeensis SM3e-1b was isolated [83]. Bioassay-guided fractionation of the extracts from the culture medium and mycelium
led to the isolation of coriloxine (17) and two quinone derivatives, 18 and fumiquinone B (97). In addition, four semisynthetic compounds from 17 were prepared (17a – 17d) ([Fig. 16]). All compounds showed a phytotoxic effect in a concentration-dependent manner on
germination, elongation of the root, and oxygen uptake (respiration) of seedlings
from Trifolium pratense L., Medicago sativa L., A. hypochondriacus, and Panicum miliaceum L. In general, the IC50 values for all these activities ranged between 0.1 – 1.1 mM, which were comparable
to the values obtained for the commercial herbicides Rival and ExterPro [83]. Of all the compounds, only 17a and 17d inhibited ATP synthesis, basal and uncoupled electron transport, and enhanced the
phosphorylating electron transport and Mg2+-ATPase enzymatic activity when tested in a series of photosynthetic light reactions
in freshly lysed spinach chloroplasts. Overall, these results indicated that 17a acted as an uncoupler and a weak Hillʼs reaction inhibitor, whereas 17d acted as a Hillʼs reaction inhibitor at the photosystem II [84]. Thus, coriloxine derivatives represented new leads for the development of novel
herbicides.
Fig. 16 Secondary metabolites isolated from endophyte X. feejeensis SM3e-1b.
Xylaria sp. PB3f3
The endophyte Xylaria sp. PB3f3 was isolated from leaves of H. brasiletto collected at the REBIOSH. Based on phylogenetic studies, the genera Muscodor and Xylaria are anamorphs. In a multiple antagonism bioassay during 40 days, the VOCs produced
by Xylaria sp. PB3f3 significantly inhibited the radial growth of two oomycetes, P. aphanidermatum (78.3%) and P. capsici (48.3%), and two fungi, A. solani (24.5%), and F. oxysporum (24.2%) [85]. Furthermore, Xylaria sp. PB3f3, in simple direct antagonism bioassays, produced a significant inhibitory
effect at distance of 90% over the growing of the four plant pathogens. On the other
hand, the VOCs from 30- and 20-day cultures significantly inhibited the root growth
of A. hypochondriacus (27.6%) and S. lycopersicum (53.2%), respectively. From the mixtures obtained at days 10 and 30 of culturing,
2-methyl-1-butanol and 2-methyl-1-propanol were identified as the most abundant components.
Both compounds strongly inhibited root growth and respiration in the seedlings of
A. hypochondriacus and S. lycopersicum, with IC50 values ranging between 4.6 – 48.2 µg/mL and 23.2 – 130.0 µM, respectively [85].
Nodulisporium and Hypoxylon
The genus Hypoxylon and its anamorph Nodulisporium are well-known producers of antimicrobial VOCs [86]. From healthy leaves of G. sepium collected in the REBIOSH, the endophyte Nodulisporium sp. GS4d2II1a was isolated. VOCs and diffusible metabolites of Nodulisporium sp. showed anti-oomycete (P. aphanidermatum, P. capsici, Phythophtora palmivora, P. cinnamomi, P. parasitica,
Pythium ultimum, and Pythium polytylum) and antifungal (A. solani and F. oxysporum) activities when evaluated using three types of antagonism bioassays, and monitored
at different ages of the fungus (3 to 6 days of growth culture) and interaction conditions.
The growth of the oomycetes and fungi was inhibited in all cases, and, as visually
observed in the petri dish, their mycelia were partially or completely replaced. In
addition, VOCs inhibited the growth of three oomycetes (P. parasitica, 29.7%; P. cinnamomi, 18.1%; and P. capsici, 25.7%) and one fungus (F. oxysporum, 21.6%) after 3 days of antagonism using divided plates. VOCs were not active after
5 and 7 days of the fungal culture. Finally, the VOCs resulting from Nodulisporium sp. grown for 3 and 5 days in PDA at 28 °C, and from the interaction of Nodulisporium sp. with P. aphanidermatum and Nodulisporium sp. with Nodulisporium sp., were analyzed by GC-MS (70 VOCs detected). The results revealed four different
volatile profiles, however, the main metabolites were terpenes, which represent over
60% of the total, with eucalyptol, limonene, and ocimene being among the most predominant.
The remaining compounds were benzene derivatives, esters, ketones, alcohols, and carboxylic
acids, including 2-methyl-1-butanol, and 3-methyl-1-butanol as the most relevant [86].
Hypoxylon anthochroum
The endophyte H. anthochroum Blaci was isolated from leaves of Bursera lancifolia collected at REBIOSH [87]. The VOCs produced by this endophyte exhibited a significant phytotoxic effect on
seed germination, root elongation, and seedling respiration of A. hypochondriacus, P. miliaceum, T. pratense, and M. sativa, the majority with inhibition values over 40%. Furthermore, the VOCs slightly affected
the growth of the plant pathogenic fungi A. solani and F. oxysporum, and the oomycetes P. ultimum and P. capsici. Once more, eucalyptol was the most predominant compound in the VOCs analyzed. On
the other hand, the culture medium and mycelium extracts of H. anthochroum showed a high phytotoxic activity on the four test plants. From these, phenylethyl
alcohol, 2-methyl-1-butanol, eucalyptol, and terpinolene were the most predominant
metabolites. Pure phenylethyl alcohol and eucalyptol were also evaluated in the same
vegetal processes, displaying IC50 values between 21.4 µg/mL and > 500.0 µg/mL, and 11.6 µg/mL and > 500.0 µg/mL, respectively.
The IC50 values of the positive control Rival were found between 14.8 µg/mL and > 500.0 µg/mL
[87].
Recently, six pure compounds (phenylethyl alcohol, 2-methyl-1-butanol, 3-methyl-1-butanol,
eucalyptol, ocimene, and terpinolene) present in the VOCs of Nodulisporium sp. GS4d2II1 and H. anthochroum were evaluated in vivo and in vitro against F. oxysporum
[88]. In vivo studies were performed inoculating the pathogen into cherry tomatoes. All compounds,
and particularly the mixture of the six, significantly inhibited the growth of the
mycelium on the wounds of infected tomatoes in a concentration-dependent manner. The
in vitro assay, using agar dilution and gas phase methods, revealed that the VOCs mixture
significantly affected the respiration and cell membrane permeability of the target
fungus. The damage rate caused by individual compounds and the VOCsʼ mixtures on the
cell membrane of F. oxysporum was evaluated through the measurement of electrolytes leak (% relative conductivity).
After 8 h of treatment, the best effect was observed with 2-methyl-1-butanol (45.4%),
phenylethyl alcohol (43.1%), and the VOCsʼ mixture (42.9%). According to a microscopic
analysis of mycelia fresh preparations, the pure compounds and the six compoundsʼ
mixture at 1000 µg/mL induced important ultrastructure morphological changes in F. oxysporum hyphae. Ocimene and the mixture induced a vacuolization process and thickening and
thinning on the middle and the tips, respectively. Finally, an inhibitory effect on
the respiration were observed with all treatments, which could be attributed to the
proliferation of vacuoles and the severe vacuolization process provoked by the rupture
of hyphae [88]. VOCsʼ mixture showed the greatest oxygen consumption inhibition (97.1%) after 8 h
incubation (IC50 = 183.9 µg/mL). These results revealed that VOCs are promising candidates for the
development of biopesticides, having great potential for tomatoes postharvest treatment
against diseases caused by F. oxysporum.
Coprophilous Penicillium sp. G1-a14
Penicillium sp. G1-a14 was also isolated from bat dung obtained in the Chontalcoatlán cave in
the State of Guerrero [89]. This fungus was selected for bioassay-directed fractionation on the basis of its
phytogrowth inhibitory activity against seedlings of A. hypochondriacus and E. crus-galli (IC50 = 46.2 and 184.7 µg/mL, respectively). Extensive chromatography of the active extract
led to the isolation of a new eremophilane sesquiterpene (98), along with three known analogues, namely, isopetasol (99), sporogen AO-1 (100), and dihydrosporogen AO-1 (101) ([Fig. 17]). Only compounds 100 and 101 revealed a concentration-dependent inhibition on radicle elongation against A. hypochondriacus (IC50 = 0.17 mM for both compounds) and E. crus-galli (IC50 = 0.17 and 0.30 mM, respectively) with IC50 values similar to tricolorin A, used as a positive control [89]. The eremophilane-type sesquiterpenoid is common in Penicillium, Aspergillus, and Xylaria species, and a few of them have also shown important phytotoxic activity.
Fig. 17 Secondary metabolites isolated from coprophilous Penicillium sp. G1-a14.
Concluding Remarks and Outlook
Concluding Remarks and Outlook
In the last 20 years, more than 100 secondary metabolites from various classes have
been identified from fungi from different substrates throughout Mexico. Some of these
natural products possessed new scaffolds and have attracted the attention of synthetic
chemists, such as in the case of herbarumins I – III (34 – 36) and malbrancheamides (75 – 78). Most of these fungal compounds displayed diverse bioactivities of medicinal and/or
agrochemical interest. Thus, a few showed potential as antidiabetic drugs with α-glucosidase inhibitory activity. Preclinical pharmacological evidence indicated that
most of them were more active than acarbose, and some, like benzomalvin A (25), also possess antihyperalgesic properties of utility for treating diabetic neuropathies.
Another, malbrancheamide (75), is a vasorelaxant compound with a maximum effect of almost 100%, which is unusual
for a natural product. The effect is mediated by CaM inhibitory properties throughout
a positive modulation of eNOS and a perturbation of the Ca2+-hCaM-MLCK complex. A series of dioxomorpholine derivatives (81, 83, and 84) were cytotoxic in a panel of human cancer cell lines, with a different functional
status for p53, and against a cell line (A549/doxorubicin) that overexpressed Pgp
and developed resistance to doxorubicin. A few were CaM inhibitors, valuable for the
study of CaM-mediated physiological processes or pharmacological properties in mammals
and plants. Among the isolates, the prenylated xanthones 52 and 57 and acremoxanthone C (71) were the best CaM inhibitors. In the agrochemical area, many active antifungal,
anti-oomycetes, and phytotoxic agents more active than commercial products were discovered.
The VOCs of several species, including those from the new species M. yucatanensis, showed promise as pest control agents. In some cases, their mode of action was also
established. A few new fungal species were discovered, and a new genus, Guanomyces, was created to accommodate G. polythrix. The fact that Mexican fungi are still understudied shows that many more new scaffolds
and several drug and agrochemical leads are likely to be discovered in the future.
Thus, bioprospecting for structurally unique and bioactive metabolites from Mexican
fungi has great potential for the discovery of new drug candidates in various applications
in clinics and the agrochemical area.
