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Abstract

Fungi in the genus of Tolypocladium are often discovered as endophytes, soil inhabitants, and insect pathogens. Many biologically active secondary metabolites have been discovered from them in the past half-century, including medicinally important oligopeptide cyclosporine A, and other compounds like terpenes, polyketides, polyketide-non-ribosomal peptides. This review has summarized the chemistry and pharmacology of natural products discovered from Tolypocladium genus fungi, to provide a full record of all Tolypocladium secondary metabolites.

Keywords

cyclosporin meroterpenes bioactivity cyclic oligopeptides nonribosomal pepetides

Introduction

The genus Tolypocladium was first established in 1970s, when 3 species of fungi were isolated from soil samples by Grams,¹ and was synonymized with Chaunopycnis and Elaphocordyceps by genetic phylogenetic analyses.² Distribution of this genus is wide, despite the original discovery from soil, most members in this genus are discovered as endophytic fungi, and they were possibly favored by plants for their abilities of producing insecticidal substances.

Fungi of this genus are medicinally and biologically important for their secondary metabolites (SMs), many of which are pharmacologically potent. Cyclosporin A, an oligopeptide discovered from T. inflatum, has been developed as a good immunosuppressant. Thus, metabolites in Tolypocladium sp. are worth exploring for their structural diversity and biological significance. Terpenes, polyketides (PKs) and PK-non-ribosomal peptides (PK-NRPs) and peptides including cyclosporines, have been discovered from fungus of Tolypocladium sp. Currently, there are review articles on taxonomy of Tolypocladium and research on cyclosporin A,^3–5 but chemical and pharmacological properties of Tolypocladium SMs have never been summarized in full details. Thus, in this review, we have retrieved all papers on the chemistry and bioactivities of SMs isolated from fungi of Tolypocladium genus, and summarized structures and pharmacology of all the compounds, in order to show what we already discovered from this genus, and what to expect from it.

Secondary Metabolites

In the past half century, approximately 100 new Tolypocladium SMs have been found, they are mostly meroterpenoids, oligopeptides, PK-NRP and so forth. In this section, we summarized all the compounds isolated from Tolypocladium spp., and they are listed according to their structure types.

Terpenes and Meroterpenoids

Most commonly found terpenes in Tolypocladium sp. are diterpenes and terpenoid indole alkaloids. In 2011, Lin et al⁶ first obtained 4 diterpenes from T inflatum and named them as aphidicolins (1-4, Figure 1, Table 1). Compounds 1 and 2 are to some degree toxic to 8 human cancer cell lines (see Table S1), IC₅₀ of 2 to Hep3B-2 was 7.3 μM. Nogawa et al⁷ discovered a compound named metarhizin C (5) which showed IC₅₀ up to 0.79 μg/mL against MG-63 human osteosarcoma cells in a glucose-free medium and > 30 μg/mL in normal medium. Xu et al^6,8 discovered 12 new terpenoid indole alkaloids, tolypocladin A-L (6-17) and a known compound terpendole L (18). These compounds are suspected to be originated from tryptophan. Tolypocladin A (6) showed significant antifungal effect against 6 strains of plant pathogens (Alternaria fragariae, Corynespora cassiicola, Alternaria alternata, Botrytis cinereal Pers., Cercospora personata, Verticillium dahlia Kleb, and Sclerotinia sclerotiorum), all MICs were below 25 µg/mL, also it was toxic to multiple cancer cell lines and patient-derived tumor cells (Table S1). Tolypocladin H (13) showed MICs below 25 µg/mL against all tested bacteria (Bacillus cereus, methicillin-resistant Staphylococcus aureus, Micrococcus lysodeikticus, B paratyphosum, B subtilis, Enterobacter aerogenes, Salmonella typhi, and Proteus vulgaris). Terpendole L (18) inhibited Micrococcus luteus with MIC value of 6.25 µg/mL. Lin et al⁹ reported isolation of inflatin G (19), a diterpene structurally similar to aphidicolins. Terpendole E (20) was discovered by Motoyama et al¹⁰ from Chaunopcnis alba and it was a kinesin Eg5 inhibitor. On the basis of gene knock-out experiments, they proposed that terpendole E was a biosynthetic intermediate of indole-diterpene.

Figure 1.

Terpenes and meroterpenes isolated from fungi of the Tolypocladium genus.

Table 1.

Terpenes and Meroterpenoids and Their Origins.

No.	Name	Origins	References
1	Inflatin A	T inflatum	⁶
2	Inflatin B	T inflatum	⁶
3	Aphidicolin	T inflatum	⁶
4	Aphidicolin-17-monoacetate	T inflatum	⁶
5	Metarhizin C	T album RK17-F0007	⁷
6	Tolypocladin A	T. sp. XL115	⁶
7	Tolypocladin B	T. sp. XL115	⁶
8	Tolypocladin C	T. sp. XL115	⁶
9	Tolypocladin D	T. sp. XL115	⁶
10	Tolypocladin E	T. sp. XL115	⁶
11	Tolypocladin F	T. sp. XL115	⁶
12	Tolypocladin G	T. sp. XL115	⁶
13	Tolypocladin H	T. sp. XL115	⁶
14	Tolypocladin I	T. sp. XL115	⁶
15	Tolypocladin J	T. sp. XL115	⁶
16	Tolypocladin K	T. sp. XL115	⁸
17	Tolypocladin L	T. sp. XL115	⁸
18	Terpendole L	T. sp. XL115	⁸
19	Inflatin G	T inflatum	⁹
20	Terpendole E	Chaunopycnis alba	¹⁰

Polyketides

A quite diversified collection of PKs can be produced by species of Tolypocladium, including pyrones, benzophenones, coumarins, etc, though mostly not active in bio-assays (Table 2 and Table S1). Structures and tested activities are illustrated in Figure 2 (Table 2 and Table S1).

Figure 2.

Polyketides isolated from fungi of the Tolypocladium genus.

Table 2.

Polyketides Discovered from Fungi in Tolypocladium Genus.

No.	Name	Origins	References
21	Tolypocladin	T inflatum DSM 915	¹¹
22	Inflatin C	T inflatum	⁶
23	Inflatin D	T inflatum	⁶
24	Inflatin E	T inflatum	⁶
25	Inflatin F	T inflatum	⁶
26	Isochlamydosporol	T inflatum	⁶
27	Cylindromicin	T. sp. SCSIO 40433	¹²
28	Epi-citrinin H1	T. sp. SCSIO 40433	¹²
29	Dicitrinin A	T. sp. SCSIO 40433	¹²
30	Citreorosein	T. sp. SCSIO 40433	¹²
31	Phenol A	T. sp. SCSIO 40433	¹²
32	Phenol A acid	T. sp. SCSIO 40433	¹²
33	tolypocladone A	T. sp. (4259a)	¹³
34	Tolypocladone B	T. sp. (4259a)	¹³
35	Glycine, n-(2,3-dihydroxybenzoyl)-methyl ester	T. sp. (4259a)	¹³
36	2h-pyran-2-one, 4-methyoxy-6-(1,3-pentadienyl)	T. sp. (4259a)	¹³
37	Gulypyrone A	T inflatum	⁹
38	3,8-dihydroxy-4-(4-hydroxyphenyl)-6-methylcoumarin	T cylindrosporum	¹⁴
39	Malettinin A	T. sp. Sup5-1 (T2)	¹⁵
40	Malettinin B	T. sp. Sup5-1 (T2)	¹⁵
41	Malettinin F	T. sp. Sup5-1 (T2)	¹⁵
42	Malettinin G	T. sp. Sup5-1 (T2)	¹⁵
43	Malettinin H	T. sp. Sup5-1 (T2)	¹⁵

Aza-anthraquinones are not commonly discovered as natural products. Tolypocladin (21), an aza-anthraquinone was discovered in 1990 from T inflatum.¹¹ The authors suspected that it was a hydrogen acceptor for maintaining normal physiological functions of T inflatum during oxygen limitation. Lin et al⁶ discovered 5 chlamydosporol derivatives from T inflatum, namely inflatins C-F (22-25) and isochlamydosporol (26). Inflatin C was a chlamydosporol monomer while the latter 3 were dimers, all of which were inactive in either antimicrobial or cytotoxic assays. In the research of Khan et al,¹² 2 phenols, 1 pyrone, 2 benzophenones and anthraquinones (27-32) were isolated from T cylindrosporum, whereas Hu et al¹³ also obtained 2 new pyrones (33 and 34) and 2 known compounds (35 and 36) from Tolypocladium sp. (4259a). The discovery of Du et al¹⁵ indicated that malettinins (39-43), a family of antibacterial PKs originally isolated from Cladosporium sp.,¹⁶ were also produced by Tolypocladium.

Polyketide-Non-Ribosomal Peptides

Tolypocladium-originated PK-NRPs are mostly tyrosine or alanine-derived pyridones and pyrrolidones, the latter is often referred to as tetramic acids. Tolypoalbin (44) discovered from Tolypocladium Album TAMA 479 can induce cellular differentiation of ST-31 preadipocytes to mature adipycytes which produced adiponectin.¹⁷ The authors suspected that the effect could be related to PPARγ activation, and a reporter gene assay was conducted to show that 44 can activate PPARγ. A similar effect was not shown by the other derivative, F-14329 (45). Iqalisetins A and B (46 and 47) were 2 tetramic acids isolated by Grunwald et al¹⁸ from a Tolypocladium sp., an arctic marine sediment-originated fungal species. Interestingly, judging from the structures, only iqalisetins A and B were derived from alanine-lovastatin whereas most PK-NRPs isolated from Tolypocladium spp. were biosynthetically originated from tyrosine and methylated fatty acids.

Li et al¹⁴ discovered 6 PK-NRPs (48-53, Figure 3 and Table 3) from an endolichen fungus strain T cylindrosporum which inhabits Lethariella zahlbruckneri. Pyridoxatin (52) was significantly toxic to 4 human cancer cell lines (MDA-MB-231, A549, A2780, and K562, Table S1). Furthermore, 52 can cause cell cycle arrest at G0/G1 phase in A549 cells.

Figure 3.

Polyketide-non-ribosomal peptides isolated from fungi of the Tolypocladium genus.

Table 3.

Polyketide-Non-Ribosomal Peptides Discovered from Fungi in Tolypocladium Genus.

No.	Name	Origins	Reference
44	Tolypoalbin	T album TAMA 479	¹⁷
45	F-14329	T album TAMA 479	¹⁷
46	Iqalisetin A	T. sp. (RKAG373)	¹⁸
47	Iqalisetin B	T. sp. (RKAG373)	¹⁸
48	Tolypocladenol A₁	T cylindrosporum	¹⁴
49	Tolypocladenol A₂	T cylindrosporum	¹⁴
50	Tolypocladenol B	T cylindrosporum	¹⁴
51	Tolypyridone A	T cylindrosporum	¹⁴
52	Pyridoxatin	T cylindrosporum	¹⁴
53	Tolypyridone B	T cylindrosporum	¹⁴
54	Tolypyridone C	T. sp. 49Y*	¹⁹
55	Tolypyridone D	T. sp. 49Y*	¹⁹
56	Tolypyridone E	T. sp. 49Y	¹⁹
57	Tolypyridone F	T. sp. 49Y	¹⁹
58	Tolypyridone G	T. sp. 49Y	¹⁹
59	Tolypyridone H	T. sp. 49Y	¹⁹
60	Chaunolidine A	Chaunopycnis sp. (CMB-MF028)	²⁰
61	Chaunolidine B	Chaunopycnis sp. (CMB-MF028)	²⁰
62	Chaunolidine C	Chaunopycnis sp. (CMB-MF028)	²⁰
63	Chaunolidone A	Chaunopycnis sp. (CMB-MF028)	²⁰
64	Tolypyridone I	T album dws120	²¹
65	Tolypyridone J	T album dws120	²¹
66	Tolypyridone K	T album dws120	²¹
67	Tolypyridone L	T album dws120	²¹
68	Tolypyridone M	T album dws120	²¹

*Obtained from heterologous expression of gene clusters of T. sp. 49Y in Aspergillus oryzae NSAR1.

Zhang et al¹⁹ heterologously expressed a pyridone biosynthetic gene cluster from Tolypocladium sp. 49Y in Aspergillus oryzae NSAR1 and obtained 2 new compounds, tolypyridones C and D (54, 55), the former inhibited growth of Cryptococcus neoformans (MIC 6.25 μM, 2.22 μg/mL). Besides, 4 new pyridones tolypyridones E-H (56-59) were isolated from the rice ferments of the host strain of this gene cluster. This research also confirmed that tolypyridones originated from a tenellin-like biosynthetic pathway, the tolypyridone synthase gene (tolA) shared high homology with tenellin synthase gene (tenS), the construction of biosynthetic gene cluster was also same to that of tenellin's (ten gene cluster).²² Biosynthesis of these compounds involves cyclization by condensation between the amino acid and PK chain²² and free-radical-mediated ring expansion from pyrrolidone to pyridone.²³

From a Chaunopycnis sp., Shang et al²⁰ discovered 4 new PK-NRPs (60-63). Chaunolidine C (62) was mildly antibacterial, whereas chaunolidone A (63) was potently and selectively inhibitory to human non-small cell lung carcinoma cells (NCI-H460). They also mentioned these pyrrolidones can form metal chelates.

Recently, 5 new pyridone derivatives (tolypyridones I-M, 64-68) were isolated from rice medium ferments of T album dws120 by Wu et al,²¹ and they revealed tolypyridone I (64) can protect liver cells against ethanol-induced injury.

NonRibosomal Peptides

Nonribosomal peptides (NRPs) are the most structurally diversified among all Tolypocladium SMs, over 50 cyclic or linear oligopeptides have been discovered till now. NRPs discovered in this genus are usually linear, cyclic, or branched oligopeptides consisted of at most 22 amino acid units which are sometimes acylated, glycosylated or N-methylated. This family of compounds is medicinally important. Cyclosporine A (amino acid sequence shown in Table 4, and structure in Figure 4), a cyclopeptide consists of 11 amino acid moieties, is now a first-line immunosuppressant (Sandimmun, Novartis) that is commonly used in transplantation and autoimmune diseases (eg psoriasis, rheumatoid arthritis, etc).²⁴

Figure 4.

Structure of cyclosporin A.

Table 4.

Amino Acid Sequences of Cyclic Peptides in Tolypocladium sp. (Cyclosporines B-Z were from Traber²⁵).

Name	1	2	3	4	5	6	7	8	9	10	11
A²⁶	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
B	MeBmt	Ala	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
C	MeBmt	Thr	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
D	MeBmt	Val	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
E	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	Val
F	Desoxy-MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
G	MeBmt	Nva	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
H	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	D-MeVal
I	MeBmt	Val	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	Leu	MeVal
K	Desoxy-MeBmt	Val	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
L	Bmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
M	MeBmt	Nva	Sar	MeLeu	Nva	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
N	MeBmt	Nva	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	Leu	MeVal
O	MeLeu	Nva	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
P	Bmt	Thr	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
Q	MeBmt	Abu	Sar	Val	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
R	MeBmt	Abu	Sar	MeLeu	Val	Leu	Ala	D-Ala	MeLeu	Leu	MeVal
S	MeBmt	Thr	Sar	Val	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
T	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	Leu	MeVal
U	MeBmt	Abu	Sar	MeLeu	Val	Leu	Ala	D-Ala	MeLeu	MeLeu	MeVal
V	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Abu	D-Ala	MeLeu	MeLeu	MeVal
W	MeBmt	Thr	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	Val
X	MeBmt	Nva	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	Leu	MeLeu	MeVal
Y	MeBmt	Nva	Sar	MeLeu	Val	Leu	Ala	D-Ala	MeLeu	MeLeu	MeVal
Z	Methylamino-octanoic acid	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
MeIle²⁷	MeBmt	Abu	Sar	MeIle	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
4-Leu A²⁸	MeBmt	Abu	Sar	Leu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
J²⁸	MeLeu	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
D hydro-peroxide²⁹	Xaa	Val	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
FR190293³⁰	DiOHOrn-R	Thr	OHPro	DiOHTyr	OHGln	OHMePro

Abbreviations: Bmt, (2S, 3R, 4R, 6E)-2-amino-3-hydroxy-4-methyl-6-octenoic acid; MeBmt, N-methyl Bmt; Xaa, 3-hydroxy-7-hydroperoxy-4-methyl-2-methyl-amino-5E-octenoic acid; R, 10,12-dimethyl myristic acid; Nva, norvaline.

Cyclic Oligopeptides

Cyclosporin was first isolated from Trichoderma polysporum (Link ex Pers) (later redesignated as T inflatum Grams)³¹ and identified in 1970s by Petcher et al.²⁶ It was a sarcosine-containing cyclo-undecapeptide (Figure 4). Then Traber et al^25,32–34 discovered over 20 cyclosporins from T niveum, namely cyclosporins B-Z (Table 4). Structures ofcyclosporins were featured with unnatural amino acid moieties, (2S, 3R, 4R, 6E)-2-amino-3-hydroxy-4-methyl-6-octenoic acid (Bmt) and Bmt derivatives, and it was believed that this moiety was critical for immunosuppression, alternation of Bmt (deoxidation, replacement by other amino acids) caused loss of activity. They also found anti-HIV cyclosporine (MeIle) from T niveum fermentations supplemented with D-threonine, this compound can inhibit HIV-1 (strain IIIB)-induced cytopathic effect in MT4 cells.²⁷ With 60 cyclosporin derivatives, Loor et al³⁵ investigated the structure-activity relationship (SAR) of cyclosporin inhibiting human MDR-1 P-glycoprotein (P-gp) ABC transporter, the results indicated that hydrophobic residues favored P-gp inhibition, while N-desmethylation mostly abolished the activity.

Biosynthesis of Cyclosporin A

As cyclosporin A is chemically unique and clinically important, it is worth to give a brief summary on the biosynthesis of cyclosporin. Currently, it is commonly recognized that 3 major steps have been involved in the biosynthesis of cyclosporin A: isomerization of l-alanine, formation of Bmt and assembly of undecapeptide chain (Figure 6).

Figure 5.

Diketopiperazines isolated from fungi of the Tolypocladium genus.

Figure 6.

Biosynthesis of cyclosporin A involves 3 steps: (1) l-Ala is converted into d-Ala by a racemase; (2) the backbone of Bmt is synthesized by a polyketide synthase; (3) CsSyn is both multidomain and multifunctional, each amino acid unit is activated by adenylation, transferred to the thiolation domain (PCP) or linked to the amino group of adjacent moiety with the help of condensation domain; methylation occurs when there is a methyltransferase domain. Abbreviations: PCP, peptidyl carrier protein; CsSyn, cyclosporin A synthase; C, condensation domain; A, adenylation domain; T, thiolation domain; MT, methyltransferase domain.

The unnatural amino acid d-alanine was yielded by alanine racemase acting upon l-alanine. Hoffman et al³⁶ isolated this alanine racemase from T niveum, and revealed that it can catalyze the reversible racemase of l- and d- alanines. The Bmt building block was a threonine derivative synthesized by oxidation and transamination of a PK.³⁷ Isotope labelling^38,39 indicated that T inflatum can incorporate ¹³C in glucoses into the Bmt moiety, and Offenzeller et al³⁷ showed that in T niveum NRRL 8044 a carbon chain was synthesized by a PK synthase and then was transformed and released as amino acid coenzyme A thioester.

Assembly of the undecapeptide cycle was the most sophisticate step and it was catalyzed by a NRP synthase,⁴⁰ cyclosporin synthase (CySyn). CySyn, encoded by a 45.8 kb open reading frame (assigned as simA) in T niveum NRRL 8044, was a giant multimodule enzyme that consisted 11 sets of condensation-adenylation-thiolation domains (the C-A-T domains). Adenylation domain activated amino acids by adding adenyl group onto the amino group, thiolation domain with a free thiol group acted as a carrier of activated amino acids and peptidyls, and condensation domain catalyzed the formation of peptide bonds.^41,42 Seven of the 11 modules contained a methyltransferase (MT) domain, and their function was catalyzation of N-methylation on the corresponding amino acid units (Bmt-1, Sar-3, Leu-4, Leu-6, Leu-9, Leu-10, and Ala-11).⁴⁰

Linear Oligopeptides

Efrapeptins are linear oligopeptides that are featured by their characteristic constituents α-amino-isobutyric acid (Aib) and pipecolic acid (Pip) (Table 5). Efrapeptins A-G were isolated from T niveum.^43–45 Efrapeptins D and F were insecticidal (LC₅₀ were 18.9 and 8.4 ppm) against Colorado potato beetle.

Table 5.

Sequences of Linear Peptides Discovered from Tolypocladium Species.

Name	Sequences	Origins	References
LP237-F8	Lol-Ala-Gln-EtNor-Aib-Gln-Gln-Aib-Phe-Pro-Aib-Oc	T geodes	⁴⁶
LP237-F5	Lol-Ala-Gln-EtNor-Aib-Gln-Gln-Aib-Tyr-Pro-Aib-Oc	T geodes	⁴⁷
LP237-F7	Lol-Ala-Gln-Aib-Aib-Gln-Gln-Aib-Phe-Pro-Aib-Dec	T geodes	⁴⁷
Efrapeptin C	AcPip-Aib-Pip-Aib-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Gly-Leu-Aib-HPP	T inflatum	⁴⁸
Efrapeptin D	AcPip-Aib-Pip-Aib-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Gly-Leu-Iva-HPP	T inflatum	⁴⁸
Efrapeptin E	AcPip-Aib-Pip-Iva-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Gly-Leu-Iva-HPP	T inflatum	⁴⁸
Efrapeptin F	AcPip-Aib-Pip-Aib-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Ala-Leu-Iva-HPP	T inflatum	⁴⁸
Efrapeptin G	AcPip-Aib-Pip-Iva-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Ala-Leu-Iva-HPP	T inflatum	⁴⁸
Efrapeptin J	AcPip-Aib-Pip-Aib-Leu-β-Ala-Gly-Aib-Aib-Pip-Aib-Pip-Aib-Ala-Leu-Aib-HPP	Tolypocladium sp.	⁴⁹
Efrapeptin K	AcPip-Aib-Pip-Aib-Leu-β-Ala-Gly-Aib-Aib-Pip-Aib-Pip-Aib-Ala-Leu-Iva-HPP	Tolypocladium sp.	⁴⁹
Efrapeptin L	AcPip-Aib-Pip-Aib-Leu-β-Ala-Gly-Aib-Aib-Pip-Aib-Pip-Aib-Ala-Leu-Iva-HPP	Tolypocladium sp.	⁴⁹
Gichigamin A	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin B	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Iva-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin C	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin D	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Iva-Aib-Leu-bAla	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin E	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Aib-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin F	AcAib-Pro-Aib-Pro-Phe-Aib-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin G	AcAib-Pro-Aib-Pro-Phe-Aib-Pro-Ala-Aib-bAla-Ala-Aib-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain A	Dec-Aib-Pro-Leu-Aib-Gln-Gln-Aib-Aib-Gln-Ala-Lol	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain B	Dec-Aib-Pro-Tyr-Aib-Gln-Gln-Aib-Aib-Gln-Ala- Lol	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain C	Dec-Aib-Pro-Tyr-Aib-Gln-Gln-Aib-Iva-Gln-Ala- Lol	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain D	Dec-Aib-Pro-Phe-Aib-Gln-Gln-Aib-Iva-Gln-Ala- Lol	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain E	Non-Aib-Pro-Leu-Aib-Gln-Gln-Aib-Aib-Gln-Ala- Lol	T. sp. Sup5-1 (T2)	¹⁵
Tolypocladamide A	Dec-Aib-Pro-Bmt-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide B	Dec-Iva-Pro-Bmt-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide C	Dec-Aib-Pro-Bmt-Iva-Gln-Gln-Aib-Iva-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide D	Oct-Aib-Pro-Bmt-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide E	Dec-Aib-Pro-Bmt-Aib-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide F	Oct-Aib-Pro-Leu-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide G	Dec-Iva-Pro-Leu-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide H	Dec-Aib-Pro-Bmt-Aib-Gln-Gln-Aib-Aib-Gln-Val-Lol	T inflatum	⁵¹
Tolypocaibol A	Dec-Aib-Pro-Phe-Aib-Gln-Gln-Aib-Aib-Gln-Leu-Valol	Tolypocladium sp.	⁵²
Tolypocaibol B	Dec-Aib-mPro-Phe-Aib-Gln-Gln-Aib-Aib-Gln-Leu-Valol	Tolypocladium sp.	⁵²

Abbreviations: AcAib, acetyl-2-aminoisobytyric acid; GlyOH, 2-hydroxyethylamine; Dec, decanoic acid; Non, nonanoic acid; Oc, octanoic acid; Lol, D-leucinol; Valol, valinol; mPro, 4-methyl proline, bAla, D-alanine.

Gichigamins are a family of 21 and 22-residue peptides, Du et al¹⁵ comprehensively investigated the structures of gichigamins A-G by NMR, mass spectroscopy, electronic circular dichroism (ECD) or single-crystal x-ray diffraction. Gichigamin A was cytotoxic in vitro (GI₅₀ 0.55 ± 0.04 μM) and it showed antitumor activity in tumor xenograft mice as well. Gichigamin A can disrupt mitochondrial functions. In their semisynthetic structural modification research, by linking a coumarin to its C-terminus, antitumor potency increased to 60-fold of the prototype. Along with these peptides, the author also isolated 6 new 11-residue lipopeptaibols named dakwaabakains A-E and LP237-F7 along with 5 PKs.

In 2022, an array of new 11-residue peptaibols named tolypocladamides were discovered from T inflatum by 2 groups of researchers separately.^50,51 Tolypocladamides A-G were tested for their Ras/Raf inhibitory activities, and tolypocladaminde C showed the lowest IC₅₀ at 0.56 μM.

Recently in 2023, an endophytic Tolypocladium sp. from marine alga Spongomorpha arcta was investigated by Morehouse et al⁵² Isolation led to the discovery of 2 antibacterial peptides named tolypocaibols A and B and an NRPS-PK-shikimate metabolite mixture maxinmiscin. Both peptides showed MIC values at 4 to 16 μM against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and other pathogenic bacteria.

Besides, diketopiperazines and other small-molecule peptides have been discovered alongside (69-73, Fig. 5 and Table 6).^53–56 Bioactivities of the compounds mentioned above were listed in the Supplemental Materials (Table S2).

Table 6.

Diketopiperazines and Other Small-Molecule Peptides.

No.	Name	Origins	References
64	Sch54794	Tolypocladium sp.	⁵³
65	Sch54796	Tolypocladium sp.	⁵³
66	Sch56396	Tolypocladium sp.	⁵⁴
67	1-Alaninechlamydocin	Tolypocladium sp.	⁵⁵
68	Tolyprolinol	T. sp. FKI-7981	⁵⁶

Discussion and Conclusions

Over the past half century since the genus Tolypocladium has been described and established, much has been done to understand its chemistry and discover pharmacologically active natural products from species in this genus. Gratifyingly, a potent immunosuppressant, cyclosporin A, is found and developed into a useful medicine.

Though species of this genus can be found from different sources including plants and insects, small molecules like PK-NPRs are rather similar (eg tolypyridones vs chaunolidones) among strains. This indicated that they are biosynthetically similar, and secondary metabolic gene clusters of small molecules are conserved between strains.

We believe that Tolypocladium genus is still a good resource of new natural products, especially oligopeptides. With the help of bio-informative methods and gene manipulation techniques, more cryptic biosynthetic gene clusters can hopefully be disclosed in the future. Noteworthily, pharmacology of many of the compounds, especially the oligopeptides, is somehow inadequately investigated, thus it is necessary to revisit these compounds for more useful bioactivities.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231225538 - Supplemental material for Diversity of Secondary Metabolites from Fungi of the Ascomycete Genus Tolypocladium

Supplemental material, sj-docx-1-npx-10.1177_1934578X231225538 for Diversity of Secondary Metabolites from Fungi of the Ascomycete Genus Tolypocladium by Ting-Ting Niu, Wei-Qiong Chen, Wen-Xing Li, Li Zhang, Ying-Qiao Shan and Li-Wei Ren in Natural Product Communications

Footnotes

Author Contributions

Investigation and Project administration were done by T.-T. N., L.-W. R.. Supervision was done by L. Z., L.-W. R. Data collection was done by T.-T. N., Y.-Q. S. Writing—original draft was done by T.-T. N, W.-Q. C. Writing—review & editing was done by W.-X. L, L.-W. R.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science, Technology and Innovation Commission of Shenzhen Municipality, Shenzhen Key Medical Discipline Construction Fund (grant numbers JCYJ20230807112402005, SZXK059).

ORCID iD

Ting-Ting Niu

Supplemental Material

Supplemental material for this article is available online.

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