Verticillins: fungal epipolythiodioxopiperazine alkaloids with chemotherapeutic potential

Abstract

Covering: 1970 through June of 2023Verticillins are epipolythiodioxopiperazine (ETP) alkaloids, many of which possess potent, nanomolar-level cytotoxicity against a variety of cancer cell lines. Over the last decade, their in vivo activity and mode of action have been explored in detail. Notably, recent studies have indicated that these compounds may be selective inhibitors of histone methyltransferases (HMTases) that alter the epigenome and modify targets that play a crucial role in apoptosis, altering immune cell recognition, and generating reactive oxygen species. Verticillin A (1) was the first of 27 analogues reported from fungal cultures since 1970. Subsequent genome sequencing identified the biosynthetic gene cluster responsible for producing verticillins, allowing a putative pathway to be proposed. Further, molecular sequencing played a pivotal role in clarifying the taxonomic characterization of verticillin-producing fungi, suggesting that most producing strains belong to the genus Clonostachys (i.e., Bionectria), Bionectriaceae. Recent studies have explored the total synthesis of these molecules and the generation of analogues via both semisynthetic and precursor-directed biosynthetic approaches. In addition, nanoparticles have been used to deliver these molecules, which, like many natural products, possess challenging solubility profiles. This review summarizes over 50 years of chemical and biological research on this class of fungal metabolites and offers insights and suggestions on future opportunities to push these compounds into pre-clinical and clinical development.

Fig. 1. The basic structure of dimeric epipolythiodioxopiperazine (ETP) alkaloids, illustrating their biosynthetic origins. The color coding is used to illustrate the building blocks of these molecules. In red is tryptophan (Trp), common to all three dimeric structural classes. For verticillins, the “blue” substituents dictate the appendages at C-3 and C-3′. Unlike chaetocins and leptosins, the constituents at these positions vary and are typically derived from alanine (Ala), threonine (Thr), and in some rare cases, serine (Ser). The N-methylation, common to all three classes, likely derives from S-adenosylmethionine. For chaetocins and leptosins, biosynthesis often uses Ser (purple) and/or Val (green). Moreover, in these two classes, the sulfur bridge is often greater than two atoms (n = 2–4), whereas in the verticillins, the bridge is typically, but not always, a disulfide.

Fig. 2. Structures of verticillin analogues (2–27) discovered after A (1) as of June 2023 listed in chronological order and the biosynthetically related ETP alkaloid, gliotoxin (28). Note, while gliocladin A (11) and bionectin C (21) appear to have the same structure, they were reported in two separate publications with slightly different NMR data.

Fig. 3. Various structural drawings of verticillin A (1). Verticillin A (1) can be represented several different ways. The representation shown in the middle is seen widely in literature and was how Minato and colleagues first presented the molecule. However, the dash line between the two halves of the molecule (i.e., 10b to 10b′) is problematic, as it is connecting two asymmetric centers. Alternatively, the drawing of the molecule on the left displays the same information (i.e. 3S, 5aR, 10bS, 11S, 11aS, 3′S, 5a′R, 10b′S, 11′S, 11a′S) in a non-ambiguous manner. Interestingly, Minato and colleagues subsequently drew 1 as shown on the right, where they defined the absolute configuration via circular dichroism; these assignments were confirmed via X-ray crystallography in 2006.

Fig. 4. Two microscopic images of the phialides of Clonostachys rogersoniana (strain MSX59553). This fungus biosynthesizes the following verticillins: verticillin A (1), Sch 52900 (4), Sch 52901 (5), verticillin D (6), 11′-deoxyverticillin A (9), gliocladicillin C (26), and verticillin H (27). The morphology of the phialides (which have a ‘flask shape’) are characteristic to species of the Clonostachys, Gliocladium, and Verticillium and can cause confusion for non-experts. As such, it is conceivable that fungi used in previous natural products chemistry studies that led to the isolation of some of the analogues shown in Fig. 2 and Table 1, many of which were identified based on morphology, were misidentified. Scale bars = (A) 10 μm and (B) 20 μm.

Fig. 5. (A) Biosynthetic gene cluster for 11′-deoxyverticillin A (9) from C. rogersoniana. (B) Proposed biosynthetic pathway for verticillin A (1) based on knowledge from gliotoxin. (C) Possible route for the late stage verticillin A (1) biosynthetic steps.

Scheme 1. Conceptual strategies for formation of bis-pyrroloindoline containing compounds via (A) reductive or (B) oxidative transformations.

Scheme 2. Total synthesis of (+)-bionectins A (19) and C (21) by Coste et al. Conditions: (a) TiCl(OEt)₃, NEt₃, CH₂Cl₂, 0 °C, 81% (58% desired diastereomer); (b) TBSOTf, 2,6-lutidine, CH₂Cl₂, 0 °C, 72%; (c) 2 N HCl, THF, 81%; (d) N-Boc-sarcosine, EDC·HCl, HOBt, CH₂Cl₂, 23 °C, 98%; (e) TFA, CH₂Cl₂, 23 °C; AcOH, morpholine, t-BuOH, 80 °C, 97%; (f) Br₂, MeCN, 0 °C; anisole, 94%, 9 : 1 dr; (g) DMAP, THF, 23 °C, 74%; (h) AgBF₄, DTBMP, EtNO₂, 0 °C, 68%; (i) 6 N HCl, THF, 80 °C, 58%; (j) Boc₂O, DMAP, CH₂Cl₂, 23 °C, 92%; (k) Py₂AgMnO₄, CH₂Cl₂, 23 °C, 45%; (l) NaBH₄, MeOH, −20 °C, 75%; (m) PivCl, DMAP, CH₂Cl₂, 23 °C, 83%; (n) 4-mercapto-2-butanone, TFA, MeNO₂, 80%, 3 : 1 dr; (o) 350 nm, 1,4-dimethoxynaphthalene, l-ascorbic acid, sodium l-ascorbate, H₂O, MeCN, 25 °C, 56%; (p) pyrrolidine, EtSH, THF, 23 °C; KI₃, Py, CH₂Cl₂, 81%; (q) NaBH₄, MeI, Py, MeOH, 0 °C, 97%.

Fig. 6. Examples of biosynthetic and semisynthetic generation of verticillin analogues.

Fig. 7. (A) In colon cancer, verticillin A (1) inhibits H3K9me3 levels via selective inhibition of HMTases, specifically G9a, SUV39H1 and SUV39H2, leading to restoration of Fas expression. This, in turn, leads to suppression of immune evasion and overcoming chemoresistance to 5-fluorouracil (5-FU). (B) In pancreatic cancer, verticillin A (1) inhibits MLL1, resulting in a decrease of H3K4me3 levels, consistent with a decrease of expression of PD-L1, and activates the immune response against tumor cells. (C) Verticillin A (1) causes global methylation and acetylation histone modifications in ovarian cancer, which generates oxidative stress mediated DNA damage and apoptosis.