Metabolomics-guided Discovery, Isolation, Structure Elucidation, and Bioactivity of Myropeptins C-E from Myrothecium inundatum

Abstract

The saprotrophic filamentous fungus Myrothecium inundatum represents a chemically underexplored ascomycete with a high number of putative biosynthetic gene clusters in its genome. Here, we present new linear lipopeptides from non-genetic gene activation experiments using nutrient and salt variations. Metabolomics studies revealed four myropeptins and structural analyses by NMR, HRMS, Marfey’s analysis, and ECD assessment for their helical properties established their absolute configuration. A biosynthetic myropeptin gene cluster in the genome was identified. The myropeptins exhibit general non-specific toxicity against all cancer cell lines in the NCI-60 panel, larval zebrafish with EC₅₀ concentrations of 5–30 μM, and pathogenic bacteria and fungi (MICs of 4–32 μg/mL against multidrug-resistant S. aureus and C. auris). In vitro hemolysis, cell viability, and ionophore assays indicate that the myropeptins target mitochondrial and cellular membranes, inducing cell depolarization and cell death. The toxic activity is modulated by the length of the lipid side chain which provides valuable insight into their structure-activity relationships.

Graphical Abstract

The fungal genus Myrothecium (family Stachybotriaceae, order Hypocreales) includes a diversity of endophytic, saprophytic, and pathogenic species.^{1, 2} Myrothecium inundatum was first described by Tode in 1790,¹ but is chemically under-explored although a small number of secondary metabolites, including bioactive di- and triterpenes, were recently reported from an endolichenic M. inundatum strain.³ The genome of M. inundatum CBS120646 was sequenced and made publicly available by the Department of Energy Joint Genome Institute (JGI, https://mycocosm.jgi.doe.gov/Myrin1/Myrin1.home.html).⁴ It contains over 80 annotated biosynthetic gene clusters (BGCs), suggesting many dormant secondary metabolites compared to the small number of metabolites that have been discovered thus far. Generally, most BGCs are not expressed under common laboratory conditions,⁵ but exposing a strain to varied biotic and abiotic culture conditions, including different media nutrients, temperatures, light, pH, or co-cultivation can activate repressed BGCs.^6–8 Here, by changing the culture conditions we isolated highly toxic lipopeptides: myropeptin A₁, previously described from a M. roridum strain,⁹ and three new myropeptins with a single amino acid change, or different length fatty acid chain. We report the isolation, structure elucidation, and in vitro and in vivo activity of myropeptins E-C (1–3), and the full spectroscopic assignment and bioactivity of the known myropeptin A₁ (4)⁹ from M. inundatum. In addition, we identify a putative biosynthetic gene cluster containing a 20 module non-ribosomal peptide synthetase (NRPS).

RESULTS & DISCUSSION

M. inundatum was cultured in four media conditions: malt extract-peptone (M2), tomato juice (V8), potato extract-dextrose (PDA), and starch-yeast-peptone-seawater (SYP-SW). The metabolites produced were detected with high-resolution LC-MS and analyzed with multivariate statistics and principal component analysis (PCA). The PDA derived metabolome was the most distinct from that produced in the media M2, V8, and SYP-SW (Figure 1A), however this was due to few, low abundant mass spectrometric features. We focused on the metabolome derived from seawater-based media which revealed significantly different, and more abundant metabolites (Figure 1B).

Figure 1:

(A) PCA scores plot and (B) PCA loadings plot of M. inundatum metabolome after cultivation for two weeks, 26 °C in different media (M2, V8, PDA, and SYP-SW, n=3, 95% confidence interval of normalized m/z intensities from LC-HRMS, positive ionization mode). PCA loadings plot (B) depicts one of the significantly distinct (ANOVA, accompanied by Fishers Least Significant Difference test and an FDR correction) features found in SYP-SW extract.

The fungal metabolome, grown in SYP-SW medium, exhibited unique and highly abundant mass spectrometric features with large m/z values at four different retention times. Related mass features eluted at specific times (7.2/7.3 min m/z 774.51 & 1052.61; 7.8 min m/z 774.51 & 1066.62; 8.0/7.9 min m/z 802.54 & 1052.61; 8.6 min m/z 802.54 & 1066.62 (Figure 2).

Figure 2:

Heatmap visualization abundance of mass spectrometric features for M. inundatum cultivated in SYP-SW, V8, M2, and PDA media for two weeks, 26 °C. The blue color represents low abundance, red represents high abundance.

Structure elucidation

Four compounds of M. inundatum cultured in SYP-SW medium were targeted for isolation based on the mass spectrometric features from the metabolomic profiling. The structure determination of the lipopeptides was performed using a combination of MS/MS and extensive NMR analyses. Myropeptin C (1) was isolated as a white amorphous powder. Electrospray ionization coupled with high-resolution mass spectrometry (ESI-HRMS) confirmed the molecular mass and formula of myropeptin C (1) of C₈₇H₁₄₈N₂₀O₂₂ (Figure 4B). Additionally, isotope labeling experiments with ¹⁵N-ammonium chloride resulted in the incorporation of 20 ¹⁵N-atoms, corroborating the presence of 20 amino acids in total (Figure 3C).

Figure 4:

(A) QTOF-MS² spectra of myropeptin C (1) of in-source fragment 1052.6097 [M+H]⁺ and (B) in-source fragment 774.5138 [M+H]+, collision-induced dissociation (35 eV), dashed lines show generated b-ions.

Figure 3:

(A) MS¹ spectra of myropeptin C (1), in positive ionization mode, (B) in negative ionization mode, and (C) in negative ionization mode after ¹⁵N-labeling.

The MS² patterns of the two in-source fragments from positive ionization resembled peptides. Indeed, ten b-ions were produced upon fragmentation of the m/z 1052 fragment and neutral losses derived from respective amino acids were assigned (Figure 4A). The m/z 774 portion was more challenging to analyze, but several b-ions, internal b-ions, and an a-ion were detected (Figure 4B).

The ¹H-NMR data in DMF-d₇ revealed 17 amide protons from 7.28–8.70 ppm. From the recorded ¹³C NMR spectrum, 19 carbonyl carbons from 172.2–177.0 ppm and 11 aliphatic non-protonated carbons from 56.3 to 56.9 ppm were observed (Table 1, Figure S22). HMBC correlations between a bulk of methyl protons (1.42–1.60 ppm) and carbons between 56.3 to 56.9 ppm confirmed the presence of 11 Aib residues (Figure 5A, Figure S15–S19. Analysis of protons ranging from 3.31–4.38 ppm (Table 1, Figure S21) in combination with 2D-NMR data including HSQC (Figure S23), TOCSY Figure S24), and HMBC (Figure S25–26) suggested that (1) contains three alanine (Ala), three proline (Pro) residues, and two β-alanine (βala), supported by the HRMS fragmentation pattern (Figure 4). The number of methylene groups (¹H 1.22–1.37 ppm, ¹³C 22.6–35 ppm), their TOCSY interactions, and one HMBC correlation to a carbonyl group, indicated a saturated fatty acid chain in the molecule (Figure 5A). MS² fragmentation of the m/z 774 in-source fragment ion resulted in generation of (internal) b-ions (Figure 4B) and accurate m/z values of respective fragments confirmed the presence of a decanoyl (C₁₀) chain attached to the C-terminus. NOESY correlations verified the sequence of the 20-mer: C₁₀-Aib-Pro-Aib-Aib-Pro-Aib-Aib-Pro-Ala-Aib-βala-Ala-Aib-Aib-βala-Aib-Aib-Aib-Ala-βala (Figure 5A).

Table 1:

NMR data of myropeptin C (1) in DMF-d₇ (600 and 150 MHz, δ in ppm, * signal overlapped, ** signal not detected).

Residue	δ13C, type	δ1H, mult., (J in Hz)	Residue	δ13C, type	δ1H, mult., (J in Hz)	Residue	δ13C, type	δ1H, mult., (J in Hz)
C10	13.8, CH3	0.87, t (6.8)	Aib 6 , NH		7.62, s*	Aib 14 , NH		7.62, s*
C9	22.6, CH2	1.22–1.37*	α	56.9, C		α	56.8, C
C8	31.9, CH2	1.22–1.37*	β1	23.3, CH3	1.63, m	β1	23.5–27.7, CH3	1.47, m*
C7	29.3, CH2	1.22–1.37*	β2	23.4, CH3	1.51, m*	β2	23.5–27.7, CH3	1.47, m*
C6	29.5, CH2	1.22–1.37*	C=O	177.0, C		C=O	175.7, C
C5	29.4, CH2	1.22–1.37*	Aib 7 , NH		8.02, s	βala 15 , NH		7.71, s
C4	29.2, CH2	1.22–1.37*	α	56.8, C		α	37.9, CH2	2.45; 2.40, m
C3	24.5, CH2	1.61*	β1	23.4, CH3	1.59, m*	β	36.3, CH2	3.63; 3.31, m
C2	35.0*, CH2	2.36; 2.33, m	β2	25.9, CH3	1.44, m*	C=O	172.7, C
C1, C=O	173.5, C		C=O	173.5, C		Aib 16 , NH		8.29, s
Aib 1 , NH		8.70, s	Pro8 α	63.3, CH	4.38, m	α	56.5, C
α	56.3, C		β	28.9*, CH2	2.28; 1.81, m	β1	23.5–27.7, CH3	1.42–1.60, m*
β1	25.3, CH3	1.53, m	γ	25.7, CH2	1.94, m	β2	23.5–27.7, CH3	1.42–1.60, m*
β2	23.4, CH3	1.46, m	δ	48.8, CH2	4.04; 3.73, m	C=O	175.7, C
C=O	174.0, C		C=O	173.4, C		Aib 17 , NH		8.56, s
Pro2 α	64.1, cH	4.19, m	Ala 9 , NH		7.92, s	α	56.8, C
β	28.8, CH2	2.29; 1.70, m	α	50.7, CH	4.16, m	β1	23.5–27.7, CH3	1.42–1.60, m*
γ	25.3, CH2	1.99; 1.92, m	β	16.6, CH3	1.45, m	β2	23.5–27.7, CH3	1.42–1.60, m*
δ	48.9, CH2	3.95; 3.45, m	C=O	173.1, C		C=O	176.2, C
C=O	172.2, C		Aib 10 , NH		7.52, s*	Aib 18 , NH		7.93, s*
Aib 3 , NH		7.62, s*	α	56.8, C		α	56.8, C
α	56.9, C		β1	26.0, CH3	1.49, m	β1	23.5–27.7, CH3	1.42–1.60, m*
β1	23.3, CH3	1.60, m	β2	26.0, CH3	1.51, m	β2	23.5–27.7, CH3	1.42–1.60, m*
β2	23.5–27.7, CH3	1.50, m	C=O	175.0, C		C=O	174.9, C
C=O	176.2, C		βala 11 , NH		7.28, t (6.0)	Ala 19 , NH		7.79, d, (7.9)
Aib 4 , NH		7.94, s*	α	36.6, CH2	2.47; 2.41, m	α	49.7, CH	4.19, m
α	56.6, C		β	36.4, CH2	3.57; 3.33, m	β	17.1, CH3	1.42, m
β1	23.4, CH3	1.46, m	C=O	172.5 C		C=O	173.0, C
β2	23.4, CH3	1.59, m	Ala 12 , NH		8.27, s	βala 20 , NH		7.52, s*
C=O	173.7, C		α	51.5, CH	4.09, m	α	35.0*, CH2	2.54; 2.51, m
Pro5 α	64.3, CH	4.19, m	β	16.6, CH3	1.40, m	β	36.3, CH2	3.41; 3.40, m
β	28.9*, CH2	2.27; 1.74, m	C=O	174.5, C		C=O	**, C
γ	25.8, CH2	1.98; 1.93, m	Aib 13 , NH		8.49, s
δ	48.9, CH2	3.76; 3.89, m	α	56.6, C
C=O	172.5, C		β1	23.5–27.7, CH3	1.42–1.60, m*
			β2	23.5–27.7, CH3	1.42–1.60, m*
			C=O	174.6, C

Figure 5:

(A-C) Key 2D NMR correlations (TOCSY, COSY, NOESY, HMBC) of myropeptins C-E (1–3) in DMF-d₇.

Compound (2) featured a similar unique set of in-source fragmentation ions with m/z values of 774.5138 and 1066.6269 (7.8 min) but shared the same m/z 774 fragment as seen in 1 (Supplementary Information, Figure S2+S5). The second fragment mass increase of 14 Da from m/z 1052 to m/z 1066 suggested a congener with an additional methyl(ene) group. Indeed, analysis of the MS² fragmentation pattern of m/z 1066 uncovered a different b₁₀-ion (Supplementary Information, Figure S5). Instead of a Pro-Ala fragment (m/z 169.0976) found in myropeptin C, m/z 183.1129 was detected for Pro-Aib. All other neutral losses were identical to 1, hence the compound was named myropeptin D (2). The molecular formula of 2 was determined by ESI-HRMS to be C₈₈H₁₅₀N₂₀O_22, m/z 918.5544, [M-2H]²⁻ calc. for, 918.5544 (Supplementary Information, Figure S2). As expected, protons from 4.10–4.22 ppm were found in ¹H-NMR and HSQC spectra (Table 2, Supplementary Information, Figure S29) for two Ala moieties instead of three, while the three Pro residues remained. NOESY and HMBC correlations (Supplementary Information, Figure S31–34) supported the peptide sequence to be C₁₀-Aib-Pro-Aib-Aib-Pro-Aib-Aib-Pro-Aib-Aib-βala-Ala-Aib-Aib-βala-Aib-Aib-Aib-Ala-βala (Figure 5B).

Table 2:

NMR data of myropeptin D (2) in DMF-d₇ (600 and 150 MHz, δ in ppm, * signal overlapped, ** signal not detected).

Residue	δ13C, type	δ1H, mult., (J in Hz)	Residue	δ13C, type	δ1H, mult., (J in Hz)	Residue	δ13C, type	δ1H, mult., (J in Hz)
C10	13.6, CH3	0.88, t, (6.9)	Aib 6 , NH		7.58, s	Aib 14 , NH		7.61, s*
C9	22.6, CH2	1.22–1.37*	α	57.0, C		α	56.8, C
C8	31.9, CH2	1.22–1.37*	β1	23.4, CH3	1.51, m	β1	23.3–27.7, CH3	1.40–1.63, m*
C7	29.3, CH2	1.22–1.37*	β2	23.8, CH3	1.51, m	β2	23.3–27.7, CH3	1.40–1.63, m*
C6	29.5, CH2	1.22–1.37*	C=O	176.9, C		C=O	175.7, C
C5	29.4, CH2	1.22–1.37*	Aib 7 , NH		8.02, s	βala 15 , NH		7.68, s
C4	29.2, CH2	1.22–1.37*	α	56.7, C		α	37.9, CH2	2.43; 2.41, m
C3	25.3, CH2	1.62, m	β1	23.3–27.7, CH3	1.40–1.63, m*	β	36.3, CH2	3.61; 3.31, m
C2	34.9, CH2	2.36; 2.33, m	β2	23.3–27.7, CH3	1.40–1.63, m*	C=O	172.5, C
C1, C=O	174.4, C		C=O	173.4, C		Aib 16 , NH		8.29, s
Aib 1 , NH		8.68, s	Pro 8	63.9, CH	4.22, m	α	56.5, C
α	56.3, C		β	28.8, CH2	2.28; 1.81, m	β1	23.3–27.7, CH3	1.40–1.63, m*
β1	26.0, CH3	1.53, m	γ	25.7, CH2	1.94, m	β2	23.3–27.7, CH3	1.40–1.63, m*
β2	23.9, CH3	1.46, m	δ	48.7, CH2	4.03; 3.73, m	C=O	175.6, C
C=O	174.7, C		C=O	174.1, C		Aib 17 , NH		8.54, s
Pro2 α	64.1, CH	4.20, m	Aib 9 , NH		7.87, s	α	56.8, C
α	28.8, CH2	2.29; 1.70, m	α	56.6, C		β1	23.3–27.7, CH3	1.40–1.63, m*
β1	26.0, CH2	1.99; 1.92, m	β1	23.3–27.7, CH3	1.40–1.63, m*	β2	23.3–27.7, CH3	1.40–1.63, m*
β2	48.9, CH2	3.95; 3.45, m	β2	23.3–27.7, CH3	1.40–1.63, m*	C=O	176.1, C
C=O	172.2, C		C=O	176.0, C		Aib 18 , NH		7.92, s*
Aib 3 , NH		7.63, s*	Aib 10 , NH		7.50, s*	α	56.8, C
α	57.0, C		α	56.8, C		β1	23.3–27.7, CH3	1.40–1.63, m*
β1	23.5, CH3	1.60, m	β1	23.3–27.7, CH3	1.40–1.63, m*	β2	23.3–27.7, CH3	1.40–1.63, m*
β2	23.3–27.7, CH3	1.40–1.63, m*	β2	23.3–27.7, CH3	1.40–1.63, m*	C=O	174.9, C
C=O	176.1, C		C=O	175.0, C		Ala 19 , NH		7.79, s
Aib 4 , NH		7.93, s*	βala 11 , NH		7.32, s	137	49.7, CH	4.20, m
	56.7, C		α	36.5, CH2	2.47; 2.41, m	139	17.1, 03	1.40, m
	23.3–27.7, CH3	1.40–1.63, m*	β	36.2, CH2	3.32, m	C=O	173.0, C
β2	23.3–27.7, CH3	1.40–1.63, m*	C=O	172.5 C		βala 20 , NH		7.51, s*
C=O	173.6, C		Ala 12 , NH		8.26, d, (4.5)	α	34.5, CH2	2.47, m
Pro 5	64.3, CH	4.19, m	α	51.5, CH	4.10, m	β	35.8, CH2	3.32; 3.54, m
β	28.9, CH2	2.27; 1.74, m	β	16.5, CH3	1.40, m	C=O	**, C
γ	25.7, CH2	1.98; 1.93, m*	C=O	174.5, C
δ	48.8, CH2	3.76; 3.90, m	Aib 13 , NH		8.54, s
C=O	172.4, C		α	56.7, C
			β1	23.3–27.7, CH3	1.40–1.63, m*
			β2	23.3–27.7, CH3	1.40–1.63, m*
			C=O	174.5, C

The remaining two related peptides that eluted at RT 8.0 and 8.6 min shared characteristic MS² feature of m/z 802 and unique mass fragments of m/z 1052 and m/z 1066, respectively. An increase of 28 Da from the m/z 774 feature of the myropeptins C and D to m/z 802 suggested the addition of two methyl(ene) groups. With no remarkable changes in the ¹H NMR and identical neutral amino acids losses in the MS² compared to 1 and 2, we hypothesized the additional methylene groups to be in the fatty acid moiety. Indeed, the MS² fragmentation pattern (Supplementary Information, Figure S6) as well as the observed 11 aliphatic carbon NMR shifts, instead of nine, support a dodecanoyl (C₁₂) chain (Table 3, Supplementary Information, Figure S36). The new peptide with a molecular formula of C₉₀H₁₅₄N₂₀O₂₂ (HRMS (ESI) m/z 932.5701, [M-2H]²⁻ calc. 932.5701, Supplementary Information, Figure S3) and sequence of C₁₂-Aib-Pro-Aib-Aib-Pro-Aib-Aib-Pro-Aib-Aib-βala-Ala-Aib-Aib-βala-Aib-Aib-Aib-Ala-βala was named myropeptin E (3).

Table 3:

NMR data of myropeptin E (3) in DMF-d₇ (800 and 201 MHz, δ in ppm, * signal overlapped).

Residue	δ13C, type	δ1H, mult., (J in Hz)	Residue	δ13C, type	δ1H, mult., (J in Hz)	Residue	δ13C, type	δ1H, mult., (J in Hz)
C12	13.9, CH3	0.88, t, (7.2)	Aib 6 , NH		7.57, s	Aib 14 , NH		7.63, s
C11	22.7, CH2	1.24–1.36*	α	57.1, C		α	56.9, C
C10	32.0*, CH2	1.24–1.36*	β1	23.5, CH3	1.64, s	β1	23.5–27.2, CH3	1.42–1.51, m*
C9	29.3, CH2	1.24–1.36*	β2	23.6–27.2, CH3	1.42–1.51, m*	β2	23.5–27.2, CH3CH3	1.42–1.51, m*
C8	29.6, CH2	1.24–1.36*	C=O	176.3, C		C=O	175.5, C
C7	29.5, CH2	1.24–1.36*	Aib 7 , NH		7.97, s	βala 15 , NH		7.70, t (6.0)
C6	29.3, CH2	1.24–1.36*	α	57.0, C		α	37.8, CH2	2.29, 2.30, m
C5	29.4, CH2	1.24–1.36*	β1	23.4, CH3	1.59, s	β1	36.5, CH2	3.37, m*
C4	29.3, CH2	1.24–1.36*	β2	23.6–27.2	1.42–1.51, m*	β2	172.7, C*
C3	25.2, CH2	1.62, m*	C=O	173.4, C		Aib 16 , NH		8.53, s
C2	34.8, CH2	2.34; 2.37, m	Pro8, α	63.9, CH2	4.25, m	α	56.7, C
C1, C=O	174.0, C		β	29.1, CH2	2.28; 1.75, m*	β1	23.5–27.2, CH3	1.42–1.51, m*
Aib 1 , NH		8.66, s	γ	26.1, CH2	1.99; 1.92, m*	β2	23.5–27.2, CH3	1.42–1.51, m*
α	56.5, C		δ	48.9, CH	3.96; 3.77, m	C=O	175.5, C
β1	25.9, CH3	1.54, s	C=O	173.7, C		Aib 17 , NH		8.78, s
β2	24.4, CH3	1.42–1.51, m*	Aib 9 , NH		7.84, s	α	56.7, C
C=O	174.7, C		β	56.8, CH		β1	23.5–27.2, CH3	1.42–1.51, m*
Pro2, α	64.2, CH	4.21, m	γ	23.5–27.2, CH3	1.42–1.51, m*	β2	23.5–27.2, CH3	1.42–1.51, m*
β	28.9, CH2	2.28; 1.71, m	δ	23.5–27.2, CH3	1.42–1.51, m*	C=O	175.8, C
γ	26.2, CH2	1.99; 1–92, m*	C=O	175.8, C		Aib 18 , NH		7.87, s
δ	49.0, CH2	3.87; 3.46, m	Aib 10 , NH		7.47, s	α	56.9, C
C=O	172.3, C		α	56.9, C		β1	23.5–27.2, CH3	1.42–1.51, m*
Aib 3 , NH		7.70, s	β1	23.5–27.2, CH3	1.42–1.51, m*	β2	23.5–27.2, CH3	1.42–1.51, m*
α	57.1, C		β2	23.5–27.2, CH3	1.42–1.51, m*	C=O	174.8, C
β1	23.6, CH3	1.61, s	C=O	175.0, C		Ala 19 , NH		7.76, d (7.9)
β2	27.8, CH3	1.42–1.51, m*	βala 11 , NH		7.41, t (5.6)	α	49.8, CH	4.20, m
C=O	176.1, C		α	36.5, CH2	2.49; 2.42*	β1	17.4, CH3	1.40, m*
Aib 4 , NH		7.93, s	β1	36.3 CH2	3.37*	β2	172.9, C
α	56.9, C		β2	172.4, C		βala 20 , NH		7.50, t (5.8)
β1	23.5–23.8, CH3	1.55, s	Ala 12 , NH		8.41, s	α	34.6, CH2	2.42, m*
β2	23.5–27.2, CH3	1.42–1.51, m*	α	51.3, CH	4.16, m	β	35.7, CH2	3.37, m*
C=O	173.7, C		β1	16.9, CH3	1.39, m*	C=O	176.3, C
Pro5, α	64.4, CH2	4.21, m	β2	174.2, C
β	29.0, CH2	2.28; 1.75, m*	Aib 13 , NH		8.72, s
γ	26.1, CH2	1.99; 1.92, m*	α	56.7, C
δ	49.0, CH	3.77; 3.89, m*	β1	23.5–27.2, CH3	1.42–1.51, m*
C=O	172.7, C*		β2	23.5–27.2, CH3	1.42–1.51, m*
			C=O	174.6, C

Lastly, based on MS² and 2D-NMR analyses, the peptide eluting at 8.0 min with the mass spectroscopic in-source fragments m/z 802 and 1052 was determined to be myropeptin A₁⁹ (4) with the molecular formula of C₈₉H₁₅₂N₂₀O₂₂ (Figure S4). The structural assignment and sequence were supported by MS² (Figure S7), NMR experiments including ¹⁵N-HSQC and selected HMBC correlations (Table S2, Figure S8–20) to be C₁₂-Aib-Pro-Aib-Aib-Pro-Aib-Aib-Pro-Ala-Aib-βala-Ala-Aib-Aib-βala-Aib-Aib-Aib-Ala-βala. Myropeptin A₁ was first reported by Yoshimura and co-workers in 2019 as an inseparable mixture together with myropeptin A₂ from M. roridum.⁹ Here we add detailed spectroscopic data for purified myropeptin A₁, and expanded bioactivity testing. For all four myropeptins, we employed advanced Marfey’s analysis¹⁰ and determined the amino acids to be l-configured, additionally the presence of βala and Aib was confirmed (Figure S41). With chemical shift differences between the β- and the γ-carbons (ΔδC_β-δC_γ) between 2.7–3.5 ppm for the three Pro residues, their trans conformation was deduced.¹¹

Peptides featuring a large number of Aib residues tend to form helical structures.^{12, 13} We recorded circular dichroism (CD) spectra for the myropeptins (1-4), which display helical behavior with a positive band around 190 nm and negative bands at 208 nm and 230 nm (Figure 6). Specifically, we have indication of a 3₁₀-helix in solution, as reported for myropeptin A₁ (4)⁹ and gichigamin A,¹⁴ based on the weak positive band at 192 nm and the negative band at 207 nm accompanied by a shoulder band at 225 nm (absorption ratio R=mdeg₂₂₅/mdeg₂₀₇ of 0.4).¹⁵

Figure 6:

Circular dichroism (CD) spectra of lipopeptides 1-4 in acetonitrile-water (1/1, v/v) at 100 μg/mL.

Genome Analysis of Myrothecium inundatum CBS 120646

The genome of M. inundatum CBS 120646 was sequenced, assembled, and made publicly available by JGI (project ID: 1019495, genome version 01).⁴ After annotation of the genome with fungiSMASH (version 5.1.2)¹⁶ we identified 12 NRPS-PKS hybrid clusters, with six matching for a lipopeptide made by a NRPS and type-I PKS hybrid (T1PKS) (c.f. Supplementary Information, Figure S42). Specifically, scaffold_2_c8 – region 1 (region on contig edge, total of 121,819 nt) encoded a T1PKS cluster (782,386 – 788,169, total: 5508 nt, excluding introns) adjacent to a 20-amino acid encoding NRPS module 782,386 – 788,169, (total: 5508 nt, excluding introns) (Figure 7) that matched the structures of the myropeptins, which contain 20 amino acids.

Figure 7:

(A) Gene organization with open reading frames (ORF) of the putative biosynthetic gene cluster of myropeptins from M. inundatum CBS 120646. Domain structures of the type I-polyketide synthase (T1PKS module consists of a beta-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), ketoreductase (KR) and phosphopantetheine acyl carrier protein group (PP)) and the 20-module non-ribosomal peptide synthetase (NRPS, modules consist of condensation (C), adenylation (A) and peptidyl-carrier protein (PCP) domains) are shown. i) indicates the fungiSMASH prediction of loaded substrates (Iva = isovaline, Pro = proline, ? = no consensus), ii)-v) display the observed loaded substrates experimentally found for myropeptin C (ii), D (iii), E (iv), and A₁ (v); (B) Stachelhaus specificity codes from A domains responsible for Ala loading (top row), Aib loading (bottom row), and loading of both Ala and Aib (middle row), bottom motif was generated with WebLogo3.¹⁷

The PKS is putatively responsible for the formation of the decanoyl and dodecanoyl lipid chains and is a highly reducing iterative PKS with the domain order KS-AT-DH-KR-ACP. The PKS does not encode an enoyl reductase (ER) domain and we were unable to identify an ER in proximity to the BGC; we propose that ER activity is supplied by another protein encoded at disparate genomic loci. The fatty acid is likely transferred from the PKS to the initial T domain of the NRPS and then coupled with the downstream Aib residue. There are 19 elongation modules consisting of C-A-T domains and a C-terminal condensation domain, similar to previously described fungal NRPS.^{18, 19} The adenylation (A)-domains can be used to infer the amino acid specificity (Stachelhaus prediction, Figure 7, Table S3)^{20, 21} via NRPSpredictor2²² (integrated into fungiSMASH version 5.1.2) (Figure 7A). Prolines were predicted with high confidence, but no predictions were possible at several positions where beta-alanine or alanine was found. Isovaline is predicted to be incorporated by most modules (1, 3, 4, 6, 7, 10, 13, 14, 16, and 17), however, we observed Aib being incorporated into the myropeptins at these positions. Interestingly, the Stachelhaus code predicting the amino acid loading at position 9 is unique compared to the others that encode for Aib and Ala (Figure 7B), and this is responsible for the sequence differences between myropeptin C/A₁ and D/E.

Biological activity

Antimicrobial activity

Lipopeptides 1-4 were active against Enterococcus faecium, Staphylococcus aureus (methicillin-susceptible, methicillin-resistant, and multidrug-resistant) and Candida auris (Table 6, Supplementary Information, Table S4). Myropeptin A₁ (4) exhibited the most potent activity with an MIC value of 4 μg/mL (2 μM) against S. aureus and C. auris, myropeptins C and D (1-2) demonstrated moderate MIC values of 16 μg/mL (8 μM), and myropeptin E showed less activity with MIC values from 4–16 μg/mL (2–8 μM). (Figure S43). Total growth inhibition was observed for myropeptins C, D, and A₁ around 64–128 μg/mL (32–64 μM), while myropeptin E induced a stunned growth phenotype (c.f. Supplementary Information, Figure S43).

Table 6.

Minimum inhibitory concentration (MIC) assessment of lipopeptides 1-4 against the pathogens S. aureus and C. auris in μg/mL (microbroth dilution assay). Kanamycin, vancomycin and caspofungin were used as positive controls, respectively.

	S. aureus ATCC 25923	S. aureus ATCC BAA-41 (methicillin resistant)	S. aureus ATCC BAA-44 (multidrug resistant)	C. auris CDCB11903
myropeptin C (1)	16	16	16	16
myropeptin D (2)	16	16	16	16
myropeptin E (3)	4	8	8	16
myropeptin A1 (4)	4	4	4	4
kanamycin	1	-	-	-
vancomycin	-	1	1	-
caspofungin	-	-	-	2

In vitro cytotoxicity

Myropeptin A₁ (4) was cytotoxic to HCT116 human colon carcinoma and MDA-MB-231 human breast cancer cells with average IC₅₀ values of 1 μM to both cell lines (Figure 8, Table 5).²³ Myropeptins C (1), D (2) and E (3) exhibited slightly higher IC₅₀ values ranging from 5 – 11 μM. Notably, the dose-response curves for these lipopeptides were extremely steep, especially for myropeptin A₁, a characteristic also seen in the National Cancer Institute’s (NCI) 60 cell line panel (Supplementary Information, Figure S44). Myropeptin A₁ (4) exhibited strong inhibitory activity against all tested cancer cell lines in the five-dose assay, conducted at two different occasions. Only four leukemia cell lines and the ovarian cell line OVCAR-8 are less responsive to myropeptin A₁ with LC₅₀ values of > 16 μM (c.f. Supplementary Information, Figure S44).

Figure 8:

Concentration-response curves for the antiproliferative effects of myropeptins 1-4 and mensacarcin as positive control in HCT116: human colon carcinoma and MDA-MB-231: human breast cancer after 24 h. Results represent biological triplicates and two technical replicates as mean ± SEM. Corresponding antiproliferative effects [μM] of myropeptins 1-4 and control mensacarcin against cancer cell lines in an MTT assay after 24 h, ± nd=could not be determined by GraphPad Prism due to the high slope factor and no data points in the steepest part of the dose-response curve.

In vitro hemolytic activity and in vivo toxicity (zebrafish assay)

To study the interactions of lipopeptides with membranes, hemolysis experiments were carried out with sheep erythrocytes and the release of hemoglobin was used to quantify the membrane-damaging properties.⁹ Interestingly, the dodecanoyl containing myropeptins E (3) and A₁ (4) exhibited lower IC₅₀ values (2.0 and 1.5 μM, respectively) compared to the decanoyl myropeptins C (1) and D (2) (7 and 10 μM, respectively) (Figure 9A). We hypothesized that the length of the fatty acid chain modulates hemolysis, which was supported by in vivo toxicity data in a zebrafish assay. Here, compound 4 with the C₁₂ fatty acid chain induced 100% mortality in zebrafish embryos at 10 μM, while compound 1 with a C₁₀ fatty acid side chain required a 5-fold higher concentration (50 μM) for the same effect (Figure 9), which agrees with the observed hemolytic effects of 1–4. Notably, in both assays the dose response curves were exceptionally steep and similar to the cytotoxicity assay curves.

Figure 9:

(A) Hemolytic activities of lipopeptides 1–4. Sheep erythrocytes were incubated with different concentrations at 37 °C for 1 h and OD₄₅₀ was measured. Data are the average of three independent experiments. Error bars represent the standard deviations; (B/C) effects of 4 and 1 on zebrafish embryo mortality at concentrations of 2–25 μM (8 embryos (72 hours post-fertilization (hpf) per concentration).

Ionophoric activity

Due to the amphipathic nature and helical structures of the myropeptins, their effect on membrane stability was examined. In our Fluo-4-AM calcium imaging assay,²⁴ myropeptin A₁ (4) acts as a Ca²⁺-ionophore, as does the positive control ionomycin (Figure 10). Ionomycin induced Ca²⁺-influx starting at 1 μM with a rapid increase of fluorescence onset at higher concentration (4 μM), which resembles the IC₅₀ value of 3.8 μM (Supplementary Information, Figure S45). Myropeptin A₁ (4) causes slight Ca²⁺-influx at 4 μM and rapid influx at 8μM, cells swell instantly once perfusion with 4 begins and many cells rupture; at 16 μM, we most likely observe the lysis of cells which results in the loss of Fluo-4, which might explain the rapid onset but also decline in fluorescence (Figure 10B, Figure S46).

Figure 10:

(A) Fluo-4 fluorescence changes (Ca²⁺-influx) in SK-Mel-5 cells after perfusion of cells with ionomycin, and (B) myropeptin A₁ (4). 60 s no perfusion (baseline recording), then 30 s of perfusion with respective compound treatment solution (μM, in 1× PBS, with 0.5% DMSO, 1 mM CaCl₂ and 1 mM MgCl₂), followed by 30 s of recording, n=3, shaded areas indicate bootstrap standard error.

Influence on the mitochondrial membrane potential

Gichigamin A, a peptaibol rich in βala- and Aib-residues, and its synthetic derivatives selectively disrupt mitochondrial function after penetrating intact cells.¹⁴ Therefore, an intracellular target might be affected first before pore-formation in the cellular membrane is lethal. A transmembrane potential (ΔΨm) across the mitochondrial membrane generates ATP and a voltage-indicating fluorescent dye can be used to visualize mitochondrial health.²⁵ Cells were incubated and imaged with 4, MitoOrange, and CMTMRos dye. The mitochondrial uncoupling reagent carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was used as a positive control. Untreated cells (0.1% DMSO) are healthy and accumulated MitoOrange dye in the mitochondria. FCCP treated cells exhibit less dye accumulation and show signs of apoptosis such as membrane blebbing. Cells treated with 1 μM myropeptin A₁ (4) also show less dye accumulation comparable to FCCP (50 μM), but no membrane blebbing or other signs of apoptosis (Supplementary Information, Figure S47).

Flow cytometry-based apoptosis detection

To determine if the myropeptins induce apoptosis in SK-Mel-5 cells, a flow cytometry approach with FITC-annexin V and propidium iodide (PI) was used.²⁶ Annexin V labelling indicates both early and late stages of apoptosis, whereas PI-stained cells are late phase apoptotic or necrotic. We applied concentrations lower or around the IC₅₀ value, e. g., 0.5–0.7 μM, and did not observe any FITC or PI signal increase or cellular changes after 3–4 h of incubation (Supplementary Information, Figure S48). Only at concentrations of 1 μM or higher can some signs of early apoptosis be observed for cells treated with 4, similarly to the control of 5 μM camptothecin. At this concentration, annexin-PI staining is observed after an incubation time of 10 min, accompanied with a ballooning cell morphology (similarly to gichigamin A treated cells).¹⁴ Taken together with the cell membrane disruption seen in the Fluo-4 AM assay at concentrations > 4 μM of 4, apoptosis may be a downstream event but is likely not the cause of cell death for these fast acting lipopeptides.

Experimental Section

General Experimental Procedures

Myropeptins C, D, and A₁ 1D and 2D NMR spectra were recorded at 600 MHz for ¹H NMR and 150 MHz for ¹³C NMR at 318 K on a Bruker Avance III HD equipped with a 1.7 mm TCI Cryoprobe. Myropeptin E 1D and 2D NMR spectra were recorded at 800 MHz for ¹H NMR (equipped with a 5 mm TCI cryoprobe) and 201 MHz for ¹³C NMR on a Bruker Avance III spectrometer (Billerica, MA, USA) with DMF-d₇ as solvent and internal standard 318 K in a 1.5 mm HTS probe. The solvent signals were referenced to δ_H 2.92 ppm and δ_C 163.15 ppm. Optical rotations were determined on Perkin Elmer 341 polarimeter at the sodium D line (589 nm), 20 s integration time, rotations were averaged over 6 integration periods. Circular dichroism (CD) was measured with a J-1500 spectropolarimeter (Jasco, Oklahoma City, OK, USA) at 100 μg/mL in acetonitrile/water (1/1, v/v) at 20 °C from 180–260 nm with a 1 sec DIT, 0.1 nm data interval, 1.00 nm bandwidth, and 50 nm scanning speed for 2 accumulations.

High-resolution LC-MS experiments for metabolomic profiling were conducted on an Agilent 1290 Infinity II series UPLC coupled to an Agilent 6546 QTOF mass spectrometer with an electrospray ionization (ESI) source (Agilent Technologies, Santa Clara, CA, USA). Chromatography was performed using a Kinetex^® C₁₈ column (50 × 2.1 mm, 2.6, Phenomenex, Torrance, CA, USA) oven temperature was set to 40 °C and the sample injection volume was 5 μL. A binary gradient consisting of MeCN (eluent A) and water (eluent B) (both + 0.1% formic acid (FA)) at a constant flow rate of 500 μL/min was used. The gradient was applied as following: 0–0.25 min, 10% A; 10.00 min, 95% A; 13.00 min, 95% A; 13.1 min, 10% A; 15.00 min 10% A. For data acquisition and subsequent qualitative analysis, MassHunter software (Agilent Technologies, Santa Clara, CA, USA) was used. All parameters regarding the QTOF are listed in Supplementary Information, Table S5.

Data treatment, processing, and statistical analysis for metabolomics

The obtained raw data files were first converted into mzML format using the msconvert tool provided by ProteoWizard.²⁷ The following multistage processing of the data was performed using MZmine 2²⁸ to obtain a feature list (respective mass-to-charge ratios with corresponding retention times and peak areas). Processing steps and applied parameters are given in the Supplementary Information Table S1. The processed feature list was exported into a CSV file for statistical analysis in using the export tool in MZmine 2. Features from the different growth media within the OSMAC approach were normalized (missing values set to zero) and analyzed with principal component analysis (PCA) in RStudio.²⁹ PCA loadings were tested for significance using ANOVA, accompanied by a Fishers LSD test and an FDR correction. Heatmap visualization of mass spectrometric feature abundances were averaged from triplicates and normalized to the highest and lowest intensities and z-scored, using ggplot2 v3.4.0.³⁰

Fungal strain and culture conditions

The fungal strain Myrothecium inundatum CBS 120646 was purchased from the Westerdijk Fungal Biodiversity Centre (CBS, Utrecht, The Netherlands). The strain was maintained on slants of oat-meal agar at 4 °C. A seed culture was prepared on starch-yeast-peptone-seawater (SYP-SW) agar to serve for 3-point-inoculation of 130 agar SYP-SW agar plates. The SYP-SW medium contained 10 g/L starch (modified soluble, Fisher Scientific, Waltham, MA, USA), 4 g/L yeast extract (CRITERION^™, Hardy Diagnostics, VWR, Radnor, PA, USA), 2 g/L peptone (Fisher Scientific), 33.3 g/L instant ocean^®, 15 g/L agar (VWR) and was autoclaved at 121 °C for 15 min; 100 × 15 mm petri dishes were purchased from Fisher Scientific. Inoculated agar plates were incubated for 21 days at 26 °C in an incubator under saturated humidity.

Extraction and isolation

After 21 days of incubation, the agar plates (80) were extracted with EtOAc (VWR) by shaking on a laboratory shaker over night at 110 rpm and filtered through cheesecloth (3 x). The extracts were combined and concentrated in vacuo to obtain the organic extract (2.5 g). Next, solid phase extraction (SPE) was applied by using Chromabond C₁₈ ec columns (10 g, 70 mL, Macherey-Nagel, Dueren, Germany). After equilibrating the solid phase, the organic extract was dissolved in water and loaded on the column, remaining salt from extraction was removed by washing with water. Then, elution was started by applying a MeCN-H₂O gradient from 10–100%. The myropeptins eluted with 70–80%, MeCN was evaporated, and remaining water was lyophilized to pre-concentrate the fractions. Finally, semi-preparative HPLC-UV (210 nm) with a MeCN-H₂O gradient from 60%−100% over a Kinetex^® C₁₈ column (125 × 10 mm, 5 μm, Phenomenex), and a flow rate of 4 mL/min separated the final, purified lipopeptides. Myropeptins were isolated as white amorphous powders at the following retention times and yields: 1, 5 min, 10 mg; 2, 7 min, 4 mg; 3, 9 min, 30 mg; 4, 13 min, 2 mg (after re-purification, lipophilic impurities after the first semi-preparative step).

Determination of absolute configuration of amino acids – Marfey’s analysis

Amino acid configurations of lipopeptides 1-4 were determined by acid hydrolysis and subsequent derivatization with Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, l-FDAA, Thermo Fisher Scientific).³¹ In order to perform coelution experiments with l-FDAA-derivatized amino acids standards, a slightly modified method based on Sica et al. was used.³² Briefly, 0.8–1.0 mg of isolated lipopeptides were dissolved in 500 μL 6 N HCl in a 2 mL glass vial and heated at 110 °C for 24 h. The solution was lyophilized, and the dried hydrolysate was resuspended in 25 μL H₂O, 10 μL of aqueous NaHCO₃ (1 M), 50 μL of l-FDAA (1% w/v in acetone) and stirred at 40 °C for 1 h. Then, the reaction was quenched with 5 μL of HCl (2 N) and the solutions were lyophilized and prepared for LC-DAD-HRMS analysis by dissolving in MeCN and dilution of 1:10 in starting conditions (10% MeCN + 0.1% FA). Amino acid standards (0.2–0.3 mg of l-alanine, d-alanine, β-alanine, l-proline, d-proline and α-aminoisobutyric acid, all Fisher Scientific) were prepared in 50 μL H₂O, 20 μL of NaHCO₃ (1 M) and 100 μL of l-FDAA (1% w/v in acetone) and stirred at 40 °C for 1 h. The lyophilized reaction mixture was dissolved as described above and analyzed by high-resolution LC-MS.

• Myropeptin C (1): white amorphous solid; [α]²⁰d +4.6 (c 0.8, MeOH); HRMS (ESI) m/z 911.5476, [M-2H]²⁻ calc. for C₈₇H₁₄₈N₂₀O₂₂, 911.5466; ¹H and ¹³C NMR, c.f. Table 1. UV/Vis (MeCN): λ_max = 210 nm.

• Myropeptin D (2): white amorphous solid; [α]²⁰d +3.1 (c 0.2, MeOH); HRMS (ESI) m/z 918.5544, [M-2H]²⁻ calc. for C₈₈H₁₅₀N₂₀O₂₂, 918.5544; ¹H and ¹³C NMR, c.f. Table 2. UV/Vis (MeCN): λ_max = 210 nm.

• Myropeptin E (3): white amorphous solid; [α]²⁰d +13.0 (c 0.4, MeOH); HRMS (ESI) m/z 932.5701, [M-2H]²⁻ calc. for C₉₀H₁₅₄N₂₀O₂₂, 932.5701; ¹H and ¹³C NMR, c.f. Table 3. UV/Vis (MeCN): λ_max = 210 nm.

• Myropeptin A₁ (4): white amorphous solid; [α]²⁰d +15.5 (c 1.2, MeOH); HRMS (ESI) m/z 925.5632, [M-2H]²⁻ calc. for C₈₉H₁₅₂N₂₀O₂₂, 925.5622; ¹H and ¹³C NMR, c.f Table S2. UV/Vis (MeCN): λ_max = 210 nm.

Cell culture assays

Colon cancer cells (HCT-116) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Melanoma cells (SK-Mel-5) were obtained from the National Cancer Institute (NCI) cell line repository (Frederick, MD, USA). Triple-negative breast cancer cells (MDA-MB-231) were a kind gift from Dr. April Risinger (UTHSCSA). Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffer saline (PBS), trypsin/EDTA (0.25%/2.21 mM) and penicillin/streptomycin solution were obtained from Thermo Fisher Scientific. Fetal bovine serum (FBS) was obtained from R&D systems.

HCT-116, SK-Mel-5, and MD-MB-231 cells were cultivated in DMEM, and PC-3 cells in RPMI 1640 each supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL). The cell lines were incubated at 37 °C, 5% CO₂. Cells were plated into 96-well plates at different densities (HCT-116: 7000 cells/well; SK-Mel-5: 6000 cells/well; MDA-MB-231: 8000 cells/well). The passage number for cells used in the experiments never exceeded 15. All cell lines were tested mycoplasma- negative by real time PCR (Myco Solutions mycoplasma detection kit, Akron Biotech, Boca Raton, FL, USA).

Growth inhibition and cytotoxicity in the different cell lines were measured by the reduction of the tetrazolium salt MTT by metabolically active cells. Cells were plated into 96-well plates and maintained overnight before treatment was started with the addition of mensacarcin to each well. After the designated time, MTT (5 mg/ml in PBS) was added to each well at a final concentration of 0.5 mg/ml. The plates were incubated for 2 h at 37 °C. The medium was removed, cells were lysed, and the purple formazan product was solubilized by the addition of 50 μl of DMSO. Absorbance was measured at 550 nm with a microplate reader (Synergy HTX, Biotek, Winooski, VT, USA). Metabolic activity of vehicle-treated cells (0.5% DMSO unless otherwise stated) was defined as 100% cell growth.

Antimicrobial assays

For antibacterial activity in cell-based assays, established protocols were followed.^{33, 34} Gram-positive bacteria, including Enterococcus faecium (ATCC 49032), Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (ATCC BAA-41), multidrug-resistant Staphylococcus aureus (ATCC BAA-44) as well as two Gram-negative pathogens, Pseudomonas aeruginosa (ATCC 15442) and Escherichia coli (ATCC 8739), and Candida auris (CDC B11903) were tested. Vancomycin, kanamycin, ampicillin, and caspofungin were used as positive controls. DMSO served as negative control. For single-dose assay, compounds, mixtures, and antibiotic controls were tested at 125 μg/mL. Microbial growth rates were measured after 16 h by absorbance at 620 nm using a Biotek Synergy 96-well plate reader. All human pathogens used in the study were acquired from the American Type Culture Collection (ATCC).

Hemolysis assay

Hemolytic activity was determined by using defibrinated sheep blood (VWR). 1 mL blood was washed 3 × with PBS (1×) by gentle vortexing, followed by a centrifuge step at 4000 rpm for 5 min and discarding of the supernatant. The remaining cells were diluted (1:500) to achieve ~8×10⁷ cells/mL. 0.1% triton X-100 (Fisher Scientific) served as a positive control. Lipopeptides 1–4 stock solutions were prepared in 20-fold concentrations in PBS (1×) + 0.5% DMSO. Then, 190 μL of blood suspension and 10 μL sample stock solutions were mixed and incubated for 1 h at 37 °C. Subsequently, the samples were centrifuged at 4000 rpm for 5 min and 100 μL of the supernatant was transferred to a 96-well plate and OD₄₅₀ was determined with a microplate reader (Synergy HTX, Biotek).

Zebrafish assay – in vivo toxicity

Zebrafish adults (Danio rerio, AB strain) were reared under standard conditions (28 °C, 14:10 light:dark) and crossed to produce embryos. Embryos with intact chorions were transferred into 96-well plates (1 embryo/well) at 24 hours post-fertilization (hpf) using 50 μL medium (HEPES buffered E3: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄, 1mM HEPES, titrated to 7.2 pH). Compounds myropeptins C (1) and A₁ (4) were dissolved in DMSO, serially diluted using HEPES buffered E3, and then added to well plates (final conc for (1): 0, 2.0, 5.0, 15, 25, and 50 μM in 1% DMSO; final conc for (4): 0, 2.0, 5.0, 10, 15, and 25 μM in 1% DMSO; total volume in well: 150 μL). Plates were incubated for 48 h (72 hpf; 28 °C, 14:10 light:dark) after which the number of surviving embryos were counted. Each assay examined 8 embryos per concentration, and the assay was replicated 3 times using animals from 3 clutches. The results were similar across replicates and so survivorship is plotted from pooled data, with error bars representing 68% confidence intervals (like SEM) found by fitting results for each concentration to a binomial distribution (MATLAB R2020b). EC₅₀ values were calculated by fitting unpooled survivorship values to the Hill equation, and 95% confidence intervals were calculated using a bootstrap analysis. Protocols were approved by the University of Florida Institutional Animal Care and Use Committee.

Fluo-4-AM assay for Ca²⁺-influx

SK-Mel-5 were cultured as described in the cell culture section and 20,000 cells/well were seeded in a black 96-well glass bottom plate overnight. After removing the medium, cells were incubated in the calcium indicator Fluo-4 AM (Invitrogen, Thermo Fisher Scientific; 100 μL of 3.5 μM Fluo-4 AM in 1x PBS supplemented with 1 mM CaCl₂, 1 mM MgCl₂, 0.025% DMSO, and 0.005% Pluronic F-127) for 30 min at 37 °C in the dark. The dye loading solution was removed, then cells were washed with buffer and left in buffer (100 μL 1× PBS with 1 mM CaCl₂ and 1 mM MgCl₂). Cells were imaged using epifluorescence microscopy (Olympus IX71, objective: 20x/0.45 LUCPlanFLN, camera: Hamamatsu C10600–10B-H [0.9 frame/sec, 1 sec exposure], light: Sutter LB-LS/17, excitation: 482/35nm with 1.0 ND filter, emission: 536/40nm). Baseline fluorescence was recorded for 30 s, then test compounds were perfused directly onto cells for 30 s using a rapid solution changer (BioLogic RSC-160), and then the fluorescence was recorded for an additional 30 s. Test compounds included Myropeptin A₁ (4) at 2.0, 4.0, 8.0 and 16 μM in buffer. Ionomycin (Cayman Chemicals, Ann Arbor, MI, USA) was used as a positive control and was examined at 0.050, 1.0, 4.0, and 8.0 μM in buffer with 0.5% DMSO. All experiments were run in triplicates. Responses were analyzed by manually drawing regions of interest around cells, and the ΔF/F for individual cells was computed by subtracting background fluorescence then calculating the difference between the instantaneous fluorescence and baseline fluorescence, normalized by the baseline fluorescence. Responses were similar between replicates so recordings from all cells were pooled, and the results are presented using robust statistics (median ± SEM, where the SEM is estimated using a bootstrap analysis).

Confocal microscopy

MitoOrange^™ CMTMRos (ABP Biosciences, Beltsville, MD, USA) was purchased in 1 mg/mL DMSO. Working solution was made fresh from stock prior the experiments at 200 μM in PBS (1×). Cover slips (VWR, Radnor, PA, USA) were washed with 70% ethanol, dried and coated with 0.1% poly-l-lysine solution (Fisher Scientific). After incubation of 4 h, the coating solution was removed and slips were rinsed with sterile water, dried, and sterilized under UV light for 15 min. Coated cover slips were then placed into a 6-well plate and no more than ~1 × 10⁶ cells/cm² were seeded onto the cover slip surface, cells were incubated at 37 °C, 5% CO₂ before treatment. After 24 h, the medium was removed, and cells washed once with warm PBS (1×). Then, 1 mL treatment solution was added, containing a final concentration of 2 μM MitoOrange, 0.1% Hoechst 33342 and either 0.1% DMSO (control), 50 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, positive control) or 1 μM myropeptin A₁ (4). The incubation time was 30 min, 37 °C, 5% CO₂. Afterward, the solution was removed, and cells washed twice with warm PBS (1×, 1mM CaCl₂, MgCl₂). The slip was mounted on a glass microscope slide and cells imaged with a Zeiss LSM 710 confocal microscope using a 20x objective and transmission light, MitoOrange CMTMRos, and Hoechst 33342 filter sets.

Flow cytometry analysis

For detection of apoptosis a kit was used according to the manufacturer instructions (Annexin V Apoptosis Detection Kit I, BD Biosciences, San Diego, CA, US). Melanoma (SK-Mel-5) cells were seeded (1 × 10⁶ cells) in T25 culture flasks. After 24 h incubation (37 °C, 5% CO₂), the medium was removed, and cells were treated with controls and myropeptin A₁ (4, 1 μM, 4 μM) for 3 h. Treatment solutions were prepared in cell medium, 0.5% DMSO served as negative control (vehicle) and 5 μM camptothecin as positive control for early apoptosis. After detached cells (~1.5 × 10⁶ cells) from each T25 flask after trypsin treatment. Then, the cells were centrifuged at 700 × g, 3 min, RT. The obtained cell pellet was washed with PBS (1×) and resuspended in 1 mL annexin V binding buffer (1×) and gently mixed. 100 μL of the latter solution were transferred to a new sample tube and 5 μL propidium iodide as well as 5 μL FITC annexin V were added and incubated for 15 min at RT in the dark. Afterward, 400 μL of annexin V binding buffer (1×) was added to each tube and samples were analyzed with a BD FACSMelody^™ (BD Biosciences, San Diego, CA, US) within 1 h. 20,000 events were recorded for each sample and the BD FACSChorus^™ software was used.