Meliponamycins: Antimicrobials from Stingless Bee-Associated Streptomyces sp

Abstract

Social insects establish complex interactions with microorganisms, some of which play defensive roles in colony protection. The important role of pollinators such as the stingless bee Melipona scutellaris in nature encouraged us to pursue efforts to study its associated microbiota. Here we describe the discovery of two novel cyclic hexadepsipeptides, meliponamycin A (1) and meliponamycin B (2), from Streptomyces sp. ICBG1318 isolated from M. scutellaris nurse bees. Their structures were established by interpretation of NMR and MS data, and the absolute configuration of the constituent amino acids was determined by the advanced Marfey's method. Compounds 1 and 2 showed strong activity against the entomopathogen Paenibacillus larvae and human pathogens Staphylococcus aureus and Leishmania infantum.

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Meliponamycins: Antimicrobials from Stingless Bee-Associated
Streptomyces sp.
Carla Menegatti,
⊥
Vitor Bruno Lourenzon,
⊥
Diego Rodríguez-Hernández,
Weilan Gomes da Paixão Melo, Leonardo Luiz Gomes Ferreira, Adriano Defini Andricopulo,
Fabio Santos do Nascimento, and Mônica Tallarico Pupo*
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sıSupporting Information
ABSTRACT: Social insects establish complex interactions with
microorganisms, some of which play defensive roles in colony
protection. The important role of pollinators such as the stingless
bee Melipona scutellaris in nature encouraged us to pursue efforts to
study its associated microbiota. Here we describe the discovery of
two novel cyclic hexadepsipeptides, meliponamycin A (1) and
meliponamycin B (2), from Streptomyces sp. ICBG1318 isolated
from M. scutellaris nurse bees. Their structures were established by
interpretation of NMR and MS data, and the absolute
configuration of the constituent amino acids was determined by
the advanced Marfey’s method. Compounds 1and 2showed
strong activity against the entomopathogen Paenibacillus larvae and
human pathogens Staphylococcus aureus and Leishmania infantum.
Nutritional, metabolic, and defensive symbiotic partner-
ships between insects and microbes are widespread in
nature.
1
The defensive bacterial symbionts, those that benefit
the host by protecting it against pathogens,
2
have been
investigated as sources of novel bioactive natural products from
various insects, such as fungus-growing ants, termites, and
beetles.
3,4
Bees are an important and large group of insects that
pollinate a broad range of crops and wild plants and are also
economically important for honey production.
5
Climate
change and the extensive use of pesticides in agriculture may
be responsible for the decline of bee populations.
6−8
The loss
of pollinators directly impacts worldwide biodiversity, food
production, and noncrop species.
9
Bees also engage in different
types of microbial symbiosis. The understanding of the bee
microbiome and its impact on insect fitness can contribute to
the design of preservation policies. Bacterial symbionts of
honeybee and bumble bee gut and brood cells seem to protect
the hosts against bacterial pathogens, such as Paenibacillus
larvae (which causes American foulbrood disease) and
Melissococcus plutonius (which causes European foulbrood
disease), and against the parasite Crithidia bombi.
10,11
Stingless
bees (Apidae: Meliponini) are a large group of bees with more
than 500 species described, 300 of them native to Brazil.
12
As
part of an International Cooperative Biodiversity Group
(ICBG) between Brazil and the USA,
13
we have been studying
the microbial symbionts of social insects, including stingless
bees, as sources of biologically active small molecules. We
recently described the molecular basis of the nutritional
symbiosis between the fungus Zygosaccharomyces sp. and the
stingless bee Scaptotrigona depilis. The fungus grows in brood
cells and provides a steroidal precursor for the larval
pupation.
14
Monascus ruber and Candida sp. are also found in
the brood cells of S. depilis, and their low molecular weight
metabolites modulate Zygosaccharomyces growth.
15
We have
also identified natural products involved in defensive symbiosis
between bacteria and stingless bees. The bacterium Paeniba-
cillus polymyxa was isolated from the larval food of the stingless
bee Melipona scutellaris and produced L-(−)-phenyllactic acid
together with a family of cyclic lipodesipeptides, all active
against entomopathogens Beauveria bassiana and P. larvae.
16
Actinobacteria were recovered from M. scutellaris bees.
Micromonospora sp. ICBG1321 and Streptomyces sp.
ICBG1323 produced several anthracyclines and lobophorins,
respectively, some of them with strong inhibitory activity
against P. larvae.
17
Streptomyces sp. ICBG1318 was isolated from the cuticle of
M. scutellaris nurse bees and showed activity against P. larvae in
antagonistic assays.
17
Here we describe the isolation, structural
Special Issue: Special Issue in Honor of Jon Clardy
Received: October 14, 2019
Published: February 19, 2020
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© 2020 American Chemical Society and
American Society of Pharmacognosy 610
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identification, and antimicrobial activities of two novel cyclic
hexadepsipeptides (1and 2) from this actinobacteria.
■RESULTS AND DISCUSSION
Streptomyces sp. ICBG1318 (Figure S1) was grown in ISP-2
agar and extracted with EtOAc and MeOH. The EtOAc extract
was selected for bioguided fractionation due to its higher
inhibitory activity against P. larvae ATCC9545. Antibacterial
activity was assessed against the honeybee pathogen P. larvae
since there is no natural pathogen described for the stingless
bee M. scutellaris. The bioactive SPE fraction obtained in
MeOH was further fractionated by reversed-phase HPLC,
leading to the isolation of compounds 1and 2(Figure S2).
Compound 1was isolated as a colorless powder having the
molecular formula C36H61N7O12 based on HRESIMS analysis
(Figure S3). The 1H NMR spectrum (Figure S4) indicated the
presence of six protons attached to heteroatoms (Table 1).
The 1H NMR spectrum displayed one downfield singlet
proton at δH9.21 and two amide hydrogens, one as a doublet
at δ7.44 (d, J= 9.3 Hz) and the other as a broad singlet at δH
7.21. Analysis of gHMBC (Figure S5) and gHSQC (Figure S6)
spectra allowed the deduction of seven amide or ester carbonyl
carbons, four oxygen-bearing sp3carbons, seven nitrogen-
bearing sp3carbons, and 16 aliphatic carbon signals.
Interpretation of NMR spectra assigned six amino acid-derived
substructures and one polyketide-derived substructure. These
include a piperazic acid unit (Pip-1), β-hydroxyleucine (β-OH-
Leu), N-hydroxyglycine (N-OH-Gly), N-methylglycine (N-
Me-Gly), N-methylalanine (N-Me-Ala), glycine (Gly), and a
pyran-bearing acyl side chain. Based on the predicted
molecular formula and NMR data analysis, compound 1is a
cyclic peptide. The presence of the N-OH-Gly was confirmed
by the deshielded chemical shifts of C-9 and H-9 (δC51.1 and
δH5.27 and 3.90) compared to those of glycine and by the N-
OH proton signal at δH9.21. The sequence of the amino acid
units was established by analyses of gHMBC correlations.
HMBC correlations from H-17 (δH4.86, d, J= 9.6 Hz) and
both methylene H-2 (δH4.69, d, J= 17.5 Hz and δH3.43, d, J
= 17.5 Hz) to the ester carbonyl C-1 (δC167.3) connected β-
OH-Leu and N-Me-Gly units. N-Me-Gly was linked to the Gly
unit by the HMBC correlations from N-Me (δH3.12, s) and
both H-2 (δH4.69, 3.43) and H-4 (δH5.04, 3.63) to the amide
carbonyl C-3 (δC170.9). Gly was connected to N-Me-Ala by
HMBC correlations from the amide 4-NH (δH7.21) of the
glycine residue and from H-6 (δH4.23, brd J= 5.5 Hz) to C-5
(δC172.8). The linkage between N-Me-Ala and N-OH-Gly
residues was inferred by HMBC correlations from N-Me (δH
2.78, s) and from H-9 (δH5.27 d, J= 15.5 Hz and δH3.90 d, J
= 15.5 Hz) to the amide carbonyl C-8 (δC166.8). N-OH-
glycine was linked to piperazic acid; HMBC correlations were
observed from H-11 (δH5.32, d, J= 4.7 Hz) and H-12b (δH
1.87, m) to C-10 (δC171.2). Finally, β-OH-Leu protons H-16
(δH6.09, d, J= 9.3 Hz) and H-17 (δH4.86, d, J= 9.6 Hz) and
piperazic acid 14-NH (δH4.90, d, J= 13.5 Hz) showed HMBC
correlations to the amide carbonyl C-15 (δC171.9), leading to
the construction of the 19-membered cyclic hexapeptide
fragment. The second structural feature of 1was identified
to be a polyketide side chain. The COSY spectrum (Figure S7)
aided the assignment of aliphatic protons. The two methyl
protons H-31 and H-32 (δH0.89 and 0.82) showed
correlations to H-30 (δH1.67). Methyl protons H-34 (δH
0.89) are coupled to H-33 (δH1.41). The deshielded methine
H-27 (δH3.63, d, J= 15.6 Hz) is coupled to H-26 (δH1.35)
and H-33 (δH1.41). The methyl proton H-28 (δH1.35)
showed HMBC correlations to the amide carbonyl C-21 (δC
177.5), to the oxycarbon C-22 (δC77.4), and to the hemiketal
carbon C-23 (δC98.4). The absence of a hydroxy proton signal
associated with C-27 suggested that it has an ether linkage,
which was attributed to the presence of a heterocyclic system.
The attachment of the polyketide unit to the peptide moiety
was established on the basis of HMBC correlations of the
amide 16-NH (δH7.44, d, J= 9.3 Hz) and the methyl proton
H-28 (δH1.35) to the amide carbonyl C-21 (δC177.5), leading
to the overall planar structure of meliponamycin A (1)(Figure
S8).
Compound 2was isolated as a colorless powder having the
molecular formula C37H63N7O12 based on HRESIMS analysis
(Figure S9). Analysis of 1D and 2D NMR (Figures S10−S13)
data confirmed that compound 2was very similar to 1(Table
1). The main difference of 1from 2was the substituent on the
tetrahydropyran that changes from an isobutyl to a 2-
methylbutyl residue (C-29−C-33). This was confirmed
together with the sequence of amino acid units by key
correlations observed in the gHMBC spectrum, which
completed the planar structure of meliponamycin B (2)
(Figure S14).
To determine the absolute configuration of the amino acids,
compounds 1and 2were submitted to hydrolysis in 6 N HCl
at 110 °C for 24 h followed by derivatization with the
advanced Marfey reagents (L-FDLA and L,D-FDLA).
18
Both
compounds were found to have amino acids with identical
configurations. The LC-MS analyses of the N-Me-alanine
supported this having the L-configuration since it is known that
L-FDLA derivatives elute before D-FDLA for this amino acid.
19
The products derived from piperazic acid were determined to
have L-configuration based on the retention times of reported
L-FDLA derivatives of synthetic L- and D-piperazic acids.
20
The
absolute configuration of the amino acid β-hydroxyleucine was
determined to be L-configurated based on comparison with L-
and D-derivatives of the standard (2S,3S)-3-hydroxyleucine.
Therelativeconfigurations of the acyl side chain of
compounds 1and 2were proposed based on comparison of
NMR data of similar compounds previously reported, such as
dentigerumycin, polyoxypeptin, and aurantimycins (Figure
S15).
20−22
Compounds 1and 2showed MIC values of 0.55 μM (0.43
μg/mL) and 0.54 μM(0.43μg/mL) against P. larvae,
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respectively, a higher activity than the positive control
tetracycline, MIC 7.76 μM (3.54 μg/mL) (Table 2).
Tetracyclines have been used to fight P. larvae for over 50
years, and new antibiotic alternatives are needed to control
resistant strains. Among 27 drugs screened for antibiotic
activity against P. larvae, rifampicin presented the highest
activity; however its use in agriculture is unlikely since
rifampicin is the first-line tuberculosis therapy.
23,24
The
antibacterial potential of compounds 1and 2against the
entomopathogen P. larvae could be related to a protective
symbiosis between Streptomyces sp. ICBG1318 and the
stingless bee M. scutellaris. We recently found that
Table 1. NMR Spectroscopy Data (500 MHz, CDCl3) for Compounds 1 and 2
meliponamycin A (1) meliponamycin B (2)
position δC, type δH(Jin Hz) HMBC
a
δC, type δH(Jin Hz) HMBC
a
1 167.3, C 167.3, C
2 51.7, CH24.69, d (17.5) 1, 3 51.7, CH24.69, d (17.6) 1, 3
3.43, d (17.5) 3.43, d (17.6)
2 N-CH336.6, CH33.12, s 3, 2 36.6, CH33.12, s 3, 2
3 170.9, C 170.9, C
4 42.7, CH25.04, m 3, 5 42.7, CH25.03, m 3, 5
3.63, m 3.63, m
4 NH 7.21, brs 3, 5 7.21, brs 3, 5
5 172.8, C 172.9, C
6 55.3, CH 4.23, brd (5.5) 5, 7, 8, N-CH3(6) 55.9, CH 4.23, brd (6.2) 5, 7, 8, N-CH3(6)
6 N-CH329.3, CH32.78, s 6, 8 29.3, CH32.78, s 6, 8
7 15.1, CH31.61, m 5, 6 15.2, CH31.62, m 5, 6
8 166.8, C 166.9, C
9 51.1, CH25.27, d (15.5) 8 51.1, CH25.27, d (15.4) 8
3.90, d (15.5) 3.90, d (15.4)
9 N-OH 9.21, s 9.22, s
10 171.2, C 171.2, C
11 48.4, CH 5.32, d (4.7) 10, 12, 13 48.4, CH 5.32, d (4.7) 10, 12, 13
12 24.1, CH22.17, m 10 24.0, CH22.17, m
1.87, m 1.87, m
13 21.4, CH21.48, m 21.3, CH21.47, m
1.57, m 1.58, m
14 46.9, CH22.66, m 46.9, CH22.66, m
3.13, m 3.13, m
14 NH 4.90, d (13.5) 15 4,90, d (13.3) 15
15 171.9, C 172.0, C
16 46.5, CH 6.09, d (9.3) 15 46.6, CH 6.09, d (9.2) 15
16 NH 7.44, d (9.3) 21 7.44, d (9.2) 21
17 80.1, CH 4.86, d (9.6) 15 80.1, CH 4.86, d (9.6) 15
18 30.2, CH 1.74, m 17 30.2, CH 1.74, m
19 19.2, CH30.87, brs 17, 18, 20 19.3, CH30.88, brs 17, 18, 20
20 18.4, CH31.10, d (4.9) 17, 18, 19 18.4, CH31.09, d (4.9) 17, 18, 19
21 177.5, C 177.6, C
22 77.4, C 77.5, C
22 OH 3.49, s 22 3.50, s 22
23 98.4, C 98.4, C
23 OH 6.13, s 22, 23, 24 6.13, s 22, 23, 24
24 27.3, CH21.71, m 27.7, CH21.72, m
25 24.5, CH21.66, m 23 24.5, CH21.66, m 23
1.51, m 1.52, m
26 35.1, CH 1.35, m 34.8, CH 1.37, m
27 75.3, CH 3.63, m 75.3, CH 3.63, m
28 20.0, CH31.35, s 21, 22, 23 20.1, CH31.35, s 21, 22, 23
29 40.5, CH21.07, m 31, 32 38.1, CH21.01, m 26, 30
0.94, m
30 24.3, CH 1.67, m 31, 32 30.9, CH 1.39, m
31 24.4, CH30.89, brs 30.9, CH21.19, m
32 21.3, CH30.82, d (4.3) 29, 31 18.5, CH30.80, d (4.4) 29, 31
33 23.9, CH21.41, m 11.7, CH30.86, brs 31
34 8.4, CH30.89, brs 27, 33 23.8, CH21.37, m
35 8.4, CH30.89, brs 27, 34
a
HMBC correlations, proton(s) stated to the indicated carbon.
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spirotetronate macrolides, known as lobophorins K and B,
were also strongly active against P. larvae.
17
These polyketides
were biosynthesized by Streptomyces sp. ICBG1323 associated
with M. scutellaris forager bees. Therefore, actinobacteria
associated with stingless bees represent a promising source for
the discovery of antibacterial natural products against P. larvae.
Compounds 1and 2were also tested against the protozoan
parasite Leishmania infantum and Staphylococcus aureus, as part
of our goals of finding natural product mediators of bacterial−
insect symbiosis that can also be active against human
pathogens.
13,25
Compounds 1and 2showed good activity
against human pathogenic S. aureus with MIC values of 2.20
μM (1.72 μg/mL) and 1.08 μM (0.86 μg/mL) (Table 2).
Compounds 1and 2showed high antiprotozoal activity against
intracellular amastigotes of L. infantum with IC50 values of 2.19
and 1.03 μM, respectively, comparable to the positive control
miltefosine (IC50 value of 2.40 μM). Unfortunately, com-
pounds 1and 2did not show safe selectivity indexes when
tested against host macrophages of L.infantum (Table 2).
Antifungal natural products from Streptomyces strains involved
in defensive symbiosis of fungus-growing ants also showed
antileishmanial activity with good selectivity indexes.
25
The
activity of meliponamycins against human pathogens corrob-
orates the approach of studying signaling molecules from insect
microbiomes as sources of natural products hits.
3
Meliponamycin A (1) and meliponamycin B (2), two novel
cyclic hexadepsipeptides, have similar amino acids and
polyketide-derived side chains to the related depsipeptides
aurantimycins, polyoxypeptins, variapeptin, and dentigerumy-
cin.
20−22,26
However, compounds 1and 2present an
unreported sequence of amino acids in the core structure
and only one piperazic acid unit. Dentigerumycin, produced by
Pseudonocardia sp. associated with fungus-growing ants, was
previously found to have selective antifungal activity against a
specialized fungal pathogen of Attine ants.
20
Similarly, the
antibacterial activity of 1and 2against P. larvae suggests that
Streptomyces sp. ICBG 1318 might play a symbiotic protective
role in M. scutellaris colonies.
■EXPERIMENTAL SECTION
General Experimental Procedures. 1D and 2D NMR spectra
were recorded in a 500 MHz spectrometer (DRX-500, Bruker UK,
Coventry, UK) using deuterated chloroform and Shigemi tubes. The
MS system was a quadrupole time-of-flight instrument (UltrOTOF-
Q, Bruker Daltonics, Billerica, MA, USA) equipped with an ESI ion
source. The high-resolution mass spectrometry (HRESIMS) of
compounds 1and 2was acquired in positive ion mode using a
capillary voltage of 3900 V, dry gas flow of 4 L h−1, and nitrogen as
nebulizer gas. The LC-MS was carried out on a Bruker Daltonics
high-pressure liquid chromatograph coupled to a diode array UV
detector and a mass spectrometer with electrospray ionization source
(ESI) and micrOTOF (time of flight) detector. The chromatographic
analysis was performed on a C18 analytical column (Phenomenex
Gemini, 4.6 mm, 250 mm, 5 μm) using a flow rate of 0.7 mL/min, an
injection volume of 20 μL, and a solvent system with aqueous (0.1%
formic acid) acetonitrile (5% to 100% acetonitrile for 30 min and 20%
to 70% of acetonitrile for 50 min). The oven temperature was 35 °C.
The ionization source parameters were as follows: end plate offset
voltage, 500 V; capillary voltage, 3500 V; nebulizer at 5.5 bar; drying
gas flow rate of 10.0 L/min; drying gas temperature, 220 °C; for
positive mode ionization. The spectra rate of 2.0 Hz and mass range
of 300 to 1000 m/zwere maintained throughout the analyses. The
data analysis software of the Bruker Daltonics instrument was used to
analyze the metabolic profile and UV spectrum data. The crude
extract from solid media was initially fractionated using a C18 solid-
phase extraction column (Discovery DSC18 60 mL tube, 10 g).
Fractions were eluted with 100 mL in a step gradient of H2O, and
H2O/MeOH (25%, 50%, 75%, 100%), finishing with 100% acetone
and concentrated to dryness. Fraction F-1 (34.7 mg), eluted with
100% methanol, was further purified by semipreparative HPLC. The
instrumentation for the HPLC analysis consisted of an HPLC system
(Shimadzu, Kyoto, Japan), comprising an LC-20AD solvent pump, a
CBM-20A system controller, a CTO-20A column oven, a SIL-20A
injector, a SPD-M20A diode array detector (DAD), an ELSD-LTII
detector, and the LabSolution software for data acquisition. The
HPLC analyses were performed at 1.0 mL min−1using a reversed-
phase C18 analytical column (Phenomenex Gemini, 4.6 mm, 100 mm,
2.7 μm) and a gradient solvent system with aqueous (0.1% formic
acid) acetonitrile (10% to 100% acetonitrile) for 30 min. The
instrumentation for the semipreparative HPLC consisted of an HPLC
system (Shimadzu, Kyoto, Japan), comprising an LC-6AD solvent
pump, a CBM-20A system controller, a CTO-20A column oven, a
SIL-20AF injector, an SPD-M20A DAD, an FCR-10A collector, and
LabSolution software for data acquisition. The HPLC extract
purification of compounds 1and 2was performed at 3.0 mL min−1
with a reversed-phase C18 semipreparative column (Phenomenex
Gemini, 10.0 mm, 250 mm, 5 μm) using a gradient solvent system
with aqueous (0.1% formic acid) acetonitrile (40% to 100%
acetonitrile) for 21 min. A 150 μL amount of F-1 was injected at 3
mg mL−1, and eluted samples were monitored at 210 nm.
Compounds 1and 2were eluted at 15.9 and 16.8 min, yielding 1.8
and 2.0 mg, respectively.
Bacterial Isolation, Identification, and Culturing. The
bacterium coded as ICBG1318 was isolated from M. scutellaris
nurse bees and identified as Streptomyces sp. as previously reported.
17
The DNA sequence was deposited in the NCBI-GenBank under
accession number MK608318. Permits for collection and research on
genetic resources were issued by the Brazilian government (SISBIO
authorization 46555-5, CNPq process 010936/2014-9). The actino-
bacteria was cultivated in 100 Petri dishes (150 mm diameter)
containing 70 mL of ISP-2 agar each (4 g of yeast extract, 4 g of
dextrose, 10 g of malt extract, 20 g of agar per liter of H2O) at 30 °C
for 14 days. Each plate was inoculated with 500 μL of the pure
actinobacteria preculture in ISP-2 medium.
Extraction and Isolation. After growth on solid media, the
cultured agar was cut in small pieces and soaked in EtOAc overnight.
The suspension was sonicated, filtered, and concentrated under
vacuum, yielding 307 mg of EtOAc extract. The remaining culture in
Table 2. Antimicrobial Activities of Meliponamycins A (1) and B (2)
L. infantum
a
P. larvae S. aureus
compound IC50 (μM) intracellular amastigotes CC50 (μM) THP-1
b
selectivity index
c
MIC (μg/mL) MIC (μg/mL)
12.19 ±0.25 1.05 ±0.05 0.47 0.43 1.72
21.03 ±0.08 0.70 ±0.03 0.67 0.43 0.86
miltefosine 2.40 ±0.22
doxorubicin 1.80 ±0.16
tetracycline 3.45 0.05
a
Data are shown as mean ±SD (n= 2 biological replicates).
b
THP-1 human leukemia macrophages (host cells of L.infantum).
c
Selectivity index =
CC50 THP-1/IC50 intracellular amastigotes
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agar was then extracted overnight with MeOH, sonicated, filtered, and
concentrated under vacuum, yielding 13.3 g of MeOH extract. The
EtOAc extract was first fractionated by column C18 solid-phase
extraction (SPE) in a step gradient of H2O, H2O/MeOH (25%, 50%,
75%, and 100% MeOH), and acetone, yielding six fractions. The
fraction eluted with 100% MeOH (34.7 mg) was further purified by
HPLC using gradient elution consisting of aqueous (0.1% formic
acid) acetonitrile (40% to 100% acetonitrile) to afford 1.8 mg of
compound 1and 2.0 mg of compound 2.
Compound 1:1H and 2D NMR data, Table 1; HRESIMS [M +
Na]+m/z806.4252, calcd [M + Na]+at 806.4276 (error −2.97). UV
λmax 194 nm.
Compound 2:1H and 2D NMR data, Table 1; HRESIMS [M +
Na]+m/z820.4396, calcd [M + Na]+at 820.4432 (error −4.38). UV
λmax 194 nm.
Acid Hydrolysis of Compounds 1 and 2. Solutions of
compound 1(600 μg) and compound 2(400 μg) in 6 N HCl
(500 μL) were hydrolyzed at 110 °C for 24 h and dried under
vacuum. The dry material was resuspended in 500 μL of distilled
water three times and dried to remove residual acid.
Determination of the Absolute Configuration of the Amino
Acid Units in Compounds 1 and 2. The advanced Marfey’s
method was applied using L-FDLA and L,D-FDLA (FDLA is 2,4-
dinitro-5-fluorophenyl leucineamide) synthesized as previously
reported and kindly provided by Jiaxuan Yan (Bugni’s laboratory,
UW−Madison).
27
The reagents were used to generate hydrolysate-
derived diastereomers. The hydrolysates of compounds 1and 2were
divided into two portions, and each portion was mixed with 1 N
NaHCO3(100 μL). A 50 μL amount of L-FDLA (1 mg/mL in
acetone) and L,D-FDLA (1 mg/mL in acetone) and the reaction
mixtures were heated at 80 °C for 3 min. The reactions were
neutralized with 50 μL of 2 N HCl. Aqueous acetonitrile (1:1) was
added to the solutions, which were analyzed by LC/MS. The
hydrolysate derivatives of compound 1and 2were analyzed by LC-
MS using a C18 analytical column (Phenomenex Gemini, 4.6 mm, 250
mm, 5 μm) and a flow rate of 0.7 mL/min. The solvent system with
aqueous (0.1% formic acid) acetonitrile (5% to 100% of acetonitrile)
was used to determine the absolute configuration of methyl-alanine by
comparing the retention times of L-and D-derivatives. The L-FDLA
and D-FDLA derivatives of methyl alanine (m/z398) were eluted at
retention times of 12.0 and 12.1 min, respectively. Both methyl
alanines of compound 1and 2were determined to have an L-
configuration since it is known that the L-derivative elutes first in this
condition. Another LC-MS method consisting of a solvent system
with aqueous (0.1% formic acid) acetonitrile (20% to 70%
acetonitrile) for 50 min was applied to determine the absolute
configuration of the piperazic acid unit and the β-hydroxy leucine in
both compounds. The L-FDLA and D-FDLA derivatives of piperazic
acid (m/z425) were eluted at retention times of 22.3 and 29.7 min,
respectively. According to data reported in the literature, both
piperazic acids of compound 1and 2were determined to have an R-
configuration.
20
The L-FDLA and D-FDLA derivatives of β-hydroxy
leucine (m/z442) were eluted at retention times of 18.4 and 24.8
min, respectively. L- and D-Derivatives of the standard (2S,3S)-3-
hydroxyleucine were obtained, which enabled to determine an S-
configuration for both β-hydroxy leucine units in compounds 1and 2.
Antibacterial Assays. Biological screening of Streptomyces sp.
ICBG 1318 was performed in Petri dishes using the entomopatho-
genic bacterium P. larvae ATCC9545 following a previous method-
ology with modifications.
28
Agar disks (6 mm diameter) containing
the bacterium Streptomyces sp. ICBG 1318 were inoculated in the
middle of the Petri dishes containing ISP-2 agar medium and
incubated at 30 °C. After 14 days of growth, the entomopathogen was
inoculated over the entire Petri dish area (60 mm) by applying 2 mL
of BHI (brain heart infusion) soft agar mixture with 10 μLofan
inoculum of P. larvae (0.5 Mc Farland standard). Diffusion tests with
some modifications were performed for the detection of antimicrobial
activity of extracts, fractions, and compounds.
29
An inoculum of P.
larvae was prepared by direct colony suspension in ISP-2 broth, and
turbidity was adjusted to an equivalent 0.5 McFarland standard. Next,
75 μL of bacteria was mixed with 15 mL of BHI soft agar and
inoculated over the entire Petri dish area (150 mm). Extracts,
fractions, and compounds were solubilizedinmethanolata
concentration of 1 mg/mL, and 10 μL of the solutions was applied
on the plate. An inoculum containing 10 μL of methanol was used as a
negative control.
The antibacterial assays for determination of minimum inhibitory
concentration (MIC) were performed following the broth micro-
dilution method according to the guidelines of the Clinical and
Laboratory Standards Institute (CLSI).
30
ISP-2 medium was used to
disseminate the bacterial strains P. larvae ATCC9545 and S. aureus
INCAS0039. Tetracycline was used as a positive control for both
pathogens. Compounds were added from stock solution (0.125 mg/
mL in DMSO), resulting in a highest antibiotic concentration tested
of 6.9 μg/mL. Serial 2-fold dilutions of antibiotics were prepared with
DMSO being diluted along with the compounds.
Antileishmanial and Selectivity Assays. Leishmania infantum
(MHOM/MA/67/ITMAP-263) promastigotes were grown in M199
medium (pH 7.4) supplemented with 10% heat-inactivated fetal calf
serum (FCS) at 28 °C.
31
Human leukemia THP-1 cells were grown in
RPMI-1640 (FCS 10%) at 37 °C and 5% CO2. Stock solutions of
compounds 1and 2at 10 mM were prepared in DMSO (100%) and
tested in 2-fold serial dilutions in 96-well flat-bottom microtiter plates.
THP-1 cells were seeded at 2 ×104/well (RPMI-1640, 100 μL/well),
using 20 ng/mL of phorbol 12-myristate 13-acetate (PMA) for
differentiation of the monocytes into macrophages. After 72 h of
incubation (5% CO2,37°C), medium was aspirated and late-stage
promastigotes were seeded (2 ×105/well, 100 μL/well). After 24 h,
medium was withdrawn to remove extracellular parasites, compounds
were added in serial dilutions (100 μL/well), and the plates were
incubated (5% CO2,37°C, 120 h). Negative control wells (100%
parasite growth) and miltefosine as a positive control were included in
all plates. After 120 h, medium was aspirated, and the cells were fixed
in methanol and Giemsa-stained. The average number of intracellular
amastigotes per macrophage was counted using an inverted
microscope.
32
Growth inhibition was expressed as a percentage of
the number of amastigotes per macrophage in the negative control
wells. IC50 values were automatically calculated with SigmaPlot.
Dose−response curves were fitted using log of inhibitor concentration
vs normalized response between 0 and 100% with variable slope. For
the selectivity tests, THP-1 cells were seeded at 2 ×104/well (RPMI-
1640, 100 μL/well) with 20 ng/mL of PMA.
33
After 72 h (5% CO2,
37 °C), medium was removed, compounds were added in serial
dilutions (100 μL/well), and the plates were incubated (5% CO2,37
°C, 120 h). Negative control wells and doxorubicin as a positive
control were included in all plates. After 120 h, 10 μL of Alamar Blue
was added to each well, and the plates were incubated for 3 h (5%
CO2,37°C). The plates were then read with a microplate fluorometer
(excitation wavelength of 536 nm and emission wavelength of 588
nm). Growth inhibition was expressed as a percentage of the
fluorescence of the negative control wells. IC50 values were
automatically calculated as described above for the intracellular
amastigote assay.
■ASSOCIATED CONTENT
*
sıSupporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b01011.
Streptomyces photography, workflow of bioguided
fractionation, NMR and HRESIMS spectra of com-
pounds 1and 2, key HMBC correlations of compounds
1and 2,1H and 13C chemical shifts of structurally
related cyclic hexadepsipetides (PDF)
Journal of Natural Products pubs.acs.org/jnp Article
https://dx.doi.org/10.1021/acs.jnatprod.9b01011
J. Nat. Prod. 2020, 83, 610−616
614

■AUTHOR INFORMATION
Corresponding Author
Mônica Tallarico Pupo −Departamento de Ciências
Farmacêuticas, Faculdade de Ciências Farmacêuticas de
Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão
Preto, SP, Brazil; orcid.org/0000-0003-2705-0123;
Phone: +55 16 33154710; Email: mtpupo@fcfrp.usp.br;
Fax: +55 16 33154178
Authors
Carla Menegatti −Departamento de Ciências Farmacêuticas,
Faculdade de Ciências Farmacêuticas de Ribeirão Preto,
Universidade de São Paulo, 14040-903 Ribeirão Preto, SP,
Brazil
Vitor Bruno Lourenzon −Departamento de Ciências
Farmacêuticas, Faculdade de Ciências Farmacêuticas de
Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão
Preto, SP, Brazil
Diego Rodríguez-Hernández −Departamento de Ciências
Farmacêuticas, Faculdade de Ciências Farmacêuticas de
Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão
Preto, SP, Brazil
Weilan Gomes da Paixão Melo −Departamento de Ciências
Farmacêuticas, Faculdade de Ciências Farmacêuticas de
Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão
Preto, SP, Brazil
Leonardo Luiz Gomes Ferreira −Instituto de Fi ́
sica de São
Carlos, Universidade de São Paulo, 13563-120 São Carlos, SP,
Brazil
Adriano Defini Andricopulo −Instituto de Fi ́
sica de São
Carlos, Universidade de São Paulo, 13563-120 São Carlos, SP,
Brazil; orcid.org/0000-0002-0457-818X
Fabio Santos do Nascimento −Departamento de Biologia,
Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto,
Universidade de São Paulo, 14040-901 Ribeirão Preto, SP,
Brazil
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.9b01011
Author Contributions
⊥
C. Menegatti and V. B. Lourenzon contributed equally to this
work.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
This research was supported by SãoPauloResearch
Foundation (FAPESP) grants 2013/50954-0 (MTP), 2013/
07600-3 (ADA, LLGF, MTP; CEPID-CIBFar), 2014/17620-4
(CM), 2016/15576-3 (CM); 2018/07153-0 (VBL), 2017/
01188-4 (DRH), and 2015/01001-6 (WGPM) and by the
Conselho Nacional de Pesquisa e Desenvolvimento Tecnoló-
gico (CNPq) grant 303792/2018-2 (MTP). This study was
financed in part by the Coordenação de Aperfeiçoamento de
Pessoal de Ní
vel Superior, Brasil (CAPES), Finance Code 001.
We acknowledge Claudia C. de Macedo for technical support.
We are extremely grateful to Prof. Jon Clardy for the
collaborative research in the ICBG (International Cooperative
Biodiversity Group) project involving Brazilian groups and
jointly supported by FIC-NIH (grant U19TW009872) and
FAPESP (grant 2013/50954-0). His mentorship, expertise, and
advice were instrumental in strengthening the natural products
research at Pupo’s laboratory.
■DEDICATION
Dedicated to Dr. Jon Clardy of Harvard Medical School for his
pioneering work on natural products.
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