Oxazolidines from Neocalyptrocalyx longifolium Inhibit MsrA Protein in Methicillin Resistant Staphylococcus aureus

Abstract

The emergence of antimicrobial resistance has generated global concerns regarding many pathogenic microorganisms, such as methicillin-resistant Staphylococcus aureus. The inhibition of microbial molecular targets by natural products has led to the discovery of new paths capable of reverting resistance to classical antimicrobial agents. Neocalyptrocalyx longifolium (Mart.) Cornejo & Iltis, Capparaceae, is a Brazilian medicinal plant indicated for the treatment of skin and respiratory tract bacterial infections. Nevertheless, few studies have investigated its chemical composition. In view of the current development of pathogenic microorganism resistance, the isolation and identification of efflux pumps inhibitors from the roots of N. longifolium is described herein. In addition, the elements that contribute to substrate binding and inhibition of the MsrA protein, an ABC-type transporter, were analyzed based on in silico experiments. Five substances were isolated and characterized by NMR and HRMS. Four of them exhibited interesting structural features, composed of 1,3-oxazolidine-2-thione and 1,3-oxazolidine-2-one cores. 5-Methyl-5-ethyl-oxazolidine-2-one, an undescribed natural product, inhibited the activity of the MsrA transporter and, therefore, the potency of erythromycin was increased. Docking analysis revealed specific hydrogen interactions for this inhibitor at the MsrA ATP binding site.

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Revista Brasileira de Farmacognosia
https://doi.org/10.1007/s43450-023-00422-6
SHORT COMMUNICATION
Oxazolidines fromNeocalyptrocalyx longifolium Inhibit MsrA Protein
inMethicillin Resistant Staphylococcus aureus
ThalissonAmorimdeSouza1 · JoandaPaolaRaimundoSilva1 · DamaraFreitasRodrigues2 ·
ChonnyHerrera‑Acevedo1 · RenataPriscilaBarrosdeMenezes1 · NathalieHellenBorges2 ·
JoséIranildoMirandadeMelo3 · JoséPintodeSiqueira‑Júnior2 · MarcusTulliusScotti1 · LucasSilvaAbreu4 ·
JoseanFechineTavares1 · MarceloSobraldaSilva1
Received: 5 April 2023 / Accepted: 16 June 2023
© The Author(s) under exclusive licence to Sociedade Brasileira de Farmacognosia 2023
Abstract
The emergence of antimicrobial resistance has generated global concerns regarding many pathogenic microorganisms, such
as methicillin-resistant Staphylococcus aureus. The inhibition of microbial molecular targets by natural products has led to
the discovery of new paths capable of reverting resistance to classical antimicrobial agents. Neocalyptrocalyx longifolium
(Mart.) Cornejo & Iltis, Capparaceae, is a Brazilian medicinal plant indicated for the treatment of skin and respiratory tract
bacterial infections. Nevertheless, few studies have investigated its chemical composition. In view of the current develop-
ment of pathogenic microorganism resistance, the isolation and identification of efflux pumps inhibitors from the roots of
N. longifolium is described herein. In addition, the elements that contribute to substrate binding and inhibition of the MsrA
protein, an ABC-type transporter, were analyzed based on in silico experiments. Five substances were isolated and character-
ized by NMR and HRMS. Four of them exhibited interesting structural features, composed of 1,3-oxazolidine-2-thione and
1,3-oxazolidine-2-one cores. 5-Methyl-5-ethyl-oxazolidine-2-one, an undescribed natural product, inhibited the activity of
the MsrA transporter and, therefore, the potency of erythromycin was increased. Docking analysis revealed specific hydrogen
interactions for this inhibitor at the MsrA ATP binding site.
Keywords Antimicrobial resistance· Caatinga· Erythromycin· Modulatory activity· Multidrug resistance· Oxazolidinone
Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) causes
severe infections in hospitals and communities. In recent
decades, it has become a public health concern worldwide.
Among MRSA’s mechanisms of resistance, efflux pumps
(EFPs) play a pivotal role because they act as the first-line
defense mechanism against antimicrobials (Chalmers and
Wylam 2020) by decreasing the intracellular concentration
of therapeutical antibiotics, clinical drugs, and other xeno-
biotics (Huang etal. 2022).
Efflux pump inhibitors, the so-called “magic bullets”,
have become an important frontline to circumvent bacterial
resistance. Natural products have shown substantial potential
against EFPs in Gram-positive bacteria. Regarding MRSA,
approximately 30 EFPs have been identified (Schindler and
Kaatz 2016), with many studies focusing on the inhibition
of major facilitator superfamily (MFS) transporters, such
as NorA (Lira-Ricárdez and Pereda-Miranda 2020). How-
ever, few papers have reported natural substances that are
active against the MsrA protein (Pinheiro etal. 2022). First
described in the 1990s, MsrA is a transmembrane protein with
two ATP binding motifs and 488 amino acids. This EFP is
responsible for resistance to macrolides and streptogramines,
both of which are important agents for treating Gram-positive
bacterial infections in humans (Svetlov etal. 2021).
Oxazolidinethione and oxazolidinone are 5-member
heterocyclic rings widely used in organic synthesis. The
* Josean Fechine Tavares
josean@ltf.ufpb.br
1 Instituto de Pesquisa em Fármacos e Medicamentos,
Universidade Federal da Paraíba, JoãoPessoa, PB, Brazil
2 Departamento de Biologia Molecular, Universidade Federal
da Paraíba, JoãoPessoa, PB, Brazil
3 Departamento de Biologia, Universidade Estadual da
Paraíba, CampinaGrande, PB, Brazil
4 Departamento de Química Orgânica, Universidade Federal
Fluminense, RiodeJaneiro, RJ, Brazil

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oxazolidinone scaffold and analogs are well known due to
their antibacterial properties (Tsai etal. 2022). In nature,
these compounds are found as secondary metabolites in
marine organisms and in a restricted number of terrestrial
plants, typically in those that contain glucosinolates, such as
Brassicales species (Blažević etal. 2020).
Traditionally referred to as “incó”, Neocalyptrocalyx
longifolium (Mart.) Cornejo & Iltis, Capparaceae, is a
medicinal species from the Brazilian Caatinga (Silva etal.
2022). It is used as an anti-inflammatory, emmenagogue,
and for treating fever, pneumonia, bronchitis, and skin infec-
tions (Agra etal. 2007; Albuquerque etal. 2007). Neverthe-
less, chemical and pharmacological studies regarding this
plant are still limited. In view of the clinical importance
of pathogenic microorganism resistance, we sought to iso-
late oxazolidine derivatives from the roots of N. longifolium
and evaluate their potential as EFP inhibitors in methicillin-
resistant Staphylococcus aureus (MRSA) strains (de Souza
etal. 2022).
Material andMethods
The plant material, comprising the roots of Neocalyptrocalyx
longifolium (Mart.) Cornejo & Iltis, Capparaceae (SisGen
A885D6F), was collected in Monteiro, Paraiba, Brazil at
7.8888° S, 37.1171° W and identified by Professor Dr. José
Iranildo Miranda de Melo. A voucher was then deposited at
the Herbarium Manuel de Arruda Câmara of Universidade
Estadual da Paraíba (accession number: HACAM 971).
The roots (1.5kg) were dried at 40°C, pulverized, macer-
ated with 96% ethanol (3 × for 72h) at room temperature.
The extracted solution was concentrated under reduced pres-
sure at 40°C, 110g of crude ethanolic extract was obtained.
The extract (100g) was solubilized in MeOH:H2O (7:3 v/v)
and successively partitioned with hexane, CH2Cl2, EtOAc,
and n-BuOH. The resulting solutions were concentrated
under reduced pressure. Then, CH2Cl2 soluble fraction (4g)
was chromatographed over SiO2 column, a gradient system
composed of hexane and ethyl acetate (0:1 to 1:9 increased
gradients of 10%) was applied as the mobile phase. Ten
fractions were obtained from this procedure, all of which
were analyzed by thin-layer chromatography. Fraction 4 led
to the identification of compound 1 (800mg). Fraction 6
was subjected to recrystallization, resulting in white crys-
tals identified as compound 2 (50mg). Fractions 5 and 7
were purified by HPLC, isocratic and gradient modes were
applied for elution, respectively (for HPLC instrumental
conditions, see Supplementary Information). These proce-
dures yielded compound 3 (20mg) from Fraction 5, whereas
from the Fraction 7, compounds 4 (1mg) and 5 (1mg) were
obtained. The chromatographic and nuclear magnetic reso-
nance (NMR) spectral data are provided in Supplementary
Information.
Cleomin (1): [α]22589 −8.7 (c 1.0, MeOH) 1H NMR
(Figs.S2 and S3): 3.56 (1H, d, J = 10.0Hz), 3.45 (1H, d,
J = 10.0Hz) 1.75 (2H, q, J = 7.6Hz), 1.42 (3H, s), 0.92 (3H,
t, J = 7.6Hz); 13C NMR (Fig.S4): 189.6, 90.5, 53.8, 33.1,
24.9, 7.9; HRESIMS m/z 146.0628 [M + H]+ (calculated for
C6H12NOS 146.0634; error 4.5ppm).
5-Ethyl-5-methyl-oxazolidine-2-one (2): mp 67–69°C;
[α]22589 −13.2 (c 1.0, MeOH); 1H NMR (Figs.S6 and S7):
3.38 (1H, d, J = 10.0Hz), 3.26 (1H, d, J = 10.0Hz), 1.68
(2H, J = 7.6, q), 1.36 (3H, s), 0.93 (3H, t, J = 7.6Hz); 13C
NMR (Fig.S8): 159.5, 50.9, 82.8, 33.4, 7.9, 25.2; HRESIMS
m/z 130.0859 [M + H]+ (calculated for C6H12NO2 130.0863;
error 2.8ppm).
Conringin (3): 1H NMR (Fig. S11): 3.48 (2H, s),
1.48 (6H, s); 13C NMR (Fig.S12): 190.1, 89.2, 56.4,
26.8; HRESIMS m/z 132.0474 [M + H]+ (calculated for
C5H10NOS 132.0478; error 2.4ppm).
5-Dimethyl-oxazolidine-2-one (4): 1H NMR (Fig.S14):
3.55 (2H, s), 1.48 (6H, s); 13C NMR (Fig.S15): 158.6, 80.4,
52.8, 27.2; HRESIMS m/z 116,0711 [M + H]+ (calculated
for C5H10NO2 116.0706; error −4.3ppm).
Siringic acid (5): 1H NMR (Fig.S17): 7.33 (2H, s), 3.88
(6H, s); 13C NMR (Fig.S18): 167.7, 148.3, 141.5, 121.5,
108.1, 56,6.
efflux protein (Ross etal. 1989). The strain was maintained on
blood agar base (Laboratórios Difco Ltda., Brazil) slants and,
prior to use, the cells were grown overnight at 37°C in brain
heart infusion broth (BHI, Laboratórios Difco Ltda., Brazil).
After identification, the three major compounds, cleomin
(1), 5-methyl-5-ethyl-oxazilidine-2-one (2), and conringin
(3), were tested against S. aureus RN-4220 harboring plasmid
pUL5054, which carries the gene encoding the MsrA macrolide

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Erythromycin stock solutions were prepared in 95% etha-
nol according to the Clinical and Laboratory Standards Insti-
tute (CLSI) guidelines (Weinstein etal. 2023). Stock solu-
tions of compounds 1–3 were prepared in DMSO which,
at its highest final concentration after dilution in the broth
(4%), caused no inhibition of bacterial growth. The mini-
mum inhibitory concentrations (MICs) of the erythromycin,
cleomin (1), 5-methyl-5-ethyl-oxazilidine-2-one (2), and con-
ringin (3) solutions were determined in brain heart infusion
(BHI) with a microdilution assay using a suspension of ca.
105cfu/ml and a drug concentration range of 256–4.0μg/
ml (two-fold serial dilutions). The MIC was defined as the
lowest concentration at which no growth was observed. The
detection was performed after the addition of 0.01% resa-
zurin. For the evaluation of compounds 1–3 as a modulator
of drug resistance, a “modulation assay” was used, which
has been widely applied for identifying potential EPIs (Stavri
etal. 2007) (i.e., the MICs of erythromycin were determined
in the presence of cleomin (1), 5-methyl-5-ethyl-oxazilidine-
2-one (2), and conringin (3) at a sub-inhibitory concentration
(¼ MIC).
The MsrA protein model was obtained by homology.
Docking was carried out covering the ATP-binding and
ATPase active regions, including the aminoacidic residues:
Gly 39, Asn 40, Gly 41, Thr 42, Gly 43, and Lys 44 (Guo
etal. 2022). The grid box (10Å in radius and 0.30 in reso-
lution) was applied to the center of the target site. Molegro
Virtual Docker software was used to generate 5 poses for
each molecule in the active site of the protein. The pose
with the lowest interaction energy was selected and imported
to Discovery Studio 2021 to visualize the results. Further
details for in silico methodologies are included in the Sup-
plementary Information.
Results andDiscussion
The 1H and 13C NMR chemical shifts of all compounds were
compared with previously reported data, molecular formulae
were confirmed by HRMS experiments, leading to identifi-
cation of cleomin (1), 5-methyl-5-ethyl-oxazolidine-2-one
(2), conringin (3), 5-dimethyl-oxazolidine-2-one (4), and
siringic acid (5) (Oguakwa etal. 1981; Bull etal. 1999;
Leoni etal. 1999; Öksüz etal. 2002; Elenkov etal. 2008).
The 1,3-oxazolidin-2-ones are rare in nature. Agerbirk etal.
(2018) proposed a metabolic association between oxazolidi-
nethione and oxazolidinone cores by an enzymatic reaction
in glucosinolate-containing species. Our results also indicate
the presence of a putative enzyme with a similar ability for
transferring thiol groups from oxazolidine-2-thiones.
Regarding the biological assay, compounds 1–3 did not
showed antimicrobial activity against S. aureus RN-4220.
All of them had MIC values ≥ 1024µg/ml (Supplementary
material). These findings agree with Saygili etal. (2014),
who tested 2-oxazolidinothiones against S. aureus strains
and obtained MIC values of ≥ 512µg/ml. Oxazolidinones
have been reported to be potent antibacterial agents (e.g.,
linezolid: MIC ≥ 2μg/ml). Although comprising a pharma-
cophoric group in such classes of antibiotics, the activity of
linezolid relies on N-aryl substitution and their interactions
in ribosomes; the isolated 2-oxazolidinone core and their
oxazolidine-2-thione derivatives does not show antimicro-
bial activity (Zhao etal. 2021).
The MIC evaluation of erythromycin for the resistant S.
aureus strain was performed in association with compounds
1–3 (¼ MIC). Only compound 2 exhibited modulatory activ-
ity, reducing the MIC of erythromycin from 128μg/ml to
64μg/ml (2 ×). Cleomin (1) and conringin (3) were not
active in the tested strains (Table1).
According to Dantas etal. (2018), substances with low
lipophilicity and containing electron-withdrawing groups
are active as modulators of drug resistance. Analogically, the
presence of oxygen seems to play a pivotal role in the selective
inhibition of MsrA (ABC) protein; no activity was observed
for other strains, which overexpressed TetK and NorA EFPs
(data not shown). To elucidate the molecular aspects involved
in the biological activity, docking analysis was performed.
In the molecular docking study, we evaluated the prob-
ability of interaction between the compounds tested against
the MRSA strain with the amino acid residues in ATP-bind-
ing and ATPase regions of MsrA protein. These interactions
may enable the inhibitory activity and possible mechanism
of action to be more effectively determined. Among the sub-
stances tested, compound 2 was the most promising mol-
ecule; it had the lowest energy (Moldock score = -79.18kcal/
mol) and consequently a more stable interaction with the
molecular target. Differing from cleomin (1) (Moldock
score = −76.07 kcal/mol) and conringin (3) (Moldock
score = -71.20kcal/mol), which showed mostly Van Der
Waals interactions, compound 2 exhibited a hydrogen bond
with Gly A43 (Fig.1). In the oxazolidinone core, oxygen
atom allowed a stronger intermolecular interaction than the
thione-containing analogs.
These findings corroborate the results obtained from the in
vitro analyses. The results suggested that compound 2 may act
Table 1 Minimum
inhibitory concentration
(μg/ml) of erythromycin
isolated and associated
with compounds 1, 2,
and 3 in Staphylococcus
aureus RN4220
* (fold reduction in MIC)
Antibiotic + Com-
pounds (¼ MIC) MIC (μg/ml)
Erythromycin 128
Erythromycin + 1128
Erythromycin + 264 (2 ×)*
Erythromycin + 3128

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by blocking the energy supply of the MsrA EFP, similar to luteo-
lin, which also interacts by hydrogen bonding with Gly43 (Guo
etal. 2022). The observations also provided a plausible reason
for the inactivity of 1,3-oxazolidine-2-thiones, cleomin (1) and
conringin (3), and suggest that N-substitution could increase this
activity, avoiding unfavorable interactions in the active site.
Conclusion
Five substances were isolated and described for the first
time in Neocalyptrocalyx. 5-methyl-5-ethyl-oxazolidine-
2-one (2), reported for the first time as a natural product,
increased the potency of erythromycin in twofold, suggest-
ing a novel active site for 1,3-oxazolidine-2-one’s deriv-
atives. Moreover, the presence of a carbonyl in C-2 was
essential for the inhibition of MsrA (ABC) protein in methi-
cillin resistant S. aureus. This result could be explained by
additional hydrogen-bonding interactions in the active site
of the molecular target. The current study provided valuable
insights into MsrA protein inhibition by heterocyclic com-
pounds, as well as increased the knowledge on the chemical
diversity and biological potential of N. longifolium.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s43450- 023- 00422-6.
Fig. 1 Molecular interactions of cleomin (1), 5-ethyl-5-methyl-oxazolin-2-one (2), and conringin (3) in MsrA (ABC) active site

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Acknowledgements The authors gratefully acknowledge Dr. Simon
Gibbons (The School of Pharmacy, University of London) for kindly
provide the MSRA strains.
Author Contributions TAS: contributed designing the study, analyz-
ing data and writing the manuscript first draft; JPRS: acquisition of
data; JIMM: acquisition of data (identification of botanical material);
DFR, NHB, JPS: acquisition and analysis of biological essays; CH,
RPM, MTS: analysis and interpretation of in silico data; LSA, JFT:
critically revised the manuscript; MSS: final approval of the version
to be submitted.
Funding This work was supported by Coordenação de Aper-
feiçoamento do Pessoal de Nível Superior (CAPES, Finance Code
001) and Fianciadora de Estudos e Projetos (FINEP) by means of
INCT-RENNOFITO.
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