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Figure 2. Effect of pitinoic acid B (2) (100μM) on transcript levels of
pro-inflammatory cytokines in differentiated THP-1 cells. *P-value
<0.05 (t-test); n = 3. Results are calculated relative to the endo-
genous control GAPDH. Data are presented as mean ( SD.
Figure 1. Quorum sensing inhibition by pitinoic acid A (1) in
P. aeruginosa. Lyngbyoic acid (1 mM) was used as a posi-
tive control. *P < 0.05, **P < 0.01 (t-test); n = 3. Results
are calculated relative to the endogenous control rpoD. Data are
presented as mean ( SD.
the levels of those pro-inflammatory cytokines after 4 h at
100 μM (Figure S31). This is not surprising, since it has been
shown that QS mediators could modulate the production of
pro-inflammatory cytokines in mammalian cells.24,25
macrophages. Compound 2 was able to decrease the tran-
script levels of the pro-inflammatory cytokines TNF-R and
IL-6 after 4 h of LPS stimulation at 100 μM (Figure 2).23
Additionally, the anti-inflammatory effect was also sustained
after 24 h, where the mRNA levels of IL-6, IL-1β, and IL-8
were significantly reduced (Figure 2). Notably, IL-6 was the
most affected cytokine at all time points. Cell viability under
the tested conditions was 73% based on MTT colorimetric
assay.
In conclusion, we identified a set of interesting marine
bioactivelipids. Theproducing cyanobacterium appearsto
utilize a clever modular strategy to maintain the structure
and function of those secondary metabolites. We suspect 2 to
be a prodrug that utilizes the QS-inhibitor 1 as a protecting
group to circumvent early reaction and deactivation of the
R-hydroxy acid pitinoic acid C. One possible mechanism for
the biological activity of the latter is dehydration to give a
Michael acceptor fragment, which could be the reactive
species. If true, then the protection of pitinoic acid C by
esterification, as employed by this organism, is an optimum
solution to avoid the early release of this reactive species. Also,
if hydrolysis of 2 takes place in vivo, it will also release the
antiquorum sensing acid part 1. This could highlight 2 as a
hybrid prodrug molecule with dual biological activity, which
could be effective in the management of P. aeruginosa infec-
tions and associated inflammation. Further studies will allow
for the detailed understanding of the mode of action of those
molecules and the development of more potent analogues.
Based on its structural features, we hypothesize that
the chlorinated alcohol part in the ester 2 is the major
contributor to its detected anti-inflammatory effect. This
notion is consistent with the structural similarity of this
moiety to other previously reported anti-inflammatory
compounds and to the anti-inflammatory acid part
1
in honaucins.19 Additionally, using H NMR and MS
analysis, we were able to confirm the presence of this
R-hydroxy acid in one of the minor impure HPLC fractions
obtained during the purification of 2 (Figures S29, S30).
Pitinoic acid C appeared to be the major compound in this
HPLC fraction (Figure S30), but it was unstable since
purification trials led to the loss of material. Accordingly,
the impure fraction containing pitinoic acid C was tested
for the anti-inflammatory effects. Indeed, this fraction was
also able to reduce the levels of IL-6 and TNF-R after 4 h at
15 μg/mL (Figure S31). Notably, 1 was also able to reduce
Acknowledgment. We thank Dr. Qi-Yin Chen (Department
of Medicinal Chemistry, University of Florida) for helpful
discussions regarding the synthetic procedures and Dr. Sarath
Gunasekera and Vicky Pittman (Smithsonian Marine
Station) for providing additional amounts of pitinoic
acid A. We thank J. R. Rocca (AMRIS, McKnight Brain
Institute, University of Florida) for technical assistance with
NMR data acquisition. This research was supported by
the National Institutes of Health, Grants P41GM086210
and R01CA172310. This is contribution 916 from the
Smithsonian Marine Station.
(23) It is not uncommon, especially for fatty acid compounds, to be
tested and considered effective at this high concentration. For example,
the anti-inflammatory effects of the fish oil fatty acids eicosapentaenoic
acid and docosahexaenoic acid were detected at 100 μM in LPS-
stimulated THP-1 macrophages (Weldon, S. M.; Mullen, A. C.; Loscher,
C. E.; Hurley, L. A.; Roche, H. M. J. Nutr. Biochem. 2007, 18, 250–258).
For other examples, see: Speranza, L.; Franceschelli, S.; Pesce, M.;
Reale, M.; Menghini, L.; Vinciguerra, I.; De Lutiis, M. A.; Felaco, M.;
Grilli, A. Phytother. Res. 2010, 24, 1398. Zhao, G.; Etherton, T. D.;
Martin, K. R.; Vanden-Heuvel, J. P.; Gillies, P. J.; West, S. G.; Kris-
Etherton, P. M. Biochem. Biophys. Res. Commun. 2005, 336, 909.
Boonkaewwan, C.; Toskulkao, C.; Vongsakul, M. J. Agric. Food Chem.
2006, 54, 785. Liang, Q.; Wu, Q.; Jiang, J.; Duan, J.; Wang, C.; Smith, M. D.;
Lu, H.; Wang, Q.; Nagarkatti, P.; Fan, D. J. Biol. Chem. 2011, 286, 26470.
(24) Glucksam-Galnoy, Y.; Sananes, R.; Silberstein, N.; Krief, P.;
Kravchenko, V. V.; Meijler, M. M.; Zor, T. J. Immunol. 2013, 191, 337.
(25) Kravchenko, V. V.; Kaufmann, G. F.; Mathison, J. C.; Scott,
D. A.; Katz, A. Z.; Grauer, D. C.; Lehmann, M.; Meijler, M. M.; Janda,
K. D.; Ulevitch, R. J. Science 2008, 321, 259.
Supporting Information Available. 1D and 2D NMR
spectra for 1 and 2, supporting figures including spectral data
and characterization of synthetic intermediates, experimental
procedures, and additional RT-qPCR data. This material is
The authors declare no competing financial interest.
D
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