Template for Electronic Submission of Organic Letters

Searching for new natural products as proteasome inhibitors: a high
throughput screening assay towards the evaluation of a marine natural
products library
1,2
3
3
Fernandes, A. Z. N. ; Cordeiro, A. T.¹; de Morais-Urano, R. P. ; Berlinck, R. G. S. ; Trivella, D. B. B.¹*
¹Laboratório de Química e Produtos Naturais (LQPN), Laboratório Nacional de Biociências (LNBio), Centro Nacional de
2
Pesquisa em Energia e Materiais (CNPEM), Campinas/SP, Brasil; Programa de Pós-Graduação em Biociências e
3
Tecnologia de Produtos Bioativos, BTPB – IB, Unicamp, Campinas/SP, Brasil; Grupo de Química Orgânica de
Sistemas Biológicos (QOSBio), Instituto de Química de São Carlos (IQSC), Universidade de São Paulo (USP), São
Carlos/SP, Brasil. *[email protected]
Keywords: Proteasome, Drug discovery, marine natural products, High Throughput Screening.
Introduction
The proteasome complex is a validated target for
1
anti-cancer chemotherapy . The currently available
proteasome inhibitors present several problems as
high toxicity, low absorption and distribution, as well
2
as development of resistance . Therefore, better
designed proteasome inhibitors are needed.
In this direction, this work aims the discovery of
new proteasome inhibitors from natural product
libraries. Here we report the first steps of this venue,
which are the development, implementation and
validation of a reliable High Throughput Screening
(HTS) assay towards the screen of novel natural
product libraries against the 20S proteasome core
enzyme.
Results and Discussion
Human 20S Proteasome, chemical library and the
fluorogenic
substract
Suc-LLVY-AMC
were
dispensed in 384 well plates. Enzyme, substrate
concentrations, as well as inhibitor and reaction
incubation periods were tested.
A reliable HTS assay with Z’>0.7 was achieved,
using positive and negative controls, as well as a
known proteasome inhibitor (MG-132) as standard.
Then, a small natural product library (n=194)
containing extracts, enriched fractions and isolated
compounds derived from marine macro-organisms
and micro-organisms was tested to check the assay
sensitivity and reproducibility with natural product
samples.
The intrinsic fluorescence of natural products at the
wavelengths used to measure product release by
the enzyme was minimized using strategic
approaches. These include library concentration,
optimization of wavelength band width, blank control
measurements (t0) - for HTS end point assays - and
kinetic measures for hit confirmation.
We were able to identify 10 hits in the primary
assay (hit cut-off = 50% of enzyme inhibition). These
hits were further validated in concentration response
curves with kinetic measurements, revealing 4 top
hits with IC50 < 5 μg/ml. Further investigations of
inhibition mechanisms and active compound
isolation (in the case of active extracts and fractions)
are ongoing.
Conclusions
The results obtained revealed that adjusting the
acquisition parameters and assay conditions it is
possible to screen natural products using the
commercially
available
coumarin
fluorogenic
substrate. However, extra steps of hit validation,
e.g.: kinetic measurements and alternative
fluorophores, would be necessary to confirm hits
identified in the primary assay. The hits so far
identified are promising for new proteasome inhibitor
discovery.
Acknowledgements
This work is supported by FAPESP (grants
2014/10753-9 and 2013/50228-8) and CAPES
(AZNF CAPES-CNPEM a master’s degree
fellowship). Authors thank LNBio-CNPEM and
IQSC-USP for infrastructure and technical support.
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Kisselev, A. F.; van der Linden, W. A., et al. Chem
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10317-10327.