Title:Synthesis of N-acyl Derivatives of Aminocombretastatin A-4 and Study of their Interaction with Tubulin and Downregulation of c-Myc
Volume: 17
Issue: 10
Author(s): Raül Agut, Eva Falomir*, Juan Murga, Celia Martín-Beltrán, Raquel Gil-Edo, Alberto Pla, Miguel Carda*, J. Fernando Díaz, Fernando Josa-Prado and J. Alberto Marco
Affiliation:
- Dpto. de Quimica Inorganica y Organica, Universidad Jaume I, E-12071 Castellon,Spain
- Dpto. de Quimica Inorganica y Organica, Universidad Jaume I, E-12071 Castellon,Spain
Keywords:
Aminocombretastatin A-4, anti-proliferative activity, c-Myc, microtubule dynamics, tubulin, anti-mitotic.
Abstract:
Background: Six N-acyl derivatives of aminocombretastatin A-4 have been synthesized
and evaluated according to their interaction with tubulin and as c-Myc downregulators.
Aims: In search of new promising anti-cancer agents.
Objective: This study is focused on the synthesis and the biological evaluation of N-acyl derivatives
of aminocombretastatin A-4 (CA-4). Docking studies were carried out to find out whether the synthetic
derivatives could bind to tubulin at the colchicine site in a conformation similar to that of CA-
4. The synthetic derivatives' effect on the proliferation of several cancer cells and non-cancer cells
has been measured. Their effect on tubulin polymerization, cell cycle distribution, the microtubule
network and c-Myc expression has also been evaluated.
Methods: A set of six N-acyl derivatives was achieved by means of a peptide-type coupling of aminocombretastatin
A-4 and the corresponding carboxylic acid. The synthetic compounds' ability to
inhibit cell proliferation was measured by MTT assay against three human carcinoma cell lines (colorectal
HT-29, lung A549, and breast adenocarcinoma MCF-7) and one non-tumor cell line (HEK-
293). Turbidimetry time-course measurements evaluated the inhibition of tubulin polymerization.
The action of the synthetic derivatives on cell cycle distribution was measured by flow cytometry and
their effects on the microtubule network were determined by immunofluorescence microscopy. Finally,
the downregulation of the synthetic derivatives on c-Myc protein was quantified by ELISA
assay, while the effect on c-Myc gene was measured by RT-qPCR analysis.
Results: Derivatives bearing pentanoyl (compound 2), hexanoyl (compound 3), and heptanoyl (compound
4) side chains show anti-proliferative activities on the HT-29 line in the low nanomolar range,
with values similar to that exhibited by AmCA-4 but far exceeding those of CA-4. Compounds 1
(butanoyl side chain) and 2-3 inhibit tubulin polymerization in vitro in a manner similar to that of
CA-4 and AmCA-4 whereas compounds 4, 5 (octanoyl side chain) and 6 (dodecanoyl side chain)
may be considered as partial inhibitors of tubulin polymerization. While all derivatives are able to
accumulate cells in G2/M phase, compounds with the longest acyl chains (5 and 6) are the least active
ones in this particular action. Moreover, compounds 2-3 were the most active ones as c-Myc
downregulators.
Conclusion: Our studies show that the most active compounds in the disruption of the microtubule
network are also the most potent ones in the downregulation of c-Myc expression.
Other: Compounds 2 and 3 are good candidates for in vivo studies as they combine the best antimitotic
and c-Myc downregulation activities at low doses.
