A new family of small molecules to probe the reactivation of mutant p53

Article abstract of DOI:10.1021/ja045752y

Cells that express mutant p53 derived from cancers are selectively killed by a new class of small organic molecules. The protein p53 is recognized as one of the most important guardians in the body that prevents tumor development. Mutant forms of p53 are

Full text of DOI:10.1021/ja045752y

Published on Web 04/09/2005
A New Family of Small Molecules To Probe the Reactivation of Mutant p53
Michael C. Myers,| JinLing Wang,‡ Jaclyn A. Iera,§,| Jeong-kyu Bang,† Toshiaki Hara,†
Shin’ichi Saito,† Gerard P. Zambetti,‡ and Daniel H. Appella*,§
Laboratory of Bioorganic Chemistry, NIDDK, NIH, DHHS, Bethesda, Maryland 20892, Northwestern UniVersity,
EVanston, Illinois 60208, Department of Biochemistry, St. Jude Children’s Research Hospital,
Memphis, Tennessee 38105, and Laboratory of Cell Biology, NCI, NIH, Bethesda, Maryland, 20892
Received July 15, 2004; E-mail: appellad@niddk.nih.gov
The protein p53 is recognized as one of the most important
guardians in the body that prevents tumor development.1 Since its
discovery, the roles of p53 have been the focus of research geared
toward understanding the mechanisms of uncontrolled cell growth
or cancer.2 Specifically, when healthy cells are damaged, p53 levels
increase, followed by inhibition of cell growth or programmed cell
Figure 1. Organic molecules that reactivate mutant p53. (A) Structure of
death (apoptosis).3 This regulation of damaged cells is initiated by
a p53-DNA binding event. Mutated forms of p53 that lose the ability
to bind DNA cannot arrest cell growth, and the proliferation of
damaged cells results.2b Mutant forms of p53 are present in
approximately 50% of all human cancers.4 If mutant p53 can be
induced to readopt the active form of wild-type p53, tumor
suppressor function can be restored (reactivation).5 Molecules that
reactivate mutant p53 could selectively target tumor cells due to
the accumulation of mutated p53 in these cells.6
PRIMA-1. (B) General scaffold where R1 to R4 can be varied to study effects
on p53 reactivation. (C) One of the molecules that selectively kills Saos-2
cells expressing mutant p53 (referred to as molecule 7).
Table 1. Activity of Molecules on Cells Expressing Mutant p53
producta
R1
X
Y
R2
activityb
mutantb
Recently, reactivation of mutant p53 by small organic molecules
has been described.7 These studies relied on random screening of
combinatorial libraries to identify small molecule reactivators.
Among the molecules reported to reactivate mutant p53, a number
contain heteroaromatic groups that interact primarily with DNA,8
which is undesirable for drug candidates due to nonspecific binding
and possible mutagenic effects.6 In contrast, the molecule PRIMA-1
(Figure 1A) has been reported to interact directly with several p53
mutants, reactivating the protein to restore wild-type function.9,10
Currently, the molecular mechanism describing how a small organic
molecule can refold a mutated protein remains unclear.
In our research on p53 reactivation, we have initiated a program
to develop a synthetically accessible class of molecules that can
be easily modified to examine structural activity relationships
(SARs) and mechanism of biological activity or to optimize for
anticancer activity. Our first goal was to develop a class of
molecules with spatial arrangements of functional groups that
loosely resemble PRIMA-1, but within an easily modifiable
framework (Figure 1B). In particular, we sought to develop a new
family of molecules that lacks the bicyclic core of PRIMA-1 and
contains one hydroxymethyl side chain.
A variety of compounds were prepared bearing the general
structure shown in Figure 1B. Initially, our molecules were prepared
starting from 2-aminoacetophenone hydrochloride. From this start-
ing material, compounds with one or two hydroxymethyl side chains
were prepared (all molecules with one side chain were racemic)
(Table 1, 1-7, 10-12).10,11
This initial series of molecules was then studied for their ability
to arrest cell growth in Saos-2 cells expressing a mutant p53.11
While most compounds did not show the desired activity, molecule
7 (Figure 1C) was particularly effective at selectively killing cells
1
2
3
4
5
6
7
8
Boc CH2OH CH2OH Ph
Boc CH2OAc Ch2OAc Ph
NA
NA
NA
10 µM
NA
NA
281
281
281
281
281
281
H
H
CH2OH CH2OH Ph
CH2OAc CH2OAc Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
Ph
CH2OH Ph
CH2OAc Ph
CH2OAc Ph
CH2OAc Ph
5-50 µM 175, 281
5-50 µMc 175, 281
5-50 µMd 175, 281
9
H
10
11
12
13
14
15
16
17
43
44
45
46
Ac
Ac
Ac
H
H
H
Me
H
H
H
H
H
Ph
NA
175, 281
175, 281
175, 281
175, 281
281
CH2OH Ph
CH2OAc Ph
CH2OAc Et
CH2OAc OMe
CH2OAc morph.
CH2OAc Ph
TXe
TXe
NA
NA
NA
281
TXe,f
NA
175, 281
175, 281
175, 281
175, 281
175, 281
175, 281
CH2OAc Ph
CH2OAc p-F-Ph
CH2OAc p-Me-Ph
TXd,e
100 µMd
CH2OAc p-OMe-Ph NAd
CH2OAc â-naphthyl NAd
H
a All compounds are (+,-) mixtures unless otherwise noted; entries 3-9
and 13-46 were isolated as HCl salts. b PRIMA-1 is active at 75 µM. See
Supporting Information for cellular assay details; NA ) no activity and no
toxicity. c (S)-Enantiomer. d (R)-Enantiomer. e Compounds are toxic to all
cells. f Compound was 86% ee favoring the (S)-enantiomer.
expressing R175H and R281G p53 mutants at concentrations lower
than that of PRIMA-1 (Figure 2). Molecule 4 also showed
selectivity for R281G mutants. The positive results obtained with
racemic 7 underlined the need to produce both enantiomers of 7 (8
and 9). Therefore, an asymmetric synthesis was developed to
produce enantiomerically pure material.11 Interestingly, (S)- and
(R)-7 showed activity comparable to that of (()-7, indicating that
chirality does not play a role in controlling the desired activity
(Table 1, entries 7-9).
| Northwestern University.
‡ St. Jude Children’s Research Hospital.
§ Laboratory of Bioorganic Chemistry, NIH.
† Laboratory of Cell Biology, NIH.
Our synthetic strategy can be readily modified to afford numerous
other analogues of 7. Therefore, molecules 11-17 were prepared
9
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10.1021/ja045752y CCC: $30.25 © 2005 American Chemical Society

C O M M U N I C A T I O N S
mutant p53 by other mechanisms such as directing p53 to the
mitochondria14 or by blocking its gain-of-function activity, which
protects against cell death.15 Future studies will be needed to
determine whether mutant p53 is the actual target.
Acknowledgment. We gratefully acknowledge financial sup-
port from DHHS, NIH, NIDDK, and Northwestern University;
NIH/NCI grants CA63230 and CA71907 (G.P.Z.), NIH/NCI Cancer
Center Support CORE Grant CA21765, and the American Lebanese
Syrian Associated Charities of St. Jude Children’s Research Hospital
(ALSAC).
Supporting Information Available: Experimental procedures and
characterization data for all new compounds. Cellular assay procedure
and data (PDF). This material is available free of charge via the Internet
at http://pubs.acs.org
Figure 2. Cellular assay with Saos-2 cells expressing no p53 (control) or
mutant p53 at positions 175 and 281 with 50 µM of compound 7 (PRIMA-1
induces similar results at 75 µM). Each panel represents the same
magnification (20×).
References
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nitrogen of 7 (molecule 16) or a methyl group to the R-carbon of
7 (molecule 17) eliminates the desired activity and in some cases
results in nonspecific toxicity.
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ring were explored for effects on the desired biological activity.
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