Profiling small molecule inhibitors against helix-receptor interactions: The Bcl-2 family inhibitor BH3I-1 potently inhibits p53/hDM2

Article abstract of DOI:10.1039/c0cc02969f

We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein-protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction.

Full text of DOI:10.1039/c0cc02969f

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Profiling small molecule inhibitors against helix–receptor interactions:
the Bcl-2 family inhibitor BH3I-1 potently inhibits p53/hDM2wz
Jason R. Porter,a Mark R. Helmers,a Ping Wang,a Jennifer L. Furman,a Stephen T. Joy,b
Paramjit S. Arora*b and Indraneel Ghosh*a
Received 2nd August 2010, Accepted 21st August 2010
DOI: 10.1039/c0cc02969f
We validate a practical methodology for the rapid profiling of
small molecule inhibitors of protein–protein interactions. We
find that a well known BH3 family inhibitor can potently inhibit
the p53/hDM2 interaction.
mimics the complexity of a cellular environment to a first
approximation. We explore the application of this methodology
towards a set of relevant helix/receptor PPIs that have been
targeted by small molecule inhibitors, leading to surprising results.
Helix–receptor interactions are generally characterized by
the binding of an alpha-helical domain to a relatively hydro-
phobic groove of a larger protein domain (Fig. 1b). This small
yet dense interface has been particularly amenable to the
development of small-molecule inhibitors. Specifically, two
similar yet unrelated groups of helix–receptor interactions
were the first to yield to potent small molecule inhibitors;
the interaction between hDM2 with the activation domain of
p53 and interactions amongst pro-apoptotic BH3 only and
anti-apoptotic members of the Bcl-2 family of proteins
(Fig. 2).10 To interrogate this class of PPIs we chose the
fragmented luciferase reporter over other split-proteins that
also provide simple read-outs,11 as this reporter was found to
be more sensitive than split-lactamase and split-GFP under
cell free conditions.9,11 Appropriate fusions were created in
Protein–protein interactions are involved in almost all bio-
logical processes.1 As a result, reagents capable of disrupting
these interactions or stabilizing these interactions are highly
sought after both as probes for dissecting biology and
for therapeutic leads. A variety of elegant methods have been
developed to disrupt protein–protein interactions (PPIs),
including antibodies, peptides, and miniature proteins.2
Synthetic approaches have more recently found success, and
include foldamers,3 terphenyl scaffolds,4 stabilized helices,5
small-molecule fragment engineering, in silico engineering, and
compound library screening.6 Many of the small molecule
approaches have honed in on helix–receptor PPIs and thus an
important question arises: how specific are inhibitors for their
intended targets? Answering a similar question in the field of
protein kinase inhibition has resulted in a paradigm shift, where
large scale profiling approaches have demonstrated unintended
promiscuity or polypharmacology of known inhibitors.7 This
promiscuity can be potentially beneficial by targeting several
kinases of interest, or harmful. Herein we provide a potentially
scalable methodology for the rapid and simple profiling for the
helix/receptor class of PPIs and demonstrate that known small
molecule inhibitors can display potent off-target effects.
Currently PPIs and their inhibitors are routinely interrogated
by quantitative SPR or fluorescence based methods.8 These
methods rely upon purified and often chemically modified
components, which can be resource intensive and thus challenging
for the routine profiling of larger panels of PPIs. We have
recently described a split-protein methodology (also referred
to as protein complementation) (Fig. 1a), which potentially
provides an avenue for rapidly profiling PPIs and their
inhibitors.9 This approach does not require cell culture,
purified proteins, or chemical modification steps and perhaps
a Department of Chemistry and Biochemistry, University of Arizona,
1306 East University Blvd, Tucson, AZ 85721, USA.
E-mail: ghosh@u.arizona.edu; Fax: +1 520 621 8407;
Tel: +1 520 621 6345
b Department of Chemistry, New York University,
100 Washington Square East New York, NY 10003, USA.
E-mail: arora@nyu.edu; Tel: +1 212 998 8470
w This article is part of the ‘Enzymes and Proteins’ web-theme issue for
ChemComm.
z Electronic supplementary information (ESI) available: Details of
experimental procedures, synthetic procedures, and FP assays. See
DOI: 10.1039/c0cc02969f
Fig. 1 Specificity of helix–receptor interactions. (a) Schematic for the cell
free interrogation of helix–receptor interactions resulting in interaction
dependent luminescence. (b) (i) BIM (dark grey)/Bcl-xL (light grey)
(ii) BIM (dark grey)/BFL (light grey), (iii) p53 (dark grey)/hDM2 (light
grey), and (iv) Hif-1a (dark grey)/p300 (light grey). (c) Luminescence of
co-translated helix–Nfluc and Cfluc–receptor interactions for all 18 pairs.
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6 helix–receptor interactions, along with the interactions
between the coiled-coils Fos/Jun. In order to ensure that
compounds do not inhibit luciferase activity, a tethered
luciferase containing a covalent N- and C-terminal linkage
designed to mimic post-reassembled split-luciferase was also
included in the panel. The three compounds tested, significantly
inhibited their known PPI targets. In addition to significant
inhibition of p53/hDM2, (ꢀ)-nutlin-3 showed modest inhibition
of the interaction between the BIM BH3 domain and Bcl-2. In
the case of ABT-737 and its analogue ABT-263, both showed
the most potency against interactions between BH3 with Bcl-2,
Bcl-XL, and Bcl-w but not BFL6e,12,13 as previously observed.
Interestingly, ABT-263 showed significant inhibition of the
p300/Hif-1a interaction when compared to ABT-737, which
we will interrogate further in future studies. Notably, modifica-
tions at positions remote from the pharmacophore for increasing
bioavailability, may potentially lead to unanticipated changes in
inhibition profiles when tested against larger PPI panels (Fig. 3).
Finally, the well-studied Bcl-2 family inhibitor BH3I-16d,e
was interrogated. BH3I-1, while inhibiting its reported target
Bcl-2/Bim and Bcl-xL/Bim, showed significant inhibition of
both the p53/hDM2 and p300/Hif-1a interactions (Fig. 4a).
This surprising promiscuity, displayed by a well studied
Fig. 2 Interrogation of inhibitors of helix–receptor interactions.
(a) Inhibition of the interaction of p53 with hDM2 and hDM4 by
enantiomers of nutlin-3. (b) Inhibition of the interaction of Bcl-2
and Bcl-xL with the BIM BH3 domain upon addition of free BIM
peptide.
which a series of helix/receptor pairs were appended to the
N- and C-terminal fragments creating the fusions ‘‘helix-NFluc’’
and ‘‘CFluc-receptor’’ respectively (Table S1, ESIz). Using
this panel we first interrogated the interaction specificity of a
set of 18 helix/receptor combinations (Fig. 1c). Importantly,
we found the split-luciferase method recapitulated the affinity
of BIM for the Bcl-2 family of receptors, whereas none of the
four tested receptors bound p53 or Hif1-a. Similarly, neither
hDM2 nor p300 were found to bind the BIM peptide, thus
showing that the native helix/receptor pairs in our panel are
orthogonal.
Having a helix–receptor panel capable of reporting upon
interaction specificity, we next sought to interrogate its
suitability for PPI inhibitor profiling. This is particularly
relevant as the constellation of residues implicated in binding
their respective receptors are grossly similar for p53 (Phe19,
Trp23, and Leu26) and BIM (Trp147, Ile155, Phe169), while
those implicated for Hif1-a/p300 are primarily aliphatic
(Leu795, Cys800, Leu818, and Leu822). To test whether
differences in specificity could be evaluated for inhibitors of
the p53/receptor interactions we first evaluated the ability of
the (+) and (ꢀ) enantiomers of the p53/hDM2 specific
inhibitor nutlin-3 to inhibit the interaction of p53 with
hDM2 and hDM4 (Fig. 2a). Consistent with previous studies,
addition of 2.5 mM (ꢀ) nutlin-3 resulted in the specific
disruption of the reassembled p53/hDM2 complex while the
same concentration of (+) nutlin-3 showed minimal inhibition
for either the p53/hDM2 or p53/hDM4 interaction. Next, to
test inhibitors of Bcl-2 family interactions, the inhibition of the
interaction of BIM with Bcl-2 and Bcl-XL was evaluated
following the addition of BIM BH3 peptide (residues 142–161)
(Fig. 2b), demonstrating disruption of the interaction between
BIM/bcl-2 and BIM/Bcl-XL interaction. The ability to com-
petitively inhibit protein–protein interactions is an advantage
for the split-luciferase based systems as split-GFP based
systems result in an irreversible complex.
Thus with a viable method in hand to report upon the
specificity of inhibitors of helix–receptor interactions we next
investigated the specificity of nutlin as well as two well-studied
inhibitors of interactions between the pro- and anti-apoptotic
Bcl-2 family domains, specifically ABT-737 and its bioavailable
analog ABT-263 (for synthesis details please see ESIz). Each
compound was tested for its ability to inhibit the panel of
Fig. 3 Inhibition profile of (ꢀ) Nutlin, ABT-737, and ABT-263
against a panel of 6 helix/receptor interactions as well as the
Fos/Jun leucine zipper and luciferase controls. All inhibition experi-
ments were performed at 100 mM of the indicated compound.
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(b) Fluorescence polarization experiment with fluorescein labeled
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resulting in a Kd = 5.3 mM.
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BH3I-1 a well known inhibitor of the Bcl2 family further
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This research was partially supported by NIH (GM077403)
and NSF (CHE-0548264) to I. G and NIH (GM073943) to
P. S. A. We thank Dr Neal Zondlo for the His-MDM2 construct.
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