C O M M U N I C A T I O N S
Figure 2. (a) Isoxazolidines with varying spatial orientations of polar and hydrophobic functional groups. (b) Synthetic scheme for isoxazolidines 3, 5, and
6. An analogous series of reactions were used for the preparation of 4 and 7.5 (c) Results from in vitro transcription assays. The DBD is the fusion protein
LexA-DHFR; the high affinity interaction between DHFR and methotrexate localizes the Mtx-tagged small molecules (50 nM) to DNA.11 Each activity is
the average of at least three independent experiments, with the indicated error (SDOM). The maximal activation is 7-fold relative to background. See
Supporting Information for additional details.
the data indicate that precise positioning of functional groups is
not the most important determinant of activator function in our
system.
were supported by the UM CBI training program (GM08597).
A.K.M. is an Alfred P. Sloan Fellow. We thank Prof. G. Glick for
helpful discussions.
The conserved activity across amphipathic, isomeric isoxazo-
lidines 3-7 parallels the functional behavior of the endogenous
amphipathic ADs that this molecular class was originally designed
to mimic. For example, the activity of 3 and 4 is consistent with
an earlier report that the D and L-enantiomers of the natural AD
ATF29 stimulate similar transcription levels in a cell-free system.7
Among peptidic ADs, a variety of combinations of polar and
hydrophobic amino acids function as ADs, but a hydrophobic/polar
balance is conserved.2,3a-c Further, like our small molecules,
endogenous ADs share a common structural motif; for natural ADs,
structural studies suggest that formation of a helix occurs upon
binding to a number of transcriptional machinery targets, although
other secondary structures may play a role.8 Also similar to the
isoxazolidines,5 mutations in natural ADs that disrupt the hydro-
phobic surface significantly decrease activation potential.9 One
remaining question is whether the similarities between the small
molecules and natural ADs extend to the binding surfaces within
the transcriptional machinery. Although the aggregate data are
suggestive of an affirmative answer, cross-linking experiments will
be required to provide a more definitive conclusion.
In sum, our data suggest that isoxazolidines are unlikely to be
the only suitable scaffolds for the construction of small molecule
transcriptional activation domains. Rather, a variety of appropriately
functionalized conformationally constrained small molecules should
also function well, a prediction currently under investigation. This
strategy obviates the need to identify high affinity ligands for single
protein targets and takes advantage of the remarkable functional
flexibility of the endogenous transcriptional regulatory system.
Given the increased interest in small molecule ADs as mechanistic
probes and therapeutic agents,10 this approach may find wide
application.
Supporting Information Available: Synthetic details for com-
pounds 3-7 and complete refs 1b and 1c. This material is available
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Acknowledgment. The Burroughs Wellcome Fund and the
March of Dimes provided support for this work. A.R.M. and S.J.B.
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