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mediate affinity 3,4-methylenedioxyphenyl analogue 1g,
which exhibits a 14-fold reduced affinity for F36V-
FKBP, similarly proved as effective as AP1889 (1f),
although induced expression levels in stably transfected
cells were lower. All the dimerizers remained selectively
active on cells expressing F36V-FKBP fusion proteins
compared to wild-type FKBP fusions, demonstrating
that the modifications did not affect the binding specifi-
city of the engineered ‘bump’ (data not shown).
F36V, potently activates Fas-mediated apoptosis and
other signalling processes in appropriately engineered
cells.9,11 However the molecule is surprisingly inefficient
at activating transcription, only functioning in tran-
siently transfected cells where F36V-transcription factor
fusion proteins are present at high levels. The analysis
presented here indicates that while dimerizer potency in
signalling appears largely driven by binding affinity, the
structure activity relationship for transcription is more
subtle, with kinetic and/or geometric factors also con-
tributing. Indeed, we found that reducing the binding
affinity of AP1903 dramatically enhanced activity, sug-
gesting that tight binding prevents the efficient and
dynamic assembly of functional dimerized complexes.
These insights led to the design of novel, simpler
dimerizers such as AP1889 (1f) and AP1966 (1k) that
potently activate targeted gene expression in both tran-
siently and stably transfected cells expressing F36V-
FKBP fusion proteins. These homodimeric ligands,
engineered to bind minimally to endogenous FKBPs,
offer an alternative approach to the rapamycin-based
heterodimerizer system3,6 for inducing gene expression
in stable cell lines and transgenic animals.
With the availability of a dimerizer able to induce tran-
scription in stably transfected cells, we investigated the
activity of a heterodimeric compound 1i incorporating
one monomer unit of AP1889 (1f) and one of AP1903
(1b). We speculated that if slow dissociation of dimer-
ized protein complexes accounts for AP1903’s poor
activity, replacement of even one monomer with a
weaker binding moiety should still improve potency,
since non-productive homodimeric species could be
converted to productive ones by dissociation of only
one fusion protein followed by rebinding of another.
Indeed, 1i proved to be more potent than either AP1903
(1b) or AP1889 (1f) in both transiently and stably
transfected cells. Its EC50 of 2 nM in stably transfected
cells is the most potent to date of any dimerizer for the
activation of gene transcription. To assess how weak a
protein-ligand interaction is compatible with efficient
transcription, the AP1889 (1f) half of 1i was replaced
with the drastically weaker monomer of 1h (monomer
IC50: 1530 nM) to afford dimerizer 1j. Surprisingly, 1j
exhibited good, though 7-fold weaker, activity in tran-
siently transfected cells versus AP1903 (1b), and
retained some activity in stable cell lines. 1j also dis-
played a unique dose–response curve. Whereas increas-
ing doses of other dimerizers induced a peak level of
expression and then leveled off, 1j quickly dropped to
baseline after achieving its peak. This is most probably
due to an excess of the AP1903 subunit of 1j serving as a
self-antagonist through the accumulation of monomeric
(rather than dimeric) complexes.
References and Notes
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ular weight, we replaced the 2-arylbutyric acid bottom
portion of AP1903 with radically different structures. Of
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the trimethoxyphenylbutyric acid bottom was replaced
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type FKBP had been reported in the literature,13 due to
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mer of 1k exhibited far greater selectivity for F36V-
FKBP than wt-FKBP (64 nM vs 14 mM). The resulting
dimerizer exhibited an EC50 only 2–3-fold reduced from
that of AP1889 (1f) in both transient and stable assays,
despite its radically simplified structure, and peak
expression levels were also greater than those for
AP1889 in both settings.
7. Amara, J. F.; Clackson, T.; Rivera, V. M.; Guo, T.;
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Conclusion
AP1903 (1b), a dimerizer with engineered specificity and
tight binding affinity for the designed FKBP mutant