Interestingly, any changes to the secondary tert-butyl amine of UNC2170 resulted
in a complete loss of affinity for 53BP1 [146]. Noteworthy, the tertiary amine
derivative UNC2892 (Fig. 9a) was chosen as a negative control for follow-up
studies, due to its structural similarity to UNC2170 and its complete lack of binding
affinity for 53BP1. In order to confirm that the complete loss of activity for
the analogs was due to the lack of the secondary tert-butyl amine, a methyllysinebinding site mutant of 53BP1 (D1521A) was prepared. Removal of the aspartate
is known to disrupt the salt bridge and hydrogen bond between 53BP1 and the
dimethylammonium of H4K20me2 [96]. UNC2170 did not bind to the 53BP1
mutant, providing strong evidence that the secondary tert-butyl amine was
interacting with the Kme2-binding region [146]. The only tolerated substitutions
resulted from the replacement of the bromine atom in the C-3 position of the
aromatic moiety with bulky, lipophilic substituents, such as iodine, isopropyl,
and trifluoromethyl, which all yielded selective and moderate inhibition [146].
Further analysis of UNC2170 confirmed its potential as a chemical probe. Pulldown
assays with H4K20me2 and p53K382me2 demonstrated that UNC2170 binds
competitively to the TT domain of a His-53BP1 fusion protein. UNC2170 was
able to competitively displace p53K382me2 in a concentration-dependent manner
(IC 50 ¼ 30 μM). Within a cellular context, UNC2170 was nontoxic and highly
permeable with no significant measurable efflux as determined by a bidirectional
Caco-2 cell permeability assay. No significant decrease in 53BP1 foci formation was
observed in U2OS and γH2AX cells before and after ionizing radiation, possibly
due to the bivalent nature of 53BP1 readers also recognizing H2AK15ub at DSB
sites [147]. UNC2892 was inactive in each of these cellular assays, providing
strong evidence that the cellular effects seen with UNC2170 are due to inhibition
of 53BP1 and highlighting the potential of a potent 53BP1 antagonist.
Targeting UHRF1
Histone and DNA binding by UHRF1 are regulated by long-range interdomain
and linker-domain interactions within the full-length protein [148–150]. It has
been showed that a 15-residue polybasic region (PBR-UHRF1643–657) in the linker
between the SRA and RING (really interesting and new gene) domains regulates
the transition between PHD- and TTD-mediated histone reader states. This
occurs through its reversible binding to the TTD groove or the phospholipid PI5P
that in full-length UHRF1 results in the failure of UHRF1 to recognize H3K9me3
caused by a transition from TTD-mediated to PHD-mediated histone binding [148].
The PHD has also been reported to interact with the SRA domain in a UHRF1
state where histone binding is restricted [149, 150]. These studies suggest that
disruption of interdomain interactions could be a mechanism to target UHRF1
pharmacologically.
A small-molecule fragment library containing 2,040 compounds was screened
against isolated TTD (UHRF1121–UHRF1286) using a fluorescence polarization
(FP) assay that tracked the displacement of an N-terminally tagged H3K9me3
360
G. Sbardella
in a complete loss of affinity for 53BP1 [146]. Noteworthy, the tertiary amine
derivative UNC2892 (Fig. 9a) was chosen as a negative control for follow-up
studies, due to its structural similarity to UNC2170 and its complete lack of binding
affinity for 53BP1. In order to confirm that the complete loss of activity for
the analogs was due to the lack of the secondary tert-butyl amine, a methyllysinebinding site mutant of 53BP1 (D1521A) was prepared. Removal of the aspartate
is known to disrupt the salt bridge and hydrogen bond between 53BP1 and the
dimethylammonium of H4K20me2 [96]. UNC2170 did not bind to the 53BP1
mutant, providing strong evidence that the secondary tert-butyl amine was
interacting with the Kme2-binding region [146]. The only tolerated substitutions
resulted from the replacement of the bromine atom in the C-3 position of the
aromatic moiety with bulky, lipophilic substituents, such as iodine, isopropyl,
and trifluoromethyl, which all yielded selective and moderate inhibition [146].
Further analysis of UNC2170 confirmed its potential as a chemical probe. Pulldown
assays with H4K20me2 and p53K382me2 demonstrated that UNC2170 binds
competitively to the TT domain of a His-53BP1 fusion protein. UNC2170 was
able to competitively displace p53K382me2 in a concentration-dependent manner
(IC 50 ¼ 30 μM). Within a cellular context, UNC2170 was nontoxic and highly
permeable with no significant measurable efflux as determined by a bidirectional
Caco-2 cell permeability assay. No significant decrease in 53BP1 foci formation was
observed in U2OS and γH2AX cells before and after ionizing radiation, possibly
due to the bivalent nature of 53BP1 readers also recognizing H2AK15ub at DSB
sites [147]. UNC2892 was inactive in each of these cellular assays, providing
strong evidence that the cellular effects seen with UNC2170 are due to inhibition
of 53BP1 and highlighting the potential of a potent 53BP1 antagonist.
Targeting UHRF1
Histone and DNA binding by UHRF1 are regulated by long-range interdomain
and linker-domain interactions within the full-length protein [148–150]. It has
been showed that a 15-residue polybasic region (PBR-UHRF1643–657) in the linker
between the SRA and RING (really interesting and new gene) domains regulates
the transition between PHD- and TTD-mediated histone reader states. This
occurs through its reversible binding to the TTD groove or the phospholipid PI5P
that in full-length UHRF1 results in the failure of UHRF1 to recognize H3K9me3
caused by a transition from TTD-mediated to PHD-mediated histone binding [148].
The PHD has also been reported to interact with the SRA domain in a UHRF1
state where histone binding is restricted [149, 150]. These studies suggest that
disruption of interdomain interactions could be a mechanism to target UHRF1
pharmacologically.
A small-molecule fragment library containing 2,040 compounds was screened
against isolated TTD (UHRF1121–UHRF1286) using a fluorescence polarization
(FP) assay that tracked the displacement of an N-terminally tagged H3K9me3
360
G. Sbardella