These hits were further validated by affinity pull-down and fluorescence polarization
(FP) assays [44].
Disulfiram inhibits acetaldehyde dehydrogenase by covalently modifying cysteine residues and ejecting a zinc ion. Since PHD fingers contain a conserved Cys3His-Cys4 motif bound to zinc ions, it was hypothesized that inhibition occurred by
the release of zinc ions [45]. Indeed, it was found that disulfiram ejects zinc from
JARID1A-PHD3 (IC 50 ¼ 10 μM), thus suggesting that it is a covalent inhibitor
for JARID1A-PHD3 as well as other PHD finger domains. Disulfiram’s alkylating
nature also results in inhibition of other proteins such as dopamine beta-hydroxylase,
viral nucleocapsid protein, DNA methyltransferase I, and histone methyltransferases
[46]. On the contrary, amiodarone HCl and tegaserod maleate were unable to release
zinc. To further probe the structural requirements for amiodarone inhibition of
the JARID1A-PHD3, a series of derivatives with varying amine chain lengths and
methylation states (compounds WAG-003–WAG-006, Fig. 3) as well as known
metabolites, desethylamiodarone, and di-N-desethylamiodarone were synthesized
and evaluated. The results led to the identification of inhibitors with a tenfold higher
potency than amiodarone, including di-N-desethylamiodarone (IC 50 : 26 μM) and
trimethylamiodarone (WAG-003, IC 50 : 30 μM). Docking studies suggest that
amiodarone analogs bind to JARID1A-PHD3 primarily in the surface peptide
groove and H3R2-binding pocket, but do not interact in the Kme-binding region.
Mutational studies supported these docking results; however, they were unable
to determine clearly the definitive orientation of the amiodarone scaffold when
it interacts with JARID1A-PHD3 [44]. More recently, a few amiodarone derivatives were observed to inhibit catalysis of JARIF1a/KDM5A in a PHD-fingerindependent manner, thus suggesting a nonunique mode of action [47].
3.2.2 Targeting Pygo PHD Fingers
A useful starting point for the development of new PHD-finger ligand lead structures
was reported in 2014. The potent oncogene β-catenin is a key effector of Wnt
signaling and is recruited to target genes through interaction with TCF/LEF transcription factors to activate cell differentiation [48]. Activating mutations in the
β-catenin gene have been proven in many types of cancer, and this fact makes
β-catenin a potential drug target. Yet, it is rather challenging to target this protein
directly with small molecules successfully. In fact, there are no enzymes required
for its activity that could be inhibited, and its interface with TCF factors involves
most of its structured domain, the armadillo repeat domain (ARD), which is extensive and also constitutes the interface for its negative regulators, including APC and
Axin, whose interaction with the ARD overlaps that of TCF [49]. Unsurprisingly,
attempts to block specifically the interaction between β-catenin and TCFs have
met with little success and failed to uncover any promising leads [48].
Another emerging strategy to target β-catenin signaling is by indirectly affecting
its ability to promote H3K4me-mediated transcription. The N-terminus of the ARD
346
G. Sbardella
(FP) assays [44].
Disulfiram inhibits acetaldehyde dehydrogenase by covalently modifying cysteine residues and ejecting a zinc ion. Since PHD fingers contain a conserved Cys3His-Cys4 motif bound to zinc ions, it was hypothesized that inhibition occurred by
the release of zinc ions [45]. Indeed, it was found that disulfiram ejects zinc from
JARID1A-PHD3 (IC 50 ¼ 10 μM), thus suggesting that it is a covalent inhibitor
for JARID1A-PHD3 as well as other PHD finger domains. Disulfiram’s alkylating
nature also results in inhibition of other proteins such as dopamine beta-hydroxylase,
viral nucleocapsid protein, DNA methyltransferase I, and histone methyltransferases
[46]. On the contrary, amiodarone HCl and tegaserod maleate were unable to release
zinc. To further probe the structural requirements for amiodarone inhibition of
the JARID1A-PHD3, a series of derivatives with varying amine chain lengths and
methylation states (compounds WAG-003–WAG-006, Fig. 3) as well as known
metabolites, desethylamiodarone, and di-N-desethylamiodarone were synthesized
and evaluated. The results led to the identification of inhibitors with a tenfold higher
potency than amiodarone, including di-N-desethylamiodarone (IC 50 : 26 μM) and
trimethylamiodarone (WAG-003, IC 50 : 30 μM). Docking studies suggest that
amiodarone analogs bind to JARID1A-PHD3 primarily in the surface peptide
groove and H3R2-binding pocket, but do not interact in the Kme-binding region.
Mutational studies supported these docking results; however, they were unable
to determine clearly the definitive orientation of the amiodarone scaffold when
it interacts with JARID1A-PHD3 [44]. More recently, a few amiodarone derivatives were observed to inhibit catalysis of JARIF1a/KDM5A in a PHD-fingerindependent manner, thus suggesting a nonunique mode of action [47].
3.2.2 Targeting Pygo PHD Fingers
A useful starting point for the development of new PHD-finger ligand lead structures
was reported in 2014. The potent oncogene β-catenin is a key effector of Wnt
signaling and is recruited to target genes through interaction with TCF/LEF transcription factors to activate cell differentiation [48]. Activating mutations in the
β-catenin gene have been proven in many types of cancer, and this fact makes
β-catenin a potential drug target. Yet, it is rather challenging to target this protein
directly with small molecules successfully. In fact, there are no enzymes required
for its activity that could be inhibited, and its interface with TCF factors involves
most of its structured domain, the armadillo repeat domain (ARD), which is extensive and also constitutes the interface for its negative regulators, including APC and
Axin, whose interaction with the ARD overlaps that of TCF [49]. Unsurprisingly,
attempts to block specifically the interaction between β-catenin and TCFs have
met with little success and failed to uncover any promising leads [48].
Another emerging strategy to target β-catenin signaling is by indirectly affecting
its ability to promote H3K4me-mediated transcription. The N-terminus of the ARD
346
G. Sbardella