peptide (H3K9me3(1–25)). From this screen, eight putative TTD-binding hits were
identified. Analysis of amide peak movement in the (
1 H15 N) HSQC spectra of the
TTD in the presence of the fragments indicates that the binding of one of them,
4-benzylpiperidine-1-carboximidamide (BPC, Fig. 9b), occurs in the groove [151].
Further characterization of BPC binding to isolated TTD using ITC and DSF
estimated a K D of 50 μm and a calculated ligand efficiency of 0.38 (which is defined
as the binding energy per heavy atom) [151]. Saturating concentrations of this highly
soluble compound induced open conformers of the TTD-PHD module and reduced
its affinity for H3K9me3 peptides. Therefore, BPC could be a useful tool for in vitro
investigations aimed to relate open TTD-PHD conformations with specific UHRF1binding modes. Due to the small size and high ligand efficiency, the compound can
be further optimized for potency, for instance, by linking it to compounds designed
for interaction with the aromatic cage, which recognizes the trimethyl lysine of
H3K9me3.
Targeting Spindlin-1
The emerging role of Spindlin-1 in transformation and cancer prompted a growing
interest in developing inhibitors of the protein-protein interactions that it mediates.
Recently, an epigenetic reader assay platform was developed to identify smallmolecule inhibitors of the Spindlin-1-H3K4me3 interaction [152]. The methodology
included a primary screen using an in vitro AlphaLISA assay, an FP-based verification assay, a fluorescent thermal shift assay (FTSA) to investigate direct binding,
a biolayer interferometry (BLI) assay, and finally cellular thermal shift assay
(CETSA). The pilot screen of a small library of bioactive compounds identified
compound A366 (Fig. 16), previously reported as selective inhibitor of lysine
methyltransferase G9a [153], as endowed with good in vitro inhibition of the
Spindlin-1-H3K4me3 interaction (IC 50 ~200 nM) as a result of H3K4me3competitive direct binding to the Tudor domains of Spindlin-1. However, the
compound showed poor cell-based activity (100 μM) [152]. Moreover, similarly to
what was previously reported against G9a [153], even minor structural modifications
of A366 led to significant reduction of SPIN1-binding affinity. In fact, the structurally related compound YX-11-102 (Fig. 10) did not show any activity in either the
AlphaLISA or FP assay [152]. More recently, the same group used an iterative
virtual screening approach to generate pharmacophore models based on the crystal
structure of Spindlin-1 in complex with H3K4me3 peptide and, subsequently, screen
databases of commercially available compounds. After docking the identified potential hits in the crystal structure of Spindlin-1 to verify the occurrence of significant
interactions with the residues of the binding site, structural analogs of the validated
hits were synthesized and tested employing an AlphaLISA assay. Two molecular
scaffolds, 4-aminoquinazoline and quinazolinethione, were identified as promising
lead structures (Fig. 10) [154]. However, all the derivatives synthesized displayed
only low-to-moderate activity in vitro. Interestingly, the studies highlighted that an
appropriate lysine mimetic group is crucial for inhibitory activity [154].
Methyl-Readers and Inhibitors
361
identified. Analysis of amide peak movement in the (
1 H15 N) HSQC spectra of the
TTD in the presence of the fragments indicates that the binding of one of them,
4-benzylpiperidine-1-carboximidamide (BPC, Fig. 9b), occurs in the groove [151].
Further characterization of BPC binding to isolated TTD using ITC and DSF
estimated a K D of 50 μm and a calculated ligand efficiency of 0.38 (which is defined
as the binding energy per heavy atom) [151]. Saturating concentrations of this highly
soluble compound induced open conformers of the TTD-PHD module and reduced
its affinity for H3K9me3 peptides. Therefore, BPC could be a useful tool for in vitro
investigations aimed to relate open TTD-PHD conformations with specific UHRF1binding modes. Due to the small size and high ligand efficiency, the compound can
be further optimized for potency, for instance, by linking it to compounds designed
for interaction with the aromatic cage, which recognizes the trimethyl lysine of
H3K9me3.
Targeting Spindlin-1
The emerging role of Spindlin-1 in transformation and cancer prompted a growing
interest in developing inhibitors of the protein-protein interactions that it mediates.
Recently, an epigenetic reader assay platform was developed to identify smallmolecule inhibitors of the Spindlin-1-H3K4me3 interaction [152]. The methodology
included a primary screen using an in vitro AlphaLISA assay, an FP-based verification assay, a fluorescent thermal shift assay (FTSA) to investigate direct binding,
a biolayer interferometry (BLI) assay, and finally cellular thermal shift assay
(CETSA). The pilot screen of a small library of bioactive compounds identified
compound A366 (Fig. 16), previously reported as selective inhibitor of lysine
methyltransferase G9a [153], as endowed with good in vitro inhibition of the
Spindlin-1-H3K4me3 interaction (IC 50 ~200 nM) as a result of H3K4me3competitive direct binding to the Tudor domains of Spindlin-1. However, the
compound showed poor cell-based activity (100 μM) [152]. Moreover, similarly to
what was previously reported against G9a [153], even minor structural modifications
of A366 led to significant reduction of SPIN1-binding affinity. In fact, the structurally related compound YX-11-102 (Fig. 10) did not show any activity in either the
AlphaLISA or FP assay [152]. More recently, the same group used an iterative
virtual screening approach to generate pharmacophore models based on the crystal
structure of Spindlin-1 in complex with H3K4me3 peptide and, subsequently, screen
databases of commercially available compounds. After docking the identified potential hits in the crystal structure of Spindlin-1 to verify the occurrence of significant
interactions with the residues of the binding site, structural analogs of the validated
hits were synthesized and tested employing an AlphaLISA assay. Two molecular
scaffolds, 4-aminoquinazoline and quinazolinethione, were identified as promising
lead structures (Fig. 10) [154]. However, all the derivatives synthesized displayed
only low-to-moderate activity in vitro. Interestingly, the studies highlighted that an
appropriate lysine mimetic group is crucial for inhibitory activity [154].
Methyl-Readers and Inhibitors
361