282 nM, respectively) as potent inhibitors of L3MBTL1/H3K9me1 interactions.
A specific binding of these compounds to the labeled L3MBTL1-His protein was
confirmed by follow-up studies. Yet, further reports on these compounds have
not been disclosed due to unclearness of the exact mechanism of action.
4.4.2.1 Targeting L3MBTL1
A virtual screening campaign performed on a large library of commercially available
compounds containing such moieties led to the identification of several hits, which
were predicted to bind with high affinity to the H4K20Me2 pocket on L3MBTL1
[175]. Among them, a few compounds (in particular VS Hits #1, 2, and 13; Fig. 11)
that contained a substituted aromatic moiety linked to an alkylated amine through
a variety of different tethers were identified as modest inhibitors of L3MBTL1 in
the AlphaScreen™ assay (IC 50 values ¼ 14–17 μM). Docking studies with VS
Hit #13 in the MBT domain suggested that the pyrrolidine nitrogen mimics the
Kme and forms an ionic bond with Asp355, while the sulfonamide forms hydrogen
bonds with the side chains of Tyr386 and Asn358.
A combined ligand- and structure-based approach (using the co-crystal structure
of the L3MBTL1-H4K20me2 complex; PDB: 2PQW [166]) led to the design of a
series of peptidomimetic inhibitors targeting L3MBTL1 [176]. These compounds
included a histidine residue to promote interactions between the small molecule
and Thr385 on the protein surface, while interactions with the methyllysine-binding
pocket of the MBT2 domain were probed by the addition of various alkylated
diamines. Not surprisingly based on the hits from the virtual screening study,
the pyrrolidine peptidomimetic structure #6 (Fig. 11) demonstrated significantly
improved binding affinity (K D ¼ 37 μM) compared to several acyclic mono-, di-,
or trimethylated amines. Furthermore, the compound occupied the anticipated
MBT2 domain site as confirmed by the loss of binding to an L3MBTL1-D355A
mutant [176]. To simplify the peptidomimetic structure and determine whether
substituted aromatic moieties with improved drug-like properties (cell permeability,
ligand efficiency) could enhance the scaffold, a large library of analogs was synthesized and evaluated. While much of the structure-activity relationship (SAR)
for this region of the scaffold was initially flat, nicotinamide derivative UNC280
(Fig. 11) did demonstrate moderate-binding affinity (K D ¼ 26 μM) and a fourfold
increase in ligand efficiency when compared to the native peptide, H4K20me1 9-mer
(residues 17–25), in ITC studies. UNC280 was not selective for L3MBTL1
but rather demonstrated modest potency against L3MBTL3 and PHF13 domains
(IC 50 values of 28 μM and 48 μM, respectively). The incorporation of the sulfonamide moiety from VS Hit #13 into the UNC280 scaffold led to the pyrrolidine
containing sulfonamide derivatives #10 (Fig. 11), which demonstrated a comparable
binding affinity to L3MBTL1 (K D ¼ 25 μM). Both the substitution of the pyrrolidine
ring of this derivative and the addition of an oxygen atom to the linker produced
significantly reduced or inactive compounds. By contrast, substituting the alkyl
linker with a bulky and inflexible piperidine resulted in compound UNC669
366
G. Sbardella
A specific binding of these compounds to the labeled L3MBTL1-His protein was
confirmed by follow-up studies. Yet, further reports on these compounds have
not been disclosed due to unclearness of the exact mechanism of action.
4.4.2.1 Targeting L3MBTL1
A virtual screening campaign performed on a large library of commercially available
compounds containing such moieties led to the identification of several hits, which
were predicted to bind with high affinity to the H4K20Me2 pocket on L3MBTL1
[175]. Among them, a few compounds (in particular VS Hits #1, 2, and 13; Fig. 11)
that contained a substituted aromatic moiety linked to an alkylated amine through
a variety of different tethers were identified as modest inhibitors of L3MBTL1 in
the AlphaScreen™ assay (IC 50 values ¼ 14–17 μM). Docking studies with VS
Hit #13 in the MBT domain suggested that the pyrrolidine nitrogen mimics the
Kme and forms an ionic bond with Asp355, while the sulfonamide forms hydrogen
bonds with the side chains of Tyr386 and Asn358.
A combined ligand- and structure-based approach (using the co-crystal structure
of the L3MBTL1-H4K20me2 complex; PDB: 2PQW [166]) led to the design of a
series of peptidomimetic inhibitors targeting L3MBTL1 [176]. These compounds
included a histidine residue to promote interactions between the small molecule
and Thr385 on the protein surface, while interactions with the methyllysine-binding
pocket of the MBT2 domain were probed by the addition of various alkylated
diamines. Not surprisingly based on the hits from the virtual screening study,
the pyrrolidine peptidomimetic structure #6 (Fig. 11) demonstrated significantly
improved binding affinity (K D ¼ 37 μM) compared to several acyclic mono-, di-,
or trimethylated amines. Furthermore, the compound occupied the anticipated
MBT2 domain site as confirmed by the loss of binding to an L3MBTL1-D355A
mutant [176]. To simplify the peptidomimetic structure and determine whether
substituted aromatic moieties with improved drug-like properties (cell permeability,
ligand efficiency) could enhance the scaffold, a large library of analogs was synthesized and evaluated. While much of the structure-activity relationship (SAR)
for this region of the scaffold was initially flat, nicotinamide derivative UNC280
(Fig. 11) did demonstrate moderate-binding affinity (K D ¼ 26 μM) and a fourfold
increase in ligand efficiency when compared to the native peptide, H4K20me1 9-mer
(residues 17–25), in ITC studies. UNC280 was not selective for L3MBTL1
but rather demonstrated modest potency against L3MBTL3 and PHF13 domains
(IC 50 values of 28 μM and 48 μM, respectively). The incorporation of the sulfonamide moiety from VS Hit #13 into the UNC280 scaffold led to the pyrrolidine
containing sulfonamide derivatives #10 (Fig. 11), which demonstrated a comparable
binding affinity to L3MBTL1 (K D ¼ 25 μM). Both the substitution of the pyrrolidine
ring of this derivative and the addition of an oxygen atom to the linker produced
significantly reduced or inactive compounds. By contrast, substituting the alkyl
linker with a bulky and inflexible piperidine resulted in compound UNC669
366
G. Sbardella