A different approach used a library of biotin-tagged analogs of the smallmolecule probe UNC1215, a nonselective inhibitor of MBT domains (see below),
to screen a protein domain microarray that harbored about 100 GST fusion proteins,
including 41 Tudor domains, 31 chromodomains, and representative PHD, BHA,
MBT, PWWP, ANK, AGENET, and HEAT domains. This “library-on-library”
approach led to the identification of EML405 (Fig. 10) as a lead compound capable
to interact with Spindlin-1 [20]. Structural studies revealed that EML405 engages
both the first and second Tudor domains of Spindlin-1 and also revealed a large
unoccupied negatively charged pocket [20]. With the aim to engage this pocket,
a series of derivatives were designed and synthesized and revealed an improved
binding constant with respect to EML405 (3 μM for EML631 vs 14 μM for
EML405). Moreover, the novel compounds displayed a significantly weaker
binding affinity for the Tudor domain-containing proteins 53BP1 and PHF20
and for the MBT domain-containing proteins L3MBTL1 and L3MBTL3, thus
showing a dramatic improvement of selectivity for Spindlin-1. Competition assays,
chemiprecipitation experiments, and cellular thermal shift assay (CETSA) experiments demonstrated that the most specific compound identified in this study,
EML631 (Fig. 10), was capable to engage Spindlin-1 in cells, block its ability
to recognize H3K4me3 marks and inhibit its transcriptional coactivator activity.
Interestingly, the crystal structure of the complex between Spindlin-1 and
EML631 revealed an unpredicted interaction of the additional pyrrolidine group
with a negatively charged groove that lies between the first two Tudor domains.
Recently, EML631 was successfully used as a chemical probe to characterize
A366
YX-11-102 (negative control)
N
O
O
H 3 C
N
NH 2
N
CH 3
O
O
H 3 C
N
CH 3
N
H
N
O
S
N
CH 3
CH 3
O
O
H 3 C
N
H
N
HN
S
O
O
H 3 C
N
4-aminoquinazolinethione derivative 1k
thioquinazoline derivative 4q
EML405
NH
N
O
N
N
N
O
NH
N
O
N
N
N
O
O
N
EML631
Fig. 10 Small-molecule inhibitors of Spindlin-1
362
G. Sbardella
to screen a protein domain microarray that harbored about 100 GST fusion proteins,
including 41 Tudor domains, 31 chromodomains, and representative PHD, BHA,
MBT, PWWP, ANK, AGENET, and HEAT domains. This “library-on-library”
approach led to the identification of EML405 (Fig. 10) as a lead compound capable
to interact with Spindlin-1 [20]. Structural studies revealed that EML405 engages
both the first and second Tudor domains of Spindlin-1 and also revealed a large
unoccupied negatively charged pocket [20]. With the aim to engage this pocket,
a series of derivatives were designed and synthesized and revealed an improved
binding constant with respect to EML405 (3 μM for EML631 vs 14 μM for
EML405). Moreover, the novel compounds displayed a significantly weaker
binding affinity for the Tudor domain-containing proteins 53BP1 and PHF20
and for the MBT domain-containing proteins L3MBTL1 and L3MBTL3, thus
showing a dramatic improvement of selectivity for Spindlin-1. Competition assays,
chemiprecipitation experiments, and cellular thermal shift assay (CETSA) experiments demonstrated that the most specific compound identified in this study,
EML631 (Fig. 10), was capable to engage Spindlin-1 in cells, block its ability
to recognize H3K4me3 marks and inhibit its transcriptional coactivator activity.
Interestingly, the crystal structure of the complex between Spindlin-1 and
EML631 revealed an unpredicted interaction of the additional pyrrolidine group
with a negatively charged groove that lies between the first two Tudor domains.
Recently, EML631 was successfully used as a chemical probe to characterize
A366
YX-11-102 (negative control)
N
O
O
H 3 C
N
NH 2
N
CH 3
O
O
H 3 C
N
CH 3
N
H
N
O
S
N
CH 3
CH 3
O
O
H 3 C
N
H
N
HN
S
O
O
H 3 C
N
4-aminoquinazolinethione derivative 1k
thioquinazoline derivative 4q
EML405
NH
N
O
N
N
N
O
NH
N
O
N
N
N
O
O
N
EML631
Fig. 10 Small-molecule inhibitors of Spindlin-1
362
G. Sbardella