Substrate-inspired peptides have also been employed by the group of Dirk
Schwarzer for affinity capture of HDACs and their interacting partners.
Chemoproteomic studies of HDAC-containing protein complexes were first developed at Cellzome, relying on the analysis of cell lysate samples after being exposed
to beads functionalized with HDAC inhibitors [96, 97]. The proteomic analysis was
performed for samples containing a competing HDAC inhibitor in solution, which
would avoid the removal of HDACs and their interacting partners. Such differences
in enrichment were measured in the presence of different inhibitors in order to
evaluate their potency as well as to identify their selectivity toward specific isotypes
or HDAC-containing complexes. Data from these experiments highlighted the
importance of studying HDAC1–HDAC3 in a biological environment rather than
isolated, since both affinity and selectivity of the inhibitors differed from previous
in vitro studies [96]. Schwarzer and coworkers implemented hydroxamic acidcontaining peptides as chemical probes for the enrichment instead of small molecule
inhibitors [64, 98]. First, a set of short peptides (Fig. 10, structure 10.1) was
synthesized and immobilized on agarose resin by reaction of a C-terminal cysteine
residue. Some of these peptides, especially the one containing Asuha (n ¼ 4), were
able to enrich class I, IIa, and IIb HDACs, as well as members of the CoREST
complex.
Then, two new probes were prepared based on the sequence of the tumor
suppressor protein p53 (p53-K382ac, which is a known substrate for HDACs) and
the nuclear transport factor 2 (NTF2-K4ac). In both cases, Kac was replaced by
Asuha, as shown in Fig. 10 (peptides 10.2 and 10.3). These new probes were able to
enrich HDACs with a different selectivity profile when compared to 10.1. Enrichment of class IIb HDACs dropped dramatically when using peptide 10.2, together
with HDAC8, whereas HDAC4 and members of NuRD and Sin3 complexes gained
Fig. 9 (a) Chemical structure of a hydroxamic acid-containing peptide HDAC inhibitor (9.1,
H4K16Asuha) and (b) crystal structure of the HDAC1:MTA1 interaction, assisted by a molecule
of inositol hexaphosphate (InsP 6 ) and in complex with such inhibitor (PDB code: 5ICN) [62]
46
C. Moreno-Yruela and C. A. Olsen
Schwarzer for affinity capture of HDACs and their interacting partners.
Chemoproteomic studies of HDAC-containing protein complexes were first developed at Cellzome, relying on the analysis of cell lysate samples after being exposed
to beads functionalized with HDAC inhibitors [96, 97]. The proteomic analysis was
performed for samples containing a competing HDAC inhibitor in solution, which
would avoid the removal of HDACs and their interacting partners. Such differences
in enrichment were measured in the presence of different inhibitors in order to
evaluate their potency as well as to identify their selectivity toward specific isotypes
or HDAC-containing complexes. Data from these experiments highlighted the
importance of studying HDAC1–HDAC3 in a biological environment rather than
isolated, since both affinity and selectivity of the inhibitors differed from previous
in vitro studies [96]. Schwarzer and coworkers implemented hydroxamic acidcontaining peptides as chemical probes for the enrichment instead of small molecule
inhibitors [64, 98]. First, a set of short peptides (Fig. 10, structure 10.1) was
synthesized and immobilized on agarose resin by reaction of a C-terminal cysteine
residue. Some of these peptides, especially the one containing Asuha (n ¼ 4), were
able to enrich class I, IIa, and IIb HDACs, as well as members of the CoREST
complex.
Then, two new probes were prepared based on the sequence of the tumor
suppressor protein p53 (p53-K382ac, which is a known substrate for HDACs) and
the nuclear transport factor 2 (NTF2-K4ac). In both cases, Kac was replaced by
Asuha, as shown in Fig. 10 (peptides 10.2 and 10.3). These new probes were able to
enrich HDACs with a different selectivity profile when compared to 10.1. Enrichment of class IIb HDACs dropped dramatically when using peptide 10.2, together
with HDAC8, whereas HDAC4 and members of NuRD and Sin3 complexes gained
Fig. 9 (a) Chemical structure of a hydroxamic acid-containing peptide HDAC inhibitor (9.1,
H4K16Asuha) and (b) crystal structure of the HDAC1:MTA1 interaction, assisted by a molecule
of inositol hexaphosphate (InsP 6 ) and in complex with such inhibitor (PDB code: 5ICN) [62]
46
C. Moreno-Yruela and C. A. Olsen