Probe		Negative control
L-Moses		D-Moses

p300/CBP-associated factor (PCAF/KAT2B) and general control non-derepressible 5 (GCN5/KAT2A) are members of subfamily 1 of the bromodomain phylogenetic tree. These multi-domain proteins that have been implicated in retroviral infection, inflammation pathways and cancer development. However, outside of viral replication, little is known about the dependence of these effects on the C-terminal bromodomain.  L-Moses is as a chemical probe for the PCAF/GCN5 bromodomain and D-Moses is the enantiomeric negative control. Rational inhibitor design and biophysical characterization led to the discovery of L-Moses. The probe was optimized from the non-selective pan-bromodomain inhibitor, bromosporine to generate a potent, selective (>4500-fold selective over BRD4), permeable and cell-active PCAF/GCN5 bromodomain chemical probe.

Potency

PCAF K_i 47 nM in a HTRF binding competition assay using PCAF bromodomain and a biotin tagged bromodomain ligand.

PCAF K_D 48 nM in a BROMOscan assay run at DiscoverX.
GCN5 K_D 220 nM in a BROMOscan assay run at DiscoverX.

PCAF K_D 126 nM (ITC) using PCAF bromodomain.
GCN5 K_D 600 nM (ITC) using GCN5 bromodomain.

Non-family targets

GPCR/Eurofins Panel: In an panel of 130 potential off targets, L-Moses showed no binding (>60% at 10 μM) to all targets except the opioid receptors (mu 100 nM, OPRL1 840 nM, kappa 1,100 nM,) and the 5-HT transporter (220 nM).

Cellular Potency

PCAF:  IC₅₀ 220 nM in Promega NanoBRET assay, measuring displacement of NanoLuc-tagged truncated bromodomain PCAF from Halo-tagged histone H3.3 in HEK293 cells.
IC₅₀ 1.2 μM in NanoBRET assay, measuring displacement of NanoLuc-tagged full-length PCAF from Halo-tagged histone H3.3 in HEK293 cells.

IC₅₀ 660 nM for competing pull-down of full-length PCAF from cell lysates using immobilized L-Moses

GCN5: IC₅₀ 220 nM for competing pull-down of full-length PCAF from cell lysates using immobilized L-Moses.

Cytoxicity assay:

Toxicity of D-Moses and L-Moses was assessed on peripheral blood mononuclear cells  (PBMC) obtained   from   5   healthy   donors.   PBMC   were   cultured   either   with D-Moses or L-Moses at concentrations of 0.1, 1 and 10 μM or with a control (DMSO) for 24 hours. Viability of PBMC were then checked using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (ThermoFisher Scientific). No observed cytotoxicity was observed at any concentration.

Probe		Negative control
GSK4027		GSK4028

p300/CREB binding protein associated factor (PCAF/KAT2B) and general control non-derepressible 5 (GCN5/KAT2A) are multidomain proteins that have been implicated in retroviral infection, inflammation pathways and cancer development. However, outside of viral replication, little is known about the dependence of these effects on the C-terminal bromodomain.  GSK4027 is as a chemical probe for the PCAF/GCN5 bromodomain and  GSK4028 is the enantiomeric negative control. The probe was optimized from a weakly potent, non-selective pyridazinone hit to deliver high potency for the PCAF/GCN5 bromodomain, high solubility, cellular target engagement and ≥18000-fold selectivity over the BET family, together with ≥70 fold selectivity over the wider bromodomain families.

Potency

PCAF IC₅₀ 40 nM in a TR-FRET binding competition assay using truncated PCAF bromodomain and a fluorescently tagged bromodomain ligand.

PCAF Ki 1.4 nM in a BROMOscan assay run at DiscoverX.

GCN5 Ki 1.4 nM in a BROMOscan assay run at DiscoverX.

3D structure

Co-crystal structure with GCN5: PDB ID 5MLJ

Selectivity Bromodomains

>70 fold selectivity over other bromodomain targets including BRPF3 (100 nM), BRD1 (110 nM), FALZ (130 nM) and BRPF1 (140 nM) In BROMOscan assay run at DiscoverX.

Non-family targets

GSK internal enhanced, cross-screening panel (eXP); a full curve against 53 biochemical and phenotypic assays did not reveal any off-target binding <3 µM.

Cellular Potency 

IC₅₀ 60 nM in Promega NanoBRET assay, measuring displacement of NanoLuc-tagged full length PCAF from Halo-tagged histone H3.3 in HEK293 cells.

Cytoxicity assay: 

Cellular health assay looking at mitochondrial integrity, nuclear size and membrane permeability found no changes up to 200 µM.

Probe		Negative control
BAY-6035		BAY-444

A collaboration between Bayer AG and the SGC has resulted in the discovery of BAY-6035, a potent, peptide-competitive chemical probe for SMYD3. BAY-6035 has a unique chemotype relative to the published SMYD3 inhibitor, EPZ031686. BAY-6035 inhibits in vitro methylation of MEKK2 peptide with IC50 = 88 nM and has more than 100-fold selectivity over other histone methyltransferases. BAY-6035 inhibits the methylation of MEKK2 in cells with IC50 = 70 nM. A control compound, BAY-444, has also been developed. BAY-444 inhibits the in vitro methylation of MEKK2 with IC50 = 23 micromolar.

https://doi.org/10.1177/24725552211019409