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

Probe		Negative control
FM-381		FM-479

Biology of the JAK3 kinase

Janus kinases (JAKs) belong to the family of cytoplasmic tyrosine kinases. In human, the JAK family itself consist of 4 members (JAK1, JAK2, JAK3 and TYK). JAK family members are multi-domain proteins of about 130 kDa, which are highly homologous with respect to their domain structure and residue conservation within their structured domains.


All JAK family members possess a catalytic kinase domain (KD) located at the C-terminus, an adjacent pseudokinase domain (PKD) flanked by a Src homology 2 (SH2) domain.  The N-terminal FERM (four-point-one, ezrin, radixin and moesin homology) domain (B41) serves to mediate the interaction between the JAK and the cytokine receptor [1]. 

JAK kinases are effectors of the JAK-STAT signaling pathway, which is triggered by ligand binding to a cognate receptor resulting in activation of JAK by phosphorylation of key tyrosine residues within the catalytic domain. After activation, tyrosine resides in the receptor intracellular region are also phosphorylated which triggers recruitment and phosphorylation of the principal downstream effectors, the STATs. Phosphorylated STATs dimerize and translocate to the nucleus where they initiate transcription of specific responsive genes [1, 2, 3].
Contrary to the other JAK family members, JAK3 expression is restricted to the hematopoietic system, where it plays a specific role in the development of immune-competent cells [4]. This key function of JAK3 has been confirmed by loss-of-function mutations of JAK3, which cause severe combined immunodeficiency syndrome (SCID) [5].  Aberrant activation in kinase domain has been described in lymphoproliferative disorders (T-ALL; T-PLL). JAK3 is required for cytokine signalling downstream of receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 and it acts via activation of the gamma chain receptors (γc). JAK1 and JAK3 co-localise on cytokine receptor dimers suggesting that dual JAK1 and JAK3 inhibition may be required for efficient suppression of cytokine signaling [6].  In order to study JAK3 specific functions we developed in collaboration with the laboratory of Stefan Laufer a JAK3 specific reversible covalent inhibitor.
 

We recommend using FM-381 at 100-300 nM for specific JAK3 inhibition. The inactive control FM-479 has no activity on JAK3 or other kinases when used at similar concentration.
As a non-covalent control inhibitor we recommend NIBR3049 (Novartis) at 1 µM (this potent reversible compound is selective for JAK3 within the JAK family, but shows also potent inhibition of other kinases such as GSK3, PKCα, PCKθ).

FM-381: A CHEMICAL PROBE FOR JAK3

An additional non-covalent control compound (NIBR3049) has been donated by Novartis [7]. This inhibitor is highly selective for JAK3 within the JAK kinase family but inhibits also other kinases with considerable activity including GSK3, PKCα, PKCθ. NIBR3049 is therefore not suitable as an independent JAK3 specific probe. 

Co-crystal structure

Details of the co-crystal structure of FM-381 with the kinase domain of JAK3 kinase in its reversible binding mode, click on the 'Co-Crystal structures' tab above for more details.

Potency Against Target Family

FM-381 is a potent covalent reversible inhibitor of JAK3 targeting the unique Cys909 at the gatekeeper (GK) position +7 in JAK3. FM-381 exhibited apparent JAK3 IC₅₀ values of 0.154 nM, with 410, 2700 and 3600-fold selectivity over JAK1, JAK2 and TYK2, respectively.

^aIC₅₀ values were calculated from the results of a radiometric assay. Data were obtained as 5-dose singlicate IC₅₀ with 10-fold serial dilution starting at 1 µM. [ATP] = 10 µM

FM-381 shows good selectivity over other kinases with a Cys in a similar position (GK+7)


 

Selectivity within the kinase family

Selectivity of FM-381 was assessed in an activity assay against a panel of 410 kinases (ProQinase) at 100 nM and the compound was found to be exquisitely selective (800 fold selective over nearest kinase). Some weak interactions were observed in vitro at higher compound concentration (500 nM)

Selectivity profile of FM-381 assessed against 410 protein kinases (ProQinase). No significant activity was detected at 100 nM concentration (left panel). At 500 nM inhibitor concentration weak activity was detected (between 50 and 90% inhibition, as indicated by spheres).

Selectivity Beyond Target Famil

FM-381 was found to be inactive in a selectivity panel of frequently hit BRDs (BRD4, BRPF, CECR, FALZ, TAF1, BRD9). Strongest hits was 500 nM for TAF1@2

Cellular Activity

FM-381 shows an apparent EC₅₀ of 100 nM in a dose dependent BRET assay and blocks IL2 stimulated (JAK3/JAK1 dependent) STAT5 phosphorylation at 100 nM, but not JAK3 independent IL6  (JAK1/2/TYK dependent) stimulated STAT3 signalling in Human CD4⁺ T cells up to 1 µM.