L-Moses

Protein target name:

PCAF, GCN5

Mechanism of action:

Antagonist

Recommended Concentration for use in cells:

1-5 uM

Probe Information

Target and Probe Information

Target Details

Target class:

Epigenetics

Subclass:

Bromodomain

HUGO ID:

HGNC:8638 HGNC:4201

Entrez gene ID:

8850 2648

UNIPROT ID:

Q92831 Q92830

Target alias:

Lysine acetyltransferase 2B, CAF, P/CAF, KAT2B, p300 associated factor, Lysine acetyltransferase 2A, KAT2A, General Control Nonderepressible 5, GCN5L2, PCAF

Probe Details

Chemical Probes Portal ID:

CPP1289

PubChem CID:

123132211

SMILES string:

CC1=NN=C2N1N=C(N[C@@H](C)[C@@H](N(C)C)C3=CC=CC=C3)C4=CC=CC=C42

InChi Key:

MSFPLTWUFWOKBX-IFXJQAMLSA-N

Control compound:

D-Moses

Orthogonal probe:

GSK4027

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

50 mM (DMSO), >200 μM

Potential reactivities:

N/A

NMR:

1H-NMR (400 MHz, DMSO-d6): δH 8.40 (d, 1H, J= 7.8 Hz), 8.28 (d, 1H, J= 8.3 Hz), 7.93 (m, 1H), 7.82 (m, 1H), 7.37 (m, 3H), 7.22 (d, 2H, J= 7.2 Hz), 7.07 (d, 1H, J= 5.7 Hz, NH), 4.70 (m, 1H), 3.71 (d, 1H, J= 8.6 Hz), 2.61 (s, 3H), 2.11 (s, 6H), 1.11 (d, 3H, J= 6.4 Hz)

13C-NMR (100 MHz, DMSO-d6): δC 150.7 (C-1), 146.1 (C-4), 141.1 (C-5), 135.7 (C-14), 132.8 (C-8), 130.3 (C-7), 129.4 (C-15), 127.8 (C-16), 127.3 (C-17), 124.1 (C-9), 123.4 (C-3), 122.6 (C-6), 118.4 (C-2), 71.7 (C-12), 46.6 (C-10), 41.8 (C-13), 16.5 (C-11), 9.4 (C-18)

Storage Recommendations

Storage (dry powder):

4 °C

Storage (solution):

-20 °C

Storage sensitivities:

N/A

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

48 nM

Potency assay:

BROMOscan (DiscoverX; https://www.discoverx.com/technologies-platforms/competitive-binding-technology/bromoscan-technology-platform). Potency of L-Moses against GCN5 was 220 nM in the same assay. Potency was also confirmed by a PCAF HTRF assay giving a Ki=47 nM. Binding affinity by ITC with PCAF Bromodomain construct gives KD=126 nM. Binding affinity by ITC with GCN5 Bromodomain construct gives KD=550 nM

PDB ID for probe-target interaction (3D structure):

5TPX

Structure-activity relationship:

SAR data are available and has been published: http://onlinelibrary.wiley.com/doi/10.1002/anie.201610816/full

Off target activity

Probe Selectivity in Vitro:

A selectivity thermal shift assay panel of 48 of the 61 expressed human Bromodomains was set up using expressed constructs. No observed activity was seen for targets other than PCAF/GCN5.

In cell validation

On target activity in cells

Potency in cells:

EC50

256 nM

Target engagement assay (cells):

PCAF Bromodomain NanoBRET assay (Promega Corporation; https://www.promega.co.uk/products/protein-purification-and-interactions/protein-interactions/nanobret-technology-for-protein-interactions/). L-Moses permeates cells and engages a Nano-Luc fusion PCAF Bromodomain construct at nM concentration, providing evidence of cell permeability and target engagement. This was also verified in a competitive cellular pull-down experiment with a derivative compound conjugated onto beads (activity was seen at <1 μM).

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

L-Moses shows no observable cytotoxicity when exposed to Peripheral Mononuclear Blood Cells at 10 μM for up to 72 hours.

In vivo validation

Dose:

10 mg/kg

Route of delivery:

Oral

Plasma half life:

49 minutes

Systemic clearance:

Cmax:

758 nM

Tmax:

30 minutes

Primary Reference

Reference (doi):

10.1002/anie.201610816

Reference (PMID):

27966810

SAB Rating

Rating for use in Cells

5 review(s)

Rating for use in Model Organisms

5 review(s)

SAB Members

SAB Comments

This is a good inhibitor developed against the PCAF bromodomain. Reasonably potent (Kd=100 nM by ITC) and selective against other BRDs by DSF. Although in this reviewer's opinion, this assay should ideally be run by BromoScan, which is the benchmark for bromodomain selectivity panel. Crystal structure confirmed binding mode (although not on the domain from humans), and cellular activity was confirmed by NanoBRET. Some DMPK parameters were reported. The chemistry was well done, and the compound appears of reasonably good quality to warrant use in cells. More cellular data are certainly warranted in future.

Mar 20 2017 - 9:20am

This is a newly discovered probe molecule and as such there is not much data yet. The publication shows that the compound has good potency and selectivity, and the cellular target engagement assay shows it can permeate into cells and engage with the target in that complex. It would be good to see a concentration-response curve for the cellular assay, and confirmation that the compound can act at the target in a more physiologically-relevant context. But I expect this will follow in future work around this target. From the data presented I am happy to recommend the use of this probe in cellular work. Studies should also use the inactive enantiomer (D-45) presented in the paper as a control.

Mar 28 2017 - 7:19pm

L-Moses is an ~200 nM inhibitor of the bromodomains present in PCAF and GCN5, which are 78% identical, but it does not inhibit/bind to other bromodomain-containing proteins. In cells, L-Moses displaced the PCAF bromodomain from histone H3.3. When tested against full length protein, the response was not robust and the IC50 is estimated to be in the low micromolar range. The compound was non-toxic in PBMCs, but there is limited information on other biomarker readouts for this compound, making its overall utility unclear at this point. Nonetheless, this should be a useful tool to probe the role of the bromodomains in PCAF and GCN5 with the caveat that it will be difficult to distinguish between those two enzymes.

Apr 11 2017 - 11:57am

The cellular activity for this probe is significantly weaker than for GSK4027, which currently represents the best probe for PCAF-Brd/GCN5 due to greater confidence in its selective on-target-mediated mode of action in cells.

Apr 17 2017 - 8:20pm

The data in the primary publication are insufficient to meet the criteria for a high-quality chemical probe. Selectivity within the bromodomain family is only determined by DSF, rather than a more sensitive technique such as bromoscan, or for the BET family, which is an off-target of particular concern, ITC. Additionally, the cellular potency in a nanoBRET assay is only presented at 5 uM, rather than as an IC50. It isn't clear that the potency in cells is less than 1 uM. No general pharmacology panel selectivity data was provided. This is especially important for cell surface GPCRs and ion channels that might be engaged by a lipophilic base, such as L-Moses. GSK4027 is a more potent and better characterized probe and should be used instead.

Apr 2 2020 - 7:50pm

L-Moses

Probe Information

Target Details

Lysine acetyltransferase 2B, CAF, P/CAF, KAT2B, p300 associated factor, Lysine acetyltransferase 2A, KAT2A, General Control Nonderepressible 5, GCN5L2, PCAF

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

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L-Moses

Probe Information

Target Details

Lysine acetyltransferase 2B, CAF, P/CAF, KAT2B, p300 associated factor, Lysine acetyltransferase 2A, KAT2A, General Control Nonderepressible 5, GCN5L2, PCAF

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?