ML323

Reagent Authentication Certificate:

Chemical Probes Portal ML323.pdf

Protein target name:

USP1, WDR48

Mechanism of action:

Allosteric inhibitor

Recommended Concentration for use in cells:

250-20,000 nM

Probe Availability

Cayman (1572414-83-5)

MilliporeSigma (1572414-83-5)

MedChem Express (1572414-83-5)

Probe Information

Target and Probe Information

Target Details

Target class:

UAF1, KIAA1449, P80, SPG60, UBP, ubiquitin specific peptidase 1, WD repeat domain 48

Probe Details

Chemical Probes Portal ID:

CPP732

Probe alias:

ML323; ML-323; 1572414-83-5; CHEMBL3182437; ML 323

PubChem CID:

60167849

ChEMBL ID:

CHEMBL3182437

SMILES string:

CC1=CN=C(N=C1NCC2=CC=C(C=C2)N3C=CN=N3)C4=CC=CC=C4C(C)C

InChi Key:

VUIRVWPJNKZOSS-UHFFFAOYSA-N

Control compound:

NCGC-959

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

10 mM

Potential reactivities:

N/A

NMR:

1H NMR (400 MHz, CDCl3) δ 8.82 (dd, J = 3.4, and 7.9 Hz, 1 H), 8.02−7.93 (m, 2
H), 7.76 (d, J = 1.4 Hz, 1 H), 7.64−7.56 (m, 2 H), 7.51−7.33 (m, 5
H), 7.25−7.17 (m, 1 H), 4.88 (d, J = 5.9 Hz, 2 H), 3.19 (p, J = 6.8 Hz,
1H), 2.22 (s, 3 H), 1.10 (d, J = 1.2 Hz, 3 H) and 1.09−1.07 (m, 3 H).

13C NMR (100 MHz, CDCl3) δ 162.02, 160.18, 148.00, 140.63,
138.03, 136.14, 134.35, 134.28, 131.89, 130.34, 129.25, 129.00, 126.61,
126.04, 122.04, 121.99, 120.74, 113.81, 44.62, 29.43, 23.95, 13.71 and
13.67.

Storage Recommendations

Storage (dry powder):

Storage (solution):

-20C

Storage sensitivities:

The compound is stable, but as standard practice, avoid excessive exposure to light and freeze-thaw cycles when stored as a DMSO solution (to avoid water uptake).

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

76 nM

Potency assay:

USP1/UAF1 activity was monitored in a fluorometric ubiquitin−rhodamine110 assay. ML323 also showed IC50s of 174 nM and 820 nM in orthogonal gel-based assays using K63-linked diubiquitin (di-Ub) and monoubiquitinated PCNA (Ub-PCNA) as substrates, respectively.

Structure-activity relationship:

Yes, see J. Med. Chem publication for details.

Off target activity

Potency assay (off target):

ML323 showed no notable activity against the USPs (USP2, USP5, USP7, USP8, USP10, USP11, USP14 and USP21). ML323 displayed no notable against DUBs in the ubiquitin C-terminal hydrolase (UCH), ovarian tumor protease (OTU) and Machado-Joseph domain (MJD) families and did not inhibit the deSUMOylase SENP1 or the deneddylase, NEDP1.

Selectivity against non-family targets:

ML323 off-target activity was assessed by determining the inhibitory effect of ML323 against 70 unrelated proteases and 451 kinases in in vitro assays; no notable activity was detected.

In cell validation

On target activity in cells

Potency assay (cells):

Activity-based profiling with hemagglutinin (HA)-tagged ubiquitin vinyl methyl ester (HA-Ub-VME) in HEK293T cells revealed that ML323 could selectively and dose-dependently inhibit the labeling of USP1. ML323 also increased mUb of PCNA and FANCD2 in H596 cells, recapitulating results obtained with shRNA targeting USP1.

Target engagement assay (cells):

Yes, activity based assay

Off target activity in cells

Potency assay, off target (cells):

ML323 did not inhibit the labeling of the DUBs, USP8, USP7, CYLD, USP5, USP14, UCH-L3 and UCH-L1, tested by HA-Ub-VME.

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

ML323 was tested in H596 cells and showed minimal cytotoxicity as a single agent (colony formation assay). However, when used in combination with cisplatin apparent synergy was observed. In H1299 cytotoxicity was observed with ML323 as a single agent (colony formation assay) in a dose dependent manner.

Primary Reference

Reference (doi):

10.1021/jm5010495 doi: 10.1038/nchembio.1455.

Reference (PMID):

25229643 24531842

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

no rating

SAB Members

SAB Comments

ML323 is an allosteric, selective inhibitor of the USP1/UAF1 complex (also called USP1/WDR48). Note that ML323 is much less effective in inhibiting USP1 alone in biochemical assays. The full implications or relevance of this differential activity in cells has not been explored.

ML323 is a selective USP1/UAF1 inhibitor that has been shown to have good Caco-2 permeability and to be efficacious in cellular assays at ≥5 µM. A compound in the same series (compound 77 in J. Med. Chem., 2014, 8099-8110) can be used as an inactive control. ML323 was found to have rapid clearance and has not been proposed for use in vivo.

ML-323 is a unique, cell-permeable and highly selective inhibitor of the USP1/UAF1 complex. A notable discordance between biochemical and cellular potency for ML-323 is observed, and should be further examined. Furthermore, a more direct, biophysical assessment of intracellular target engagement would be valuable to explore the mechanism of the observed IC50 offset.

ML323

Probe Availability

Probe Information

Target Details

UAF1, KIAA1449, P80, SPG60, UBP, ubiquitin specific peptidase 1, WD repeat domain 48

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

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ML323

Probe Availability

Probe Information

Target Details

UAF1, KIAA1449, P80, SPG60, UBP, ubiquitin specific peptidase 1, WD repeat domain 48

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

Interested in supporting the portal?