ML323

Catalog No.S7529

For research use only.

ML323 displays reversible, nanomolar inhibitory activity and excellent selectivity toward USP1/UAF1 with IC50 of 76 nM.

ML323 Chemical Structure

CAS No. 1572414-83-5

Selleck's ML323 has been cited by 11 Publications

Purity & Quality Control

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Biological Activity

Description ML323 displays reversible, nanomolar inhibitory activity and excellent selectivity toward USP1/UAF1 with IC50 of 76 nM.
Features USP1-UAF1-selective inhibitor.
Targets
USP1-UAF1 [1]
(Cell-free assay)
76 nM
In vitro

ML323 inhibits the deubiquitination of PCNA and FANCD2 by inhibiting USP1–UAF1 activity in H596 cells. Moreover, ML323 potentiates cisplatin cytotoxicity in NSCLC H596 cells and U2OS osteosarcoma cells by targeting two major DNA damage response pathways (TLS and FA). [1]

Protocol (from reference)

Kinase Assay:

[1]

  • High-throughput screening:

    For HTS, USP1-UAF1 activity is monitored using ubiquitin-rhodamine 110 as a substrate, where hydrolysis of the amide bond between the C-terminal glycine of ubiquitin and rhodamine results in an increase in fluorescence. The assay is miniaturized to a 4 μL volume in a 1,536-well format and is used to screen approximately 402,701 compounds in quantitative HTS mode, with each compound tested over a range of four to five concentrations. The assay shows robust performance with an average Z’factor of 0.8 throughout the screen.

Cell Research:

[1]

  • Cell lines: H596 cells
  • Concentrations: ~30 μM
  • Incubation Time: 7-12 days
  • Method:

    For the colony-forming assay, cells are seeded at a density of 300–500 cells per well in six-well plates and grown overnight. Cells are then treated with ML323 alone, cisplatin alone or a combination of cisplatin and ML323 (1:1 or 1:4) at the indicated concentrations. Cells treated with an equal volume of DMSO and saline are used as control. After 48 h of treatment, fresh growth medium is added, and cells are incubated for an additional 5-10 d to allow for colony formation. For UV combination treatment, the cells are treated with ML323 at the indicated concentrations or an equal volume of DMSO. After 48 h, the medium is removed, and cells are irradiated at 254 nm at the indicated dosage. Fresh growth medium is added, and the cells are incubated for an additional 5-10 d to allow for colony formation. The cells without UV irradiation but treated with ML323 or an equal volume of DMSO are used as controls and designated as 100%. After the formation of the colonies, cells are fixed with methanol and stained with 0.5% crystal violet. Colonies consisting of >50 cells are scored. The number of colonies is determined from triplicate plates. The dose-response curves are generated using GraphPad Prism and analyzed by using CalcuSyn to calculate the combination index, which is determined for the fraction of cells affected after the addition of fixed ratios of cisplatin and the USP1-UAF1 inhibitor.

  • (Only for Reference)

Solubility (25°C)

In vitro

DMSO 76 mg/mL
(197.66 mM)
Ethanol 38 mg/mL
(98.83 mM)
Water Insoluble

* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.

Chemical Information

Molecular Weight 384.48
Formula

C23H24N6

CAS No. 1572414-83-5
Storage 3 years -20°C powder
2 years -80°C in solvent
Smiles CC1=CN=C(N=C1NCC2=CC=C(C=C2)N3C=CN=N3)C4=CC=CC=C4C(C)C

In vivo Formulation Calculator (Clear solution)

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Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Molarity Calculator

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Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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