KZR-504

Reagent Authentication Certificate:

Chemical Probes Portal KZR-504.pdf

Protein target name:

PSMB9

Mechanism of action:

Covalent Inhibitor

Recommended Concentration for use in cells:

300 nM - 1 uM

Probe Information

Target and Probe Information

Target Details

Target class:

Protein metabolism

Subclass:

Proteasome subunit

HUGO ID:

HGNC:9546

Entrez gene ID:

5698

UNIPROT ID:

P28065

Target alias:

Proteasome subunit beta 9, LMP2, PSMB6i, RING12, beta1i

Probe Details

Chemical Probes Portal ID:

CPP1636

PubChem CID:

117611788

SMILES string:

C[C@@]1(CO1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CO)NC(=O)C3=CC=CC(=O)N3

InChi Key:

QYDASSGXBXGRBS-CKJXQJPGSA-N

Physico-chemical properties

Potential reactivities:

Unknown

NMR:

1H NMR (400 MHz, DMSO-d6): δ 11.23 (s, 1H), 8.58-8.56 (m, 1H), 8.31-8.29 (m, 1H), 7.79 (br s, 1H), 7.50 (br s, 1H), 7.27-7.16 (m, 5H), 6.84 (br s, 1H), 5.10-4.90 (m, 1H), 4.65-4.60 (m, 1H), 4.53-4.48 (m, 1H), 3.63-3.61 (m, 2H), 3.20 (d, J = 5.2 Hz, 1H), 3.00 (d, J = 5.2 Hz, 1H), 2.93 (dd, J = 14.0, 4.8 Hz, 1H), 2.70 (dd, J = 14.0, 9.6 Hz, 1H), 1.36 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ 208.2, 170.2, 141.1, 137.6, 129.6, 128.8, 127.1, 62.4, 59.5, 54.9, 52.9, 52.1, 36.0, 16.7.

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

51 nM

Potency assay:

In a MOLT-4 (human T cell leukemia) cellular lysate, a proteasome constitutive/immunoproteasome subunit ELISA (ProCISE) was employed for quantitative assessment of subunit-specific activity.

Structure-activity relationship:

Yes, please see ACS Med Chem paper for details..

Off target activity

Potency (off target):

IC50

Off target protein and potency:

Beta1, MECL1, Beta2: >250 uMLMP7, Beta5: >6 uM

Potency assay (off target):

In a MOLT-4 (human T cell leukemia) cellular lysate, a proteasome constitutive/immunoproteasome subunit ELISA (ProCISE) was employed for quantitative assessment of subunit-specific activity of all human and mouse proteasome catalytic subunits.

In cell validation

On target activity in cells

Potency in cells:

IC50

136 nM

Potency assay (cells):

Utilizing MOLT-4 (human T cell leukemia) whole (live) cells, a proteasome constitutive/immunoproteasome subunit enzyme-linked immunosorbent (ProCISE) ELISA assay was employed for quantitative assessment of subunit-specific activity.

Target engagement assay (cells):

A proteasome constitutive/immunoproteasome subunit enzyme-linked immunosorbent (ProCISE) ELISA assay for quantitative assessment of subunit-specific activity.

Off target activity in cells

Potency in cells, off target:

IC50

Potency assay, off target (cells):

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

>25 uM in PMBCs using CellTiter-Glo.

In vivo validation

Organism:

Female BALBc mice

Dose:

1 mg/kg and 30 mg/kg

Route of delivery:

Intravenous

Ratio of probe in organ to plasma (O:P):

1:1 in all tissues except brain (1 mg/kg)

Organ of Interest

Organ (O):

Blood, Heart, Kidney, Liver, Brain

Target engagement assay:

Target inhibition in tissues was measured by ProCISE. In all tissues except brain, a 1 mg/kg dose yielded target inhibition >50%. In brain, a 30 mg/kg dose yielded target inhibition to 40%.

Primary Reference

Reference (doi):

10.1021/acsmedchemlett.6b00496

Reference (PMID):

28435528

Endorsed probe

The Chemical Probes Portal endorses this compound as chemical probe for use in cells and in model organisms.

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

The epoxide KZR-504 appears to be a suitable chemical probe of the LMP2 subunit of the immunoproteasome. It is a selective and potent inhibitor, as assessed in the ProCISE platform, which was also used to measure pharmacology in live cells (IC50=136 nM). No direct evidence was provided that confirmed covalent inhibition, although this could be inferred. The probe possesses poor permeability in MDCK as one would expect based on the physicochemistry of the molecule. An IV bolus in mouse produced the expected pharmacodynamic effect. Selectivity and potency is comparable to a previously disclosed probe called LU-001i, which is also a peptidic keto-epoxide, although the latter was not assessed in vivo.

Jul 5 2017 - 3:14pm

KZR-504 is a selective inhibitor of LMP2 and, therefore, of the immunoproteasome. It has been optimized for in vivo use, which has led to some decrease in cellular permeability. Based on the published selectivity and lack of cytotoxicity, as well as the demonstrated pharmacology in mice, this is a good probe for selective inhibition of LMP2. No assessment has been made of what other targets this compound may bind to or inhibit. Given the understood pharmacology of compounds in this class, significant pharmacology from unknown other targets is a relatively low risk at appropriate doses. The ability to use this same molecule in vitro and in vivo is an advantage and a reason to recommend this molecule over other analogs.

Jul 13 2017 - 12:56pm

KZR-504 is a potent and highly selective inhibitor for the LMP2 proteosome subunit with ca 50 nM IC50 and >100-fold selectivity over other subunits (80-fold for mouse LMP7) and fair cell permeability and metabolic stability. The compound selectively inhibits LMP2 in mouse in all tissues except brain at 1 mg/kg (IV, 1-hr post dose). Unlike non-selective proteosome inhibitors, such as ONX-0914, KZR-504 has no effect on release of inflammatory cytokines released from human PBMCs treated with LPS.

Jul 19 2017 - 12:13pm

KZR-504

Probe Information

Target Details

Proteasome subunit beta 9, LMP2, PSMB6i, RING12, beta1i

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?

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KZR-504

Probe Information

Target Details

Proteasome subunit beta 9, LMP2, PSMB6i, RING12, beta1i

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?