ABP-A3

Protein target name:

UBA1, UBA3

Mechanism of action:

Covalent inhibitor of ubiquitin and NEDD8 conjugation

Recommended Concentration for use in cells:

3-5 uM for 16h

Probe Information

Target and Probe Information

Target Details

Target class:

NEDD8 E1, Ubiquitin E1, NAE, A1S9, A1S9T, A1ST, AMCX1, CFAP124, GXP1, POC20, SMAX2A, UBE1, UBE1X, UBA1, NAE2, UBE1C, hUBA3

Probe Details

Chemical Probes Portal ID:

CPP584

PubChem CID:

126961737

SMILES string:

[C@H]1([C@H]([C@H]([C@@H](O1)[N]2C=NC3=C2N=CN=C3N(C4=CC(=CC=C4)C#C[H])[H])O[H])O[H])CO[S](N([H])[H])(=O)=O

InChi Key:

LZNFQRSVDZCPTR-SCFUHWHPSA-N

Control compound:

N/A

Orthogonal probe:

MLN7243

Physico-chemical properties

Solubility - preferred solvent:

DMSO

Solubility - concentration:

10-20 mM

Potential reactivities:

Not available

Storage Recommendations

Storage (dry powder):

-20

Storage (solution):

-20, 2 years

Storage sensitivities:

Potential sensitivity to freeze-thaw cycle

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

43 nM

Potency assay:

Different doses of ABP A3 were incubated with UBA1 (0.5 uM), ATP (50 uM) and Ub (50 uM), for 2 hours at room temperature. The amount of formed ABP A3-Ub covalent adduct is then quantified using click chemistry. IC50 is the concentration of ABP A3 at which half maximal amount of ABP A3-Ub covalent adduct is formed.

Structure-activity relationship:

Eight analogues of ABP A3 were prepared: 3 have 3 substituents at the N6-adenine position, and 5 have different nucleophiles capable of reacting with E1~Ub thioester.

Off target activity

Probe Selectivity in Vitro:

ABP A3 is an AMP mimic and therefore can inhibit protein kinases. Its close analogue MLN4924 was profiled against the panel of protein kinases and was shown to be inactive (Nature. 2009 Apr 9;458(7239):732-6).

In cell validation

On target activity in cells

Potency in cells:

IC50

2.5 uM A549 cells80 nM in MM1S cells

Potency assay (cells):

CellTiterGlo viability assay. A549/MM1S cells were treated with ABP A3 for 48h.

Target engagement assay (cells):

We also evaluated if ABP A3 inhibits closely related analogues SUMO E1, ISG15 E1, Ufm1 E1. To do so we treated A549 cells with ABP A3 and evaluated the amount of covalently labeled UBL proteins using click chemistry methods. Thus, A549 cells were treated with each ABP (100 uM) for 1 hour, followed by cell lysis, conjugation to fluorescent dye via copper-mediated [3 + 2] cycloaddition reaction, and in-gel fluorescence scanning. Under these conditions we did not observe SUMO labeling with ABP A3. Furthermore semi quantitative masspectrometry showed that ABP A3 potently labeled ubiquitin and Nedd8, but not SUMO1-3, Ufm1. Furthermore, western blotting was used to evaluate the effect of ABP A3 on the intracellular levels of UBL conjugates. Intracellular selectivity of ABP A3 on UBL conjugation. A549 cells were treated with different concentrations of ABP A3 or ABP1 for 16 hours, lysed, and immunoblotted using anti-Ub, Nedd8, SUMO1, SUMO2/3 and Ufm1 antibodies. Under these conditions ABP A3 inhibited the conjugation of Ub and Nedd8 to their intracellular substrates, and activated SUMO1-3 and Ufm1 conjugation. Activation of SUMO and Ufm1 pathways is due to the stress response. Importantly, the length of the cell treatment with the probe affects the potency. For example, treatment of A549 cells with ABP A3 for 1 h leads to complete inhibition of Nedd8 conjugation at 6.3 uM of ABP A3, but ubiquitn conjugation is not changed. 16 h treatment of A549 cells with ABP A3 leads to inhibition of Nedd8 and Ub conjugation at ~3.2 uM. At 1 h treatment, complete inhibition of Ub conjugation requires ~25 uM of ABP A3. In A549 cells, ~3 uM ABPA3 treatment for 16h caused an accumulation of p53, p21, induction of cleaved PARP (apoptosis) and activation of unfolded protein response (induction of CHOP, BiP, pPERK, and pelF2alpha). Furthermore, as expected ABP A3 did not cause an aggresome formation in A549 cells due to the inhibition of ubiquitin conjugation, while proteasome inhibitor MG132 did.

Off target activity in cells

Potency assay, off target (cells):

Cells were treated with ABP A3 and covalent ABP A3-Ubl adducts were visualized using in gel scanning fluorescence, or masspectrometry.

Probe Selectivity in Cell:

We also evaluated if ABP A3 inhibits closely related analogues SUMO E1, ISG15 E1, Ufm1 E1. To do so we treated A549 cells with ABP A3 and evaluated the amount of covalently labeled UBL proteins using click chemistry methods. Thus, A549 cells were treated with each ABP (100 uM) for 1 hour, followed by cell lysis, conjugation to fluorescent dye via copper-mediated [3 + 2] cycloaddition reaction, and in-gel fluorescence scanning. Under these conditions we did not observe SUMO labeling with ABP A3. Furthermore semi quantitative masspectrometry showed that ABP A3 potently labeled ubiquitin and Nedd8, but not SUMO1-3, Ufm1. Furthermore, western blotting was used to evaluate the effect of ABP A3 on the interacellular levels of UBL conjugates. Intracellular selectivity of ABP A3 on UBL conjugation. A549 cells were treated with different concentrations of ABP A3 or ABP1 for 16 hours, lysed, and immunoblotted using anti-Ub, Nedd8, SUMO1, SUMO2/3 and Ufm1 antibodies. Under these conditions ABP A3 inhibited the conjugation of Ub and Nedd8 to their intracellular substrates, and activated SUMO1-3 and Ufm1 conjugation. Activation of SUMO and Ufm1 pathways is due to the stress response. Importantly, the length of the cell treatment with the probe affects the potency. For example treatment of A549 cells with ABP A3 for 1h leads to complete inhibition of Nedd8 conjugation at 6.3 uM of ABP A3, but ubiquitn conjugation is not changed (Figure S9). But at 16h treatment of A549 cells with ABP A3 leads to inhibition of Nedd8 and Ub conjugation at ~3.2 uM. At 1h treatment complete inhibition of Ub conjugation requires ~25 uM of ABP A3.

Toxicity

Cytoxicity assay:

Yes

Notes on cytotoxicity:

Cell titer glo assay, IC50 2.5 uM for A549 cells, and 80 nM for MM1S. In A549 cells, ~3 uM ABPA3 treatment for 16 h caused an accumulation of p53, p21, induction of cleaved PARP (apoptosis) and activation of unfolded protein response (induction of CHOP, BiP, pPERK, and pelF2alpha). Furthermore, as expected ABP A3 did not cause an aggresome formation in A549 cells due to the inhibition of ubiquitin conjugation, while proteasome inhibitor MG132 did.

Primary Reference

Reference (doi):

10.1039/C5SC01351H 10.1021/ja4099643

Reference (PMID):

241384

SAB Rating

Rating for use in Cells

3 review(s)

Rating for use in Model Organisms

3 review(s)

SAB Members

SAB Comments

This compound is a non-specific, covalent inhibitor of ubiquitinylation and Nedd8 and should therefore be used with caution as a single agent, as this polypharmacolgy will almost certainly confound any attempt to use the tool to dissect specific actions of single E1 enzymes. As such, experiments with this compound must be carefully designed with appropriate follow up in order to have any meaningful consequence. However, one possible use, suggested by the authors, is to use this probe to investigate selectivity of other, next-generation E1 inhibitors - by dosing first with a putatively selective E1 inhibitor, then adding in an appropriate dose of ABPB3. Any non-bound E1 proteins will be covalently modified with the probe and can be detected by mass spectroscopy. The conclusion would then be that any non-labeled E1 enzymes were bound by the putative new selective ligands, whereas any labeled proteins were not bound by the test probe. This experiment assumes binding at competing sites and again needs to be treated with caution and validated further but may offer some outline utility in the preliminary assessment of selective E1 inhibitors.

Sep 6 2016 - 1:57pm

The published characterization of ABPA3 rests largely on phenotypic, cell-based proteomics in A549 cells. The results, showing selective formation of covalent complexes between ABPA3 and ubiquitin and NEDD8 relative to SUMO1/2/3 or UFM1, are intriguing, as is the selective inhibition of ubiquitin and NEDD8 conjugation of native proteins in A549 cells. A variety of other experiments carried out in A549 cells demonstrate phenotypic and antiproliferative effects that are consistent with the proposed mechanism of action; however, a key limitation to this probe is its inadequate biochemical characterization. MLN-7243, whose structure has now been disclosed (please see above reference), is a compound with a similar sulfamate nucleotide-like structure with reported inhibitory activity at 1 nM on ubiquitin--protein ligase E1 and at 25 nM NEDD8-activating enzyme E1 (UAB3). IABPA3 would be a more useful probe if comparative data between it and ML-7243 were available. Also, all of the cell-based work was done in one cell line, so inferring intrinsic selectivity from only A549 cells is risky when these experiments may be context dependent. Finally, in addition to the above considerations, in the absence of PK data (which have not been reported), ML-7243 should not be considered for use in intact organisms.

Dec 28 2016 - 2:38pm

ABPA3 is an activity-based probe that covalently inhibits the ubiquitin- and Nedd8-activating E1 enzymes (UBA1/UBA3), resulting in inhibition of both Nedd8-dependent and Nedd8-independent ubiquitin conjugation and protein degradation. Importantly, in A549 cells, ABPA3 selectively decreases ubiquitin and Nedd8 conjugates, but not SUMO1-3, Ufm1, ISG15, or LC3 conjugates. However, more data is needed in order for ABPA3 to be recommended as a useful probe for inhibition of UBA1/UBA3 in general since: 1) the data available are derived from cell-based studies in only one cell line (A549), 2) no broad, biochemical selectivity profile of ABPA3 is available, and 3) preclinical properties for ABPA3 have not been reported.

Jul 14 2017 - 3:16pm

ABP-A3

Probe Information

Target Details

NEDD8 E1, Ubiquitin E1, NAE, A1S9, A1S9T, A1ST, AMCX1, CFAP124, GXP1, POC20, SMAX2A, UBE1, UBE1X, UBA1, NAE2, UBE1C, hUBA3

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

Interested in supporting the portal?

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ABP-A3

Probe Information

Target Details

NEDD8 E1, Ubiquitin E1, NAE, A1S9, A1S9T, A1ST, AMCX1, CFAP124, GXP1, POC20, SMAX2A, UBE1, UBE1X, UBA1, NAE2, UBE1C, hUBA3

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

Toxicity

Primary Reference

Supporting Organizations

Interested in supporting the portal?