NCT-505

Inhibitor of ALDH1A1

Structure

Information

Protein target names: ALDH1A1

Mechanism of action: Inhibitor

Recommended in-cell concentration:
up to 1 uM

In Vitro Validations

Uniprot ID: P00352
Target Class: Other
Target SubClass: aldehyde dehydrogenase
Potency: IC50
Potency Value: 7 nM
Potency Assay: ALDH Enzymatic Assays
PDB ID for probe-target interaction (3D structure): --
Structure-activity relationship: yes
Target aliases:
Aldehyde dehydrogenase 1A1, PUMB1, ALDH1, ALDC, AL ...

In Cell Validations

In Vivo Data

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SERP ratings and comments


SERP Ratings

In Cell Rating

Comments:

The probe is a potent and isozyme selective single-digit nanomolar enzyme inhibitor of aldehyde dehydrogenase isozyme 1A1 (IC50 = 7 nM) that also inhibits ALDH 1A1 activity in cells (OV-90, HT-29 and MIA PaCa-2) at a similar level (12–30 nM) measured using an Aldefluor phenotypic assay. However, cell viability using the model OV-90 cells in 3D culture was not affected until the concentration reached 3 µM, which is nearly 100-fold higher than required to inhibit 50% of ALDH 1A1 activity in cells using the Aldefluor phenotypic assay. Moreover, very weak growth inhibition (EC50 ~ 50 µM) was observed with OV-90 cells in 2D culture. The authors suggest the 3D culture is more sensitive because it expresses higher levels of ALDH 1A1, but the Western blot shown in Figure S1 of the cited publication indicates perhaps 2-fold greater expression levels. The large difference in activity between the antiproliferative activity and the cellular inhibition of ALDH 1A1 suggests either the target is not particularly vulnerable and requires stoichiometric inhibition or that the observed antiproliferative activity is off-target. Further experiments to correlate target expression levels and antiproliferative activity are warranted using alternate cell lines or knockdown approaches. The target engagement studies using CETSA demonstrate the probe only showed engagement at micromolar concentrations, which are approximately 100-fold above the levels required to cause ALDH 1A1 inhibition in cells using the phenotypic assay. Further proteome-wide CETSA analysis would help to assess probe selectivity (if the authors suggest using the probe at 10 µM, then CETSA studies should be performed at this level). Targeted metabolomics assessing products of known dehydrogenases would also provide support for probe selectivity.

(last updated: 7 Jul 2021 )

SERP Ratings

In Cell Rating
In Model Organisms

Comments:

While CETSA clearly confirms target engagement in cells, the calculated KD values are far in excess of the low nanomolar inhibition of cell growth. While the selectivity is impressive in a panel of other ALDH isozymes, it would be helpful to have a broader selectivity profile across other target classes to confirm the lack of off-target activity that may be driving observed cell growth inhibition.

(last updated: 23 Sept 2021 )