NCT-505

Protein target name:

ALDH1A1

Mechanism of action:

Inhibitor

Recommended Concentration for use in cells:

~~up to 10 uM~~

Reviewer suggested Concentration for use in cells:

up to 1 uM

Probe Availability

MedKoo Biosciences, Inc.

Probe Information

Target and Probe Information

Target Details

Target class:

Other

Define other target class:

Metabolic enzyme

Subclass:

aldehyde dehydrogenase

HUGO ID:

HGNC:402

Entrez gene ID:

216

UNIPROT ID:

P00352

Target alias:

ALDC; ALDH1; HEL-9; HEL12; PUMB1; ALDH11; RALDH1; ALDH-E1; HEL-S-53e

Probe Details

Chemical Probes Portal ID:

CPP2060

Probe alias:

PubChem CID:

13482168

ChEMBL ID:

CHEMBL4206892

SMILES string:

N#CC1(C2=CC=CC=C2)CCN(C3=C(C(N4CCN(S(=O)(C)=O)CC4)=O)C=NC5=CC=C(F)C=C35)CC1

InChi Key:

XMNLWOQHMQRHDV-UHFFFAOYSA-N

Physico-chemical properties

NMR:

1H NMR (400 MHz, DMSO - d6) δ 8.63 (d, J = 3.4 Hz, 1H), 8.07 (dd, J = 9.2, 5.5 Hz, 1H), 7.89 (dd, J = 10.2, 2.8 Hz, 1H), 7.73 (td, J = 8.8, 2.8 Hz, 1H), 7.69 – 7.62 (m, 2H), 7.52 – 7.43 (m, 2H), 7.43 – 7.35 (m, 1H), 3.96 – 2.97 (m, 12H), 2.90 (s, 3H), 2.57 - 2.21 (m, 4H)

Validation in Cells and Model Organisms

In vitro validation

On target activity

Potency:

IC50

7 nM

Potency assay:

ALDH Enzymatic Assays

Structure-activity relationship:

yes

Off target activity

Probe Selectivity in Vitro:

Screened against a panel of enzymes including ALDH1A subfamily (ALDH1A2 and ALDH1A3), ALDH isozymes (ALDH2, and ALDH3A1) and other dehydrogenases such as 15-hydroxyprostaglandin dehydrogenase (HPGD) and type-4 hydroxysteroid dehydrogenase (HSD17β4): all showed IC50 >20 uM

In cell validation

On target activity in cells

Potency in cells:

IC50

30 nM MIA PaCa-233 nM OV-90123 nM HT-19

Potency assay (cells):

Aldefluor Cell-Based Assays.

Target engagement assay (cells):

Target engagement in a cellular thermal shift assay (CETSA)

In vivo validation

Organism:

male CD-1 mice

Dose:

2 mg/Kg IV10 mg/Kg PO

Route of delivery:

OralIntravenous

Plasma half life:

0.3 h IV 1.6h PO

Systemic clearance:

30 (mL/min/kg) IV

Fraction of probe bound to plasma protein:

44% PO

Cmax:

2350 ng/mL IV1234 ng/mL PO

Primary Reference

Reference (doi):

10.1021/acs.jmedchem.8b00270

Reference (PMID):

29767973

SAB Rating

Rating for use in Cells

2 review(s)

Rating for use in Model Organisms

2 review(s)

SAB Members

SAB 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.

Jul 7 2021 - 12:19pm

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.

Sep 23 2021 - 9:55am

NCT-505

Probe Availability

Probe Information

Target Details

ALDC; ALDH1; HEL-9; HEL12; PUMB1; ALDH11; RALDH1; ALDH-E1; HEL-S-53e

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

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Search form

NCT-505

Probe Availability

Probe Information

Target Details

ALDC; ALDH1; HEL-9; HEL12; PUMB1; ALDH11; RALDH1; ALDH-E1; HEL-S-53e

Probe Details

Physico-chemical properties

In vitro validation

In cell validation

In vivo validation

Primary Reference

Supporting Organizations

Interested in supporting the portal?