Fig 1: VAP-1 dependent activity of ABS-752 in cellular experiments.a CTG viability results for Hep3B cell line after 73-h incubation (1 h pre-treatment with 10 µM concentration of PXS-4728A or DMSO and 72 h co-treatment with tested compounds—ABS-752 and CC-90009 concentration range 0.1 nM–30 µM). Data points are means with standard deviation (technical duplicates). b Western blot results for GSPT1 and NEK7 degradation in the presence or absence of 10 µM concentration of PXS-4728A in Hep3B line supplemented with 10% FBS. The molecular weight of corresponding standards is indicated on the left. c CTG viability results for Hep3B cell line supplemented with 10% FBS or 10% human serum after 72-h treatment with tested compounds–ABS-752 and CC-90009 concentration range 0.1 nM–30 µM. Data points are means with standard deviation (technical duplicates). d Western blot results for GSPT1 and NEK7 degradation after 24-h post-treatment with ABS-752 and CC-90009 in Hep3B cell line supplemented with 10% FBS or 10% human serum. e Western blot results for GSPT1 and NEK7 degradation in the presence or absence of 10 µM concentration of PXS-4728A in Hep3B line supplemented with 10% human serum. f CTG viability results for Hep3B cell line supplemented with 10% human serum after 72-h post-treatment with tested compounds (ABS-752 and CC-90009 concentration range 0.1 nM–30 µM) in the presence or absence of 0.3 µM recombinant human VAP-1 protein. Data points are means with standard deviation (technical triplicates).
Fig 2: ABS-752 conversion to aldehyde by VAP-1 enzyme.a Structures and presumed mechanism of formation of ABS-752 metabolites. b Identification of the enzyme catalyzing the oxidation of compound ABS-752. Results of LC-MS/MS quantification of changes in the concentration of ABS-752 and its putative metabolites during incubation with and without recombinant monoamine oxidases (mouse VAP-1 (mVAP-1), human MAO-A and MAO-B). In the case of mVAP-1, metabolite formation was also checked in the presence of the VAP-1 inhibitor, PXS-4728A (0.25 µM). Data for the mixture with no enzyme are mean with standard deviation from two independent experiments, the remaining points are a single result. Legend indicates the time since the addition of ABS-752. c LC-MS/MS quantification results in the whole human tissue lysates for formation of presumed ABS-752 metabolites as a function of pro-drug concentration. ABS-752 was incubated with samples containing lysates of cirrhotic human liver or heart aorta for 1 h at 37 °C. Data shown are the mean with standard deviation for specific VAP-1 activity in lysates (based on 2 independently prepared samples). Presented Michaelis-Menten constants (Km) were determined based on formation rates of aldehyde, ABT-971. d Determination of ABS-752 deamination kinetics catalyzed by recombinant human VAP-1 monoamine oxidase. Observed ABS-752 oxidation rates as a function of pro-drug concentration. Data points are means with standard deviation from Amplex Red-based fluorogenic assay. e Representative CTG viability results for Hep3B cell line expressing wild type GSPT1 and GSPT1 G575N variant. Both cell lines were treated with ABS-752 and its presumed metabolites in a dose-dependent manner (compound concentration range 1 nM–50 µM). In the case of Hep3B expressing GSPT1 G575N, compound ABT-002 was tested in the range of 1 nM–25 µM. f Inhibition of intracellular Tracer/NanoLuc-CRBN complex formation by ABS-752 and its presumed metabolites. Representative inhibition dose-response curves, points are means with standard deviation calculated from technical duplicates.
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