Analysis of selective target engagement by small-molecule sphingosine kinase inhibitors using the Cellular Thermal Shift Assay (CETSA)

Abstract

The recently renewed interest in scientific rigor and reproducibility is of critical importance for both scientists developing new targeted small-molecule inhibitors and those employing these molecule in cellular studies, alike. While off-target effects are commonly considered as limitations for any given smallmolecule inhibitor, the ability of a given compound to distinguish between enzyme isoforms is often neglected when employing compounds in cellular studies. To call attention to this issue, we have compared the results of an assay for "direct target engagement", the Cellular Thermal Shift Assay (CETSA), to the published isoform selectivity of 12 commercially available sphingosine kinase 1 and 2 (SphK 1 and SphK2) inhibitors. Our results suggest that, at the concentrations commonly employed in cellular assay systems, none of the tested SKIs can be considered isoform selective. Thus, caution and complimentary assay strategies must be employed to fully discern isoform selectivity for the SphKs. Moreover, caution must be employed by the scientific community as a whole when designing experiments that aim to discern the effects of one enzyme isoform versus another to ensure that the concentration ranges used are able to distinguish isoform selectivity.

Sphingosine Kinase Inhibition Enhances Dimerization of Calreticulin at the Cell Surface in Mitoxantrone-Induced Immunogenic Cell Death

“…PF-543 and ABC) that enhanced cell death in response to MTX, we next determined whether the combination of MTX+SKIs enhanced the cell surface exposure of CRT. Both PF-543 and ABC were employed at concentrations that would inhibit both SphK1 and SphK2, as we have previously determined by direct target engagement assays in whole cells (Hengst et al, 2020). As shown in Figure 3A, PF-543 alone had no effect on the cell surface exposure of CRT in DLD-1 cells, compared to vehicle treated cells, as determined by flow-cytometry employing the above described assay.…”

“…cell surface expressed CRT, and secretion of ATP and high mobility group box 1; HMGB1) in enucleated cancer cells indicating that the DNA damaging activity of MTX is not required for ICD induction (Obeid et al, 2007b). To examine the effects of altered sphingolipid metabolism, we first treated the mouse CRC cell line CT-26 (routinely used for ICD experiments (Kepp et al, 2014)) with multiple SphK inhibitors including, PF-543 (Schnute et al, 2012), ABC294640 (ABC) (French et al, 2010) and SK1 We recently examined the isoform selectivity of multiple SKIs using the Cellular Thermal Shift Assay (CETSA), a whole cell assay of target engagement (Hengst et al, 2020). These studies revealed that at the concentrations commonly employed in the literature the SKIs tested, including PF-543 (SphK1-selective), ABC (SphK2-selective) and SK1-I (SphK1-selective), target engaged both SphK1 and SphK2.…”

Celastrol exerts a neuroprotective effect by directly binding to HMGB1 protein in cerebral ischemia–reperfusion

“…For CETSA, experiments were performed according to the previously published protocols with minor modifications [ 25 ]. The primary neurons in 100 mm dishes were collected with PBS containing protease inhibitor.…”

Celastrol Exerts Neuroprotective Effect via Directly Binding to Hmgb1 Protein in Cerebral Ischemic Reperfusion

“…Target engagement assay of celastrol with HMGB1 was performed the same with previous article with minor modify [25]. The primary neurons in 100 mm dishes were collected with PBS containing protease inhibitor.…”