In PMA-stimulated HEK-293 cells, BI-D1870 reduced GSK3α/β and LKB1 phosphorylation with an approximate cellular IC50 of 1 μM. ERK1/2, CREB and ribosomal S6 phosphorylation were largely maintained in the same experiment. These observations describe the tested signaling conditions and do not establish universal cellular selectivity. [1]
BI-D1870 combined with lenalidomide produced synergistic MTT viability responses in KMS11, XG1, MM1.S and JJN3 myeloma cells after six days. The normalized isobolograms also contained combinations above the additivity line, so synergy was not uniform across all dose pairs. These are combination effects and do not establish clinical efficacy. [2]
In stimulated Jurkat T cells, BI-D1870 attenuated lenalidomide-enhanced IL-2 production and did not itself reproduce lenalidomide-associated IKZF1 downregulation. Thus, the myeloma combination findings should not be generalized to enhanced T-cell stimulation. [2]
RSK biochemical inhibition and selectivity profiling
Biochemical assay · Purified RSK1/RSK2, active N-terminal RSK2 fragment, and kinase profiling panel. [1]
BI-D1870 inhibited the RSK N-terminal kinase domain and showed ATP-dependent potency. Table 2 separately reports RSK1–4 IC50 estimates. Profiling also found inhibition outside RSK, so specificity is conditional on enzyme and assay conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- Table 2 dose-response range: 1 nM to 100 μM; Fig. 1 varies inhibitor and ATP concentrations.
- Exposure duration
- Main RSK assay: 10 min at 30 °C.
- Method
- Radiometric substrate-peptide phosphorylation assay; activity compared with no-inhibitor controls.
- Vehicle
- Biochemical vehicle composition not established from reviewed main assay paragraph.
- Reported limits
- Supplemental panel protocols not retrieved; main Methods detail RSK1, RSK2 and fragment assays. Do not transfer that substrate protocol to all panel enzymes.
Methods and source locations
Compound reported in the paper: BI-D1870
Results concern the paper’s racemic BI-D1870, without validation of an Adooq lot or an isolated enantiomer.
Fig. 1A–D; Tables 1–2 and captions; Materials; Protein kinase assays; Results: potent inhibitor and specificity
RSK1, RSK2, RSK3 and RSK4 inhibition at 100 μM ATP: Table 2 means±S.E.M. of two separate experiments, each assay condition in triplicate; estimates extrapolated from concentration-response data. — Table 2: RSK1–4 rows and caption
RSK1/RSK2 IC50 shifts with ATP concentration: Values explicitly stated in Results accompanying Fig. 1; Fig. 1 caption: average of two experiments, each determination in triplicate, error for each point <5%. — Fig. 1B–D/caption; Results: BI-D1870 is a potent inhibitor of RSK isoforms
Kinase profiling outside the RSK family: Actual Table 1 panel entries and Table 2 off-target rows checked; no universal RSK-only selectivity claim. — Tables 1–2; Results: BI-D1870 is a specific inhibitor of RSK isoforms
PMA-stimulated RSK-substrate phosphorylation in HEK-293 cells
Cell-based study · Serum-deprived human HEK-293 cells stimulated with PMA. [1]
BI-D1870 reduced PMA-induced GSK3α/β and LKB1 phosphorylation. ERK1/2, CREB and ribosomal S6 phosphorylation were largely maintained under these conditions. These cellular readouts require micromolar exposure rather than the nanomolar enzyme IC50 range.
Literature-Reported Treatment Conditions
- Treatment concentration
- Fig. 2A: 10 μM; Fig. 2B: 0.01–10 μM.
- Exposure duration
- Fig. 2A pretreatment: 15–240 min; Fig. 2B: 30 min; followed by 20 min PMA stimulation.
- Method
- Immunoblotting after serum deprivation for 16 h; PMA 400 ng/mL; total-protein blots and unstimulated/stimulated controls.
- Vehicle
- 0.1% DMSO; equivalent DMSO control.
- Co-treatment
- PMA stimulation; no co-inhibitor in the adopted Fig. 2 experiment.
- Reported limits
- Two separate experiments reported; no exact numerical uncertainty for cellular IC50 provided.
Methods and source locations
Compound reported in the paper: BI-D1870
Results concern the paper’s racemic BI-D1870, without validation of an Adooq lot or an isolated enantiomer.
Fig. 2A–B and caption; Cell culture, stimulation and cell lysis; immunoblotting; associated Results
PMA-induced GSK3α/β and LKB1 phosphorylation: Reported approximate cellular estimate in Results accompanying Fig. 2B; immunoblots and GSK3 densitometry. No decimal values reconstructed from curves. — Fig. 2B; Results: Evidence that BI-D1870 inhibits RSK activity specifically in HEK-293 cells
PMA-induced ERK1/2, CREB and ribosomal S6 phosphorylation: Fig. 2A–B phospho- and total-protein controls; similar results in two separate experiments. — Fig. 2A–B and caption
BI-D1870 and lenalidomide combination responses in myeloma cells
Cell-based study · KMS11, XG1, MM1.S and JJN3 human myeloma cell lines. [2]
Combination responses were assessed by MTT and normalized isobolograms. Synergistic combinations were observed in all four lines, but the image also contains points above the additivity line, including XG1; the result does not establish synergy for every tested pair.
Literature-Reported Treatment Conditions
- Treatment concentration
- Multiple BI-D1870/lenalidomide combinations; absolute dose pairs not supplied by the normalized Fig. 3A axes.
- Exposure duration
- 6 days (Fig. 3 caption).
- Method
- MTT viability readout; normalization to vehicle-treated controls; CalcuSyn combination-index/isobologram analysis.
- Vehicle
- Vehicle-treated control; vehicle composition not specified in adopted description.
- Co-treatment
- Lenalidomide; single-agent responses provide the reference doses for normalized isobolograms.
- Reported limits
- Exact concentration matrix and replication/uncertainty for Fig. 3A not established; normalized dose coordinates are not μM treatment concentrations.
Methods and source locations
Compound reported in the paper: BI-D1870; Compound label in the paper: BI
Named-compound evidence; neither Adooq sourcing nor equivalence of a particular lot/form is established.
Fig. 3A/caption; Small molecule RSK2 inhibitor studies; Results: RSK2 small molecule inhibitors synergize with lenalidomide
Interaction of BI-D1870 with lenalidomide on MTT viability: Six-day MTT assay, vehicle normalization and CalcuSyn. Qualitative inspection of actual Fig. 3A; no exact combination index inferred from points. — Fig. 3A and caption
Effect on lenalidomide-enhanced IL-2 production in Jurkat cells
Cell-based study · PMA/ionomycin-stimulated human Jurkat T cells. [2]
BI-D1870 attenuated the lenalidomide-associated increase in IL-2-positive cells. BI-D1870 alone did not reproduce lenalidomide-associated IKZF1 downregulation. The antimyeloma combination effect therefore should not be generalized to enhanced immune stimulation.
Literature-Reported Treatment Conditions
- Treatment concentration
- BI-D1870 0.25 μM in Fig. 5A; 0.25 and 0.5 μM in Fig. 5B; lenalidomide 1 μM.
- Exposure duration
- 1 h drug pretreatment, then PMA/ionomycin stimulation for 40 h.
- Method
- Intracellular IL-2–APC flow cytometry; IKZF1 immunoblot with β-actin control.
- Vehicle
- Vehicle control; chemical identity/concentration not specified in this experiment.
- Co-treatment
- Lenalidomide 1 μM; PMA 1 ng/mL and ionomycin 1 μg/mL stimulation.
- Reported limits
- No replicate count or uncertainty specified for Fig. 5A–B; displayed flow percentages are not pooled effect estimates.
Methods and source locations
Compound reported in the paper: BI-D1870; Compound label in the paper: BI
Named-compound evidence; neither Adooq sourcing nor equivalence of a particular lot/form is established.
Fig. 5A–B/caption; ELISA analysis of TNFα and staining for intracellular IL-2 production; Western blot; Results: Effects on immune-modulatory activity
Lenalidomide-enhanced intracellular IL-2 response: Directly viewed Fig. 5A flow histograms; vehicle, stimulant-only, lenalidomide-only, BI-D1870-only and combination conditions. — Fig. 5A and caption
IKZF1 protein expression: Directly viewed Fig. 5B; β-actin loading control. — Fig. 5B and associated Results
