Identifying requirements for RSK2 specific inhibitors

Abstract

Identifying isoform-specific inhibitors for closely related kinase family members remains a substantial challenge. The necessity for achieving this specificity is exemplified by the RSK family, downstream effectors of ERK1/2, which have divergent physiological effects. The natural product, SL0101, a flavonoid glycoside, binds specifically to RSK1/2 through a binding pocket generated by an extensive conformational rearrangement within the RSK N-terminal kinase domain (NTKD). In modelling experiments a single amino acid that is divergent in RSK3/4 most likely prevents the required conformational rearrangement necessary for SL0101 binding. Kinetic analysis of RSK2 association with SL0101 and its derivatives identified that regions outside of the NTKD contribute to stable inhibitor binding. An analogue with an n -propyl-carbamate at the 4” position on the rhamnose moiety was identified that forms a highly stable inhibitor complex with RSK2 but not with RSK1. These results identify a SL0101 modification that will aid the identification of RSK2 specific inhibitors.

Therapeutic targeting of p90 ribosomal S6 kinase

“…SL0101 has an unusual binding mechanism in which the NTKD undergoes a structural rearrangement that creates a hydrophobic pocket to accommodate SL0101, and as a result disrupts the ATP-binding pocket ( Utepbergenov et al, 2012 ) (compare Figures 6A,B ). Computational modeling of the NTKDs of the other RSK family members are consistent with the ability of the RSK1 and RSK2 NTKDs to form the SL0101 binding pocket whereas this pocket is sterically inhibited from forming in the RSK3 and RSK4 NTKDs ( Wright et al, 2021 ). In a screen of 71 kinases, SL0101 (10 μM) inhibited RSK1 and RSK2 but also inhibited Aurora B and PIM3 ( Bain et al, 2007 ).…”

“…It is also possible that crystallization of the individual RSK isoforms may reveal binding pockets outside the NTKD that would inhibit kinase activity. Support for this hypothesis is provided by biochemical evidence obtained from an analogue of SL0101, which shows preferential binding to RSK2 versus RSK1( Wright et al, 2021 ). This analogue contains an n-propyl-carbamate at the 4” position of the rhamnose.…”

“…In a rigorous test of target specificity in cell-based assays C5″ n -propyl SL0101 was unable to further inhibit proliferation of the TNBC cell line, MDA-MB-231, when RSK1 and RSK2 were knocked down ( Ludwik et al, 2016 ). Analysis of the kinetics of C5″ n -propyl SL0101 showed that its off rate is much lower than that of SL0101 ( Wright et al, 2021 ). Therefore, C5″ n -propyl SL0101, theoretically, should have an improved pharmacokinetic profile compared to SL0101 ( Mrozowski et al, 2012 ).…”

“…SL0101 is not a pan-RSK inhibitor as has been mistakenly referred to in literature reports. SL0101 specifically inhibits RSK1 and RSK2 NTKD activity with no effect on RSK3 and RSK4 NTKD activity ( Wright et al, 2021 ). SL0101 has an unusual binding mechanism in which the NTKD undergoes a structural rearrangement that creates a hydrophobic pocket to accommodate SL0101, and as a result disrupts the ATP-binding pocket ( Utepbergenov et al, 2012 ) (compare Figures 6A,B ).…”

“…Structure-activity-relationship (SAR) studies using SL0101 as the lead compound were used to identify the analogue C5″ n -propyl SL0101 ( Figure 5A ) ( Smith et al, 2006 ; Smith et al, 2007 ; Hilinski et al, 2012 ; Mrozowski et al, 2012 ; Mrozowski et al, 2014 ; Li et al, 2015 ; Ludwik et al, 2016 ; Li et al, 2017 ; Li et al 2020a ; Li et al 2020b ; Wright et al, 2021 ) ( Supplementary Tables S2, S3 ). This analogue has an improved potency of two-fold in RSK2 in vitro kinase assays (IC 50 –392 nM vs. 183 nM (10 μM ATP)) and five-fold in cell-based assays compared to SL0101 (IC 50 50 μM vs. 8 μM in an MCF-7 cell line).…”