Enhanced activity of the Ser/Thr protein kinase, RSK, is associated with transformation and metastasis, which suggests that RSK is an attractive drug target. The natural product, SL0101 (kaempferol 3-O-(3″,4″-di-O-acetyl-α-L-rhamnopyranoside), has been shown to be a RSK selective inhibitor. However, the Ki for SL0101 is 1 μM with a half-life of less than 30 min in vivo. To identify analogues with improved efficacy we designed a set of analogues based on the crystallographic model of SL0101 in complex with the RSK2 N-terminal kinase domain. We identified an analogue with a 5″-n-propyl group on the rhamnose that has > 40-fold improved affinity for RSK relative to SL0101 in an in vitro kinase assay. This analogue preferentially inhibited the proliferation of the human breast cancer line, MCF-7, versus the normal untransformed breast line, MCF-10A, which is consistent with results using SL0101. However, the efficacy of the 5″-n-propyl analogue to inhibit MCF-7 proliferation was only two-fold better than for SL0101, which we hypothesize is due to limited membrane permeability. The improved affinity of the 5″-n-propyl analogue for RSK will aid in the design of future compounds for in vivo use.

The Ser/Thr protein kinase, RSK, is important in the etiology of tumor progression including invasion and motility. The natural product kaempferol-3-O-(3″,4″-di-O-acetyl-α-Lrhamnopyranoside), called SL0101, is a highly specific RSK inhibitor. Acylation of the rhamnose moiety is necessary for high affinity binding and selectivity. However, the acetyl groups can be cleaved by esterases, which accounts for the poor in vitro biological stability of SL0101. To address this problem a series of analogues containing acetyl group replacements were synthesized and their in vitro stability evaluated. Monosubstituted carbamate analogues of SL0101 showed improved in vitro biological stability while maintaining specificity for RSK. These results should facilitate the development of RSK inibitors derived from SL0101 as anticancer agents. Keywords SL0101; RSK inhibitor; RSK-specific; Breast cancer; Protein kinaseThe members of the p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases have been shown to play a role in a number of different cancers as key drivers of proliferation and metastasis. [1][2][3][4][5][6][7][8] These discoveries have been enabled in part by our report of the identification and isolation of the RSK inhibitor SL0101 (1, Figure 1). 9 SL0101 is a flavonoid glycoside (kaempferol 3-O-(3″,4″-di-O-acetyl-α-L-rhamnopyranoside)) isolated from Forsteronia refracta, a variety of dogbane found in the South American rainforest. SL0101 is highly specific for RSK, inhibiting RSK1/2 but not unrelated kinases nor the closely related kinases MSK1 and p70S6K1. 2,9,10 SL0101 inhibits the proliferation of breast and prostate cancer lines but not their normal counterparts even though it inhibits RSK activity in all the lines. 1,5,9 Thus it appears that some cancer cells have become addicted to RSK, which suggests that RSK may be a potential new target for cancer therapeutics. SL0101, owing to its exquisite specificity, is a compelling lead compound from which to begin the process of identifying drug-like RSK inhibitors. © 2012 Elsevier Ltd. All rights reserved. * Corresponding author. Tel: 434-924-1152; fax: 434-924-1236; mh6cu@virginia.edu. Supplementary Material: Experimental procedures and compound characterization for all new compounds can be found online at Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will under go copy editing, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. We and others have reported the total synthesis and biological evaluation of SL0101 and a number of analogues, with the ultimate goal of developing an anticancer drug that targets RSK. [11][12][13][14][15] These analogues have provided key information about t...

In an effort to improve upon the in vivo half-life of the known ribosomal s6 kinase (RSK) inhibitor SL0101, C4″-amide/C6″-alkyl substituted analogues of SL0101 were synthesized and evaluated in cell-based assays. The analogues were prepared using a de novo asymmetric synthetic approach, which featured Pd-π-allylic catalyzed glycosylation for the introduction of a C4″-azido group. Surprisingly replacement of the C4″-acetate with a C4″-amide resulted in analogues that were no longer specific for RSK in cell-based assays.

The Ser/Thr protein kinase, RSK, is associated with oncogenesis, and therefore, there are ongoing efforts to develop RSK inhibitors that are suitable for use in vivo. SL0101 is a natural product that demonstrates selectivity for RSK inhibition. However, SL0101 has a short biological half-life in vivo. To address this issue we designed a set of eight cyclitol analogues, which should be resistant to acid catalyzed anomeric bond hydrolysis. The analogues were synthesized and evaluated for their ability to selectively inhibit RSK in vitro and in cell-based assays. All the analogues were prepared using a stereodivergent palladium-catalyzed glycosylation/cyclitolization for installing the aglycon. The l-cyclitol analogues were found to inhibit RSK2 in in vitro kinase activity with a similar efficacy to that of SL0101, however, the analogues were not specific for RSK in cell-based assays. In contrast, the d-isomers showed no RSK inhibitory activity in in vitro kinase assay.