Indole RSK inhibitors. Part 2: Optimization of cell potency and kinase selectivity

Kirrane, Thomas M.; Boyer, Stephen J.; Burke, Jeanmarie R.; Guo, Xin; Snow, Roger J.; Soleymanzadeh, Lida; Swinamer, Alan; Zhang, Yunlong; Madwed, Jeffery B.; Kashem, Mohammed A.; Kugler, Stanley; O’Neill, Margaret M.

doi:10.1016/j.bmcl.2011.10.029

Cited by 31 publications

(23 citation statements)

References 9 publications

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“…To overcome this limitation, several inhibitors have been developed, which block either the Rsk N-terminal kinase such as SL0101 [12] and BI-D1870 [13] or the Rsk C-terminal kinase such as fmk (fluoromethylketone) [14]. Recently, another Rsk inhibitor, BIX02565 (hereafter BIX), has been described [15] which like BI-D1870 (hereafter D1870), acts as an ATP competitor. This cell-permeable compound was reported to be a highly specific RSK inhibitor with an in vitro IC 50 of 1–2 nM.…”

Section: Introductionmentioning

confidence: 99%

A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870

et al. 2014

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Protein kinase inhibitors frequently have interesting effects that cannot be fully ascribed to the intended target kinase(s) but identifying additional targets that might explain the effects is not straightforward. By comparing two different inhibitors of the Rsk (p90 ribosomal S6 kinase) kinases, we found that the increasingly used compound BI-D1870 had biological effects in murine DCs (dendritic cells) that could not be solely ascribed to Rsk or other documented targets. We assessed the ability of BI-D1870 and a second Rsk inhibitor, BIX 02565 to protect enzyme active sites from reaction with biotinylated nucleotide acyl phosphates. Using SILAC (stable isotope labelling by amino acids in cell culture)-labelled DC lysates as a source of enzyme targets, we identify several kinases that interact with BI-D1870 but not with BIX 02565. We confirmed that these kinases, including Slk, Lok and Mst1, are inhibited by BI-D1870 but to a much lesser extent by BIX 02565 and that phosphorylation of some of their substrates is blocked by BI-D1870 in living cells. Our results suggest that the BI-D1870 inhibitor should be used with caution. The SILAC-based methodology we used should be useful for further comparative unbiased profiling of the target spectrum of kinase inhibitors with interesting biological effects under conditions that closely mimic those found in cells.

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Section: Introductionmentioning

confidence: 99%

A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870

et al. 2014

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“…We previously reported the discovery of BIX 02565, a potent RSK2 inhibitor (IC 50 ϭ 1.1 nM) targeted for the treatment of heart failure Kirrane et al, 2011). However, in the present study, when the selectivity of BIX 02565 was assessed in a broad biochemical selectivity screen (68 targets, including receptors, ion channels, and G proteincoupled receptors; Table 1), the compound also elicited offtarget inhibition of radioligand binding at multiple adrenergic receptor subtypes (␣ 1A , ␣ 1B , ␣ 1D , ␣ 2A , and ␤ 2 ) and at the imidazoline I 2 receptor (IC 50 values between 0.052 and 1.820 M), many of which are important in the regulation of cardiac function and vascular tone.…”

Section: Discussionmentioning

confidence: 99%

“…Ribosomal S6 kinase (RSK) is an NHE-activating factor and, therefore, a potential point for therapeutic intervention in ischemia-mediated cardiac damage (Avkiran et al, 2008). Indeed, we have previously reported the discovery of (R)-5-Methyl-1-oxo-2,3,4,5-tetrahydro-1H- [1,4]diazepino[1,2-a] indole-8-carboxylic acid [1-(3-dimethylaminopropyl)-1H-benzoimidazol-2-yl]-amide (BIX 02565), a potent RSK2 inhibitor (IC 50 ϭ 1.1 nM) targeted for the treatment of heart failure secondary to myocardial infarction through indirect NHE inhibition Kirrane et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Mitigation of Off-Target Adrenergic Binding and Effects on Cardiovascular Function in the Discovery of Novel Ribosomal S6 Kinase 2 Inhibitors

Fryer¹,

Muthukumarana²,

Chen³

et al. 2011

J Pharmacol Exp Ther

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We previously reported the discovery of a novel ribosomal S6 kinase 2 (RSK2) inhibitor, (R)-5-Methyl-1-oxo-2,3,4,5-tetrahydro-1H- [1,4], with high potency (IC 50 ϭ 1.1 nM) targeted for the treatment of heart failure. In the present study, we report that despite nanomolar potency at the target, BIX 02565 elicits off-target binding at multiple adrenergic receptor subtypes that are important in the control of vascular tone and cardiac function. To elucidate in vivo the functional consequence of receptor binding, we characterized the cardiovascular (CV) profile of the compound in an anesthetized rat CV screen and telemetry-instrumented conscious rats. Infusion of BIX 02565 (1, 3, and 10 mg/kg) in the rat CV screen resulted in a precipitous decrease in both mean arterial pressure (MAP; to -65 Ϯ 6 mm Hg below baseline) and heart rate (Ϫ93 Ϯ 13 beats/min). In telemetry-instrumented rats, BIX 02565 (30, 100, and 300 mg/kg p.o. QD for 4 days) elicited concentration-dependent decreases in MAP after each dose (to Ϫ39 Ϯ 4 mm Hg on day 4 at T max ); analysis by Demming regression demonstrated strong correlation independent of route of administration and influence of anesthesia. Because of pronounced off-target effects of BIX 02565 on cardiovascular function, a high-throughput selectivity screen at adrenergic ␣ 1A and ␣ 2A was performed for 30 additional RSK2 inhibitors in a novel chemical series; a wide range of adrenergic binding was achieved (0 -92% inhibition), allowing for differentiation within the series. Eleven lead compounds with differential binding were advanced to the rat CV screen for in vivo profiling. This led to the identification of potent RSK2 inhibitors (cellular IC 50 Ͻ0.14 nM) without relevant ␣ 1A and ␣ 2A inhibition and no adverse cardiovascular effects in vivo.

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“…The detailed specificity and potency of BIX02565 have been tested and reported in a separate article. 19 Anti-phospho-NHE1 (S703) antibody detecting sequence “TMSRARIGSpD-PLAYE” 14 was kindly provided by Boehringer-Ingelheim.…”

Section: Methodsmentioning

confidence: 99%

The RSK Inhibitor BIX02565 Limits Cardiac Ischemia/Reperfusion Injury

O’Neill

MacDonnell

et al. 2015

J Cardiovasc Pharmacol Ther

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Aims During ischemia/reperfusion (I/R), ribosomal S6 kinase (RSK) activates Na+/H+ exchanger 1 (NHE1) by phosphorylating NHE1 at serine 703 (pS703-NHE1), which promotes cardiomyocyte death and injury. Pharmacologic inhibition of NHE1 effectively protects animal hearts from I/R. However, clinical trials using NHE1 inhibitors failed to show benefit in patients with acute myocardial infarction (MI). One possible explanation is those inhibitors block both agonist-stimulated activity (increasing I/R injury) and basal NHE1 activity (necessary for cell survival). We previously showed that dominant-negative RSK (DN-RSK) selectively blocked agonist-stimulated NHE1 activity. Therefore, we hypothesized that a novel RSK inhibitor (BIX02565) would blunt agonist-stimulated NHE1 and protect hearts from I/R. Methods and Results Serum/angiotensin II-stimulated pS703-NHE1 was significantly decreased by BIX02565 in cultured cells. Intracellular pH recovery assay showed that BIX02565 selectively inhibited serum-stimulated NHE1 activity. Ischemia/reperfusion decreased left ventricular–developed pressure (LVDP; inhibited) to 8.7% of the basal level in non-transgenic littermate control (NLC) mouse hearts, which was significantly improved (44.6%) by BIX02565. Similar protection was observed in vehicle-treated, cardiac-specific DN-RSK-Tg mice (43%). No additional protective effect was seen in BIX02565-treated DN-RSK-Tg hearts. BIX02565 also improved LVDP in cardiac-specific wild-type (WT)-RSK-Tg mouse hearts (7.4%-40.9%, P < .01). Finally, Western Blotting results confirmed DN-RSK and BIX02565 significantly decreased I/R-induced pS703-NHE1. Conclusion The RSK plays a crucial role in I/R-induced activation of NHE1 and cardiac injury. The RSK inhibition may provide an alternative target for patients with MI.

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Indole RSK inhibitors. Part 2: Optimization of cell potency and kinase selectivity

Cited by 31 publications

References 9 publications

A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870

A combination of SILAC and nucleotide acyl phosphate labelling reveals unexpected targets of the Rsk inhibitor BI-D1870

Mitigation of Off-Target Adrenergic Binding and Effects on Cardiovascular Function in the Discovery of Novel Ribosomal S6 Kinase 2 Inhibitors

The RSK Inhibitor BIX02565 Limits Cardiac Ischemia/Reperfusion Injury

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