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

Abstract

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.

Identification of RSK and TTK as Modulators of Blood Vessel Morphogenesis Using an Embryonic Stem Cell-Based Vascular Differentiation Assay

“…To validate RSK as a hit, we performed a dose-response curve using the inhibitors identified by our screen, BIX-RSK2 (referred to as compound 15 in the study by Fryer et al., 2012 ) ( Figure 3 A) and BI-D1870 ( Figure 3 B). These inhibitors have previously been shown to be selective ( Fryer et al., 2012 , Sapkota et al., 2007 ).…”

“…To validate RSK as a hit, we performed a dose-response curve using the inhibitors identified by our screen, BIX-RSK2 (referred to as compound 15 in the study by Fryer et al., 2012 ) ( Figure 3 A) and BI-D1870 ( Figure 3 B). These inhibitors have previously been shown to be selective ( Fryer et al., 2012 , Sapkota et al., 2007 ). It is important to note that BI-D1870 at high concentrations (10 μM or above) can have off-target effects, although this inhibitor was shown to be selective when used at 2.5 μM and lower ( Roffe et al., 2015 , Sapkota et al., 2007 ).…”

G Protein–Coupled Bile Acid Receptor 1 Stimulation Mediates Arterial Vasodilation through a KCa1.1 (BKCa)–Dependent Mechanism

“…The off-target profile of each GPBAR1 agonist was assessed across prototypical BA nuclear receptors as well as 29 additional receptors, ion channels, and other proteins in a selectivity screen at a standard concentration of 10 mM (Eurofins Panlabs, Taipei, Taiwan) and as previously described (Fryer et al, 2012b). The methods for the radioligand binding studies, specific to each assay performed can be found at http://www.…”

“…The first was an anesthetized rat hemodynamic model with demonstrated utility and predictivity to detect cardiovascular liabilities for the purpose of both structure-activity relationship-based research (Kym et al, 2006;Fryer et al, 2012b) as well as translation to dogs and humans (Fryer et al, 2012a). Second, a comprehensively instrumented acute model in dogs demonstrated to be predictive of effects in humans using positive controls across a wide range of mechanisms such as inhibitors of cholesterylester transfer protein (CETP), and phosphodiesterase 3 (PDE3), L-type Ca…”

Development of a RSK Inhibitor as a Novel Therapy for Triple-Negative Breast Cancer

“…However, the currently available NTKD inhibitors are not RSK specific (21, 23–26) or demonstrate poor pharmacokinetics (27, 28). Covalent inhibitors of the RSK CTKD (29–31), targeting autoactivation, are also available and have limited off-target effects.…”