Pharmacological characterization of a highly selective Rho kinase (ROCK) inhibitor and its therapeutic effects in experimental pulmonary hypertension

Abstract

Studies on the role of Rho-associated protein kinase (ROCK) in experimental pulmonary artery hypertension (PAH) relies mainly on the use of pharmacological inhibitors. However, interpreting these data is hampered by the lack of specificity of commonly utilized inhibitors. To fill this gap, we have selected and characterized a novel ROCK inhibitor, Compound 3, previously described in a patent. Inhibitory potency of Compound 3 against enzymatic activity of ROCK-1 and 2 (IC 50 = 10 ± 3.1 and 7.8 ± 0.5 nM, respectively) was accompanied by a strong vasodilating effect in phenylephrine pre-contracted isolated rat pulmonary artery rings (IC 50 = 51.7 ± 9.1 nM) as well as in aortic rings (IC 50 = 45.5 ± 1.1 nM). Compound 3 showed a remarkable selectivity towards ROCK 1 and 2 when tested against a large panel (> 400) of human kinases. A partial explanation for its selectivity is provided from docking simulations within ROCK-1. Pharmacokinetic studies showed that Compound 3 is suitable for a twice daily administration without significant accumulation upon repeated dosing. In rats with monocrotaline (MCT)-induced pulmonary hypertension, therapy with Compound 3, (1 and 3 mg/kg, s.c., b.i.d.), started 14 days after induction of the disease, attenuated right ventricle systolic pressure (RVSP) increase. Morphometric histological analysis showed that Compound 3, at both doses, counteracted MCT-induced medial thickening of lung distal arterioles with an effect comparable to macitentan (10 mg/kg, p.o., q.d.). Compound 3 is a potent and highly selective ROCK inhibitor that ameliorates hemodynamic parameters and counteracts pulmonary vascular remodeling in experimental PAH.

Rho-Kinase 1/2 Inhibition Prevents Transforming Growth Factor-β-Induced Effects on Pulmonary Remodeling and Repair

“…Accordingly, the design of isoform selective inhibitors has until now been very challenging. Unfortunately, both these first generation ROCK inhibitors have poor ROCK inhibition potency, and are additionally unselective against a range of other kinases, especially those in the AGC family (Tumbarello and Turner, 2006;Hallgren et al, 2012;Pireddu et al, 2012;Rath and Olson, 2012;Vigil et al, 2012;Xueyang et al, 2016;Huang et al, 2018;Cantoni et al, 2019). To fill this gap, we selected, as tool compounds, two ROCK inhibitors previously described in two distinct patents, a ROCK 1 and 2 inhibitor (compound 31), and a ROCK2 selective inhibitor (compound A11).…”

“…ROCK enzymatic activity inhibition was measured as described previously (Cantoni et al, 2019). Glutathione S-transferase (GST)-tagged 1-535 human ROCK1 and GST-tagged 1-552 human ROCK2 (Fisher Scientific United Kingdom Ltd., Loughborough, Leicestershire, United Kingdom) were diluted into assay buffer containing 40 mM Tris pH7.5, 20 mM MgCl2 0.1 mg/ml BSA, 50 μM DTT and 2.5 μM peptide substrate (myelin basic protein).…”

Characterization of Contractile Machinery of Vascular Smooth Muscles in Hypertension

“…Mean pulmonary artery pressure or systolic pulmonary artery pressure were decreased after receiving intravenous or inhaled fasudil treatment in patients with high-altitude PAH, congenital heart diseases or connective tissue diseases associated with PAH [ 66 ]. In addition to the inhibition of RhoA-ROCK directly by fasudil, there are many other potential approaches to inhibit the RhoA-ROCK axis in PAH, such as the aminofurazan derivative drug, SB-772077-B, simvastatin [ 64 ], resveratrol [ 67 ], Compound 3 (trans-6-((4-aminocyclohexyl)amino)-5-fluoro-2-methoxynicotinamide) [ 68 ] and fasudil dichloroacetate [ 69 ]. The roles of RhoA/Rho-kinase signaling in PH and treatment have been reviewed [ 60 , 64 , 70 , 71 , 72 , 73 ].…”

Kinases as potential targets for treatment of pulmonary hypertension and right ventricular dysfunction

“…showed similar effects in hypoxia and MCT-induced PH but also reduced right ventricular hypertrophy . Very recently, a new inhibitor, named compound 3, has been shown to be more potent and highly selective for ROCK-1 and 2 to improve haemodynamic parameters and counteract pulmonary vascular remodelling in experimental PAH (Cantoni et al, 2019 Michelakis et al have intensively studied the role of PDH in PAH (Michelakis et al, 2002). In their recent work, they investigated the therapeutic use of dichloroacetate (DCA), a PDH kinase inhibitor, in PAH patients (Michelakis et al, 2017).…”

“…Azaindole‐1 showed similar effects in hypoxia and MCT‐induced PH but also reduced right ventricular hypertrophy (Dahal, Kosanovic, et al, 2010). Very recently, a new inhibitor, named compound 3, has been shown to be more potent and highly selective for ROCK‐1 and 2 to improve haemodynamic parameters and counteract pulmonary vascular remodelling in experimental PAH (Cantoni et al, 2019). Thus, fasudil and other ROCK inhibitors deserve further investigation with regards to their effects on the right ventricle in PAH.…”