Investigational Rho Kinase Inhibitors for the Treatment of Glaucoma

Figures & data

Figure 1 Cross section of an eye illustrating aqueous humor (AH) pathways (left) and site of action of antiglaucoma medications (right). AH formation occurs in the ciliary body and flows from the posterior chamber through the pupil to the anterior chamber angle. The drainage of AH is mainly facilitated by the conventional [trabecular meshwork (TM), Schlemm’s canal and episcleral veins] pathway and the non-conventional (uveoscleral-uveovortex) pathway. The current glaucoma hypotensive medications and their sites of action are shown on the right.

Abbreviation: ROCK, rho kinase.

Figure 2 Rho kinase (ROCK) structure and mechanisms of activation. (A) Two isoforms of ROCKs have been identified: ROCK 1 and ROCK 2. They consist of a kinase domain, a coiled-coil region (CCR) containing the rho-binding domain (RBD), and the carboxyl terminus. The carboxyl terminus has a pleckstrin-homology (PH) domain with an internal cysteine-rich domain (CRD). The amino acid sequences of the two ROCK isoforms show the highest similarity at the kinase domain (92%). (B) In the inactive ROCK, both PH domain and RBD domain can bind independently to the kinase region forming an auto inhibitory loop. The GDP-bound RhoA is kept inactive by sequestration with guanine nucleotide dissociation inhibitors (GDI). The guanine nucleotide exchange factor (GEF) converts the inactive GDP-bound RhoA to active GTP-bound RhoA. In contrast, GTPase activating protein (GAP) converts the active RhoA to its inactive form. Binding of the GTP-bound RhoA to RBD results in an open conformation of the kinase and frees its catalytic activity. Similarly, ROCK can be activated by arachidonic acid, which binds to its PH domain. ROCK 1 can be activated by caspase-3-mediated cleavage near the carboxyl-terminus while ROCK 2 is activated by caspase-2 and granzyme B-mediated cleavage. Adapted with permission  from Wirth A. Rho kinase and hypertension. Biochim Biophys Acta.2010;1802(12):1276–1284. Copyright © 2010 Elsevier B.V. All rights reserved.¹⁰

Abbreviations: C2, Caspase 2; C3, Caspase 3; GB, granzyme B; ROCK, rho kinase; CCR, coiled-coil region; RBD, rho-binding domain; PH, pleckstrin-homology; CRD, cysteine-rich domain; GDP, guanosine diphosphate; GTP, guanosine triphosphate; P, phosphate; GDI, guanine nucleotide dissociation inhibitor.

Figure 3 ROCK targets. Rho proteins can be activated by guanine nucleotide exchange factors (GEFs). GEFs are themselves activated and regulated via engagement of various receptors at the plasma membrane. The active GTP-bound Rho subsequently activates ROCK 1 and ROCK 2 that phosphorylate various substrates resulting in diverse cellular responses. The barred-line notation indicates inhibition and the arrows show the cascade of the molecular response. Adapted from Hartmann S, Ridley AJ, Lutz S. The function of rho-associated kinasesROCK1 and ROCK2 in the pathogenesis of cardiovascular disease.Front Pharmacol. 2015;6(276):1–16. Copyright © 2015 Hartmann, Ridley and Lutz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) https://creativecommons.org/licenses/by/4.0/.¹⁴

Abbreviations: CPI-17, kinase C-potentiated phosphatase inhibitor of 17 kDa; CRMP-2, collapsin response mediator protein 2; ECM, extracellular matrix; ERM, ezrin-radixin-moesin; FHOD1, formin homology 2 domain-containing 1; GAP, GTPase activating protein; GPCRs, G protein-coupled receptors; LIMK, LIM-kinase; LPA, lysophosphatidic acid; MLC, myosin II regulatory light chain; MYPT1, myosin phosphatase target subunit 1; NHE, Na+/H+ exchanger; PTEN, phosphatase and tensin homolog deleted on chromosome 10; RTK, receptor tyrosine kinases; GEF, guanine nucleotide exchange factor; GDI, guanine nucleotide dissociation inhibitor; P, phosphate; GTP, guanosine triphosphate; RhoA, Ras homolog family member A.

Figure 4 Therapeutic effects of Rho kinase (ROCK) inhibitors in treatment of glaucoma. They can increase aqueous humor (AH) outflow and decrease intraocular pressure (IOP) by targeting the cytoskeleton of the trabecular meshwork (TM) and Schlemm’s canal (SC) cells. ROCK inhibitors also have a neuroprotective effect by increasing blood flow to the retina and optic nerve, promoting axonal regeneration, and increasing retinal ganglion cell (RGC) survival. In addition, the inhibitory effect on Tenon fibroblasts decreases the fibrosis after filtration surgery. Finally, on the corneal endothelium they enhance cellular proliferation, promote adhesion, and suppress apoptosis. Accordingly, they provide a greater potential for the regeneration of damaged corneal endothelium and restoration of corneal transparency. The photos shown were taken at the Departments of Ophthalmology at UT Southwestern Medical Center Dallas, Texas, USA and King Hussein Medical Center Amman, Jordan. Adapted with permission from Rao PV, Pattabiraman PP, Kopczynski C. Role of the rho GTPase/rho kinase signaling pathway in pathogenesis and treatment of glaucoma:bench to bedside research. Exp Eye Res. 2017;158:23–32. © 2016 Elsevier Ltd. All rights reserved.⁸

Abbreviation: ECM, extracellular matrix.

Figure 5 Timeline map showing the years the investigational agents were introduced (Table 1).

Table 1 Current Investigational Rho Kinase Inhibitors

Agent and Reference	Chemical Formula and Structure	ROCK1 IC50 (nM)	ROCK2 IC50 (nM)	Effect on Other Receptors	Year Introduced	Current Phase
HA-1077^Citation25^–^Citation28 C14H17N3O2S		NA	NA	PKA, PKG, PKC	1995	Phase 1
Y-27632^Citation2 C14H12N3O.2HCL		42	71	PKA, PKC, MLCK	1997	Preclinical
H-1152P^Citation33^–^Citation35 C16H21N3O2S		NA	12	PKC, AMPK	1999	Preclinical
INS-115644^Citation36,Citation37 C20H29NO5S		5	14	NA	2004	Phase 1
Y-39983^Citation38^–^Citation40 C16H17C1N4O		3.6	3.6	NA	2005	Phase 2
SB772077BS^Citation41,Citation42 C15H18N8O2		5.6	5.6	NA	2007	Preclinical
LX7101^Citation43^–^Citation46 C23H29N7O3		69	32	LIMK, Akt1	2007	Phase 1/Phase 2
AR-12286^Citation47^–^Citation52 C17H17N3O2S		NA	NA	NA	2009	Phase 2
AMA-0076^Citation44^–^Citation54	NA	3.7	2.3	NA	2011	Phase 2
RKI 1447^Citation55,Citation56 C16H14N4O2S		14.5	6.2	NA	2012	Preclinical
AR-13533^Citation57	NA	NA	NA	NET	2013	Preclinical
AMA0526^Citation58,Citation59	NA	5.7	3.1	NA	2013	Preclinical
H-1337^Citation60^–^Citation62		NA	NA	PKA, PKN2, PKD1, LRRK2	2015	Phase 2
ITRI-E-212^Citation63	NA	NA	3.2	NA	2017	Preclinical
ITRI-E-247^Citation64	Unavailable	NA	10.9	NA	2017	Preclinical

Note: IC₅₀ is the half-maximal inhibitory concentration (the amount of drug needed to inhibit, in vitro, a given biological process or biological component by 50%).

Abbreviations: nM, nanomolar; NA, not available; PKA, protein kinase A; PKC, protein kinase C; MLCK, myosin light chain kinase; NET, norepinephrine transporter; LIMK, LIM kinase; GRK, G-protein coupled receptor kinase; LRRK, Leucine-rich repeat kinase 2; AMPK, 5ʹ adenosine monophosphate-activated protein kinase.

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