RHO KINASE, A PROMISING DRUG TARGET FOR NEUROLOGICAL DISORDERS

vito · 05-06-2005, 02:59 PM

http://www.nature.com/cgi-taf/DynaPa...rd1719_fs.html

Bernhard K. Mueller, Helmut Mack & Nicole Teusch about the authors

Abbott GmbH & Co. KG, Knollstrasse 50, D-67061 Ludwigshafen, Germany

correspondence to: Bernhard K. Mueller bernhard.mueller@abbott.com

Rho kinases (ROCKs), the first Rho effectors to be described, are serine/threonine kinases that are important in fundamental processes of cell migration, cell proliferation and cell survival. Abnormal activation of the Rho/ROCK pathway has been observed in various disorders of the central nervous system. Injury to the adult vertebrate brain and spinal cord activates ROCKs, thereby inhibiting neurite growth and sprouting. Inhibition of ROCKs results in accelerated regeneration and enhanced functional recovery after spinal-cord injury in mammals, and inhibition of the Rho/ROCK pathway has also proved to be efficacious in animal models of stroke, inflammatory and demyelinating diseases, Alzheimer's disease and neuropathic pain. ROCK inhibitors therefore have potential for preventing neurodegeneration and stimulating neuroregeneration in various neurological disorders.

One of the best-characterized effectors of the small GTP-binding proteins of the Rho subfamily (Rho GTPases) is Rho-associated coiled-coil-containing protein kinase (hereafter simply referred to as ROCK). RhoGTPases, a subfamily of the Ras superfamily of GTPases, function as molecular devices that control multiple signalling pathways in a very precise and coordinated way by switching between a biochemically inactive (GDP-bound) and an active (GTP-bound) state1-3. The cycling between GDP- and GTP-bound states is controlled by two classes of proteins: GTPase-activating proteins (GAPs), which enhance intrinsic GTPase activity; and guanine nucleotide-exchange factors (GEFs), which catalyse the exchange of GDP to GTP4. Furthermore, a third set of regulatory proteins, the guanine nucleotide-dissociation inhibitors (GDIs), sequester GTPases in the cytosol in the inactive, GDP-bound state.

In the active, GTP-bound state, RhoGTPases activate numerous downstream effectors. In this review, we concentrate on one of these effectors, ROCK. The current understanding of the pathophysiological consequences of ROCK activation in the central nervous system (CNS) is summarized and we highlight the potential therapeutic use of ROCK inhibitors for the treatment of various neurological disorders, including spinal-cord injury, Alzheimer`s disease, stroke, multiple sclerosis and NEUROPATHIC PAIN.

ROCK isoforms and tissue distribution

ROCK is a serine/threonine (Ser/Thr) protein kinase that was identified about ten years ago as a RhoGTP-binding protein with a molecular mass of 160 kDa5-7. Two isoforms encoded by two different genes of ROCK have been described: ROCKI (also known as ROK or p160ROCK) and ROCKII (which is also known as ROK)8. These two proteins share an overall sequence similarity at the amino-acid level of 65% and in their kinase domains of 92%9, 10. ROCKs are most homologous to other members of the group of AGC KINASES, such as myotonic dystrophy kinase (DMPK), myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) and citron kinase. In general, the catalytic domain of all these kinases is located at the amino terminus, followed by a coiled-coil-forming region and a pleckstrin-homology domain with a cysteine-rich repeat at the carboxyl terminus. In the case of ROCK, the carboxy-terminal coiled-coil region also encompasses the Rho-binding (RBD) domain. For illustrative purposes, the predicted functional domains of both full-length ROCKs are shown in Fig. 1.

Figure 1 | Molecular structure of Rho kinase (ROCK) I and II.

The two isoforms share an overall sequence identity at the amino-acid level of approximately 60%. Their kinase domains are more than 90% identical. The catalytic domain is located at the amino terminus, followed by a coiled-coil-forming region that encompasses the Rho-binding domain (RBD), and a pleckstrin-homology domain (PH) with a cysteine-rich repeat domain (CRD) at the carboxyl terminus10.

Despite the striking similarity of the protein sequences of the two ROCK isoforms, significant differences regarding their respective tissue distribution have been reported, which indicates distinct functions of each isoform in vivo. ROCKII is preferentially expressed in brain, whereas ROCKI shows the highest expression levels in non-neuronal tissues, including heart, lung and skeletal muscles. ROCKII expression in bovine brain was mainly observed in the pyramidal neurons of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum11. Interestingly, during postnatal development of the mouse brain, ROCKII expression levels gradually increased12.

Regulation of ROCK activity

It has been previously demonstrated that the C terminus of ROCK negatively regulates its kinase activity13. Similarly to DMPK and MRCK14, 15, the C-terminal domain of ROCK folds back onto the kinase domain, thereby forming an auto-inhibitory loop that maintains ROCK in an inactive state. Binding of GTP-bound Rho to the RBD is believed to disrupt the negative regulatory interaction between the catalytic domain and the auto-inhibitory C-terminal region, which results in activation of the enzyme in response to extracellular signals. The activation mechanism of ROCK is illustrated schematically in Fig. 2. Rho binds to ROCK only in the biochemically activated, GTP-bound form3. Recently, the crystal structure of RhoA bound to the RBD has been investigated by two groups16, 17, and it indicates the formation of a parallel coiled-coil dimer of the RBD domain and provides strong evidence that the full-length kinase is also a dimer. Consistent with these conclusions, it has been shown that protein oligomerization might regulate ROCK activity18, possibly through N-terminal transphosphorylation19. Other direct activators include intracellular second messengers such as arachidonic acid20 and sphingosylphosphorylcholine21 which can activate ROCK independently of Rho. Furthermore, ROCKI activity can also be induced during apoptosis. Cleavage of the auto-inhibitory C terminus of ROCKI by caspase 3 gives rise to a constitutively active ROCKI22, 23. During apoptosis, caspase cleavage occurs specifically to ROCKI, although other apoptosis-related mechanisms can also modify ROCKII. In conclusion, the complexity of the regulation mechanisms of the kinase activity could represent a crucial feature for the maintenance of a proper balance of ROCK function in vivo.

Milano.

[This message was edited by Wise Young on 05-09-05 at 01:24 AM.]

Wise Young · 05-06-2005, 06:55 PM

Yes, rho kinases (ROCKS) are a very important target for spinal cord injury. A clinical trial is already going on right now in Canada and the United States testing Cethrin (a Rho inhibitor) on acute spinal cord injury.
http://www.t2c2capital.com/comm/soci..._20040606.html

Wise.

05-06-2005, 02:59 PM	#1
vito Senior Member Join Date: Feb 2005 Location: Texas Posts: 155	RHO KINASE, A PROMISING DRUG TARGET FOR NEUROLOGICAL DISORDERS http://www.nature.com/cgi-taf/DynaPa...rd1719_fs.html Bernhard K. Mueller, Helmut Mack & Nicole Teusch about the authors Abbott GmbH & Co. KG, Knollstrasse 50, D-67061 Ludwigshafen, Germany correspondence to: Bernhard K. Mueller bernhard.mueller@abbott.com Rho kinases (ROCKs), the first Rho effectors to be described, are serine/threonine kinases that are important in fundamental processes of cell migration, cell proliferation and cell survival. Abnormal activation of the Rho/ROCK pathway has been observed in various disorders of the central nervous system. Injury to the adult vertebrate brain and spinal cord activates ROCKs, thereby inhibiting neurite growth and sprouting. Inhibition of ROCKs results in accelerated regeneration and enhanced functional recovery after spinal-cord injury in mammals, and inhibition of the Rho/ROCK pathway has also proved to be efficacious in animal models of stroke, inflammatory and demyelinating diseases, Alzheimer's disease and neuropathic pain. ROCK inhibitors therefore have potential for preventing neurodegeneration and stimulating neuroregeneration in various neurological disorders. One of the best-characterized effectors of the small GTP-binding proteins of the Rho subfamily (Rho GTPases) is Rho-associated coiled-coil-containing protein kinase (hereafter simply referred to as ROCK). RhoGTPases, a subfamily of the Ras superfamily of GTPases, function as molecular devices that control multiple signalling pathways in a very precise and coordinated way by switching between a biochemically inactive (GDP-bound) and an active (GTP-bound) state1-3. The cycling between GDP- and GTP-bound states is controlled by two classes of proteins: GTPase-activating proteins (GAPs), which enhance intrinsic GTPase activity; and guanine nucleotide-exchange factors (GEFs), which catalyse the exchange of GDP to GTP4. Furthermore, a third set of regulatory proteins, the guanine nucleotide-dissociation inhibitors (GDIs), sequester GTPases in the cytosol in the inactive, GDP-bound state. In the active, GTP-bound state, RhoGTPases activate numerous downstream effectors. In this review, we concentrate on one of these effectors, ROCK. The current understanding of the pathophysiological consequences of ROCK activation in the central nervous system (CNS) is summarized and we highlight the potential therapeutic use of ROCK inhibitors for the treatment of various neurological disorders, including spinal-cord injury, Alzheimer`s disease, stroke, multiple sclerosis and NEUROPATHIC PAIN. ROCK isoforms and tissue distribution ROCK is a serine/threonine (Ser/Thr) protein kinase that was identified about ten years ago as a RhoGTP-binding protein with a molecular mass of 160 kDa5-7. Two isoforms encoded by two different genes of ROCK have been described: ROCKI (also known as ROK or p160ROCK) and ROCKII (which is also known as ROK)8. These two proteins share an overall sequence similarity at the amino-acid level of 65% and in their kinase domains of 92%9, 10. ROCKs are most homologous to other members of the group of AGC KINASES, such as myotonic dystrophy kinase (DMPK), myotonic dystrophy kinase-related CDC42-binding kinase (MRCK) and citron kinase. In general, the catalytic domain of all these kinases is located at the amino terminus, followed by a coiled-coil-forming region and a pleckstrin-homology domain with a cysteine-rich repeat at the carboxyl terminus. In the case of ROCK, the carboxy-terminal coiled-coil region also encompasses the Rho-binding (RBD) domain. For illustrative purposes, the predicted functional domains of both full-length ROCKs are shown in Fig. 1. Figure 1 \| Molecular structure of Rho kinase (ROCK) I and II. The two isoforms share an overall sequence identity at the amino-acid level of approximately 60%. Their kinase domains are more than 90% identical. The catalytic domain is located at the amino terminus, followed by a coiled-coil-forming region that encompasses the Rho-binding domain (RBD), and a pleckstrin-homology domain (PH) with a cysteine-rich repeat domain (CRD) at the carboxyl terminus10. Despite the striking similarity of the protein sequences of the two ROCK isoforms, significant differences regarding their respective tissue distribution have been reported, which indicates distinct functions of each isoform in vivo. ROCKII is preferentially expressed in brain, whereas ROCKI shows the highest expression levels in non-neuronal tissues, including heart, lung and skeletal muscles. ROCKII expression in bovine brain was mainly observed in the pyramidal neurons of the hippocampus and cerebral cortex, and in the Purkinje cells of the cerebellum11. Interestingly, during postnatal development of the mouse brain, ROCKII expression levels gradually increased12. Regulation of ROCK activity It has been previously demonstrated that the C terminus of ROCK negatively regulates its kinase activity13. Similarly to DMPK and MRCK14, 15, the C-terminal domain of ROCK folds back onto the kinase domain, thereby forming an auto-inhibitory loop that maintains ROCK in an inactive state. Binding of GTP-bound Rho to the RBD is believed to disrupt the negative regulatory interaction between the catalytic domain and the auto-inhibitory C-terminal region, which results in activation of the enzyme in response to extracellular signals. The activation mechanism of ROCK is illustrated schematically in Fig. 2. Rho binds to ROCK only in the biochemically activated, GTP-bound form3. Recently, the crystal structure of RhoA bound to the RBD has been investigated by two groups16, 17, and it indicates the formation of a parallel coiled-coil dimer of the RBD domain and provides strong evidence that the full-length kinase is also a dimer. Consistent with these conclusions, it has been shown that protein oligomerization might regulate ROCK activity18, possibly through N-terminal transphosphorylation19. Other direct activators include intracellular second messengers such as arachidonic acid20 and sphingosylphosphorylcholine21 which can activate ROCK independently of Rho. Furthermore, ROCKI activity can also be induced during apoptosis. Cleavage of the auto-inhibitory C terminus of ROCKI by caspase 3 gives rise to a constitutively active ROCKI22, 23. During apoptosis, caspase cleavage occurs specifically to ROCKI, although other apoptosis-related mechanisms can also modify ROCKII. In conclusion, the complexity of the regulation mechanisms of the kinase activity could represent a crucial feature for the maintenance of a proper balance of ROCK function in vivo. Milano. [This message was edited by Wise Young on 05-09-05 at 01:24 AM.]

Thread Tools
Show Printable Version Email this Page
Display Modes
Linear Mode Switch to Hybrid Mode Switch to Threaded Mode

05-06-2005, 06:55 PM	#2
Wise Young Administrator Join Date: Jul 2001 Location: New Brunswick, NJ, USA Posts: 37,975	Yes, rho kinases (ROCKS) are a very important target for spinal cord injury. A clinical trial is already going on right now in Canada and the United States testing Cethrin (a Rho inhibitor) on acute spinal cord injury. http://www.t2c2capital.com/comm/soci..._20040606.html Wise.