The Isoquinoline-Sulfonamide Compound H-1337 Attenuates SU5416/Hypoxia-Induced Pulmonary Arterial Hypertension in Rats.

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure and right heart failure. Selective pulmonary vasodilators have improved the prognosis of PAH; however, they are not able to reverse pulmonary vascular remodeling. Therefore, a search for new treatment agents is required.

IOP-lowering effect of isoquinoline-5-sulfonamide compounds in ocular normotensive monkeys.

Rho-associated coiled coil-formed protein kinase (ROCK) inhibitors are under development as a new class of antiglaucoma agents. Based on the potent ROCK inhibitor H-1152, previously developed by us, we explored the possibility of related compounds as antiglaucoma agents and synthesized seven types of H-1152-inspired isoquinoline-5-sulfonamide compounds (H-0103-H-0107, H-1001, H-1005). Although all of these compounds potently inhibited ROCK (IC50=18-48 nM), only H-0104 and H-0106 exerted strong intraocular pressure (IOP)-lowering effects into the eyes of monkeys.

K-134, a phosphodiesterase 3 inhibitor, prevents brain damage by inhibiting thrombus formation in a rat cerebral infarction model.

Background: K-134 is a more potent antiplatelet drug with a selective inhibitory effect on phosphodiesterase 3 (PDE3) compared with its analogue, cilostazol.

Objectives: This study was performed to compare the ameliorating effects of K-134 and cilostazol on brain damage in an experimental photothrombotic cerebral infarction model.

Methods And Results: We investigated the effects of oral preadministration of PDE3 inhibitors in a rat stroke model established by photothrombotic middle cerebral artery (MCA) occlusion.

The effect of the H-1152P, a potent Rho-associated coiled coil-formed protein kinase inhibitor, in rabbit normal and ocular hypertensive eyes.

Purpose: We wanted to determine the intraocular pressure (IOP)-lowering effect of H-1152P by utilizing a rabbit ocular hypertension-glaucoma model and normal eyes. H-1152P is a potent, Rho-associated, coiled, coil-forming protein kinase (ROCK) inhibitor.

Methods: IOPs were monitored by a pneumatonometer in New Zealand White rabbits that were given topically administered H-1152P or vehicle alone.

The effect of the Rho-associated protein kinase inhibitor, HA-1077, in the rabbit ocular hypertension model induced by water loading.

Purpose: The aim of this study was to investigate the intraocular pressure (IOP)-lowering effect of a new anti-glaucoma drug, the Rho-associated protein kinase (ROCK) inhibitor, HA-1077, in a rabbit ocular hypertension model.

Methods: Experiments were carried out in 18 male New Zealand white rabbits, with ocular hypertension induced by water loading. Animals were divided into three groups followed by topical administration of 1 mM, 2 mM, and 3 mM HA-1077 in the left eye.

Investigation of binding proteins for anti-platelet agent K-134 by Drug-Western method.

K-134 ((-)-6-[3-[3-cyclopropyl-3-[(1R, 2R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone) is a novel anti-platelet agent with anti-hyperplastic activities. We found previously that K-134 is a potent phosphodiesterase-3 (PDE3) inhibitor. In the present study, we found other K-134-binding proteins by Drug-Western method.

Investigation of the pronounced synergism between prostaglandin E2 and other constrictor agents on rat femoral artery.

This study investigates the pronounced synergism between the weak contractile action of prostaglandin E(2) (PGE(2)) and strong actions of phenylephrine, U-46619 and K(+) on rat isolated femoral artery. The potency ranking for synergism was SC-46275 (prostanoid receptor agonist selectivity: EP(3)>>EP(1))=sulprostone (EP(3)>EP(1))>17-phenyl PGE(2) (EP(1)>EP(3)). The novel EP(3) antagonist L-798106 (0.

PKC phosphorylates MARCKS Ser159 not only directly but also through RhoA/ROCK.

It is well recognized that phorbol 12,13-dibutyrate (PDBu)-activated PKC directly phosphorylates myristoylated alanine-rich C kinase substrate (MARCKS), whose phosphorylation is used as a marker of PKC activation. However, in SH-SY5Y neuroblastoma cells, Western blotting analyses revealed that Rho-associated coiled-coil kinase (ROCK)-specific inhibitor H-1152 inhibited PDBu-induced phosphorylation, and that a small G-protein inhibitor, toxin B, also inhibited MARCKS phosphorylation. Furthermore, in GST pull-down assays, PDBu induced RhoA activation in SH-SY5Y cells, and this activation was inhibited by PKC inhibitor Ro-31-8220.

Development of specific Rho-kinase inhibitors and their clinical application.

Hexahydro-1-(isoquinoline-5-sulfonyl)-1H-1,4-diazepine, HA-1077, is a known selective inhibitor of Rho-kinase. Although its IC(50) value against Rho-kinase is more than 10 times lower than those for kinases such as PKA, PKB, PKC, PKG, MLCK, CaMKII and others, the molecule still retains relative potent inhibition activities against these kinases. In order to produce highly specific Rho-kinase inhibitors, several HA-1077 analogs were synthesized and their kinase inhibition properties evaluated.

Rho-kinase mediates spinal nitric oxide formation by prostaglandin E2 via EP3 subtype.

Prostaglandin E2 (PGE2), the principal pro-inflammatory prostanoid, is known to play versatile roles in pain transmission via four PGE receptor subtypes, EP1-EP4. We recently demonstrated that continuous production of nitric oxide (NO) by neuronal NO synthase (nNOS) following phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and NMDA receptor NR2B subunits is essential for neuropathic pain. These phosphorylation and nNOS activity visualized by NADPH-diaphorase histochemistry were blocked by indomethacin, a PG synthesis inhibitor.

Changes in cyclic nucleotide phosphodiesterase activity and calmodulin concentration in heart muscle of cardiomyopathic hamsters.

Cyclic nucleotides (cAMP and cGMP) phosphodiesterase (PDE) activities and expression are altered in the cardiac muscle of cardiomyopathic heart failure, and PDE inhibitors improve the abnormal muscle condition through changing the cyclic nucleotide concentration. These observations prompted us to investigate the role of calmodulin (CaM) in the regulation of cyclic nucleotide PDE activities, and moreover to study the modulation of the PDE isozymes in heart failure, using cardiac muscles of cardiomyopathic hamster. The CaM concentrations in the heart muscle of the normal control and cardiomyopathic hamsters (each of three to four hamsters) varied with cell fraction and with the age of the animal.

Protein kinase A in complex with Rho-kinase inhibitors Y-27632, Fasudil, and H-1152P: structural basis of selectivity.

Protein kinases require strict inactivation to prevent spurious cellular signaling; overactivity can cause cancer or other diseases and necessitates selective inhibition for therapy. Rho-kinase is involved in such processes as tumor invasion, cell adhesion, smooth muscle contraction, and formation of focal adhesion fibers, as revealed using inhibitor Y-27632. Another Rho-kinase inhibitor, HA-1077 or Fasudil, is currently used in the treatment of cerebral vasospasm; the related nanomolar inhibitor H-1152P improves on its selectivity and potency.

In vivo antitumor activity of a novel sulfonamide, HMN-214, against human tumor xenografts in mice and the spectrum of cytotoxicity of its active metabolite, HMN-176.

The cytotoxic effects of HMN-176 ((E)-4-[[2-N-[4-methoxybenzenesulfonyl] amino] stilbazole] 1-oxide; a newly synthesized compound, were evaluated and compared with those of the clinically used antitumor agents cis-platinum, adriamycin, etoposide, taxol, and vincristine in 22 human tumor cell lines isolated from various organs. HMN-176 exhibited potent cytotoxicity with IC(50) values in the nM range, and the variance of its cytotoxic efficacy was remarkably small. Drug-resistant cell lines also showed low cross-resistance to HMN-176 corresponding to overall resistance indices of less than 14.

HMN-176, an active metabolite of the synthetic antitumor agent HMN-214, restores chemosensitivity to multidrug-resistant cells by targeting the transcription factor NF-Y.

HMN-176 ((E)-4-[[2-N-[4-methoxybenzenesulfonyl]amino]stilbazole]1-oxide) is an active metabolite of HMN-214 ((E)-4-[2-[2-(N-acetyl-N-[4-methoxybenzenesulfonyl]amino)stilbazole]]1-oxide), which has a potent antitumor activity in mouse xenograft models. In this study, we show that HMN-176 circumvents multidrug resistance in a K2 human ovarian cancer subline selected for Adriamycin resistance (K2/ARS). Upon treatment of K2/ARS cells with 3 microM HMN-176, the GI(50) of Adriamycin for the cells decreased by approximately 50%.

The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathway.

We have developed several kinds of protein kinase inhibitors, which are classified as isoquinolinesulfonamides and characterized as ATP competitive inhibitors of Ser/Thr protein kinases. These include H9, H89, KN62, and 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077) against protein kinase C (PKC), protein kinase A, Ca(2+)/calmodulin-dependent protein kinase II, and Rho-kinase, respectively, and they have been used widely to confirm the involvement of the target protein kinase in biological or physiological reaction(s). In some cases, inhibitors have predicted the involvement of the target protein kinase in cell or tissue before its precise mechanism or its effector was defined.

Hormonal regulation of DNA polymerase beta activity and expression in rat adrenal glands and testes.

We investigated whether DNA polymerase beta activity and expression in rat adrenal glands and testes are controlled by the cAMP dependent protein kinase (A-kinase) phosphorylation system in addition to anterior pituitary hormones. DNA polymerase beta mRNA expression in rat testes was decreased by hypophysectomy and recovered with administration of gonadotropic hormone, suggesting that this enzyme is controlled at the mRNA level by this pituitary hormone. In addition, DNA polymerase beta activity in the adrenal glands and testes and the amount of mRNA in the testes increased when cAMP was administered to the normal rat.

Inhibition of rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor.

The functions of small G protein Rho-associated kinase (Rho-kinase) have been determined in muscle and non-muscle cells, but, particularly in neuronal cells, its effector(s) has not been well known. Recently, we preliminarily reported that Rho-kinase phosphorylates the Ser159 residue in myristoylated alanine-rich C kinase substrate (MARCKS) in vitro, but it remains obscure in vivo. To further clarify this point, we developed an isoquinolinesulfonamide derivative, H-1152, that is a more specific, stronger and membrane-permeable inhibitor of Rho-kinase with a Ki value of 1.