Identification and characterization of a phenyl-thiazolyl-benzoic acid derivative as a novel RAR/RXR agonist.

OBJECTIVE: To identify an agonist of RXRα and RARα with reduced undesired profiles of all-trans retinoic acid for differentiation-inducing therapy of acute promyelocytic leukemia (APL), such as its susceptibility to P450 enzyme, induction of P450 enzyme, increased sequestration by cellular retinoic acid binding protein and increased expression of P-glycoprotein, a virtual screening was performed. RESULTS AND CONCLUSION: In this study, a phenyl-thiazolyl-benzoic acid derivative (PTB) was identified as a potent agonist of RXRα and RARα. PTB was characterized in nuclear receptor binding, reporter gene, cell differentiation and cell growth assays. PTB bound directly to RXRα and RARα, but not to PPARα, δ(ß) or γ. PTB fully activated reporter genes with enhancer elements for RXRα/RXRα, and partially activated reporter genes with enhancer elements for RARα/RXRα, PPARδ(ß) and PPARγ. Furthermore, PTB induced differentiation and inhibited the growth of human APL cells. Thus, PTB is a novel dual agonist of RXRα and RARα and works as both a differentiation inducer and a proliferation inhibitor to leukemic cells.

Blood Pressure-Associated Genetic Variants in the Natriuretic Peptide Receptor 1 Gene Modulate Guanylate Cyclase Activity.

BACKGROUND: Human genetic variation in the NPR1 (natriuretic peptide receptor 1 gene, encoding NPR-A, atrial natriuretic peptide receptor 1) was recently shown to affect blood pressure (BP). NPR-A catalyzes the intracellular conversion of guanosine triphosphate to cGMP (cyclic 3',5'-guanosine monophosphate) on binding of ANP, BNP (atrial or brain natriuretic peptide). Increased levels of cGMP decrease BP by inducing natriuresis, diuresis, and vasodilation. METHODS: We performed a meta-analysis of low-frequency and rare NPR1 variants for BP association in up to 491 584 unrelated individuals. To examine whether the identified BP-associated variants affect NPR-A function, the cGMP response to ANP and BNP was measured in cells expressing wild-type NPR1 and cells expressing the NPR1 variants. RESULTS: In this study, we identified BP associations of 3 amino acid altering variants of NPR1. The minor alleles of rs35479618 (p.E967K, gnomAD non-Finnish European allele frequency 0.017) and rs116245325 (p.L1034F, allele frequency 0.0007) were associated with higher BP (P=4.0×10-25 and P=9.9×10-8, respectively), while the minor allele of rs61757359 (p.G541S, allele frequency 0.003) was associated with lower BP (P=1.8×10-9). Cells transiently expressing 967K or 1034F NPR-A displayed decreased cGMP production in response to ANP and BNP (all P<10-6), while cells expressing 541S NPR-A produced more cGMP compared with cells expressing wild-type NPR-A (P≤4.13×10-5 for ANP and P≤4.24×10-3 for BNP). CONCLUSIONS: In summary, the loss or gain of guanylate cyclase activity for these NPR1 allelic variants could explain the higher or lower BP observed for carriers in large population-based studies.

Pharmacological profiling of a dual FAK/IGF-1R kinase inhibitor TAE226 in cellular and in vivo tumor models.

OBJECTIVE: A dual inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor 1 receptor (IGF-1R), TAE226, was evaluated in a panel of cancer cell lines, MIA PaCa-2 human pancreatic tumor and 4T1 murine breast tumor models. The profiling data were generated during the drug discovery research prior to the first publication of TAE226 appeared in 2007 (Liu et al. in Mol Cancer Ther 6:1357-1367, 2007; Shi et al. in Mol Carcinog 46(6):488-496, 2007; Halder et al. in Cancer Res 67(22):10976-10983, 2007). RESULTS: In a panel of 37 cancer cell lines, TAE226 showed a mean GI50 value of 0.76 µmol/L. In the MIA PaCa-2 model, TAE226 inhibited phosphorylation of Y397-FAK and phosphorylation of S473-Akt as IGF-1R signaling in the cell culture in vitro and the tumor in mice. Oral administration of TAE226 induced tumor stasis at 30 mg/kg and tumor regression at 100 mg/kg in the subcutaneous tumor, and inhibited the orthotopic tumor growth in a dose-dependent manner. Similarly in the 4T1 model, TAE226 inhibited phosphorylation of Y397-FAK and S473-Akt in the cell culture in vitro and the tumor in mice. Oral administration of TAE226 inhibited the orthotopic tumor growth and metastasis to the lung in a dose-dependent manner. Thus, TAE226 represents a novel class of selective and small molecule kinase inhibitor with a potent in vivo activity.

Discovery of a small molecule RXFP3/4 agonist that increases food intake in rats upon acute central administration.

The relaxin family peptide receptors have been implicated in numerous physiological processes including energy homeostasis, cardiac function, wound healing, and reproductive function. Two family members, RXFP3 and RXFP4, are class A GPCRs with endogenous peptide ligands (relaxin-3 and insulin-like peptide 5 (INSL5), respectively). Polymorphisms in relaxin-3 and RXFP3 have been associated with obesity, diabetes, and hypercholesterolemia. Moreover, central administration of relaxin-3 in rats has been shown to increase food intake, leading to body weight gain. Reported RXFP3 and RXFP4 ligands have been restricted to peptides (both endogenous and synthetic) as well as a low molecular weight positive allosteric modulator requiring a non-endogenous orthosteric ligand. Described here is the discovery of the first potent low molecular weight dual agonists of RXFP3/4. The scaffold identified is competitive with a chimeric relaxin-3/INSL5 peptide for RXFP3 binding, elicits similar downstream signaling as relaxin-3, and increases food intake in rats following acute central administration. This is the first report of small molecule RXFP3/4 agonism.

Discovery of 1-((6-Aminopyridin-3-yl)Methyl)-3-(4-Bromophenyl)Urea as a Potent, Irreversible Myeloperoxidase Inhibitor.

Myeloperoxidase (MPO) is a leukocyte-derived redox enzyme that has been linked to oxidative stress and damage in many inflammatory states, including cardiovascular disease. We have discovered aminopyridines that are potent mechanism-based inhibitors of MPO, with significant selectivity over the closely related thyroid peroxidase. 1-((6-Aminopyridin-3-yl)methyl)-3-(4-bromophenyl)urea (Aminopyridine 2) inhibited MPO in human plasma and blocked MPO-dependent vasomotor dysfunction ex vivo in rat aortic rings. Aminopyridine 2 also showed high oral bioavailability and inhibited MPO activity in vivo in a mouse model of peritonitis. Aminopyridine 2 could effectively be administered as a food admixture, making it an important tool for assessing the relative importance of MPO in preclinical models of chronic inflammatory disease.

In vitro and in vivo pharmacological characterization of the main metabolites of nalfurafine hydrochloride.

Pharmacological characterization of the main metabolites of nalfurafine hydrochloride ((E)-N-[17-(cyclopropylmethyl)-4,5α-epoxy-3,14-dihydroxymorphinan-6ß-yl]-3-(furan-3-yl)-N-methylprop-2-enamide monohydrochloride; a selective κ-opioid receptor agonist and an antipruritic for uremic pruritus in hemodialysis patients in Japan) such as 17-decyclopropylmethylated nalfurafine (de-CPM), 3-glucuronide of nalfurafine (NFA-G) and 3-glucuronide of 17-decyclopropylmethylated nalfurafine (de-CPM-G) was performed in vitro (human opioid receptor radioligand binding assay and forskolin-stimulated cyclic adenosine monophosphate (cAMP) assay) and in vivo (substance P-induced scratching behavior in mice). These main metabolites of nalfurafine showed the low affinities for human κ-, µ- and δ-opioid receptors except for the affinity of de-CPM to κ-opioid receptor (inhibition constant (Ki) values: 5.95nmol/l), which was 24 times lower than that of nalfurafine. Moreover, the main metabolites of nalfurafine had much lower agonistic activities than that of nalfurafine for three opioid receptors in forskolin-stimulated cAMP assays. In the substance P-induced mouse scratching behavior, the subcutaneous administration of each metabolite did not statistically significantly reduce the scratching behavior at doses up to 1000µg/kg which was 100 times higher than the effective dose of nalfurafine. These findings suggest that the main metabolites of nalfurafine do not make any contribution to its pharmacological actions including antipruritic effects in vivo.

Silencing of focal adhesion kinase by tumor direct injection of small interfering RNA decreases in vivo tumor growth.

Focal adhesion kinase (FAK) is shown to be frequently correlated with malignancy of the tumor and poor prognosis of the diseases.Because FAK resides immediately downstream of the interaction of cell surface adhesion molecules and extracellular matricies, it is considered to be critical to regulate several cellular processes including growth, differentiation, adhesion, motility and apoptosis. However, the studies on the role of FAK related to cell proliferation have been limited even in vitro. Here, in order to validate the role of FAK in in vivo tumor formation and proliferation, we employed direct intratumoral injection of short hairpin RNA (shRNA) targeting FAK with cationic liposome. Using shRNAs targeting FAK selected from the constructed shRNA library for FAK and by optimization of in vivo delivery conditions, we demonstrated different patterns of the association of FAK inhibition with in vivo tumor formation/proliferation inhibition in two models, PC3M heterotopic xenograft and 4T1 orthotopic syngraft models. These observations indicated that the roles of FAK in tumorigenesis are different among the tumor species. In addition, we showed that ERK is the critical MAP kinase in the signaling pathway down stream of FAK in in vivo proliferation of 4T1 tumor cells.

Tumor growth inhibition by synthetic and expressed siRNA targeting focal adhesion kinase.

Focal adhesion kinase (FAK) was first identified as a viral Src substrate, and substantial experimental data have significantly correlated the elevated FAK expression in human tumor cells with an increased cell adhesion and invasion potential. However, studies investigating the role of FAK in cell proliferation have been limited. Recently, a technique known as RNA interference (RNAi) was successfully adapted to mammalian cells to decrease specifically the expression of targeted cellular genes. In this study, we investigated the role of FAK in cell proliferation, adhesion, and migration by using small interfering RNA (siRNA) technique. Firstly, we constructed a plasmid library expressing short hairpin RNAs (shRNAs) targeting FAK and selected clones substantially suppressing FAK expression in HeLa and HT1080 cells. We then studied the function of FAK in the highly invasive human prostate cancer cell line, PC3M, and mouse breast cancer cell line 4T1, by using selected shRNA clones (#40 and #42) and siRNAs chemically synthesized following the target sequences of #40 and #42. We demonstrated that the decrease of FAK protein expression by treatment with shRNA/siRNA targeting FAK inhibited cell adhesion on a fibronectin/laminin-coated plate, cell migration in a haptotactic migration assay, and cell proliferation in vitro. Furthermore, it suppressed tumor growth in vivo in heterotopic/orthotopic mice models. These results support our hypothesis that FAK plays a crucial role in tumor formation and growth in vivo by regulation of cell adhesion and proliferation by FAK-dependent signals.

[Effect of TRK-820, a selective kappa opioid receptor agonist, on scratching behavior in an animal model of atopic dermatitis].

In atopic dermatitis patients, pruritus is a severe symptom that is difficult to treat. It is previously reported that TRK-820, a kappa-opioid receptor agonist, reduces murine scratching behavior induced by an intradermal injection of histamine or substance P or an intracisternal injection of morphine. It is also reported that TRK-820 ameliorates the intractable pruritus in hemodialysis patients. However, it is still unclear whether TRK-820 possesses antipruritic effects on the pruritus in dermatitis patients. Therefore, the effect of TRK-820 on scratching behavior in NC/Nga mice maintained in a conventional environment, an animal model of atopic dermatitis, was examined. Oral TRK-820 (10-100 microg/kg) inhibited the scratching behavior but did not affect the locomotor activity. On the other hand, ketotifen (3-30 mg/kg, po), an antihistamine, did not attenuate the scratching behavior. TRK-820 showed the highest selectivity and activity for kappa-opioid receptor among all human opioid receptors. Release of various inflammatory mediators from a variety of cells and activity of nitric oxide synthase were not altered by TRK-820. This compound showed much lower affinities for other receptors than that for opioid receptors. These results suggest that TRK-820 is effective against antihistamine-resistant pruritus in atopic dermatitis patients via the kappa opioid receptor.

Therapeutic efficacy of a novel focal adhesion kinase inhibitor TAE226 in ovarian carcinoma.

Focal adhesion kinase (FAK) overexpression is frequently found in ovarian and other cancers and is predictive of poor clinical outcome. In the current study, we characterized the biological and therapeutic effects of a novel FAK inhibitor, TAE226. Taxane-sensitive (SKOV3ip1 and HeyA8) and taxane-resistant (HeyA8-MDR) cell lines were used for in vitro and in vivo therapy experiments using TAE226 alone and in combination with docetaxel. Assessment of cytotoxicity, cell proliferation [proliferating cell nuclear antigen (PCNA)], angiogenesis (CD31), and apoptosis (terminal nucleotidyl transferase-mediated nick end labeling) were done by immunohistochemistry and immunofluorescence. In vitro, TAE226 inhibited the phosphorylation of FAK at both Y397 and Y861 sites, inhibited cell growth in a time- and dose-dependent manner, and enhanced docetaxel-mediated growth inhibition by 10- and 20-fold in the taxane-sensitive and taxane-resistant cell lines, respectively. In vivo, FAK inhibition by TAE226 significantly reduced tumor burden in the HeyA8, SKOV3ip1, and HeyA8-MDR models (46-64%) compared with vehicle-treated controls. However, the greatest efficacy was observed with concomitant administration of TAE226 and docetaxel in all three models (85-97% reduction, all P values <0.01). In addition, TAE226 alone and in combination with chemotherapy significantly prolonged survival in tumor-bearing mice. Even in larger tumors, combination therapy with TAE226 and docetaxel resulted in tumor regression. The therapeutic efficacy was related to reduced pericyte coverage, induction of apoptosis of tumor-associated endothelial cells, and reduced microvessel density and tumor cell proliferation. The novel FAK inhibitor, TAE226, offers an attractive therapeutic approach in ovarian carcinoma.

Inhibition of both focal adhesion kinase and insulin-like growth factor-I receptor kinase suppresses glioma proliferation in vitro and in vivo.

Multiple genetic aberrations in human gliomas contribute to their highly infiltrative and rapid growth characteristics. Focal adhesion kinase (FAK) regulates tumor migration and invasion. Insulin-like growth factor-I receptor (IGF-IR), whose expression correlates with tumor grade, is involved in proliferation and survival. We hypothesized that inhibiting the phosphorylation of FAK and IGF-IR by NVP-TAE226 (hereafter called TAE226), a novel dual tyrosine kinase inhibitor of FAK and IGF-IR, would suppress the growth and invasion of glioma cells. In culture, TAE226 inhibited extracellular matrix-induced autophosphorylation of FAK (Tyr(397)). TAE226 also inhibited IGF-I-induced phosphorylation of IGF-IR and activity of its downstream target genes such as MAPK and Akt. TAE226 retarded tumor cell growth as assessed by a cell viability assay and attenuated G(2)-M cell cycle progression associated with a decrease in cyclin B1 and phosphorylated cdc2 (Tyr(15)) protein expression. TAE226 treatment inhibited tumor cell invasion by at least 50% compared with the control in an in vitro Matrigel invasion assay. Interestingly, TAE226 treatment of tumor cells containing wild-type p53 mainly exhibited G(2)-M arrest, whereas tumor cells bearing mutant p53 underwent apoptosis. Induction of apoptosis by TAE226 was substantiated by detection of caspase-3/7 activation and poly(ADP-ribose) polymerase cleavage and by an Annexin V apoptosis assay. More importantly, TAE226 treatment significantly increased the survival rate of animals in an intracranial glioma xenograft model. Collectively, these data show that blocking the signaling pathways of FAK and IGF-IR with TAE226 has the potential to be an efficacious treatment for human gliomas.

Design and synthesis of 7H-pyrrolo[2,3-d]pyrimidines as focal adhesion kinase inhibitors. Part 2.

A series of 2-amino-9-aryl-7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized as focal adhesion kinase (FAK) inhibitors using molecular modeling in conjunction with a co-crystal structure. Chemistry was developed to introduce functionality onto the 9-aryl ring, which resulted in the identification of potent FAK inhibitors. In particular, compound 32 possessed single-digit nanomolar IC(50) and represents one of the most potent FAK inhibitors discovered to date.

Involvement of central mu-opioid system in the scratching behavior in mice, and the suppression of it by the activation of kappa-opioid system.

The role of central mu- and kappa-opioid receptors in the regulation of itch sensation was examined using pruritogen-induced mouse scratching behavior model. Intracerebroventricular administration of beta-funaltrexamine, a selective mu-opioid receptor antagonist, inhibited the scratching behavior induced by intradermal substance P, but subcutaneous administration of beta-funaltrexamine did not. Similarly, the scratching inhibitory activity of subcutaneously administered TRK-820, (-)-17-(cyclopropylmethyl)-3, 14beta-dihydroxy-4, 5alpha-epoxy-6beta-[N-methyl-trans-3-(3-furyl) acrylamido] morphinan hydrochloride, a kappa-opioid receptor agonist, was antagonized by intracerebroventricular administration of nor-binaltorphimine (10 microg/site), a kappa-opioid receptor antagonist, but was not by subcutaneous administration of nor-binaltorphimine. In addition, the scratching induced by the direct activation of central mu-opioid receptor by intracisternal morphine was significantly and dose-dependently inhibited by subcutaneous administration of TRK-820. Taken all together, it is suggested that the central mu-opioid receptors play a role in the processing of itch sensation, and the activation of central kappa-opioid receptors antagonize the central mu-opioid receptor mediated itch processing, thereby suppressing itch sensation.

Antipruritic activity of the kappa-opioid receptor agonist, TRK-820.

The effects of the kappa-opioid receptor agonist, TRK-820, (-)-17-(cyclopropylmethyl)-3, 14beta-dihydroxy-4, 5alpha-epoxy-6beta-[N-methyl-trans-3-(3-furyl) acrylamido] morphinan hydrochloride, on the itch sensation were compared with those of histamine H1 receptor antagonists, using the mouse pruritogen-induced scratching model. Peroral administration of TRK-820 reduced the numbers of substance P- or histamine-induced scratches dose dependently. No obvious suppression of the spontaneous locomotor activity was observed at the doses used for the experiments, indicating that the inhibition of scratches was not due to the effect on general behavior. Furthermore, the scratching inhibitory activity of TRK-820 was dose dependently antagonized by the specific kappa-opioid receptor antagonist, nor-binaltorphimine, suggesting that the inhibitory activity was mediated via kappa-opioid receptors. Histamine H1 receptor antagonists, chlorpheniramine and ketotifen, did not inhibit substance P-induced scratches, or did so only partially. Both antihistamines inhibited the histamine-induced scratches completely. These results suggest that TRK-820 has antipruritic activity which is mediated by kappa-opioid receptors, and is effective in both antihistamine-sensitive and -resistant pruritus.