Synthesis of L-[5-11 C]Leucine and L-α-[5-11 C]Methylleucine via Pd0 -mediated 11 C-Methylation and Microfluidic Hydrogenation: Potentiality of Leucine PET Probes for Tumor Imaging.

The efficient synthesis of L-[5-11 C]leucine and L-α-[5-11 C]methylleucine has been investigated using a continuous two-step sequence of rapid reactions consisting of Pd0 -mediated 11 C-methylation and microfluidic hydrogenation. The synthesis of L-[5-11 C]leucine and L-α-[5-11 C]methylleucine was accomplished within 40 min with a decay-corrected radiochemical yield of 15-38 % based on [11 C]CH3 I, radiochemical purity of 95-99 %, and chemical purity of 95-99 %. The Pd impurities in the injectable solution measured using inductively coupled plasma mass spectrometry met the international criteria for human use. Positron emission tomography scanning after an intravenous injection of L-[5-11 C]leucine or L-α-[5-11 C]methyl leucine in A431 tumor-bearing mice was performed. As a result, L-α-[5-11 C]methylleucine was found to be a potentially useful probe for visualizing the tumor. Tissue distribution analysis showed that the accumulation value of L-α-[5-11 C]methylleucine in tumor tissue was high [12±3% injected dose/g tissue (%ID/g)].

Novel bicyclic pyrazoles as potent ALK2 (R206H) inhibitors for the treatment of fibrodysplasia ossificans progressiva.

Mutant activin receptor-like kinase-2 (ALK2) is associated with the pathogenesis of fibrodysplasia ossificans progressiva, making it an attractive target for therapeutic intervention. We synthesized a new series of bicyclic pyrazoles and evaluated their mutant ALK2 enzyme inhibitory activities, leading to the identification of 8 as the most potent inhibitor. This compound showed moderate microsomal metabolic stability and human ether-a-go-go related gene (hERG) safety. In C2C12 cells carrying mutant ALK2 (R206H), 8 efficiently inhibited the bone morphogenetic protein (BMP)-induced alkaline phosphatase activity.

BMP signaling is a therapeutic target in ovarian cancer.

BMP signaling has been found to have tumor-promoting as well as tumor-suppressing effects in different types of tumors. In this study, we investigated the effects of BMP signaling and of BMP inhibitors on ovarian cancer (OC) cells in vitro and in vivo. High expression of BMP receptor 2 (BMPR2) correlated with poor overall survival of OC patients in the TCGA dataset. Both BMP2 and BMPR2 enhanced OC cell proliferation, whereas BMP receptor kinase inhibitors inhibited OC cell growth in cell culture as well as in a mouse model. BMP2 also augmented sphere formation, migration, and invasion of OC cells, and induced EMT. High BMP2 expression was observed after chemotherapy of OC patients in the GSE109934 dataset. In accordance, carboplatin, used for the treatment of OC patients, increased BMP2 secretion from OC cells, and induced EMT partially via activation of BMP signaling. Our data suggest that BMP signaling has tumor-promoting effects in OC, and that BMP inhibitors might be useful therapeutic agents for OC patients. Considering that carboplatin treatment augmented BMP2 secretion, the possibility to use a combination of BMP inhibitors and carboplatin in the treatment of OC patients, would be worth exploring.

Structural Basis of Activin Receptor-Like Kinase 2 (R206H) Inhibition by Bis-heteroaryl Pyrazole-Based Inhibitors for the Treatment of Fibrodysplasia Ossificans Progressiva Identified by the Integration of Ligand-Based and Structure-Based Drug Design Approaches.

Fibrodysplasia ossificans progressiva (FOP) is a rare but severe genetic disorder in which acute inflammation elicits progressive heterotopic ossification in the muscles, tendons, and ligaments. Classic FOP is caused by the R206H mutation in ALK2/ACVR1. While several activin receptor-like kinase 2 (ALK2) inhibitors were found to be efficacious in animal models of FOP, most of the ALK2 (R206H) inhibitors lacked sufficient oral bioavailability for efficacy. Previously, the synthesis of a series of novel bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitors that achieved both substantial potency and an improved ADMET profile was reported. In the present study, the detailed procedure of the in silico approach employed to identify the initial bis-heteroaryl pyrazole-based ALK2 (R206H) inhibitor RK-59638 and the analysis of the ALK2 (R206H) RK-59638 complex structure to guide the synthetic optimization of the chemical series, obtaining RK-71807 showing improved potency and metabolic stability, were described. According to the initial in silico screening, the screening efficiencies and chemical diversity of the hit compounds of both ligand-based and structure-based methods were evaluated. Then, X-ray structures of ALK2 (R206H) and the inhibitors were analyzed to assess the structure-activity relationships of the synthesized compounds. The 3D-RISM analysis indicated the existence of the additional hydrogen bond via water molecules restricting the attachment point in the pyrazole scaffold. The quantum mechanics calculation of the newly determined ALK2 (R206H) RK-71807 complex structure using a fragment molecular orbital method and pair interaction energy decomposition analysis was employed to evaluate the interaction energies between the inhibitor and each of the amino acid residues and decompose them to electrostatic, exchange-repulsion, and charge transfer energies. The pattern of decomposed interaction energies was then compared to that formed by RK-59638 and LDN-193189 to investigate the structural basis of ALK2 (R206H) inhibition.

Bis-Heteroaryl Pyrazoles: Identification of Orally Bioavailable Inhibitors of Activin Receptor-Like Kinase-2 (R206H).

Mutant activin receptor-like kinase-2 (ALK2) was reported to be closely associated with the pathogenesis of fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG), and therefore presents an attractive target for therapeutic intervention. Through in silico virtual screenings and structure-activity relationship studies assisted by X-ray crystallographic analyses, a novel series of bis-heteroaryl pyrazole was identified as potent inhibitors of ALK2 (R206H). Derived from in silico hit compound RK-59638 (6a), compound 18p was identified as a potent inhibitor of ALK2 (R206H) with good aqueous solubility, liver microsomal stability, and oral bioavailability.

Autocrine BMP-4 Signaling Is a Therapeutic Target in Colorectal Cancer.

Poor prognoses for colorectal cancer patients with metastatic lesions have driven demand for the development of novel targeted therapies. Here, we demonstrate that expression of bone morphogenetic protein 4 (BMP-4) is universally upregulated in human colorectal cancer cells and tissues, resulting in activated BMP signaling. Inhibition of endogenous BMP signaling by the BMP type I receptor inhibitor LDN-193189 elevated expression of the phosphatase DUSP5 in colorectal cancer cells, inducing apoptosis via dephosphorylation of Erk MAPK. Administering LDN-193189 to mice diminished tumor formation of colorectal cancer cells. Our findings suggest inhibition of autocrine BMP-4 as a candidate treatment strategy for colorectal cancer. Cancer Res; 77(15); 4026-38. ©2017 AACR.

Transforming growth factor ß-inhibitor Repsox downregulates collagen expression of scleroderma dermal fibroblasts and prevents bleomycin-induced mice skin fibrosis.

Inhibition of transforming growth factor (TGF)-ß1 signalling may be one of the most reliable approaches to treat skin fibrosis of scleroderma. Although there have been many basic researches of TGF-ß blockade reagents, few of them were proved to have inhibitory effects on fibrosis both in vitro and in vivo. In this study, we randomly chose four commercially available low molecular weight compounds (Repsox, LY2109761, LY364947 and K02288) from TGF-ß1 inhibitor library, and compared their antifibrotic effects in vitro and in vivo. We demonstrated that Repsox has the most potent inhibitory effects on TGF-ß-induced expression of CTGF and collagen of cultured normal dermal fibroblasts in vitro and their constitutive overexpression of scleroderma fibroblast in vitro. In addition, Repsox could attenuate skin fibrosis by bleomycin in vivo, via the downregulation of CTGF or collagen. Our results may facilitate clinical trial of Repsox against fibrotic diseases in future.

The synthesis and biodistribution of [(11)C]metformin as a PET probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1) in vivo.

In order to develop a new positron emission tomography (PET) probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1), (11)C-labelled metformin was synthesized and then evaluated as a PET probe. [(11)C]Metformin ([(11)C]4) was synthesized in three steps, from [(11)C]methyl iodide. Evaluation by small animal PET of [(11)C]4 showed that there was increased concentrations of [(11)C]4 in the livers of mice pre-treated with pyrimethamine, a potential inhibitor of MATEs, inhibiting the hepatobiliary excretion of metformin. Radiometabolite analysis showed that [(11)C]4 was not degraded in vivo during the PET scan. Biodistribution studies were undertaken and the organ distributions were extrapolated into a standard human model. In conclusion, [(11)C]4 may be useful as a PET probe to non-invasively study the in vivo function of hepatobiliary transport and drug-drug interactions, mediated by MATE1 in future clinical investigations.

A pyrrolo-pyrimidine derivative targets human primary AML stem cells in vivo.

Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rg(null) mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

A fluorescent-based high-throughput screening assay for small molecules that inhibit the interaction of MdmX with p53.

A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC(50) values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.

Pathogenic mutation of ALK2 inhibits induced pluripotent stem cell reprogramming and maintenance: mechanisms of reprogramming and strategy for drug identification.

Fibrodysplasia ossificans progressiva (FOP) is a rare congenital disorder characterized by progressive ossification of soft tissues. FOP is caused by mutations in activin receptor-like kinase 2 (ALK2) that cause its constitutive activation and result in dysregulation of BMP signaling. Here, we show that generation of induced pluripotent stem cells (iPSCs) from FOP-derived skin fibroblasts is repressed because of incomplete reprogramming and inhibition of iPSC maintenance. This repression was mostly overcome by specific suppression of ALK2 expression and treatment with an ALK2 inhibitor, indicating that the inhibition of iPSC generation and maintenance observed in FOP-derived skin fibroblasts results from constitutive activation of ALK2. Using this system, we identified an ALK2 inhibitor as a potential candidate for future drug development. This study highlights the potential of the inhibited production and maintenance of iPSCs seen in diseases as a useful phenotype not only for studying the molecular mechanisms underlying iPS reprogramming but also for identifying drug candidates for future therapies.

Whole-body distribution and radiation dosimetry of [11C]telmisartan as a biomarker for hepatic organic anion transporting polypeptide (OATP) 1B3.

INTRODUCTION: Telmisartan, a nonpeptide angiotensin II AT1 receptor antagonist used as an antihypertensive drug, is specifically taken up by the liver through the OATP1B3. PET imaging with [(11)C]telmisartan is expected to provide information about the whole body pharmacokinetics of telmisartan as well as its transport property by OATP1B3. The purpose of the study was to determine the biodistribution and radiation dosimetry of [(11)C]telmisartan in humans. METHODS: Biodistribution of [(11)C]telmisartan was measured in three rats and six healthy male human volunteers. In the rat study, a dynamic emission scan was performed for 90 min. In the human study, dynamic whole-body PET images were acquired after intravenous injection of [(11)C]telmisartan. ROIs were defined for source organs on the PET images to measure time-course of [(11)C]telmisartan uptake as percentage injected dose and the number of disintegration for each organ. Radiation dosimetry was calculated with OLINDA/EXM. RESULTS: In the rat study, most radioactivity was rapidly taken up by the liver and part of it was excreted into the biliary tract and intestine. Extrapolating from the rat data, the effective dose for the adult human being was estimated to be 3.65±0.01 microSv/MBq (n=3). In the human study, most of the tracer was taken up by the liver as well, although not as rapidly as in the rat. The activity in the gall bladder and intestine increased gradually. The effective dose for the adult human being was 4.24±0.09 microSv/MBq (n=6). CONCLUSIONS: [(11)C]Telmisartan is a safe PET tracer with a dosimetry profile comparable to other common (11)C PET tracers.

The involvement of organic anion transporting polypeptide in the hepatic uptake of telmisartan in rats: PET studies with [¹¹C]telmisartan.

Telmisartan, a selective angiotensin II receptor antagonist, is primarily excreted via hepatobiliary transport. The predominant contribution of organic anion transporting polypeptide (OATP) 1B3 in its hepatic uptake of telmisartan has been demonstrated by in vitro transport studies. In the present study, a quantitative positron emission tomography (PET) methodology was developed for in vivo kinetic assessment of hepatobiliary transport of telmisartan. Serial abdominal PET scans were performed in rats following intravenous administration of [(11)C]telmisartan as a radiotracer. PET scans revealed that [(11)C]telmisartan was localized primarily in the liver and some of the radioactivity moved to the intestine, which corresponds to biliary excretion. Radiometabolite analysis by radiometric HPLC showed that [(11)C]telmisartan was converted to its acylglucuronide, which was mainly detected in bile, but little in plasma and liver. Integration plot analysis revealed that [(11)C]telmisartan was taken up into the liver as rapidly as the hepatic blood flow rate, and the radiometabolite was subsequently excreted into the bile. When rifampicin, a typical Oatp inhibitor, was coadministered with [(11)C]telmisartan in rats, hepatic uptake clearance of [(11)C]telmisartan was significantly decreased, whereas biliary efflux clearance was not changed. Coinjection with unlabeled telmisartan (4 and 10 mg/kg) also decreased hepatic uptake clearance of [(11)C]telmisartan. On the other hand, PET imaging analysis revealed a significant increase of biliary efflux when telmisartan dose was increased to more than 4 mg/kg. These results suggested that the hepatic uptake of [(11)C]telmisartan mainly consists of a saturable process mediated by Oatps in rats, according to noninvasive real-time measurement of tissue radioactivity with the use of PET. The present study with rats is expected to provide the feasibility of PET imaging study to quantitatively estimate OATP1B3 function in humans.

Design and pharmacological evaluation of PF-4840154, a non-electrophilic reference agonist of the TrpA1 channel.

TrpA1 is an ion channel involved in nociceptive and inflammatory pain. It is implicated in the detection of chemical irritants through covalent binding to a cysteine-rich intracellular region of the protein. While performing an HTS of the Pfizer chemical collection, a class of pyrimidines emerged as a non-reactive, non-covalently binding family of agonists of the rat and human TrpA1 channel. Given the issues identified with the reference agonist Mustard Oil (MO) in screening, a new, non-covalently binding agonist was optimized and proved to be a superior agent to MO for screening purposes. Compound 16a (PF-4840154) is a potent, selective agonist of the rat and human TrpA1 channel and elicited TrpA1-mediated nocifensive behaviour in mouse.

First automatic radiosynthesis of 11C labeled Telmisartan using a multipurpose synthesizer for clinical research use.

OBJECTIVE: Telmisartan, a nonpeptide angiotensin II AT1 receptor antagonist, is an antihypertensive drug. Positron emission tomography (PET) imaging with [(11)C]Telmisartan is expected to provide information about the whole body pharmacokinetics of telmisartan as well as the transport function of hepatic OATP1B3. We developed a first automatic preparation system of [(11)C]Telmisartan to applicable clinical research using a new (11)C and (18)F multipurpose synthesizer. METHODS: Two milligrams of precursor (1) in 5 µl of 1 M KOH in 0.5 ml of dimethyl sulfoxide was reacted with [(11)C]CH(3)I for 5 min at 120°C. The resultant solution was hydrolyzed with 1 M NaOH at 100°C for 3 min. The neutralization was carried out with acetic acid, followed by purification with high-performance liquid chromatography. The desired radioactive fraction was collected and solvent was replaced by 10 ml saline containing 0.3 ml of EtOH and 0.5 ml of PEG400, and then passed through a sterile 0.22 µm filter (Millex-GV, Millipore) to a pyrogen-free vial as the final product. RESULTS: The yield of [(11)C]Telmisartan for clinical research use was 16.8 ± 2.9% EOB as decay corrected (n = 8, mean ± SD) in 32-36 min. The radiochemical purity of [(11)C]Telmisartan was >97%, and specific activity was higher than 86.3 MBq/nmol. CONCLUSIONS: We succeeded in the first synthesis of [(11)C]Telmisartan for clinical research use by appropriate quality tests.