Magnesium implantation or supplementation ameliorates bone disorder in CFTR-mutant mice through an ATF4-dependent Wnt/ß-catenin signaling.

Magnesium metal and its alloys are being developed as effective orthopedic implants; however, the mechanisms underlying the actions of magnesium on bones remain unclear. Cystic fibrosis, the most common genetic disease in Caucasians caused by the mutation of CFTR, has shown bone disorder as a key clinical manifestation, which currently lacks effective therapeutic options. Here we report that implantation of magnesium-containing implant stimulates bone formation and improves bone fracture healing in CFTR-mutant mice. Wnt/ß-catenin signaling in the bone is enhanced by the magnesium implant, and inhibition of Wnt/ß-catenin by iCRT14 blocks the magnesium implant to improve fracture healing in CFTR-mutant mice. We further demonstrate that magnesium ion enters osteocytes, increases intracellular cAMP level and activates ATF4, a key transcription factor known to regulate Wnt/ß-catenin signaling. In vivo knockdown of ATF4 abolishes the magnesium implant-activated ß-catenin in bones and reverses the improved-fracture healing in CFTR-mutant mice. In addition, oral supplementation of magnesium activates ATF4 and ß-catenin as well as enhances bone volume and density in CFTR-mutant mice. Together, these results show that magnesium implantation or supplementation may serve as a potential anabolic therapy for cystic fibrosis-related bone disease. Activation of ATF4-dependent Wnt/ß-catenin signaling in osteocytes is identified as a previously undefined mechanism underlying the beneficial effect of magnesium on bone formation.

Therapeutic strategies for diffuse midline glioma from high-throughput combination drug screening.

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi-histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.

High-throughput screening identifies candidate drugs for the treatment of recurrent respiratory papillomatosis.

Recurrent respiratory papillomatosis (RRP) is a benign neoplasm of the larynx caused mainly by human papillomavirus type 6 or 11 and its standard treatment involves repeated surgical debulking of the laryngeal tumors. However, significant morbidity and occasional mortality due to multiple recurrences occur. Conditional reprogramming (CR) was used to establish a HPV-6 positive culture from an RRP patient, named GUMC-403. High-throughput screening was performed at the National Center for Advanced Technology (NCATS) to identify potential drugs to treat this rare but morbid disease. GUMC-403 cells were screened against the NPC library of >2800 approved drugs and the MIPE library of >1900 investigational drugs to identify new uses for FDA-approved drugs or drugs that have undergone significant research and development. From the two libraries, we identified a total of 13 drugs that induced significant cytotoxicity in RRP cells at IC50 values that were clinically achievable. We validated the efficacy of the drugs in vitro using CR 2D and 3D models and further refined our list of drugs to panobinostat, dinaciclib and forskolin as potential therapies for RRP patients.

Structural optimization on a virtual screening hit of smoothened receptor.

The Hedgehog (Hh) pathway plays a critical role during embryonic development by controlling cell patterning, growth and migration. In adults, the function of Hh pathway is curtailed to tissue repair and maintenance. Aberrant reactivation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. The Smoothened (Smo) receptor, a key component of the Hh pathway which is central to the signaling transduction, has emerged as an attractive therapeutic target for the treatment of human cancers. Taking advantage of the availability of several crystal structures of Smo in complex with different antagonists, we have previously conducted a molecular docking-based virtual screening to identify several compounds which exhibited significant inhibitory activity against the Hh pathway activation (IC50 < 10 µM) in a Gli-responsive element (GRE) reporter gene assay. The most potent compound (ChemDiv ID C794-1677: 47 nM) showed comparable Hh signaling inhibition to the marketed drug vismodegib (46 nM). Herein, we report our structural optimization based on the virtual screening hit C794-1677. Our efforts are aimed to improve potency, decrease cLogP, and remove potentially metabolic labile/toxic pyrrole and aniline functionalities presented in C794-1677. The optimization led to the identification of numerous potent compounds exemplified by 25 (7.1 nM), which was 7 folds more potent compared with vismodegib. In addition, 25 was much less lipophilic compared with C794-1677 and devoid of the potentially metabolic labile/toxic pyrrole and aniline functional groups. Furthermore, 25 exhibited promising efficacy in inhibiting Gli1 mRNA expression in NIH3T3 cells with either wildtype Smo or D473H Smo mutant. These results represented significant improvement over the virtual screening hit C794-1677 and suggested that compound 25 can be used as a good starting point to support lead optimization.

Discovery of potent and novel smoothened antagonists via structure-based virtual screening and biological assays.

The Hedgehog (Hh) signaling pathway plays a critical role in controlling patterning, growth and cell migration during embryonic development. Aberrant activation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. As a key member of the Hh pathway, the Smoothened (Smo) receptor, a member of the G protein-coupled receptor (GPCR) family, has emerged as an attractive therapeutic target for the treatment and prevention of human cancers. The recent determination of several crystal structures of Smo in complex with different antagonists offers the possibility to perform structure-based virtual screening for discovering potent Smo antagonists with distinct chemical scaffolds. In this study, based on the two Smo crystal complexes with the best capacity to distinguish the known Smo antagonists from decoys, the molecular docking-based virtual screening was conducted to identify promising Smo antagonists from ChemDiv library. A total of 21 structurally novel and diverse compounds were selected for experimental testing, and six of them exhibited significant inhibitory activity against the Hh pathway activation (IC50 < 10 µM) in a GRE (Gli-responsive element) reporter gene assay. Specifically, the most potent compound (compound 20: 47 nM) showed comparable Hh signaling inhibition to vismodegib (46 nM). Compound 20 was further confirmed to be a potent Smo antagonist in a fluorescence based competitive binding assay. Optimization using substructure searching method led to the discovery of 12 analogues of compound 20 with decent Hh pathway inhibition activity, including four compounds with IC50 lower than 1 µM. The important residues uncovered by binding free energy calculation (MM/GBSA) and binding free energy decomposition were highlighted and discussed. These findings suggest that the novel scaffold afforded by compound 20 can be used as a good starting point for further modification/optimization and the clarified interaction patterns may also guide us to find more potent Smo antagonists.

Transcriptomic profiling and quantitative high-throughput (qHTS) drug screening of CDH1 deficient hereditary diffuse gastric cancer (HDGC) cells identify treatment leads for familial gastric cancer.

BACKGROUND: Patients with hereditary diffuse gastric cancer (HDGC), a cancer predisposition syndrome associated with germline mutations of the CDH1 (E-cadherin) gene, have few effective treatment options. Despite marked differences in natural history, histopathology, and genetic profile to patients afflicted by sporadic gastric cancer, patients with HDGC receive, in large, identical systemic regimens. The lack of a robust preclinical in vitro system suitable for effective drug screening has been one of the obstacles to date which has hampered therapeutic advances in this rare disease. METHODS: In order to identify therapeutic leads selective for the HDGC subtype of gastric cancer, we compared gene expression profiles and drug phenotype derived from an oncology library of 1912 compounds between gastric cancer cells established from a patient with metastatic HDGC harboring a c.1380delA CDH1 germline variant and sporadic gastric cancer cells. RESULTS: Unsupervised hierarchical cluster analysis shows select gene expression alterations in c.1380delA CDH1 SB.mhdgc-1 cells compared to a panel of sporadic gastric cancer cell lines with enrichment of ERK1-ERK2 (extracellular signal regulated kinase) and IP3 (inositol trisphosphate)/DAG (diacylglycerol) signaling as the top networks in c.1380delA SB.mhdgc-1 cells. Intracellular phosphatidylinositol intermediaries were increased upon direct measure in c.1380delA CDH1 SB.mhdgc-1 cells. Differential high-throughput drug screening of c.1380delA CDH1 SB.mhdgc-1 versus sporadic gastric cancer cells identified several compound classes with enriched activity in c.1380 CDH1 SB.mhdgc-1 cells including mTOR (Mammalian Target Of Rapamycin), MEK (Mitogen-Activated Protein Kinase), c-Src kinase, FAK (Focal Adhesion Kinase), PKC (Protein Kinase C), or TOPO2 (Topoisomerase II) inhibitors. Upon additional drug response testing, dual PI3K (Phosphatidylinositol 3-Kinase)/mTOR and topoisomerase 2A inhibitors displayed up to >100-fold increased activity in hereditary c.1380delA CDH1 gastric cancer cells inducing apoptosis most effectively in cells with deficient CDH1 function. CONCLUSION: Integrated pharmacological and transcriptomic profiling of hereditary diffuse gastric cancer cells with a loss-of-function c.1380delA CDH1 mutation implies various pharmacological vulnerabilities selective to CDH1-deficient familial gastric cancer cells and suggests novel treatment leads for future preclinical and clinical treatment studies of familial gastric cancer.

Discovery of Novel and Selective Adenosine A2A Receptor Antagonists for Treating Parkinson's Disease through Comparative Structure-Based Virtual Screening.

Among non-dopaminergic strategies for combating Parkinson's disease (PD), antagonism of the A2A adenosine receptor (AR) has emerged to show great potential. In this study, on the basis of two crystal structures of the A2A AR with the best capability to distinguish known antagonists from decoys, docking-based virtual screening (VS) was conducted to identify novel A2A AR antagonists. A total of 63 structurally diverse compounds identified by VS were submitted to experimental testing, and 11 of them exhibited substantial activity against the A2A AR (Ki < 10 µM), including two compounds with Ki below 1 µM (compound 43, 0.42 µM; compound 51, 0.27 µM) and good A2A/A1 selectivity (fold < 0.1). Compounds 43 and 51 demonstrated antagonistic activity according to the results of cAMP measurements (cAMP IC50 = 1.67 and 1.80 µM, respectively) and showed good efficacy in the haloperidol-induced catalepsy (HIC) rat model for PD at doses of up to 30 mg/kg. Further lead optimization based on a substructure searching strategy led to the discovery of compound 84 as an excellent A2A AR antagonist (A2A Ki = 54 nM, A2A/A1 fold < 0.1, cAMP IC50 = 0.3 µM) that exhibited significant improvement in anti-PD efficacy in the HIC rat model.

Antiviral activity of PHA767491 against human herpes simplex virus in vitro and in vivo.

BACKGROUND: Herpes simplex virus (HSV) is a common human pathogen that causes a variety of diseases, including oral-labial, genital lesions and life-threatening encephalitis. The antiviral nucleoside analogues such as acyclovir are currently used in anti-HSV therapies; however, clinical overuse of these drugs has led to the emergence of drug-resistant viral strains. Hence, there is an urgent need to develop new anti-HSV agents. METHODS: To identify novel anti-HSV-1 compounds, we screened the LOPAC small scale library of 1280 bioactive compounds to identify inhibitors of HSV-1-induced necroptosis. Further experiments including western blot analysis, Q-PCR analysis and immunohistochemistry were performed to explore the antiviral mechanism of the compounds. RESULTS: Here, we identified PHA767491 as a new inhibitor of HSV. PHA767491 potently blocked the proliferation of HSV in cells, as well as HSV induced cell death. Further, we found that PHA767491 strongly inhibited HSV infection post viral entry. Moreover, PHA767491 reduced the expression of viral genes required for DNA synthesis including UL30/42 DNA polymerase and UL5/8/52 helicase-primase complex. The essential immediate early (IE) genes such as ICP4 and ICP27 are critical for the expression of the early and late genes. Of note, PHA767491 inhibited the expression of all IE genes of both HSV-1 and HSV-2. Importantly, PHA767491 reduced viral titers in the tissues from the mice infected with HSV-1. Consistently, immunohistochemistry analysis showed that PHA767491 dramatically attenuated expression of viral protein gB in the livers. CONCLUSIONS: Taken together, PHA767491 has potent anti-HSV activity by inhibiting viral replication both in vitro and in mouse model. Thus, PHA767491 could be a promising agent for the development of new anti-HSV therapy.

Identification of a New Series of Potent Adenosine A2A Receptor Antagonists Based on 4-Amino-5-carbonitrile Pyrimidine Template for the Treatment of Parkinson's Disease.

Adenosine receptor A2A antagonists have emerged as potential treatment for Parkinson's disease in the past decade. We have recently reported a series of adenosine receptor antagonists using heterocycles as bioisosteres for a potentially unstable acetamide. These compounds, while showing excellent potency and ligand efficiency, suffered from moderate cytochrome P450 inhibition and high clearance. Here we report a new series of adenosine receptor A2A antagonists based on a 4-amino-5-carbonitrile pyrimidine template. Compounds from this new template exhibit excellent potency and ligand efficiency with low cytochrome P450 inhibition. Although the clearance remains moderate to high, the leading compound, when dosed orally as low as 3 mg/kg, demonstrated excellent efficacy in the haloperidol induced catalepsy rat model for Parkinson's disease.

Cryptotanshinone targets tumor-initiating cells through down-regulation of stemness genes expression.

Recent evidence indicates that tumor-initiating cells (TICs), also called cancer stem cells (CSCs), are responsible for tumor initiation and progression, therefore representing an important cell population that may be used as a target for the development of future anticancer therapies. In the present study, Cryptotanshinone (CT), a traditional Chinese herbal medicine, was demonstrated to regulate the behaviors of LNCaP prostate cells and prostate LNCaP TICs. The results demonstrate that treatment with CT alters cellular proliferation, cell cycle status, migration, viability, colony formation and notably, sphere formation and down-regulation of stemness genes (Nanog, OCT4, SOX2, ß-catenin, CXCR4) in TICs. The present study demonstrates that CT targets the LNCaP CD44+CD24- population that is representative of prostate TICs and also affects total LNCaP cells as well via down-regulation of stemness genes. The strong effect with which CT has on prostate TICs suggests that CT may potentially function as a novel natural anticancer agent that specifically targets TICs.

Augmented efficacy of brentuximab vedotin combined with ruxolitinib and/or Navitoclax in a murine model of human Hodgkin's lymphoma.

Despite relative success of therapy for Hodgkin's lymphoma (HL), novel therapeutic agents are needed for patients with refractory or relapsed disease. Recently, anti-PD1 immunotherapy or treatment with the anti-CD30 toxin conjugate brentuximab vedotin (BV) have been associated with remissions; however, the median responses of complete responses (CRs) with the latter were only 6.7 mo. To obtain curative therapy, other effective agents, based on HL biology, would have to be given in combination with BV. Hodgkin's Reed-Sternberg (HRS) cells secrete cytokines including IL-6 and -13, leading to constitutive activation of JAK/STAT signaling. In the present study the JAK1/2 inhibitor ruxolitinib reduced phosphorylation of STAT3 and STAT6 and expression of c-Myc in the HL cell line HDLM-2. These changes were enhanced when, on the basis of a matrix screen of drug combinations, ruxolitinib was combined with the Bcl-2/Bcl-xL inhibitor Navitoclax. The combination augmented expression of Bik, Puma, and Bax, and attenuated Bcl-xL expression and the phosphorylation of Bad. The use of the two-agent combination of either ruxolitinib or Navitoclax with BV or the three-agent combination strongly activated Bax and increased activities of cytochrome c and caspase-9 and -3 that, in turn, led to cleavage of poly(ADP ribose) polymerase and Mcl-1. Either ruxolitinib combined with Navitoclax or BV alone prolonged survival but did not cure HDLM-2 tumor-bearing mice, whereas BV combined with ruxolitinib and/or with Navitoclax resulted in a sustained, complete elimination of the HDLM-2 HL. These studies provide scientific support for a clinical trial to evaluate BV combined with ruxolitinib in select patients with HL.

Anti-inflammatory effects of glaucocalyxin B in microglia cells.

Over-activated microglia is involved in various kinds of neurodegenerative process including Parkinson, Alzheimer and HIV dementia. Suppression of microglial over activation has emerged as a novel strategy for treatment of neuroinflammation-based neurodegeneration. In the current study, anti-inflammatory and neuroprotective effects of the ent-kauranoid diterpenoids, which were isolated from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, were investigated in cultured microglia cells. Glaucocalyxin B (GLB), one of five ent-kauranoid diterpenoids, significantly decreased the generation of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in the lipopolysaccharide (LPS)-activated microglia cells. In addition, GLB inhibited activation of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS) in LPS-activated microglia cells. Furthermore, GLB strongly induced the expression of heme oxygenase (HO)-1 in BV-2 microglia cells. Finally, GLB exhibited neuroprotective effect by preventing over-activated microglia induced neurotoxicity in a microglia/neuron co-culture model. Taken together, the present study demonstrated that the GLB possesses anti-nueroinflammatory activity, and might serve as a potential therapeutic agent for treating neuroinflammatory diseases.

[Effect of fertilization levels on soil microorganism amount and soil enzyme activities].

Field experiments were conducted in Shangluo pharmaceutical base in Shaanxi province to study the effect of nitrogen, phosphorus and potassium in different fertilization levels on Platycodon grandiflorum soil microorganism and activities of soil enzyme, using three-factor D-saturation optimal design with random block design. The results showed that N0P2K2, N2P2K0, N3P1K3 and N3P3K1 increased the amount of bacteria in 0-20 cm of soil compared with N0P0K0 by 144.34%, 39.25%, 37.17%, 53.58%, respectively. The amount of bacteria in 2040 cm of soil of N3P1K3 increased by 163.77%, N0P0K3 increased the amount of soil actinomycetes significantly by 192.11%, while other treatments had no significant effect. N2P0K2 and N3P1K3 increased the amounts of fungus significantly in 0-20 cm of soil compared with N0P0K0, increased by 35.27% and 92.21%, respectively. N3P0K0 increased the amounts of fungus significantly in 20-40 cm of soil by 165.35%, while other treatments had no significant effect. All treatments decrease soil catalase activity significantly in 0-20 cm of soil except for N2P0K2, and while N2P2K0 and NPK increased catalase activity significantly in 2040 cm of soil. Fertilization regime increased invertase activity significantly in 2040 cm of soil, and decreased phosphatase activity inordinately in 0-20 cm of soil, while increased phosphatase activity in 2040 cm of soil other than N1P3K3. N3P0K0, N0P0K3, N2P0K2, N2P2K0 and NPK increased soil urease activity significantly in 0-20 cm of soil compared with N0P0K0 by 18.22%, 14.87%,17.84%, 27.88%, 24.54%, respectively. Fertilization regime increased soil urease activity significantly in 2040 cm of soil other than N0P2K2.

[Responses of Scutellaria baicalensis Georgi yield and root baicalin content to the fertilization rates of nitrogen, phosphorus, and potassium].

A field experiment with three-factor (N, P, and K) and quadratic saturation D-optimal design was conducted to evaluate the effects of the fertilization rates of N, P, and K on the yield and root baicalin content of Scutellaria baicalensis Georgi at harvesting time. A ternary quadratic polynomial mathematical model was built, in which, the N, P, and K fertilization rates were independent variables, and the yield and root baicalin content were the target functions. Through the analysis of the model, the optimum fertilization pattern was obtained. The results showed that the fertilization rates of N and P had significant effects on the yield of S. baicalensis. Under low fertilization level, the yield increased with increasing N and P fertilization rates; after exceeding definite fertilization range, no obvious effects were observed. N, P, and K fertilization all had significant effects on the root baicalin content. With increasing fertilization rates of N and K, the root baicalin content rates decreased after an initial increase. Under low fertilization level, the root baicalin content increased with increasing P fertilization rate first, and kept stable then. There existed interactive effects between the fertilization rates of N and P, N and K, and P and K on the yield and root baicalin content of S. baicalensis. Under our experimental conditions, the optimum fertilization model for obtaining over 4000 kg x hm(-2) of S. baicalensis yield and > 14% of root baicalin content was 90.5-104.7 kg x hm(-2) of N, 163.9-199.9 kg x hm(-2) of P2O5, and 84.1-140.8 kg x hm(-2) of K2O, with an N:P2O5:K2O ratio of approximately 1:1.86:1.15.

Walnut-like In2S3 microspheres: ionic liquid-assisted solvothermal synthesis, characterization and formation mechanism.

Walnut-like In(2)S(3) microspheres were synthesized through an ionic liquid-assisted solvothermal method for the first time. The crystal structure and morphology of the as-synthesized products were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray photoelectron spectroscopy (XPS), UV-vis diffuse reflectance spectra (DRS) and nitrogen adsorption-desorption measurement. It was found that the additional amount of ionic liquid, solvothermal temperature and time played crucial roles in controlling the structure and morphology of the In(2)S(3) microspheres. A possible formation mechanism of the walnut-like In(2)S(3) microsphere was proposed on the basis of the experimental results.

[Effect of Astragalus membranaceus var. mongholicus seed extracts on seed germination and seedling growth of different Codonopsis pilosula caltiver].

OBJECTIVE: To reveal the allelopathy effect of Astragalus membranaceus var. mongholicus seeds and provide information for the intercrop production. METHOD: The A. membranaceus. var. mongholicus seeds were soaked in distilled water for different time (12, 24, 36, 48, 60 h) , and then the seed extracts were used to study their effects on the seed germination, seedling growth and development of two Codonopsis pilosula. RESULT: The A. membranaceus var. mongholicus seeds contained some allelopathy compounds. Their soaked liquid had significantly influence on the seed germination and seedling growth of C. pilosula. The seed germination rate, germination power, germination index and vigor index of two C. pilosula calrivar were improved and then inhabited with soaking time elongation. The extract soaking for 24 h significantly improved the germination traits but the extract for 60 h appeared different degrees of inhibiting vigor. The seed extracts soaking ranging between 12 and 60 h all significantly improved the above plant growth of C. pilosula but significant inhibited their radicle growth in length. And with the soaking time elongation the facilitation effect weakened and the inhibiting effect enhanced, especially more significant in the C. pilosula caltivar (Baitiaodangshen). CONCLUSION: The A. membranaceus var. mongholicus seeds have allelopathic compounds and the endogenous inhibitor can be extracted when soaked for more than 24 h in water with intact seeds, resulting in improvement of seed germination rate. The C. pilosula could be intercropped in A. membranaceus var. mongholicus field, however, when intercroped it should notice that the intercrop proportion should vary with the caltivar.

Lead optimization of 4-acetylamino-2-(3,5-dimethylpyrazol-1-yl)-6-pyridylpyrimidines as A2A adenosine receptor antagonists for the treatment of Parkinson's disease.

4-Acetylamino-2-(3,5-dimethylpyrazol-1-yl)-pyrimidines bearing substituted pyridyl groups as C-6 substituents were prepared as selective adenosine hA2A receptor antagonists for the treatment of Parkinson's disease. The 5-methoxy-3-pyridyl derivative 6g (hA2A Ki 2.3 nM, hA1 Ki 190 nM) was orally active at 3 mg/kg in a rat HIC model but exposure was poor in nonrodent species, presumably due to poor aqueous solubility. Follow-on compound 16a (hA2A Ki 0.83 nM, hA1 Ki 130 nM), bearing a 6-(morpholin-4-yl)-2-pyridyl substituent at C-6, had improved solubility and was orally efficacious (3 mg/kg, HIC) but showed time-dependent cytochrome P450 3A4 inhibition, possibly related to morpholine ring metabolism. Compound 16j (hA2A Ki 0.44 nM, hA1 Ki 80 nM), bearing a 6-(4-methoxypiperidin-1-yl)-2-pyridyl substituent at C-6, was sparingly soluble but had good oral exposure in rodent and nonrodent species, had no cytochrome P450 or human ether-a-go-go related gene channel issues, and was orally efficacious at 1 mg/kg in HIC and at 3 mg/kg for potentiation of l-dopa-induced contralateral rotations in 6-hydroxydopamine-lesioned rats.

2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 1. Structure-activity relationships and optimization of heterocyclic substituents.

Previously we have described a novel series of potent and selective A 2A receptor antagonists (e.g., 1) with excellent aqueous solubility. While these compounds are efficacious A 2A antagonists in vivo, the presence of an unsubstituted furyl moiety was a cause of some concern. In order to avoid the potential metabolic liabilities that could arise from an unsubstituted furyl moiety, an optimization effort was undertaken with the aim of replacing the unsubstituted furan with a more metabolically stable group while maintaining potency and selectivity. Herein, we describe the synthesis and SAR of a range of novel heterocyclic systems and the successful identification of a replacement for the unsubstituted furan moiety with a methylfuran or thiazole moiety while maintaining potency and selectivity.

2-Amino-N-pyrimidin-4-ylacetamides as A2A receptor antagonists: 2. Reduction of hERG activity, observed species selectivity, and structure-activity relationships.

Previously we have described a series of novel A 2A receptor antagonists with excellent water solubility. As described in the accompanying paper, the antagonists were first optimized to remove an unsubstituted furyl moiety, with the aim of avoiding the potential metabolic liabilities that can arise from the presence of an unsubstituted furan. This effort identified a series of potent and selective methylfuryl derivatives. Herein, we describe the further optimization of this series to increase potency, maintain selectivity for the human A 2A vs the human A 1 receptor, and minimize activity against the hERG channel. In addition, the observed structure-activity relationships against both the human and the rat A 2A receptor are reported.

Nuclear receptors as negative modulators of STAT3 in multiple myeloma.

Multiple myeloma (MM) remains largely incurable despite conventional and high-dose therapies. Therefore, novel biologically based treatment approaches are urgently required. Particularly, STAT3 activated by IL-6 has a key role in preventing apoptosis and stimulating growth of multiple myeloma cells. Nuclear receptors, a distinct class of ligand-activated transcriptional factors, can interact and modify the function of transcriptional factors intrinsic to the cytokine signal transduction pathways. We have investigated regulation of two nuclear receptors, peroxisome proliferator-activated receptor gamma (PPARgamma) and estrogen receptor (ER), and their crosstalk with STAT3 in multiple myeloma. These results indicate that ligand-activated nuclear receptors can function as negative modulators of STAT3 through direct mechanisms, or in turn, by facilitating coregulators such as PIAS or SMRT. Therefore, different classes of nuclear receptors affect suppression of STAT3 functions through diverse mechanisms resulting in downregulating IL-6-mediated cell growth and gene expression. Given the importance of IL-6 in multiple myeloma, the estrogen receptor-STAT3 or PPARgamma-STAT3 interaction may have significant therapeutic implications in multiple myeloma.