GDC-0449—A potent inhibitor of the hedgehog pathway

SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5 mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC.     

Synthesis and evaluation of 2-amino-dihydrotetrabenzine derivatives as probes for imaging vesicular monoamine transporter-2

A novel series of analogs of 2-amino-dihydrotetrabenazine derivatives, 4–6, targeting the vesicular monoamine transporter have been prepared. In vitro binding was carried out in tissue homogenates prepared from rat striatal tissue homogenates with both [¹²⁵I]-iodovinyl-TBZ and [³H]DTBZ. There was a good correlation (r² = 0.925) between the affinities of the different compounds for [¹²⁵I]-iodovinyl-TBZ and [³H]-DTBZ binding. Compound 5 exhibited a better affinity for the vesicular monoamine transporter (K_i = 8.68 ± 1.26 nM and 7.01 ± 0.07 nM, respectively), which may be a good lead compound for further structural modification to develop useful probes for VMAT2.     

Hypoxia potentiates microRNA-mediated gene silencing through posttranslational modification of Argonaute2

Hypoxia contributes to the pathogenesis of various human diseases, including pulmonary artery hypertension (PAH), stroke, myocardial or cerebral infarction, and cancer. For example, acute hypoxia causes selective pulmonary artery (PA) constriction and elevation of pulmonary artery pressure. Chronic hypoxia induces structural and functional changes to the pulmonary vasculature, which resembles the phenotype of human PAH and is commonly used as an animal model of this disease. The mechanisms that lead to hypoxia-induced phenotypic changes have not been fully elucidated. Here, we show that hypoxia increases type I collagen prolyl-4-hydroxylase [C-P4H(I)], which leads to prolyl-hydroxylation and accumulation of Argonaute2 (Ago2), a critical component of the RNA-induced silencing complex (RISC). Hydroxylation of Ago2 is required for the association of Ago2 with heat shock protein 90 (Hsp90), which is necessary for the loading of microRNAs (miRNAs) into the RISC, and translocation to stress granules (SGs). We demonstrate that hydroxylation of Ago2 increases the level of miRNAs and increases the endonuclease activity of Ago2. In summary, this study identifies hypoxia as a mediator of the miRNA-dependent gene silencing pathway through posttranslational modification of Ago2, which might be responsible for cell survival or pathological responses under low oxygen stress.     

The Association between DNA Copy Number Aberrations at Chromosome 5q22 and Gastric Cancer

Background

Gastric cancer is common cancer. Discovering novel genetic biomarkers might help to identify high-risk individuals. Copy number variation (CNV) has recently been shown to influence risk for several cancers. The aim of the present study was sought to test the association between copy number at a variant region and GC.

Methods

A total of 110 gastric cancer patients and 325 healthy volunteers were enrolled in this study. We searched for a CNV and found a CNV (Variation 7468) containing part of the APC gene, the SRP19 gene and the REEP5 gene. We chose four probes targeting at APC-intron8, APC-exon9, SRP19 and REEP5 to interrogate this CNV. Specific Taqman probes labeled by different reporter fluorophores were used in a real-time PCR platform to obtain copy number. Both the original non-integer data and transformed integer data on copy number were used for analyses.

Results

Gastric caner patients had a lower non-integer copy number than controls for the APC-exon9 probe (Adjusted p = 0.026) and SRP19 probe (Adjusted p = 0.002). The analysis of integer copy number yielded a similar pattern although less significant (Adjusted p = 0.07 for APC-exon9 probe and Adjusted p = 0.02 for SRP19 probe).

Conclusions

Losses of a CNV at 5q22, especially in the DNA region surrounding APC-exon 9, may be associated with a higher risk of gastric cancer.     

Elite young soccer players have smaller inter-limb asymmetry and better body composition than non-elite players

Body composition (BC) and inter-limb anthropometric asymmetries (LA) may influence the physical performance of soccer players. This study aimed to determine differences in BC and LA among soccer across four performance levels. The study involved 110 male soccer players participating in Czech senior teams who were grouped into four different performance levels (i.e. G1: national team, G2: 1^st division, G3: 2^nd division, G4: 3^rd division). The following BC and LA parameters were compared among groups: body height, body mass, absolute fat-free mass, relative fat-free mass (FFMrel), percentage of fat mass (FM), total body water (TBW), intracellular water (ICW), extracellular water (ECW), phase angle, and bilateral muscle mass differences in the upper and lower extremities. Significant differences were observed in BC parameters among all groups (λ = 0.06, F75,246 = 5.38, p = 0.01, η_p² = 0.62). High-performance players (i.e. G1, G2) had significantly (p < 0.01) lower FM than lower performance players (i.e. G3, G4). The lowest values of FFMrel, relative TBW, relative ICW and ECW were detected in the lowest-performance players (i.e. G4). Significantly lower bilateral muscle mass differences were detected in G1 players (2.71 ± 1.26%; p < 0.01) compared with G4 players (3.95 ± 1.17%). G1 and G2 players had a higher proportion of muscle mass in the torso (p < 0.01) and upper limbs than G3 and G4 (p < 0.01). Elite and high-performance players have better BC and lower inter-limb anthropometric asymmetries compared with low-performance level players.     

Clinical adoptive chemoimmunotherapy with allogeneic alloactivated HLA-haploidentical lymphocytes: controlled induction of graft-versus-host-reactions

Summary A total of 13 cancer patients were treated with Adoptive Chemoimmunotherapy (ACIT) using alloactivated HLA haploidentical lymphocytes. Donor lymphocytes were activated in vitro using a pool of irradiated allogeneic lymphocytes (MLC-cells) and some further expanded by culturing in T-cell growth factor (TCGF-cells). The first 6 patients received i.v. cyclophosphamide (CPM) followed 24 h later by escalating doses of MLC-cells, then 7 days later they received an infusion of TCGF-cells. Minimal toxicity was seen. The next 7 patients received CPM (800 mg/m²) and a combined MLC and TCGF-cell infusion (total cell dose ranged from 0.79×10¹⁰ to 2.26×10¹⁰). Of these 7 patients, 3 developed mild graft-versus-host reaction (GVHR) which resolved without treatment, and 2 patients had progressive GVHR which was arrested by methylprednisolone (2 mg/kg). Peripheral blood lymphocytes from these 2 patients, during the GVHR, had increased activated T-cells (OKT-10+ and OK-Ia+). In vitro expansion, in TCGF, of these activated T-cells enabled HLA typing to prove they were of donor origin. Only 1 clinical antitumor response was observed in the first 6 patients. The results of this study indicate that this form of ACIT can be given to patients with acceptable toxicity. Self-limited or easily controlled GVHR may be induced and primed donor cells persisting in the circulation are probably responsible. Further testing is required to determine whether the immune response induced by this form of ACIT may be therapeutically effective.This research was supported by American Cancer Society grant CH-237B and NCI-CA 32685     

Restriction fragment length polymorphisms in diploid and allotetraploid Gossypium: assigning the late embryogenesis-abundant (Lea) alloalleles in G. hirsutum

Summary We have determined the copy number of 21 genes in an allotetraploid and several diploid species of cotton by gel and dot blot hybridization with cloned cDNAs. The legumin A, legumin B, and all 18 unique Lea (late embryogenesis-abundant) cDNA sequences isolated from the AD allotetraploid Gossypium hirsutum are present in one copy in A, D, E, and F diploid species and in two copies in G. hirsutum. Gel blot analysis of DNAs digested with EcoRI or BamHI usually detects different sized fragments in A and D diploids. Conservation of these restriction fragment length polymorphisms in G. hirsutum allows most of these fragments to be assigned to their respective subgenomes. Furthermore, both subgenomes in G. hirsutum can be distinguished from those in the interfertile allotetraploid G. barbadense. These results show that physical mapping of both sets of chromosomes in an allotetraploid should be possible by segregation analysis.     

Synthesis of (+)-(R)- and (−)-(S)-trans-8-hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)] aminotetralin: New 5-HT1A receptor ligands

(R,S)-trans-8-Hydroxy-2-[N-n-propyl-N-(3′-iodo-2′-propenyl)amino]tetralin 7 , a new radioiodinated ligand based on 8-OH-DPAT, was reported as a potential ligand for 5-HT_1A receptors. The optically active (+)-(R)- and (?)-(S)- 7 were prepared to investigate the stereoselectivity of (R,S)- 7 . Racemic intermediate 8-methoxy-2-N-n-propyltetralin was reacted with the acyl chloride of (?)-(R)-O-methylmandelic acid to form a mixture of (S,R)- and (R,R)-diastereoisomers, which were separated by flash column chromatography. After removing the N-acyl group from the diastereoisomers, the desired (+)-(R)-or (?)-(S)- 7 was obtained by adding an N-iodopropenyl group. In vitro homogenate binding studies showed the stereoselectivity of this new compound for 5-HT_1A receptors. (+)-(R)- 7 isomer displayed 100-fold higher affinity than the (?)-(S)- 7 isomer. Biochemical study indicated that (+)-(R)- 7 potently inhibited forskolin-stimulated adenylyl cyclase activity in hippocampal membranes (E_max and EC₅₀ were 24.5% and 5.4 nM, respectively), while (?)-(S)- 7 showed no effect at 1 μM. The radioiodinated (+)-(R)- and (?)-(S)-[¹²⁵I] 7 were confirmed by coelution with the resolved unlabeled compound on HPLC (reverse phase column PRP-1, acetonitrile/pH 7.0 buffer, 80/20). The active isomer, (+)-(R)-[¹²⁵I] 7 , displayed high binding affinity to 5-HT_1A receptors (K_d = 0.09 ± 0.02 nM). In contrast, the (?)-(S)- 7 isomer displayed a significantly lower affinity to the 5-HT_1A receptor (K_d > 10 nM). Thus, (+)-(R)-[¹²⁵I]trans-8-OH-PIPAT, (+)-(R)- 7 , an iodinated stereoselective 5-HT_1A receptor agonist, is potentially useful for study of in vivo and in vitro function and pharmacology of 5-HT_1A receptors in the central nervous system. © 1995 Wiley-Liss, Inc.