Potent quinoxaline-based inhibitors of PDGF receptor tyrosine kinase activity. Part 2: the synthesis and biological activities of RPR127963 an orally bioavailable inhibitor

RPR127963 demonstrates an excellent pharmacokinetic profile in several species and was found to be efficacious in the prevention of restenosis in a Yucatan mini-pig model upon oral administration of 1-5 mg/kg. The in vitro selectivity profile and SAR of the highly optimized PDGF-R tyrosine kinase inhibitor are highlighted.     

Comparison of micronuclei frequencies in mono-, bi- and poly-nucleated lymphocytes from subjects of a residential suburb and subjects living near a metallurgical plant

Spontaneous baseline frequencies of micronuclei in mono-, bi- and poly-nucleated lymphocytes were analyzed, using the cytokinesis-block technique, in 103 subjects living in a residential suburb (Genova-Nervi), and in 203 subjects living in an urban industrialized area near a metallurgical plant and a coke factory (Genova-Cornigliano). Statistical analysis showed that the average frequency of micronucleated binucleated lymphocytes (MnBNL) was significantly higher (1.42-fold) in donors of Nervi than in donors of Cornigliano living in a contaminated environment. In contrast, the average frequency of micronucleated polynucleated lymphocytes (MnPNL) was significantly higher (1.66-fold) in donors of Cornigliano than in donors of Nervi. The existence in the whole population examined of a positive correlation between frequency of MnBNL and frequency of MnPNL and the absence of a positive correlation between frequency of bi- and poly-nucleated lymphocytes and frequency of MnPNL suggest that the formation of MnPNL is a consequence of genetic damage and not of mitotic errors arising during the division of bi- and poly-nucleated cells. In agreement with previous findings the frequency of MnBNL increased with age and was significantly higher in females than in males; unexpectedly it was higher in non-smokers/non-drinkers than in smokers/drinkers.     

Receptor activator of NF-kB (RANK) expression in primary tumors associates with bone metastasis occurrence in breast cancer patients

Background

Receptor activator of NFkB (RANK), its ligand (RANKL) and the decoy receptor of RANKL (osteoprotegerin, OPG) play a pivotal role in bone remodeling by regulating osteoclasts formation and activity. RANKL stimulates migration of RANK-expressing tumor cells in vitro, conversely inhibited by OPG.

Materials and Methods

We examined mRNA expression levels of RANKL/RANK/OPG in a publicly available microarray dataset of 295 primary breast cancer patients. We next analyzed RANK expression by immunohistochemistry in an independent series of 93 primary breast cancer specimens and investigated a possible association with clinicopathological parameters, bone recurrence and survival.

Results

Microarray analysis showed that lower RANK and high OPG mRNA levels correlate with longer overall survival (P = 0.0078 and 0.0335, respectively) and disease-free survival (P = 0.059 and 0.0402, respectively). Immunohistochemical analysis of RANK showed a positive correlation with the development of bone metastases (P = 0.023) and a shorter skeletal disease-free survival (SDFS, P = 0.037). Specifically, univariate analysis of survival showed that “RANK-negative” and “RANK-positive” patients had a SDFS of 105.7 months (95% CI: 73.9–124.4) and 58.9 months (95% CI: 34.7–68.5), respectively. RANK protein expression was also associated with accelerated bone metastasis formation in a multivariate analysis (P = 0.029).

Conclusions

This is the first demonstration of the role of RANK expression in primary tumors as a predictive marker of bone metastasis occurrence and SDFS in a large population of breast cancer patients.     

Triterpene glycosides from Astragalus icmadophilus

Six cycloartane-type triterpene glycosides were isolated from Astragalus icmadophilus along with two known cycloartane-type glycosides, five known oleanane-type triterpene glycosides and one known flavonol glycoside. The structures of the six compounds were established as 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3-acetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxy cycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3,4-diacetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3-acetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane, 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane by the extensive use of 1D- and 2D-NMR experiments along with ESIMS and HRMS analysis.The first four compounds are cyclocanthogenin and cycloastragenol glycosides, whereas the last two are based on cyclocephalogenin as aglycone, more unusual in the plant kingdom, so far reported only from Astragalus spp.     

The sigma-2 receptor agonist PB28 inhibits calcium release from the endoplasmic reticulum of SK-N-SH neuroblastoma cells

In this paper we demonstrate that PB28 abolishes the Ca2+ release through the inositol 1,4,5-trisphosphate (InsP3) receptors and ryanodine receptors in SK-N-SH cells. Sigma receptors are divided into the subtypes sigma-1 and sigma-2, which are expressed in tumor cell lines and characterized by distinct pharmacological profiles. The sigma-1 receptor has been recently cloned, whereas the sigma-2 receptor is less well characterized. The endogenous ligand(s) of both subtypes remain unclear. In isolated guinea pig ileum, PB28 inhibits the contraction induced by carbachol dose dependently and in a non-competitive manner. In SK-N-SH cells PB28 challenge does not affect the intracellular Ca2+ concentration but incubation with PB28 for 45 min abolishes the cytosolic Ca2+ increases evoked by carbachol or histamine. This effect, not sensitive to cycloheximide, is caused by direct inhibition of the InsP3 receptors, since PB28 abolishes the response elicited by InsP3 administration in permeabilized SK-N-SH cells. Finally, incubation for 45 min with PB28 also abolishes the cytosolic Ca2+ increase evoked by caffeine.     

Extensive activation,tissue trafficking,turnover and functional impairment of NK cells in COVID-19 patients at disease onset associates with subsequent disease severity

The SARS-CoV-2 infection causes severe respiratory involvement (COVID-19) in 5–20% of patients through initial immune derangement, followed by intense cytokine production and vascular leakage. Evidence of immune involvement point to the participation of T, B, and NK cells in the lack of control of virus replication leading to COVID-19. NK cells contribute to early phases of virus control and to the regulation of adaptive responses. The precise mechanism of NK cell dysregulation is poorly understood, with little information on tissue margination or turnover. We investigated these aspects by multiparameter flow cytometry in a cohort of 28 patients hospitalized with early COVID-19.Relevant decreases in CD56^brightCD16+/- NK subsets were detected, with a shift of circulating NK cells toward more mature CD56^dimCD16+KIR+NKG2A+ and “memory” KIR+CD57+CD85j+ cells with increased inhibitory NKG2A and KIR molecules. Impaired cytotoxicity and IFN-γ production were associated with conserved expression of natural cytotoxicity receptors and perforin. Moreover, intense NK cell activation with increased HLA-DR and CD69 expression was associated with the circulation of CD69+CD103+ CXCR6+ tissue-resident NK cells and of CD34+DNAM-1^brightCXCR4+ inflammatory precursors to mature functional NK cells. Severe disease trajectories were directly associated with the proportion of CD34+DNAM-1^brightCXCR4+ precursors and inversely associated with the proportion of NKG2D+ and of CD103+ NK cells.Intense NK cell activation and trafficking to and from tissues occurs early in COVID-19, and is associated with subsequent disease progression, providing an insight into the mechanism of clinical deterioration. Strategies to positively manipulate tissue-resident NK cell responses may provide advantages to future therapeutic and vaccine approaches.     

Potent and selective tariquidar bioisosters as potential PET radiotracers for imaging P-gp

Compounds 8a–d have been designed as bioisosters of tariquidar for imaging P-gp expression and density by PET. The results displayed that compounds 8b and 8d could be considered potential P-gp/BCRP ligands suitable as ¹¹C and ¹⁸F radiotracers, respectively.     

Regional (north american) studbook for the Geoffroy's tamarin,Saguinus geoffroyi

A regional (North American) studbook of Saguinus geoffroyi, Geoffroy's tamarin, is presented. Supplemental information regarding range, habitat, status in the wild, taxonomy, husbandry, level of inbreeding, and international holdings is included. Living animals are cross-indexed by holding institution.     

Bovine ovarian follicular fluid vitronectin content is influenced by the follicle size

Vitronectin was quantified in the follicular fluid aspirated from bovine follicles with diameters of 3 to 15 mm (as determined by ultrasonography) using a specific enzyme-linked immunosorbent assay (ELISA) validated for bovine vitronectin. The primary antibody was raised in rabbit against vitronectin purified from bovine plasma. Vitronectin quantified in serial dilutions of bovine plasma and ovarian follicular fluid was highly correlated with the volume of each sample assayed. In addition, known amounts of purified bovine vitronectin added to samples of plasma or follicular fluid were accurately recovered. Follicular fluid concentrations of vitronectin were positively correlated with the follicle diameter (r = 0.8; P < 0.01). These data indicate that bovine follicular fluid concentration of vitronectin is influenced by the stage of follicular development.     

Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells

We have previously shown that clusters of guanine quadruplex (G4) structures can form in the human herpes simplex-1 (HSV-1) genome. Here we used immunofluorescence and immune-electron microscopy with a G4-specific monoclonal antibody to visualize G4 structures in HSV-1 infected cells. We found that G4 formation and localization within the cells was virus cycle dependent: viral G4s peaked at the time of viral DNA replication in the cell nucleus, moved to the nuclear membrane at the time of virus nuclear egress and were later found in HSV-1 immature virions released from the cell nucleus. Colocalization of G4s with ICP8, a viral DNA processing protein, was observed in viral replication compartments. G4s were lost upon treatment with DNAse and inhibitors of HSV-1 DNA replication. The notable increase in G4s upon HSV-1 infection suggests a key role of these structures in the HSV-1 biology and indicates new targets to control both the lytic and latent infection.