Discovery of 5-pyrrolopyridinyl-2-thiophenecarboxamides as potent AKT kinase inhibitors

A pyrrolopyridinyl thiophene carboxamide 7 was discovered as a tractable starting point for a lead optimization effort in an AKT kinase inhibition program. SAR studies aided by a co-crystal structure of 7 in AKT2 led to the identification of AKT inhibitors with subnanomolar potency. Representative compounds showed antiproliferative activity as well as inhibition of phosphorylation of the downstream target GSK3β.     

Assessment of ProteoExtract subcellular fractionation kit reveals limited and incomplete enrichment of nuclear subproteome from frozen liver and heart tissue

The nuclear fraction of the ProteoExtract subcellular fractionation kit was assessed using frozen rat liver and heart tissue. Fractionation was evaluated by Western blot using protein markers for various subcellular compartments and followed up with LC/MS/MS analysis of the nuclear fractions. Of the proteins identified, nuclear proteins were in the minority (less than 15%) and there was poor representation of the various nuclear substructures when compared with liver nuclear isolations using a classical density‐based centrifugation protocol. The ProteoExtract kit demonstrated poor specificity for the nucleus and offers limited promise for proteomics investigations of the nuclear subproteome in frozen tissue samples.     

A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge

Severe acute respiratory syndrome coronavirus (SARS-CoV) is an important emerging virus that is highly pathogenic in aged populations and is maintained with great diversity in zoonotic reservoirs. While a variety of vaccine platforms have shown efficacy in young-animal models and against homologous viral strains, vaccine efficacy has not been thoroughly evaluated using highly pathogenic variants that replicate the acute end stage lung disease phenotypes seen during the human epidemic. Using an adjuvanted and an unadjuvanted double-inactivated SARS-CoV (DIV) vaccine, we demonstrate an eosinophilic immunopathology in aged mice comparable to that seen in mice immunized with the SARS nucleocapsid protein, and poor protection against a nonlethal heterologous challenge. In young and 1-year-old animals, we demonstrate that adjuvanted DIV vaccine provides protection against lethal disease in young animals following homologous and heterologous challenge, although enhanced immune pathology and eosinophilia are evident following heterologous challenge. In the absence of alum, DIV vaccine performed poorly in young animals challenged with lethal homologous or heterologous strains. In contrast, DIV vaccines (both adjuvanted and unadjuvanted) performed poorly in aged-animal models. Importantly, aged animals displayed increased eosinophilic immune pathology in the lungs and were not protected against significant virus replication. These data raise significant concerns regarding DIV vaccine safety and highlight the need for additional studies of the molecular mechanisms governing DIV-induced eosinophilia and vaccine failure, especially in the more vulnerable aged-animal models of human disease.     

Epithelial Membrane Protein-2 (EMP2) Activates Src Protein and Is a Novel Therapeutic Target for Glioblastoma

Despite recent advances in molecular classification, surgery, radiotherapy, and targeted therapies, the clinical outcome of patients with malignant brain tumors remains extremely poor. In this study, we have identified the tetraspan protein epithelial membrane protein-2 (EMP2) as a potential target for glioblastoma (GBM) killing. EMP2 had low or undetectable expression in normal brain but was highly expressed in GBM as 95% of patients showed some expression of the protein. In GBM cells, EMP2 enhanced tumor growth in vivo in part by up-regulating αvβ3 integrin surface expression, activating focal adhesion kinase and Src kinases, and promoting cell migration and invasion. Consistent with these findings, EMP2 expression significantly correlated with activated Src kinase in patient samples and promoted tumor cell invasion using intracranial mouse models. As a proof of principle to determine whether EMP2 could serve as a target for therapy, cells were treated using specific anti-EMP2 antibody reagents. These reagents were effective in killing GBM cells in vitro and in reducing tumor load in subcutaneous mouse models. These results support the role of EMP2 in the pathogenesis of GBM and suggest that anti-EMP2 treatment may be a novel therapeutic treatment.