[71] Use of eukaryotic expression technology in the functional analysis of cloned genes

Despite the availability of effective antiretroviral therapies, drugs for HIV-1 treatment with new mode of action are still needed. An innovative approach is aimed to identify dual HIV-1 inhibitors, small molecules that can inhibit two viral functions at the same time. Rhubarb, originated from Rheum palmatum L. and Rheum officinale Baill., is one of the earliest and most commonly used medicinal plants in Traditional Chinese Medicine (TCM) practice. We wanted to explore TCM for the identification of new chemical scaffolds with dual action abilities against HIV-1.

R. palmatum L. and R. officinale Baill. extracts along with their main single isolated constituents anthraquinone derivatives were tested on both HIV-1 Reverse Transcriptase (RT)-associated DNA Polymerase (RDDP) and Ribonuclease H (RNase H) activities in biochemical assays. Active compounds were then assayed for their effects on HIV-1 mutated RTs, integrase (IN) and viral replication.

Both R. palmatum L. and R. officinale Baill. extracts inhibited the HIV-1 RT-associated RNase H activity. Among the isolated constituents, Sennoside A and B were effective on both RDDP and RNase H RT-associated functions in biochemical assays. Sennoside A was less potent when tested on K103N, Y181C, Y188L, N474A and Q475A mutated RTs, suggesting the involvement of two RT binding sites for its antiviral activity. Sennoside A affected also HIV-1 IN activity in vitro and HIV-1 replication in cell-based assays. Viral DNA production and time of addition studies showed that Sennoside A targets the HIV-1 reverse transcription process.

Sennoside A is a new scaffold for the development of HIV-1 dual RT inhibitors.

RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3′ by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus.

Programmed cell death plays a critical role during cerebellar development. In particular, it has been shown in vivo and in vitro that developing cerebellar granule neurons (CGN) die apoptotically. Apoptosis involves a series of morphological changes and the activation of caspases. Inhibitor of apoptosis proteins (IAPs) is implicated in negative regulation of caspase activation and apoptotic cell death. Although apoptotic death of CGN has been extensively studied, there is no information about the role of IAPs in the developing cerebellum. Here, we studied the participation of some members of IAPs in the survival of the developing rat CGN in culture and under physiological conditions. Under these conditions, we found a differential expression pattern of cIAP-1, cIAP-2, XIAP and survivin during cerebellar development in an age-dependent manner, highlighting the significant increase of XIAP levels. We also detected an interaction between XIAP and caspase 3 at postnatal day (P) 12 and 16. On the other hand, we found a significant decrease of XIAP levels in cultured CGN maintained in chronic potassium deprivation, an apoptotic condition, suggesting a possible relationship between XIAP levels and neuronal viability. Under these conditions, we also detected the interaction of XIAP with active caspase-3. The down-regulation of XIAP in CGN cultured under survival conditions (chronic potassium depolarization) induced a reduction of cell viability and an increment of apoptotic cells. These findings support the idea that IAPs could be involved in the survival of CGN and that XIAP might be critical for neuronal survival in cerebellar development and during chronic depolarization in cultured CGN through a mechanism involving caspase inhibition.

The clinical efficacy of antipsychotic drugs has been associated with a certain binding profile for a set of G protein-coupled receptors (GPCR)s. In this work, we use the structurally-related clozapine–olanzapine pair to progress in the understanding of the structural properties that determine their divergent binding profiles and, thereby, their differing therapeutic efficacy. First, we present novel site-directed mutagenesis results that confirm our previous hypothesis on the importance of ligand interaction with positions 5.42 and 5.46 in transmembrane helix 5. Then, we use refined models of ligand-receptor complexes, built from recently published GPCR crystal structures, to gain further insight into the molecular mechanisms responsible for the observed experimental outcomes. In particular, we observe that preventing or potentiating hydrogen bonding with position 5.46, could allow obtaining ligands with, respectively, clozapine or olanzapine-like affinities. Results presented in this study could guide the design of antipsychotic candidates with tailored binding profiles.

NMDA receptor (NMDAR) stimulation promotes neuronal survival during brain development. Cerebellar granule cells (CGCs) need NMDAR stimulation to survive and develop. These neurons differentiate and mature during its migration from the external granular layer to the internal granular layer, and lack of excitatory inputs triggers their apoptotic death. It is possible to mimic this process in vitro by culturing CGCs in low KCl concentrations (5 mm) in the presence or absence of NMDA. Using this experimental approach, we have obtained whole genome expression profiles after 3 and 8 h of NMDA addition to identify genes involved in NMDA-mediated survival of CGCs. One of the identified genes was Nurr1, a member of the orphan nuclear receptor subfamily Nr4a. Our results report a direct regulation of Nurr1 by CREB after NMDAR stimulation. ChIP assay confirmed CREB binding to Nurr1 promoter, whereas CREB shRNA blocked NMDA-mediated increase in Nurr1 expression. Moreover, we show that Nurr1 is important for NMDAR survival effect. We show that Nurr1 binds to Bdnf promoter IV and that silencing Nurr1 by shRNA leads to a decrease in brain-derived neurotrophic factor (BDNF) protein levels and a reduction of NMDA neuroprotective effect. Also, we report that Nurr1 and BDNF show a similar expression pattern during postnatal cerebellar development. Thus, we conclude that Nurr1 is a downstream target of CREB and that it is responsible for the NMDA-mediated increase in BDNF, which is necessary for the NMDA-mediated prosurvival effect on neurons.

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121–150 or 451–470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination.