A Strategy Combining Differential Low-Throughput Screening and Virtual Screening (DLS-VS) Accelerating the Discovery of new Modulators for the Orphan GPR34 Receptor.

The DLS-VS strategy was developed as an integrated method for identifying chemical modulators for orphan GPCRs. It combines differential low-throughput screening (DLS) and virtual screening (VS). The two cascaded techniques offer complementary advantages and allow the experimental testing of a minimal number of compounds. First, DLS identifies modulators specific for the considered receptor among a set of receptors, through the screening of a small library with diverse chemical compounds. Then, an active molecular model of the receptor is built by homology to a validated template, and it is progressively refined by rotamers modification for key side-chains, by VS of the already screened library, and by iterative selection of the model generating the best enrichment. The refined active model is finally used for the VS of a large chemical library and the selection of a small set of compounds for experimental testing. Applied to the orphan receptor GPR34, the DLS-VS strategy combined the experimental screening of 20 000 compounds and the virtual screening of 1 250 000 compounds. It identified one agonist and eight inverse agonists, showing a high chemical diversity. We describe the method. The strategy can be applied to other GPCRs.

DeltaN-p73alpha accumulates in human neuroblastic tumors.

Neuroblastic tumors (NTs), occurring in early childhood, display a wide spectrum of differentiation. Recurrent deletions involving the p73 locus are frequently observed in undifferentiated NTs. To address the question of the possible implication of p73 in neuroblastic differentiation, we investigated the status of the expression of this gene in a panel of differentiated and undifferentiated tumors. Although mutations were not found, p73 transcript profiles differed between undifferentiated and differentiated tumors. The frequency of the transcripts lacking exon 2 (species 1-3) appeared to be higher in undifferentiated than in differentiating and differentiated NTs. In contrast, products from using an alternate promoter (DeltaN-p73) were present in all NTs. In addition, only DeltaN-p73, but not full-length proteins, were detected by immunoblotting, suggesting a greater stability of N-truncated isoforms. Importantly, as in the adrenal medulla, most NTs showed p73-positive immunohistological staining with a cellular distribution and intensity varying according to the neuronal differentiation. Surprisingly, we observed redistribution of p73 from the nucleus to the cytoplasm during neuroblastic differentiation. Our data suggest that, in undifferentiated NTs, a link may exist between the accumulation of DeltaN-p73alpha variants and the "nuclear exclusion" of p53.

On the shoulders of giants: p63, p73 and the rise of p53.

The discoveries of the p53 homologs, p63 and p73, have both fueled new insights and exposed enigmas in our understanding of the iconic p53 tumor suppressor. Although the pivotal role of p53 in cancer pathways remains unchallenged, because p63 and p73 are now implicated in stem cell identity, neurogenesis, natural immunity and homeostatic control. Despite their seemingly separate tasks, there are hints that the p53 family members both collaborate and interfere with one another. The question remains, therefore, as to whether these genes evolved to function independently or whether their familial ties still bind them in pathways of cell proliferation, death and tumorigenesis.