Research Activities
High-throughput and high-content Screening Unit (SU)

We will set-up a technological platform for the identification of disease-relevant molecular targets. The platform will be based on the analysis of complex cellular phenomena, such as the activity of entire signaling pathways or the structure/function of sub-cellular compartments.

Molecular targets will be identified through phenotype screens using nucleic acid (cDNA or siRNA) or small-molecule (chemical compounds) libraries, respectively (phenotype screens).

These approaches require robotized solutions with multiplexing capability, capable of handling in the order of 105-106 samples and of simultaneously measuring multiple readout parameters. Fluorescence-based techniques are the best-suited detection approaches for high-content and high-throughput screenings (due to their high sensitivity and amenability to automation). Therefore, cell phenotypes will be investigated by direct imaging of living or fixed cells, using widefield and confocal fluorescence microscopy (2D and 3D Confocal Cell Imaging).

One critical technological challenge of the Screening Unit will be the automation and integration of the different components of the experimental and analytical process, including:

  • various robotic-transfer and -handling units (e.g. for sample preparation and loading; chemical compound or biomolecule administration etc.);
  • multi-channel automated microscopes (for image collection); and
  • additional multi-mode readers (to increase the versatility of the platform for different assay formats).

The Unit will be supported by the activity of a Wet Biology Unit for the adaptation of the various biological/biochemical assays to a high-throughput screening format.

Critical to the success of the chemical-genomics and drug-discovery programs is the access to a chemical library of high quality, in terms of size, molecular diversity and intrinsic properties of individual compounds.


The high-throughput and high-content Screening Unit will be available also for other activities, mainly connected to the Drug Discovery programs of the IFOM-IEO Campus and the IIT Central Research Lab at Genova:

  • compound profiling (cellular efficacy, specificity, selectivity, mechanism of action) and
  • fluorescence-based biochemical and radiochemical assays (single-molecule assays).
 
Target identification through chemical screens

This project will develop ad hoc strategies for identifying the cellular targets of small-molecule inhibitors. More specifically, the program will address the mechanism of action of selected subsets of small molecules that have a recognized potential to eventually become drugs. We will operate therefore in a collaborative framework with the drug screening and medicinal chemistry programs at the IFOM-IEO Campus and the IIT Central Research Lab at Morego. From a technological perspective, the project will exploit a combination of cellular assays, classical biochemistry, medicinal chemistry, and large-scale proteomics and genomics analyses. Cellular resistance/sensitivity to drugs will be exploited as the main entry point for primary and secondary target identification.

A large fraction of known drugs are small organic molecules (molecular weight <500). Small molecules are identified from a large library of compounds through two main types of screens, based, respectively, on their ability to interfere with a known protein target (molecular-target based screens) or to revert a specific phenotype in a cell or a tissue (phenotype screens). Our focus on phenotype screens of important cellular pathways will feed into a cancer drug discovery program.

A necessary complement to the phenotype-screening program is a program for the identification and validation of targets. The main limitation of phenotypic screening programs based on small molecules resides in identifying the macromolecular targets of the small-molecule inhibitors that cause the phenotype.

We will tackle this issue following three approaches:

  • affinity purification of potential cellular binders, by proteome analysis, using rational biochemical approaches;
  • identification of resistant mutants of the target of the small molecule;
  • RNAi-based genetic screens to identify those genes that, once ablated, enhance the sensitivity to the drug of interest. These genes will be part of the so-called synthetic genetic pathways and may represent potential secondary drug targets.