Some like it hot, some hotter!

Thermal Shift Assays meet HTS to discover and characterize novel protein binders

Webinar Summary:

Thermal Shift Assay (TSA), termed also Differential Scanning Fluorimetry (DSF), is a well-established biophysical technology to interrogate protein stability and ligand binding through controlled thermal denaturation. Being a label-free and an immobilization-free system which only requires the use of an environmentally-sensitive fluorogenic probe, TSA has been favorably applied with low-/mid-throughput processivity to characterize target proteins and to profile chemical series of interacting compounds. Conversely, inherent technical limitations have largely hampered the integration of TSA in early-stage drug discovery programs entailing more demanding high-throughput approaches.

This webinar will highlight recent results achieved at Axxam to transition TSA to an increasingly parallelized, higher-throughput technology. Case studies conducted on enzymatic drug targets in 384 well/plate format will be presented, describing strategies to accomplish assay configuration to tackle screening campaigns on defined compound collections using TSA, leading to the identification of novel binding compounds and repurposing lead candidates. Interestingly, tight correlation between affinity parameters determined with TSA (KD) and inhibition potency of the catalytic activity (IC50; Ki) validated TSA as an effective and innovative technology for hit identification. To end, toolbox solutions for recalcitrant proteins yielding unrecordable signals under standard conditions have been devised, to include, for example, high-throughput matrix optimization of buffer conditions, assessment of environmental-sensitive probes alternative to Sypro™ Orange, and screening of an in-house assembled dye library.


Thermal Shift Assay (TSA); Differential Scanning Fluorimetry (DSF); affinity parameters (KD); inhibition parameters (IC50, Ki); Sypro™ Orange; fluorescent probes; environmental-sensitive dyes; High-Throughput Screening (HTS); biophysical method; compound re-purposing; binding assay; drug discovery; Hit-to-Lead; compound profiling.


– Daniele Carettoni – Head of Biochemistry
– Simone Nenci – Principal Scientist, Biochemistry

Daniele Carettoni has been Head of Biochemistry at Axxam since its foundation, leading the efforts for recombinant protein expression and development of functional assays to support high-throughput screening campaigns on diverse enzyme targets and proximity assays. Previously, he was a research scientist at Bayer, working on the configuration and adaptation of uHTS enzymatic assays, and at GlaxoWellcome, where he was involved in a collaborative project aimed at the characterization and validation of novel anti-infective targets. Daniele Carettoni did his post-doctoral training in national pharmaceutical companies, working mainly on expression and functional characterization of recombinant proteins, and received his Ph.D. in Molecular and Cellular Biology from University of Milan, studying DNA-processing enzymes.

Simone Nenci is currently a Principal Scientist in the biochemistry group at Axxam, with 6 years of experience in the field of protein purification, assay development and HTS assay configuration on therapeutically relevant targets, with focused expertise in biochemical and biophysical assays. He is responsible for the strategic design of recombinant protein expression and has succeeded in developing several functional biochemical assays for HTS across a broad range of targets including different enzyme classes, hetero-multimeric protein complexes, protein-protein interaction, and protein-nucleic acid interaction. Simone has a Master’s degree in Applied Biology and a Ph.D. in Biotechnology, and is author and co-author of a number of relevant scientific publications.

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