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Sunday
Mar292015

Turning up the heat on drug binding

Small-molecule therapeutic and imaging agents should be effective and selective, binding to one (or a few) protein targets and not to others. Many techniques exist for measuring drug-target interactions in vitro, including methods that measure the thermodynamic stabilization of the target upon drug binding. In contrast, relatively few techniques directly measure drug binding in a cellular or organismal context; instead, cellular assays often measure the functional consequences of target engagement, such as downstream post-translational modifications or induction of target genes. The recently described cellular thermal shift (CETSA) assay bridges this gap and has several appealing characteristics:

  • Most soluble proteins can be assayed with minimal optimization required.

  • Drugs of interest do not have to be labeled.

  • Any cell or tissue type can be examined.

  • The function of the target of interest does not need to be known.

  • Targets with functions that are traditionally difficult to measure in cells (for example, ‘readers,’ or proteins that recognize and bind to a motif but do not have enzymatic activity) can be examined.

  • Proteome-wide readouts allow for off-targets to be identified.

The assay suffers from two major limitations: it does not apply to membrane-associated proteins, and small-molecule binding affinities cannot be extrapolated from CETSA data. Binding affinities can, however, be rank-ordered.


The nature of this second limitation becomes apparent when considering the CETSA method. Intact cells/tissue or lysate are incubated with a drug of interest, then multiple aliquots of the sample are heated to different temperatures. A temperature is reached above which a protein of interest’s native structure is less thermodynamically favorable, and the protein denatures or unfolds. The midpoint in the unfolding curve is the Tm, and represents a temperature where the free energy of the native and non-native forms of the protein are equivalent. A small molecule that binds to and stabilizes the native state of its target will increase that protein’s thermal stability. Experimentally, this is observed as an increase in the protein Tm and is proportional to both the concentration and the binding affinity of the drug. Converting changes in Tm to an accurate binding constant assumes equilibrium conditions (ie. the protein unfolds and refolds reversibly), extrapolation to a standard temperature (ie. the Kd at 37 or 24C), and knowledge of the unfolding enthalpy of the protein of interest, as measured by other thermal experiments. Proteins with a smaller enthalpy of unfolding will show a larger increase in Tm for than proteins with a larger enthalpy of unfolding when bound by a drug with equal affinities.


The CESTA assay does not directly measure Tm, but rather the related parameter Tagg, which represents midpoint of aggregation and precipitation of proteins in their unfolded states. This precipitation is used for detection in the CETSA assay, as samples are centrifuged to remove precipitated proteins and the presence of the protein of interest in the soluble fraction is quantified by western blotting or mass spectrometry. Many purified proteins precipitate irreversibly during in vitro thermal stabilization experiments. These assays inform the idea that in nonequilibrium systems, Tagg is generally very close to Tm and can be used as a relative measure to rank affinities, despite the complex, higher-order reactions that govern protein precipitation. Aggregation of proteins in a cellular context, as in the CETSA assay, is even more convoluted:

  • Melting points acquired in cell extracts versus intact cells are poorly correlated, suggesting that disruption of cellular context has diverse effects on protein stability.

  • Likewise, lysates prepared from different tissues with very different protein compositions may vary in their propensity to aggregate. Spiking a control protein into the lysate may help determine the effects of protein composition on aggregation.

  • While most cells remain unlysed as measured by Trypan blue exclusion at 65-70 C, organelles and larger protein assemblies within the cells have characteristic disintegration temperatures. Thus, mitochondrial proteins, for example, will have an apparent increase in concentration in the soluble fraction upon rupture of that subcellular structure.

  • Proteins that are post-translationally modified or assembled into complexes may exhibit context-specific enthalpies of unfolding, which will in turn modulate the magnitude of the Tm shift observed upon ligand binding.

  • In intact cells, Tm shifts may be seen in the downstream targets of the protein of interest, for example if downstream post-translational modifications are affected by drug treatment. This represents the CETSA variant of a traditional functional assay.


A ligand-induced change in Tagg as measured by CETSA is a complex parameter than cannot be rigorously converted to a binding affinity. Nonetheless, this assay is incredibly powerful in its ability to reveal engagement of endogenous proteins by unlabeled small molecules and to rank the relative affinities of drugs binding to a shared target.


-ELR


References:


Matulis, D., Kranz, J. K., Salemme, F. R., & Todd, M. J. (2005). Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry, 44(13), 5258-5266.


Molina, D. M., et al.. (2013). Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science, 341(6141), 84-87.


Savitski, M. M., et al. (2014). Tracking cancer drugs in living cells by thermal profiling of the proteome. Science, 346(6205), 1255784.

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