Most drugs bind directly to a target protein usually at a functional site and can either inhibit or activate the target protein to elicit the desired clinical effect. How effective the drug is (efficacy) is dependent on how effectively it binds to the target protein (target engagement). Target engagement is determined by the local concentration of a drug as well as its binding efficiency, both of which can be variable within and between patients. Drug efficacy has traditionally been monitored by evaluation of downstream cellular responses as drug binding has not been able to be directly measured in cells. Martinez-Molina et al., have recently developed a novel technique to measure target engagement inside cells and tissues.
Thermal shift assays (TSA) are used on purified proteins to measure the extent of unfolding of the protein over a range of temperatures. Binding of drug ligand to the purified protein can ‘shift’ the temperature at which the protein unfolds. Here, a protocol has been developed in which soluble protein fractions from cell lysates are separated into multiple aliquots and heated to different temperatures. The heated lysates are then centrifuged to separate soluble fractions from precipitates. The soluble fractions are resolved by SDS-PAGE followed by western blot analysis. Thermal melt curves for each target protein are then constructed from the data. Known drugs were added to cell lysates and shifts in melting curves of target proteins detected. Due to the similarities of this method to TSA, the authors named this technique ‘cellular thermal shift assay (CETSA)’. They correlated the CETSA data with TSA data from purified proteins, with similar melting temperatures obtained for the unliganded protein. They demonstrated thermal melt shifts of target protein with the addition of compound using both CETSA and TSA methods. They obtained dose response curves termed ‘Isothermal dose-response fingerprints’ by treating lysates with different concentrations of drug whilst keeping temperature and heat exposure time constant.
The authors were also able to acquire thermal melt dose response curves using intact cells by exposing the cells to drugs before the preparation of lysates and demonstrated relative binding differences between intact cells and lysates. The use of dye exclusion experiments confirmed that cellular membranes remained intact up to a temperature of 65˚C. Further, they established that CETSA can be used to determine target selectivity. A CDK inhibitor selectively engaged with CDK4 and CDK6 but not CDK2 or CDK9, confirming results from activity assays.
CETSA can be used to monitor the in vivo engagement of drugs with their targets. After dosing of animals with a compound inhibiting MetAP2, the group were able to demonstrate that thermal melt profiles of MetAP2 could be produced from lysates from frozen organs.
This technique therefore potentially has wide spread applications in preclinical drug development, such as assessment of target engagement and specificity, validation of clinical drug candidates and estimation of drug efficacy in patients.
Martinez Molina D, Jafari R, Ignatushchenko M, Seki T, Larsson EA, Dan C, Sreekumar L, Cao Y, Nordlund P. Science. 2013 Jul 5;341(6141):84-7. doi: 10.1126/science.1233606.