Study Of Calorimetry In The Optimization Of Bh3Mimetics

The B-cell lymphoma2(Bcl-2) family proteins are the main regulators of apoptotic processes and include anti-apoptotic and pro-apoptotic members. The balance between the relative levels of these antagonistic proteins is critical for cell fate. As such, Many groups have taken up the task to develop small-molecule inhibitors (SMIs) targeting the Bcl-2homology domain3(BH3) binding pocket, which is shared by Bcl-2family members, to induce cancer cells apoptosis. These SMIs thus act as BH3mimetics. However, there is not any BH3mimetics in clinical application to date. The most promising small molecule ABT-737is suffered by inability to hit the Bcl-2-like myeloid cell leukemia sequence1(Mcl-1) protein, which confers resistance in many cancers. As such, the research of BH3mimetics as antitumor agents still faces significant challenges.As is well-known, the binding of drugs and their targets is a process controlled by thermodynamics. The binding affinity (Ka), Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) changes are the thermodynamic parameters describing the interaction of drug and protein. Isothermal titration calorimetry (ITC) is the only experimental technique that provides a partition of the binding energy into its enthalpic and entropic components. The changes of enthalpy come from the hydrogen bonding between drug and target, thus the formation of hydrogen bonding can identified from the enthalpy gain. The enthalpy and entrory monitored during the optimization can guide the design and optimization of the small-molecule inhibitors. As such, it is an attractive strategy for ITC to using in the optimization of small molecule inhibitors.Through the ITC experiments between BH3mimetics and Mcl-1, we analysed the binding model of them to guide the optimization of other BH3mimetics.Firstly, we constructed the expression plasmid of human Mcl-1protein, optimized the expression and purification and purified the protein with the purity of95%. The circular dichroism spectra showed that Mcl-1has a stable secondary structure. The1H-15N HSQC spectra of Mcl-1alone showed well-dispersed peaks, indicating a folded and stable protein. This protein can be used in the lasting experiment.Next, the HSQC technology of protein was employed which showed that compound8bind to the BH3groove of Mcl-1. Then, ITC experiment was carried out with compound8and Mcl-1and Mcl-1(R263A), respectively. The change of enthalpy identified a hydrogen bond is formed by8with the BH3groove of Mcl-1.Based on the above research, we obtained the thermodynamic parameters for the interactions of a series of BH3mimetics with Mcl-1protein by isothermal titration calorimetry. Notably, an enthalpy gain of4.65kcal/mol identified that an additional hydrogen bond is formed by2with Mcl-1, compared with compound1. Owing to compounds1-6overcoming enthalpy-entropy compensation, the affinities of them improved gradually. Toward binding to the Mcl-1protein, compound6was deemed optimial with an obtained Kd value of238nM, which is a104fold improvement compared with1. Analysis of the enthalpy and-T△S efficiencies showed that ligand efficiencies with respect to molecular size are correlated with the enthalpic efficiencies. To our knowledge, this is the first time that Bcl-2inhibitors were optimized thermodynamically using ITC.