Surface plasmon resonance study of apoptotic regulators Bcl-2, Bcl-w, Bcl-X_L, Mcl-1 and A1 and the small molecule inhibitor (SMI) TW-37: the idea of a temporal window for drug activity

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The intrinsic apoptotic pathway is controlled through interplay of the Bcl-2 family of proteins. This family of proteins is confined to the membrane surfaces of the endoplasmic reticulum and outer mitochondrion. In the cell these proteins undergo reactions in a very small amount of time and space, which results in equilibrium measurements being inappropriate measures of protein-protein interactions. This motivated us to measure the reactions in a non-equilibrium setting, namely through using surface plasmon resonance and the Biacore 3000. We investigated protein-protein interactions between the anti-apoptotic Bcl-2 family proteins and the pro-apoptotic proteins Bid and Bim, which are BH3-only family members and contain an amphipathic α-helix. The mechanism of the non-peptide small molecule TW-37 to inhibit the interaction between anti-apoptotic and pro-apoptotic proteins was also investigated. This drug is in a new class of chemical compounds that are able to induce apoptosis in cancerous cells. TW-37 binds to the hydrophobic groove of the anti-apoptotic Bcl-2 family preventing the binding of the pro-apoptotic amphipathic α-helix, thus shifting the balance towards apoptosis.
 In order to investigate the protein-protein interactions the Bid or Bim peptides were covalently bound to a CM5 chip via the N-terminus; the proteins Bcl-2, Bcl-X_L, Mcl-1, Bcl-w, and Bfl-1/A1 were analytes. This allowed the measurement of binding on (k_a) and off (k_d) rates as well as association (K_A) and dissociation (K_D) constants. Although there is variation in the binding affinity and binding rates between the pro-apoptotic proteins, it is evident that there is a pattern emerging with certain Bcl-2 family proteins having higher affinities and longer duration of heterodimer formation, with A1 and Bcl-w having the highest affinity and slowest dissociation rates and Bcl-2 having the lowest affinity and slowest dissociation rate. Experiments were also done in which the Bcl-2 family proteins were amine coupled to a CM5 chip and the drugs TW-37 and TW-37A (an inactive form of TW-37) were analytes. In this case a different pattern emerged, with Bcl-w having the highest affinity and Bcl-2 having the lowest. Finally, an experiment was done to test the mechanism of action of TW-37, which found TW-37 is unable to disrupt already-formed heterodimers. All of these findings together lead to the idea of certain anti-apoptotic proteins being more important targets for drug inhibition for induction of apoptosis (a temporal window for drug activity). Although a certain protein may have a high affinity for TW-37, it may not be available for binding because 1) the drug is unable to disrupt heterodimers and 2) the protein may be bound to a pro-apoptotic regulator forming a heterodimer for a period of time based on the dissociation rate.
 Acknowledgements: LLS 8028 & NIH CA109389