Protein–protein interactions are involved in the control of diverse physiological and pathological processes in living organisms such as cell apoptosis and proliferation, which represent an emerging class of molecular targets for novel drug discovery.1
To monitor molecular interactions, a number of assay technologies have been developed, such as time-resolved Förster (or fluorescence) resonance energy transfer (TR-FRET), fluorescence polarization (FP), and surface plasmon resonance.2–5
These assay technologies, particularly in homogenous format, have been extensively used in high-throughput screening (HTS) campaigns for the identification of new chemical entities in the drug discovery field and new molecular probes for chemical biology studies.5
However, the application of different assay technologies often gives rise to different hit lists even when monitoring the same biochemical interaction. Because of the high cost of screening large chemical libraries, HTS campaigns are often conducted in a single-point format and investigators are forced to choose a single-assay technology. To enhance the efficiency of HTS campaigns, we have designed and developed a novel HTS technology that allows the generation of two HTS readouts from one reaction by combining FRET and FP technologies into one platform. This technology is termed dual-readout F2
assay, where F2
standing for FRET and FP. We have further miniaturized the F2
assay to a 1,536-well ultra-HTS (uHTS) format. To provide a proof of concept, this F2
uHTS assay technology was used to monitor the interaction of Mcl-1 and Noxa for the eventual goal of discovering the next generation of small molecule modulators of apoptosis.
Apoptosis, or programmed cell death, is a critical process in both development and homeostasis of multicellular organisms.6
Alterations in apoptotic pathways can disrupt the delicate balance between cell proliferation and cell death and lead to a variety of diseases.6,7
Mcl-1 belongs to the prosurvival Bcl-2 subfamily along with Bcl-XL
, Bcl-2, Bcl-w, and A1.7–9
Mcl-1 is overexpressed in many human cancers and its overexpression contributes to chemoresistance and disease relapse.10–12
Recently, a number of groups have reported the discovery of small-molecules known as BH3 mimetics, which induce apoptosis by inhibiting antiapoptotic Bcl-2 family members.13–23
This family of molecules demonstrates a wide range of both potency and selectivity for different antiapoptotic Bcl-2 proteins. However, there is still a need for developing BH3 mimetics that can efficiently and selectively target Mcl-1 protein.
One of the essential elements in discovering and identifying small-molecule Mcl-1 inhibitors is the development of a robust, quantitative, and high-throughput assay for evaluation of the binding affinities of potential small molecule inhibitors. In vitro
binding studies have demonstrated that BH3 peptides from pro-apoptotic proteins exhibit preferences in binding to anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1).24
Noxa BH3 peptide is highly selective for Mcl-1 and Bcl-2A1 proteins (within the nM range) but does not bind detectably to the other members of this family (>100
Recently published structures of Mcl-1 in complex with the Noxa and Puma BH3 domains demonstrate that Noxa specifically targets Mcl-1 and exploits a basic patch unique to the Mcl-1 sequence.25
These interactions between Mcl-1 and the Noxa BH3 peptide form the basis for the design of the dual-readout F2
assay, which can be used to screen for small molecule inhibitors that selectively disrupt the interaction of Mcl-1 protein and Noxa.