Mcl-1 protein was purified from the soluble fraction using Ni-NTA resin (Qiagen), following the manufacturer’s instructions. with F2 for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F2 assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns. Introduction ProteinCprotein 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 F?rster (or fluorescence) resonance energy transfer (TR-FRET), fluorescence polarization (FP), and surface plasmon resonance.2C5 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 L-165,041 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 A1.7C9 Mcl-1 is overexpressed in many human cancers and its overexpression contributes to chemoresistance and disease relapse.10C12 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.13C23 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. 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?M).24 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 L-165,041 be used to screen for small molecule inhibitors that selectively disrupt the interaction of Mcl-1 protein and Noxa. Materials and Methods Peptides All the peptides were synthesized at Emory Microchemistry and Proteomic Facility. The 26-mer Noxa peptide (residues 18C43: PAELEVECATQLRRFGDKLNFRQKLL-NH2) used in this study was synthesized and labeled with 5/6-carboxytetramethyl-rhodamine (TMR). The nonlabeled Noxa peptide and 21-residue Bid-BH3 peptide (residues 79C99; QEDIIRNIARHLAQVGDSMDR-NH2) were synthesized and used as peptide antagonists. Expression and Purification of Recombinant Mcl-1 Protein Human Mcl-1 cDNA was purchased from Origene. The Mcl-1 fragment, amino acid residues 171C327, was cloned L-165,041 into the pHis-TEV vector (a modified pET vector) through BL21 (DE3) cells. Cells were grown at 37C in 2xYT containing antibiotics to an OD600 of 0.6. Protein expression was induced by 0.4?mM isopropyl -D-1-thiogalactopyranoside at 37C.In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed Dual-Readout F2 assay with F2 for FP and FRET. discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F2 assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns. Introduction ProteinCprotein 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 F?rster (or fluorescence) resonance energy transfer (TR-FRET), fluorescence polarization (FP), and surface plasmon resonance.2C5 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 Rabbit Polyclonal to SFXN4 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 A1.7C9 Mcl-1 is L-165,041 overexpressed in many human cancers and its overexpression contributes to chemoresistance and disease relapse.10C12 Recently, a number of organizations possess reported the finding of small-molecules known as BH3 mimetics, which induce apoptosis by inhibiting antiapoptotic Bcl-2 family members.13C23 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 powerful, quantitative, and high-throughput assay for evaluation of the binding affinities of potential small molecule inhibitors. binding studies have shown that BH3 peptides from pro-apoptotic proteins show 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?M).24 Recently published constructions 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 display for small molecule inhibitors that selectively disrupt the interaction of Mcl-1 protein and Noxa. Materials and Methods Peptides All the peptides were synthesized at Emory Microchemistry and Proteomic Facility. The 26-mer Noxa peptide (residues 18C43: PAELEVECATQLRRFGDKLNFRQKLL-NH2) used in this study was synthesized and labeled with 5/6-carboxytetramethyl-rhodamine (TMR). The nonlabeled Noxa peptide and 21-residue Bid-BH3 peptide (residues 79C99; QEDIIRNIARHLAQVGDSMDR-NH2) were synthesized and used as peptide antagonists. Manifestation and Purification of Recombinant Mcl-1 Protein Human being Mcl-1 cDNA was purchased from Origene. The Mcl-1 fragment, amino acid residues 171C327, was cloned into the pHis-TEV vector (a revised pET vector) through BL21 (DE3) cells. Cells were cultivated at 37C in 2xYT comprising antibiotics to an OD600 of 0.6. Protein manifestation was induced by 0.4?mM isopropyl -D-1-thiogalactopyranoside at 37C for 4?h. Cells were lysed in 50?mM Tris pH 8.0 buffer containing 500?mM NaCl, 0.1% bME, and 40?L of Leupeptin/Aprotin..
Categories