Objective-Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimidePEGs to engineered cysteine analogs of Epo, and to determine whether the PEGylated Epo cysteine analogs have improved pharmacokinetic properties in vivo. Materials and Methods-Thirty-fourEpo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monoPEGylated derivatives of 12 cysteine analogs were prepared using 20 kDamaleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild type Epo (BV Epo) and a PEGylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats.Results-Biological activities of 17 purified Epo cysteine analogs and 10 purified PEGylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only PEGylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The PEGylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation.Conclusions-Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monoPEGylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed EPO proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific PEGylation appears to improve the pharmacokinetic properties of Epo.

Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes ;240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.

Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes ;240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.

Expression of transcription factor estrogen receptor (ER) drives and defines almost three-fourth of all breast cancer (BC). Endocrine therapies such as tamoxifen and aromatase inhibitors form the backbone of therapeutic regimen in treatment of ER-positive (ER+) BC. However, some ER+ BC patients do not respond well to these therapies and develop endocrine resistance. Mechanisms of endocrine resistance is multi-faceted and may require inhibition of multiple pathways. FDA-approved inhibitors of CDK4/6 and mTOR pathways have improved outcomes in ER+ metastatic BC patients. However, additional therapeutic agents targeting novel and essential nodes of endocrine resistance are needed for better management of ER+ BC. Recent research has shown that ER coregulators play a crucial role in endocrine resistance. The DNA interactions and transcriptional potential of ER rely on the pioneer factor Forkhead Box A1 (FOXA1), which plays an essential role in determining tumor growth and progression especially in the endocrine resistance setting. FOXA1 is gene amplified and/or overexpressed in multiple preclinical models and clinical samples of endocrine-resistant BC. Overexpression of FOXA1 promotes metastasis in animal models even during tamoxifen treatment, making it an attractive drug target to overcome endocrine resistance. However, no pharmacologic inhibitors of FOXA1 are currently available. To launch the drug discovery efforts to identify novel FOXA1 inhibitors, we aimed to express and purify functionally active full-length human FOXA1 protein that can be used for high-throughput drug screening in this research. We utilized various protein production strategies that we explored using baculovirus, bacterial, and mammalian expression system to achieve highly stable recombinant FOXA1 in an active form. While the baculovirus system had high protein expression, the yield, stability and purity of the recombinant FOXA1 was substandard. We maximized the yield and purity of FOXA1 with the bacterial expression system, but the protein functionality was lost due to misfolding. The mammalian expression system, which is well-known for its ability to properly recapitulate the protein posttranslational modifications, yielded stable FOXA1 protein. The recombinant FOXA1 expressed and purified in mammalian cells was functionally active as determined by the DNA-binding activity using enzymatic mobility shift assay (EMSA). However, the purity achieved was suboptimal for drug discovery application. Ongoing efforts are focused on optimizing the protein purification strategies that could ensure optimal yield of functional protein with highest purity for downstream applications. Citation Format: Hariprasad Thangavel, Yingmin Zhu, Kurt R. Christensen, Xiaoyong Fu, Dean Edwards, Rachel Schiff, Meghana V. Trivedi. Production of functionally active recombinant FOXA1: The first step towards targeted drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 930.