SBIR-STTR Award

Activators of the tissue-protective erythropoietin receptor (EPOR/?c) demonstrate encouraging pre-clinical and early clinical results for the treatment of Friedreich’s Ataxia and Parkinson’s disease and hold significant potential for expansion to additional indications. In preliminary studies, a small molecule scaffold was identified that increased the binding of EPOR extracellular 25-250 193-216 domain (EPOR ) and a fluorescently-labeled peptide corresponding to EPOR . SAR optimization of this scaffold produced a small molecule library of both “active” and “inactive” molecules based on activation of EPOR/?c, activation of canonical signaling molecules, cytoprotection in cell-based assays and ability to increase frataxin protein in vitro and in vivo. The central goal of this Phase 1 SBIR Proposal is to establish a homogeneous, high-throughput receptor-domain interaction assay capable of identifying and ranking small molecule activators of EPOR/?c. The key objectives are to (1) evaluate ®® fluorescence-polarization, FRET/HTRF and Alpha methodologies to demonstrate association of peptide/receptor and receptor domains in solution, (2) demonstrate increased association of peptide/receptor and receptor domains in the presence of validated small molecule activators of EPOR/?c and (3) demonstrate that the assay is capable of discriminating between validated “active” and “inactive” small molecules in a proprietary small molecule library. If successfully established, this assay would be used to support (1) lead-optimization of known receptor-activating scaffolds and (2) a high-throughput screen to identify new receptor activating scaffolds with follow-on optimization. The latter studies would be the subject of a future Phase II application, which would also include an assessment of small molecule binding site(s), receptor activation, cytoprotective activity and the ability of these small molecules to increase frataxin protein.

Public Health Relevance Statement:
PROJECT NARRATIVE Preclinical and early clinical studies suggest that activators of the tissue-protective erythropoietin receptor have significant potential for the treatment of Friedreich’s Ataxia and Parkinson’s disease, where the discovery of selective, orally bioavailable small molecules is urgently needed. The development of small molecule screens for erythropoietin receptor has been technically challenging and slow, while studies of new binding interactions that mediate receptor activation have demonstrated promising preliminary success for identifying drug-like “hit” compounds that supported lead optimization for selectivity in tissue protection, bioavailability in brain and other tissues, and efficacy in relevant disease models. This proposal focuses on developing a homogeneous high-throughput assay for the discovery of such molecules based on these studies.

Project Terms:
assay development; Binding; Binding Sites; Bioavailable; Biological Assay; Biological Availability; Brain; Cells; Clinical; Clinical Research; Cytoprotection; Development; Disease model; Dose; drug discovery; EPOR gene; Erythropoietin Receptor; extracellular; Extracellular Domain; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; frataxin; Friedreich Ataxia; Future; Goals; high throughput screening; improved; In Vitro; in vitro Assay; in vivo; Label; Laboratories; Lead; Libraries; Measures; Mediating; Methodology; Methods; Minnesota; Mus; Neurons; neuroprotection; Oral; Parkinson Disease; Peptide Receptor; Peptides; Performance; Permeability; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; pre-clinical; Property; Proteins; receptor; Receptor Activation; response; scaffold; screening; Series; Signaling Molecule; Small Business Innovation Research Grant; small molecule; small molecule libraries; standard measure; success; Testing; Time; Tissues; Universities; Validation

Activation of the “tissue-protective” cytokine receptor produces cytoprotective, anti-inflammatory, and anti- apoptotic effects in vitro and in vivo in preclinical studies, and has demonstrated therapeutic utility in early clinical studies of the rare neuromuscular disease, Friedreich’s ataxia. This heteromeric receptor, composed of EPOR (erythropoietin receptor) and CSF2RB (colony-stimulating factor 2 receptor beta subunit, also “beta common receptor,” and CD131), is expressed widely in non-hematopoietic tissues where it is activated by locally produced EPO, especially near sites of oxidative stress and tissue damage. Selective EPOR/CSF2RB activators include recombinant proteins, e.g., EPO derivatives, and pharmacologically active small molecules which increase EPOR and CSF2RB phosphorylation in cells and increase expression of the mitochondrial protein, frataxin, a biomarker for Friedreich’s ataxia. Development of small molecules compared to recombinant proteins provides distinct opportunities for optimization of pharmaceutical properties including receptor sub-type selectivity, pharmacokinetics, and tolerability/safety and has produced promising probe compounds. However, discovery of small molecule cytokine receptor modulators has been challenging and slow to advance, despite substantial efforts to develop technologies for studying the effects of modulators of protein-protein interactions. In Phase I, we developed an innovative proximity-based method for detecting dimerization of the EPOR/CSF2RB receptor extracellular domains using homogeneous formats which are fully compatible with high-throughput screening (“HTS”) and demonstrated modulation by EPO and small molecules. In Phase II, we will focus on three major Aims: (1) continue developing the homogeneous EPOR/CSF2RB receptor oligomerization assay to enable HTS and screen a collection of chemically diverse compounds with which we’ll identify new small molecule leads and establish a receptor-binding SAR; (2) expand homogeneous assays for three closely related cytokine receptors as counter-screens to EPOR/CSF2RB to establish target selectivity; and (3) demonstrate the activity of selective small molecules in cellular models in vitro for EPOR/CSF2RB receptor activation and frataxin increase. Through these studies, we will develop an unprecedented small molecule SAR for receptor binding and pharmacological activity, supporting identification of new candidates for Friedreich’s ataxia.

Public Health Relevance Statement:
PROJECT NARRATIVE Preclinical and early clinical studies suggest that activators of the heteromeric tissue-protective cytokine receptor, EPOR/CSF2RB, have significant potential for the treatment of Friedreich’s Ataxia as well as additional indications, where the discovery of selective, orally bioavailable small molecules is urgently needed. While the development of assay methodologies for cytokine receptors has been technically challenging and slow to advance, we established a new FRET-based method to detect dimerization of the receptor extracellular domains by homogeneous formats which are fully compatible with high-throughput screening (“HTS”). The assay is the first method to detect direct receptor binding by small molecules, and this proposal extends these methods to enable HTS and screen a collection of chemically diverse compounds to identify new small molecule leads and establish a unique SAR for receptor-binding and pharmacological activity.

Project Terms:
Anti-inflammatory; Apoptotic; assay development; base; Binding; Bioavailable; Biochemical; Biological Assay; Biological Markers; Biophysics; Cell Line; Cell model; Cells; Cellular Assay; Chemicals; Chemistry; Clinical; Clinical Research; Collection; Colony-Stimulating Factors; Complex; counterscreen; CSF2RB gene; Cytokine Receptors; Cytoprotection; Development; Dimerization; Disease; Drug Kinetics; efficacy study; EPOR gene; Erythropoietin Receptor; Extracellular Domain; Fluorescence Resonance Energy Transfer; follow-up; frataxin; Friedreich Ataxia; Funding; Future; Grant; high throughput screening; Human; In Vitro; in vitro Model; in vivo; inhibitor/antagonist; innovation; Institutes; Investments; Lead; Libraries; Methodology; Methods; Minnesota; Mitochondrial Proteins; neonatal hypoxic-ischemic brain injury; Neuromuscular Diseases; novel; Oral; Oxidative Stress; Parkinson Disease; Peripheral Blood Mononuclear Cell; Pharmacologic Substance; Pharmacology; Phase; Phosphorylation; pre-clinical; preclinical efficacy; preclinical study; Property; Protein Analysis; protein phosphatase inhibitor-2; protein protein interaction; Proteins; Reagent; receptor; Receptor Activation; receptor binding; Recombinant Proteins; Safety; scaffold; screening; Series; Site; Small Business Innovation Research Grant; small molecule; small molecule libraries; Technology; Tertiary Protein Structure; Therapeutic; Tissues; trend; Triage; Universities; Validation; Work