SBIR-STTR Award

Ubiquitin-mediated signaling plays a central role in the control of protein degradation. Ubiquitin ligases synthesize polyubiquitin chains on target proteins, which are then degraded by the proteasome. The era of PROTAC drugs (proteolysis-targeting chimeras) was launched by the emergence of new concepts in medicinal chemistry, wherein small molecules are designed to contain groups that bind to a ubiquitin ligase at one end and to the target protein at the other end, leading to proteasome-mediated degradation of the target protein. PROTACs recruit cellular protein degradation machinery for polyubiquitination and subsequent proteasomal degradation. While this concept is quite promising, there are major hurdles in developing new PROTAC drugs. Medicinal chemists rapidly synthesize a variety of PROTAC molecules, which must then be tested in laborious cell-based assays to monitor degradation of target proteins by immunoblotting. The rate of development of novel PROTAC designs has surpassed the existing capacity to validate their ability to recruit respective E3 ligase(s) and candidate substrates and to effect polyubiquitination and degradation of the target. Currently, no efficient methods are available to validate the ubiquitination ability of newly designed PROTACs. Here we propose to develop a novel high-throughput method to screen polyubiquitination and degradation properties of PROTACs. This technology will help medicinal chemists to design PROTAC drugs in a rational way and accelerate the development of PROTACs in new therapeutic areas.

Public Health Relevance Statement:
PROTACs (PROteolysis TArgeting Chimeras) are novel experimental drugs, in which a Ub E3 ligase binding molecule is linked chemically to a target protein binding molecule; when the ligase and target protein are brought together, the target is degraded for therapeutic effect. The ability to design new PROTACs that work on an expanded range of therapeutic targets is hindered by inadequate cell assays currently available to guide chemical improvement of the molecule for clinical use. Progenra proposes a new cell-based assay that increases the throughput and eliminates artifacts; this assay will enable development of new PROTACs that can treat heretofore unaddressed diseases.

Project Terms:
amino group; analog; Antineoplastic Agents; Area; Autoimmune Diseases; Autophagocytosis; base; Binding; Binding Proteins; Biological Assay; C-terminal; cell growth regulation; Cell model; Cells; Cellular Assay; Chemicals; Chemistry; Child; chimera drug; Chimera organism; Clinical; Cullin Proteins; Defect; design; Development; Disease; drug discovery; Drug Targeting; Enzymatic Biochemistry; Enzymes; Family; G-Protein-Coupled Receptors; German population; Glues; Glycine; Half-Life; Human Genome; Imides; Immunoblotting; Immunomodulators; immunoregulation; In Vitro; Investments; Knowledge; Lead; lenalidomide; Ligands; Ligase; Link; Lysine; Malignant Neoplasms; MDM2 gene; Mediating; Metabolism; Methods; Modality; Molecular; Monitor; Morning Sickness; Morphologic artifacts; Mothers; multicatalytic endopeptidase complex; mutant; Neurodegenerative Disorders; Nobel Prize; novel; novel therapeutics; Nuclear Receptors; overexpression; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Phosphotransferases; pill; Play; Polyubiquitin; Polyubiquitination; Process; Property; Protac; protein degradation; protein transport; Proteins; Proteolysis; receptor function; reconstitution; Recording of previous events; recruit; Regulation; Revlimid; Ring Finger Domain; Role; screening; Signal Transduction; small molecule; Specificity; Structure; Structure-Activity Relationship; System; Technology; Testing; Thalidomide; Therapeutic Effect; therapeutic target; Tissues; transcription factor; Ubiquitin; ubiquitin ligase; ubiquitin-protein ligase; Ubiquitination; Work

Ubiquitin (Ub) tags regulate multiple properties and functions of proteins in cells. Proteasomal degradation oftarget proteins is a well-established means whereby the Ub proteasome system (UPS) controls protein content.Enzymes called Ub E3 ligases conjugate Ub to target proteins by forming an isopeptide bond between the ε-amino group of the target protein lysine and the carboxylate of the C-terminal glycine of Ub. Conjugation ofmultiple Ubs forms poly-Ub chains at any of its seven lysines (K), and K6, K11, K27, K29, K33, K48, and K63Ub chains having various roles are present in all tissues. Recently, several groups designed small moleculesthat bind to an E3 ligase at one end and a target protein at the other, physically facilitating ubiquitylation of thetarget protein, which is then degraded. This hijacking of a ligase to ubiquitylate a desired protein has launcheda new class of drug called PROTACs (PROteolysis TArgeting Chimeras). PROTAC-based approaches fortherapeutics offer several advantages: 1) selective, catalytic degradation of the target; 2) conversion of weakbinders into selective PROTAC drugs; 3) degradation of overexpressed or mutant targets; and 4) maximaldegradation from limited target engagement. To date, cereblon and VHL ligase binders have been mostcommonly used as vehicles to ubiquitylate target proteins such as nuclear receptors, kinases, transcriptionfactors, and neuronal proteins tau and α-synuclein. A major problem has hindered development of new PROTACdrugs, however. Chemical optimization of PROTAC molecules depends on rapid evaluation of synthesizedcompounds to guide the synthetic strategy for producing drug candidate molecules. Assays currently availableare labor intensive and do not provide results to the medicinal chemists fast enough - often, a week is required.In Phase I, a facile in vitro method employing Ub ligases cereblon and HDM2 was developed to screen forpotential PROTAC drugs; PROTAC-mediated ubiquitylation of selected proteins was recapitulated in vitro in away that mimics observed PROTAC-dependent ubiquitination and degradation of these proteins in vivo,achieving the aims of Phase I. In phase II, the utility of this method will be expanded to include representativemembers of all Ub ligase families (cullin families, RING finger ligases, Hect family ligases, and SUMO ligase),increasing the biochemical and chemical space for PROTAC drug discovery. To scale up PROTAC screens, amicrotiter plate-based, high throughput method will be established to monitor in vitro PROTAC drug discovery,and biochemical and Ub mass spec proteomics will be employed to demonstrate that target protein lysinesubiquitylated in vitro are correlated with in vivo PROTAC mediated degradation of target proteins.Commercialization of the microtiter plate based PROTAC system will have a major impact on academic researchas well as PROTAC drug discovery.

Public Health Relevance Statement:
PROTACs, bifunctional small molecules that bind to a ubiquitin E3 ligase at one end and a target protein at the other, degrading the bound target protein, are a promising new drug class. To produce an optimal therapeutic molecule, it is necessary to test large numbers of PROTACs quickly, so that the best candidate can be advanced efficiently to the clinic. LifeSensors has developed a rapid test that can be performed on large numbers of molecules simultaneously; such a test will be optimized for commercial use in PROTAC drug development.

Project Terms:
<τ Proteins><20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><α-syn><α-synuclein>