Targeted protein stabilization using Protein Rescue Targeting Chimeras (PRESTACs)

Targeting the ubiquitin pathway is a potential option for treating a variety of devastating diseases, including cancers and neurodegeneration. Recent focus on heterobifunctional proteolysis targeting chimeras, (PROTACs), which recruit E3 ligases to targets of interest for ubiquitination and degradation of the target, is a promising therapeutic avenue. PROTACs have been developed to degrade a wide range of protein families, and new targets are continually being investigated. In cells, ubiquitin conjugation by ligases is reversed by de- conjugating proteases (DUBs), and the dynamics of these two enzyme classes regulates protein turnover, compartmentation, and other processes. Some cell pathologies are driven by excessive degradation of a key protein, for example, a tumor suppressor, and the pathological state can be corrected by attenuating degradation, thereby sparing the critical protein. Formally, DUBs could be recruited by a heterobifunctional molecule to deubiquitinate target proteins and prevent their degradation, and it is therefore hypothesized that heterobifunctional molecules called Protein Rescue Targeting Chimeras (PRESTACs) can be generated by chemically linking a DUB ligand to a target ligand and evaluated for their physiological function. Progenra has identified and characterized novel small molecule USP7 ligands that bind USP7 and activate its DUB function. These molecules, along with ligands for targets of interest, will be used to design and synthesize PRESTACs, which will represent the first tethering of DUB activity to disease-relevant targets and provide a novel tool for expansion of UPS-based therapies. As proof of concept, bifunctional molecules will be synthesized to recruit the DUB USP7 to stabilize the nuclear receptor PPAR, for which several agonists are known. Thiazolidinediones (TZDs) targeting PPAR treat type 2 diabetes by inducing genes that decrease insulin resistance. Ligand binding of TZDs to PPAR not only regulates transcription, but also leads to PPAR ubiquitination and proteasomal degradation. Thus, stabilizing PPAR will prolong its activation and augment the effects of these drugs. PRESTACs will be designed using Progenra’s USP7 activators and rosiglitazone, a TZD whose interaction with and effects on PPAR are well-characterized. PRESTAC candidates will be evaluated biochemically, biophysically, and in NIH 3T3 cells for their ability to form ternary complexes (USP7-PRESTAC-PPAR), to stabilize PPAR, and to affect downstream targets of PPAR activation (for example, PPAR reporter assays, levels of adiponectin).Recently, a new class of therapeutic molecule, called a PROTAC, entered clinical trial; it was a bifunctional molecule with one end binding a ubiquitin ligase and the other end binding a protein to be degraded for the therapeutic effect of eliminating a harmful protein from the cell. Progenra is proposing to develop a novel class of heterobifunctional molecule (PRESTACs), with one end binding to a ubiquitin deconjugating enzyme (DUB) and other end binding to a protein to be rescued from proteolysis for purposes of preserving a beneficial protein.