SBIR-STTR Award

PROteolysis TArgeting Chimeras (PROTACs) is a new therapeutic class comprised of small molecules binding a target protein and a ubiquitin (Ub) E3 ligase, enabling selective target degradation. PROTACs’ advantages include exquisite selectivity, tolerance of weak binders, and maximal degradation with limited target engagement. The first PROTACs employed the E3 ligase component pVHL to degrade target proteins. Around the same time, thalidomide and related analogs (IMiDs) were successfully repurposed as anti-cancer agents, and subsequently their ubiquitin ligase-associated molecular mechanism was discovered, when thalidomide was shown to bind to cereblon (CRBN), a pVHL-like subunit of a Cullin 4-type ubiquitin ligase. IMiDs promote interaction of this E3 ligase transcription factors that control T cell immunity. In cells, Ub-mediated signaling regulates protein content, location, and activity, primarily through protein degradation, and dysregulation of ubiquitin ligases is linked to numerous devastating diseases. Thus, ligases are promising drug targets and vehicles for PROTACs. Of ~700 Ub ligases, only CRBN and pVHL have been exploited for PROTAC development, a process hindered by several issues. There is a disconnect between the rapid synthesis of new PROTACs by chemists and the time- consuming, artifact-susceptible immunoblot cell assays used to evaluate them. Moreover, binding of a PROTAC to its target does not ensure degradation, owing to steric hindrance, ubiquitylation at the wrong site or in the wrong chain configuration (K63 vs K48), or metabolism inside or poor penetrance into cells. Thus, it is difficult for PROTAC chemists to develop meaningful structure activity relationships, which are essential for preclinical development. It is proposed here to develop ligase-selective, high-throughput cellular assays for PROTAC- mediated ubiquitylation of target proteins. In phase I, LifeSensors will employ unique affinity matrices called TUBEs (Tandem Ubiquitin Binding Entities) in a high throughput mode to analyze ubiquitylation patterns of endogenous proteins in cells. This approach offers the potential to expedite discovery of novel PROTACs, establish a relationship between ubiquitylation and degradation, eliminate low throughput, time-consuming western blot analysis, and lead to the timely identification and development of novel PROTAC drugs, as medicinal chemists will be able to design PROTACs efficiently and rationally, eventually encompassing both degradative and non-degradative ubiquitylation. Phase I will be accomplished by establishing a clear relationship between PROTAC-mediated ubiquitylation and degradation for CRBN and HDM2 ligase target proteins in cells and adapting LifeSensors’s TUBEs technology to monitor PROTAC ubiquitylation and degradation in a high throughput fashion in cells. In Phase II the technology will be expanded to entire ligase families.

Public Health Relevance Statement:
PROteolysis TArgeting Chimeras (PROTACs) are novel drugs that bind both disease-related proteins and the ubiquitin ligase enzymes that ultimately degrade them. Thus, they have the potential to eliminate very selectively the causes of disease with no side effects. It has been difficult to evaluate large numbers of PROTACs in cells because the assays used have been cumbersome, thereby hindering their development. LifeSensors will configure very efficient high throughput cellular assays to accomplish this using its ubiquitin binding technology and will employ these assays to relieve the current bottleneck and allow rapid development of PROTAC drugs for the clinic.

Project Terms:
Affinity; Amyloid beta-Protein; analog; Antineoplastic Agents; Autophagocytosis; Autophagosome; base; Binding; Biological Assay; cancer therapy; Cell model; Cells; Cellular Assay; chimera drug; Clinic; Communities; Consumption; Cullin Proteins; Cultured Cells; design; Development; Disease; drug discovery; Drug Targeting; Drug vehicle; Enzymes; Family; Glues; Goals; Health; Human Genome; immune function; Immunity; Immunomodulators; innovation; Lead; Ligase; Link; Location; Lysosomes; MDM2 gene; Measures; Mediating; member; Metabolism; Methods; misfolded protein; Modality; Modeling; Molecular; Monitor; Morphologic artifacts; multicatalytic endopeptidase complex; mutant; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; novel; novel strategies; novel therapeutics; overexpression; Pattern; Penetrance; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Play; Polymers; preclinical development; Process; Protac; protein aggregation; protein degradation; protein transport; Proteins; Research; Research Personnel; Revlimid; Role; side effect; Signal Transduction; Site; small molecule; Specificity; Structure-Activity Relationship; System; T-Lymphocyte; tau aggregation; tau Proteins; Technology; Testing; Thalidomide; Time; transcription factor; Ubiquitin; ubiquitin ligase; ubiquitin-protein ligase; Ubiquitination; Uncertainty; United States Food and Drug Administration; Western Blotting; Work

Novel Approaches for Tau PROTAC Drug DiscoveryAlzheimer's disease (AD), frontotemporal dementia (FTD), progressive supranuclear palsy (PSP) are a group ofneurodegenerative diseases characterized by the pathological accumulation of hyper-phosphorylated tau (P-tau)protein, in the form of intracellular paired helical filaments (PHFs) or neurofibrillary tangles (NFTs), or aggregates,within neurons and glia of affected brain regions, leading to cell death. Several approaches to remove aggregatesof pathogenic tau have failed, including antibody therapy. Novel approaches to remove aggregated proteins aredesired. Targeted protein degradation by PROteolysis TArgeting Chimeras (PROTACs) has emerged as noveltherapeutic modality. PROTACs are heterobifunctional small molecules that simultaneously bind to a targetprotein and a ubiquitin E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target.They present a new opportunity to modulate proteins in a manner independent of enzymatic or signaling activity.PROTAC drugs offer many advantages, such as: 1) target can be selectively degraded with catalytic specificity;2) weak binders can be converted into selective PROTAC drugs; 3) overexpressed or mutant targets can bedegraded; and 4) limited target engagement can lead to maximal degradation. Although most misfolded andaggregated proteins in the human proteome can be degraded by proteasomal system, some native and mutantproteins prone to aggregation, such as tau oligomers are resistant to all known proteolytic pathways thus subjectto autophagic or lysosomal pathway. Development of PROTACs that can efficiently degrade pathogenic tauaggregates is an attractive and breakthrough therapy for AD and FTD. No one, industry or academia has showncredible data for tau PROTACs to succeed in the clinic. Traditional methods for PROTAC analysis, western blotor reporter gene analysis are time consuming, cumbersome, and prone to artifacts. In this proposal we describea high throughput method for rapid development of tau PROTACs. Development of PROTACs that promote taudegradation by lysosomal/autophagic mechanism is challenging. This grant proposes strategies to screeningdifferent E3 ubiquitin ligases for tau PROTAC that will remove pathogenic (monomeric) as well as aggregatesthrough autophagic mechanism. Goal of the project is to establish rules that govern pathogenic tau degradations,such as poly-ubiquitin signatures conjugated on tau aggregates to differentiate proteasomal vs lysosomaldegradation. Development of the technology that degrades tau aggregates can be directly applied to Parkinson'sdisease, Huntingdon disease and Amyotrophic Lateral Scleroses.

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. We describe methods for rapid development of tau PROTACs drugs. We have proposed strategies to discover novel PROTACs that will remove pathogenic (monomeric) tau proteins as well as aggregates through autophagic mechanism. Development of the technology that degrades tau aggregates can be directly applied to Parkinson's disease, Huntingdon disease, and Amyotrophic Lateral Scleroses.

Project Terms:
<τ Proteins><20S Catalytic Proteasome><20S Core Proteasome><20S Proteasome><20S Proteosome><τ mutation><τ aggregation>