Kanak Raina

Small-molecule hydrophobic tagging–induced degradation of HaloTag fusion proteins

The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules that bind a bacterial dehalogenase (HaloTag protein) and present a hydrophobic group on its surface. Remarkably, hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated, and transmembrane fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting RasG12V-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models.

PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer

Significance We describe the development of a small molecule that mediates the degradation of bromodomain and extra-terminal (BET) proteins and its application in the treatment of castration-resistant prostate cancer (CRPC). Few therapeutic options exist to treat CRPC, especially CRPC tumors expressing constitutively active androgen receptor (AR) splice variants that lack the ligand-binding domain and can effect androgen-independent transactivation of target genes. Importantly, we demonstrate that targeted degradation of BET proteins using proteolysis-targeting chimera (PROTAC) technology causes cell death in cultured prostate cancer cells and results in tumor growth inhibition or regression in mouse models of CRPC, including models that express high levels of AR splice variant 7. Our work thus contains a significant potential therapeutic advance in the treatment of this cancer. Prostate cancer has the second highest incidence among cancers in men worldwide and is the second leading cause of cancer deaths of men in the United States. Although androgen deprivation can initially lead to remission, the disease often progresses to castration-resistant prostate cancer (CRPC), which is still reliant on androgen receptor (AR) signaling and is associated with a poor prognosis. Some success against CRPC has been achieved by drugs that target AR signaling, but secondary resistance invariably emerges, and new therapies are urgently needed. Recently, inhibitors of bromodomain and extra-terminal (BET) family proteins have shown growth-inhibitory activity in preclinical models of CRPC. Here, we demonstrate that ARV-771, a small-molecule pan-BET degrader based on proteolysis-targeting chimera (PROTAC) technology, demonstrates dramatically improved efficacy in cellular models of CRPC as compared with BET inhibition. Unlike BET inhibitors, ARV-771 results in suppression of both AR signaling and AR levels and leads to tumor regression in a CRPC mouse xenograft model. This study is, to our knowledge, the first to demonstrate efficacy with a small-molecule BET degrader in a solid-tumor malignancy and potentially represents an important therapeutic advance in the treatment of CRPC.

HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins

Small molecule-induced protein degradation is an attractive strategy for the development of chemical probes. One method for inducing targeted protein degradation involves the use of PROTACs, heterobifunctional molecules that can recruit specific E3 ligases to a desired protein of interest. PROTACs have been successfully used to degrade numerous proteins in cells, but the peptidic E3 ligase ligands used in previous PROTACs have hindered their development into more mature chemical probes or therapeutics. We report the design of a novel class of PROTACs that incorporate small molecule VHL ligands to successfully degrade HaloTag7 fusion proteins. These HaloPROTACs will inspire the development of future PROTACs with more drug-like properties. Additionally, these HaloPROTACs are useful chemical genetic tools, due to their ability to chemically knock down widely used HaloTag7 fusion proteins in a general fashion.

Identification of hydrophobic tags for the degradation of stabilized proteins.

New HyTs are a knockout: we previously reported that labeling HaloTag proteins with low molecular weight hydrophobic tags (HyTs) leads to targeted degradation of HaloTag fusion proteins. In this report, we employed a chemical approach to extend this hydrophobic tagging methodology to highly stabilized proteins by synthesizing and evaluating a library of HyTs, which led to the identification of HyT36.

Chemical Inducers of Targeted Protein Degradation

The functions of many cellular proteins have been elucidated by selective gene inactivation and subsequent phenotypic analysis. For example, genetic mutations, gene knock-out generation, and the use of RNA interference to target mRNA for degradation can all result in decreased production of a specific protein, yielding informative cellular phenotypes. However, these techniques each have certain inherent limitations. This minireview focuses on the recent development of new approaches to study protein function at the post-translational level, namely chemical induction of targeted protein degradation.

Targeted Protein Destabilization Reveals an Estrogen-mediated ER Stress Response

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) of eukaryotic cells leads to an unfolded protein response (UPR) that either restores homeostasis or commits the cells to apoptosis. Tools traditionally used to study the UPR are pro-apoptotic and thus confound analysis of long-term cellular responses to ER stress. Here, we describe an Endoplasmic Reticulum-localized HaloTag (ERHT) protein that can be conditionally destabilized using a small molecule hydrophobic tag (HyT36). Treatment of ERHT-expressing cells with HyT36 induces an acute, resolvable ER stress that results in transient UPR activation without induction of apoptosis. Transcriptome analysis of late-stage responses to this UPR stimulus reveals a link between UPR activity and estrogen signaling.

Novel BET protein proteolysis-targeting chimera exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm secondary (s) AML cells

The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Although the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reverse Phase Protein Array and mass cytometry ‘CyTOF’ analyses demonstrated that ARV-825 caused greater perturbations in messenger RNA (mRNA) and protein expressions than OTX015 in sAML cells. Specifically, compared with OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, p-STAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared with OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against established and PD CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.

Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that recruit an E3 ligase to a target protein to facilitate ubiquitination and subsequent degradation of that protein. While the field of targeted degraders is still relatively young, the potential for this modality to become a differentiated and therapeutic reality is strong, such that both academic and pharmaceutical institutions are now entering this interesting area of research. In this article, we describe a broadly applicable process for identifying degrader hits based on the serine/threonine kinase TANK-binding kinase 1 (TBK1) and have generalized the key structural elements associated with degradation activities. Compound 3i is a potent hit (TBK1 DC50 = 12 nM, Dmax = 96%) with excellent selectivity against a related kinase IKKε, which was further used as a chemical tool to assess TBK1 as a target in mutant K-Ras cancer cells.

BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mantle cell lymphoma cells

Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and nuclear factor-κB (NF-κB) target genes that undermines the growth and survival of mantle cell lymphoma (MCL) cells. However, BET bromodomain inhibitor (BETi) treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4 that potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1 and the NF-κB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the levels of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared with ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Cotreatment of ARV-771 with ibrutinib or the BCL2 antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior preclinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or -resistant MCL.

Protein targeting chimeric molecules specific for bromodomain and extra-terminal motif family proteins are active against pre-clinical models of multiple myeloma

Bromodomain and extraterminal (BET) domain containing protein (BRD)-4 modulates the expression of oncogenes such as c-myc, and is a promising therapeutic target in diverse cancer types. We performed pre-clinical studies in myeloma models with bi-functional protein-targeting chimeric molecules (PROTACs) which target BRD4 and other BET family members for ubiquitination and proteasomal degradation. PROTACs potently reduced the viability of myeloma cell lines in a time-dependent and concentration-dependent manner associated with G0/G1 arrest, reduced levels of CDKs 4 and 6, increased p21 levels, and induction of apoptosis. These agents specifically decreased cellular levels of downstream BRD4 targets, including c-MYC and N-MYC, and a Cereblon-targeting PROTAC showed downstream effects similar to those of an immunomodulatory agent. Notably, PROTACs overcame bortezomib, dexamethasone, lenalidomide, and pomalidomide resistance, and their activity was maintained in otherwise isogenic myeloma cells with wild-type or deleted TP53. Combination studies showed synergistic interactions with dexamethasone, BH3 mimetics, and Akt pathway inhibitors. BET-specific PROTACs induced a rapid loss of viability of primary cells from myeloma patients, and delayed growth of MM1.S-based xenografts. Our data demonstrate that BET degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed and/or refractory disease.