Targeting Nuclear Receptors with PROTAC degraders.

Nuclear receptors comprise a class of intracellular transcription factors whose major role is to act as sensors of various stimuli and to convert the external signal into a transcriptional output. Nuclear receptors (NRs) achieve this by possessing a ligand binding domain, which can bind cell permeable agonists, a DNA-binding domain, which binds the upstream sequences of target genes, and a regulatory domain that recruits the transcriptional machinery. The ligand binding alters the activation state of the NR, either by activating or inactivating its transcriptional output. Given the central role of NRs in signal transduction, many currently approved therapeutics modulate the activity of NRs. Here we discuss how PROTAC degraders afford a novel approach to abrogate the downstream signaling activity of NRs. We highlight six broad functional reasons why PROTAC degraders are preferable to the classical ligand binding pocket antagonists, with specific examples provided for each category. Lastly, as Androgen Receptor and Estrogen Receptor PROTAC degraders are being pursued as treatment for prostate cancer and breast cancer, respectively, a rationale is provided for the translational utility for the degradation of these two NRs.

Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of prostate cancer drug resistance.

The androgen receptor is a major driver of prostate cancer and inhibition of its transcriptional activity using competitive antagonists, such as enzalutamide remains a frontline therapy for prostate cancer management. However, the majority of patients eventually develop drug resistance. We propose that targeting the androgen receptor for degradation via Proteolysis Targeting Chimeras (PROTACs) will be a better therapeutic strategy for targeting androgen receptor signaling in prostate cancer cells. Here we perform a head-to-head comparison between a currently approved androgen receptor antagonist enzalutamide, and its PROTAC derivative, ARCC-4, across different cellular models of prostate cancer drug resistance. ARCC-4 is a low-nanomolar androgen receptor degrader able to degrade about 95% of cellular androgen receptors. ARCC-4 inhibits prostate tumor cell proliferation, degrades clinically relevant androgen receptor point mutants and unlike enzalutamide, retains antiproliferative effect in a high androgen environment. Thus, ARCC-4 exemplifies how protein degradation can address the drug resistance hurdles of enzalutamide.

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.

Targeted protein degradation by PROTACs.

Targeted protein degradation using the PROTAC technology is emerging as a novel therapeutic method to address diseases driven by the aberrant expression of a disease-causing protein. PROTAC molecules are bifunctional small molecules that simultaneously bind a target protein and an E3-ubiquitin ligase, thus causing ubiquitination and degradation of the target protein by the proteasome. Like small molecules, PROTAC molecules possess good tissue distribution and the ability to target intracellular proteins. Herein, we highlight the advantages of protein degradation using PROTACs, and provide specific examples where degradation offers therapeutic benefit over classical enzyme inhibition. Foremost, PROTACs can degrade proteins regardless of their function. This includes the currently "undruggable" proteome, which comprises approximately 85% of all human proteins. Other beneficial aspects of protein degradation include the ability to target overexpressed and mutated proteins, as well as the potential to demonstrate prolonged pharmacodynamics effect beyond drug exposure. Lastly, due to their catalytic nature and the pre-requisite ubiquitination step, an exquisitely potent molecules with a high degree of degradation selectivity can be designed. Impressive preclinical in vitro and in vivo PROTAC data have been published, and these data have propelled the development of clinically viable PROTACs. With the molecular weight falling in the 700-1000Da range, the delivery and bioavailability of PROTACs remain the largest hurdles on the way to the clinic. Solving these issues and demonstrating proof of concept clinical data will be the focus of many labs over the next few years.

Small-Molecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging.

Androgen receptor (AR)-dependent transcription is a major driver of prostate tumor cell proliferation. Consequently, it is the target of several antitumor chemotherapeutic agents, including the AR antagonist MDV3100/enzalutamide. Recent studies have shown that a single AR mutation (F876L) converts MDV3100 action from an antagonist to an agonist. Here we describe the generation of a novel class of selective androgen receptor degraders (SARDs) to address this resistance mechanism. Molecules containing hydrophobic degrons linked to small-molecule AR ligands induce AR degradation, reduce expression of AR target genes and inhibit proliferation in androgen-dependent prostate cancer cell lines. These results suggest that selective AR degradation may be an effective therapeutic prostate tumor strategy in the context of AR mutations that confer resistance to second-generation AR antagonists.

A bidirectional system for the dynamic small molecule control of intracellular fusion proteins.

Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds.

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.

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 to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-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.

A genome-wide screen for regulators of TORC1 in response to amino acid starvation reveals a conserved Npr2/3 complex.

TORC1 is a central regulator of cell growth in response to amino acid availability, yet little is known about how it is regulated. Here, we performed a reverse genetic screen in yeast for genes necessary to inactivate TORC1. The screen consisted of monitoring the expression of a TORC1 sensitive GFP-based transcriptional reporter in all yeast deletion strains using flow cytometry. We find that in response to amino acid starvation, but not to carbon starvation or rapamycin treatment, cells lacking NPR2 and NPR3 fail to fully (1) activate transcription factors Gln3/Gat1, (2) dephosphorylate TORC1 effector Npr1, and (3) repress ribosomal protein gene expression. Both mutants show proliferation defects only in media containing a low quality nitrogen source, such as proline or ammonia, whereas no defects are evident when cells are grown in the presence of glutamine or peptone mixture. Proliferation defects in npr2Delta and npr3Delta cells can be completely rescued by artificially inhibiting TORC1 by rapamycin, demonstrating that overactive TORC1 in both strains prevents their ability to adapt to an environment containing a low quality nitrogen source. A biochemical purification of each demonstrates that Npr2 and Npr3 form a heterodimer, and this interaction is evolutionarily conserved since the human homologs of NPR2 and NPR3 (NPRL2 and NPRL3, respectively) also co-immunoprecipitate. We conclude that, in yeast, the Npr2/3 complex mediates an amino acid starvation signal to TORC1.

Superoxide anions regulate TORC1 and its ability to bind Fpr1:rapamycin complex.

The small natural product rapamycin, when bound to FKBP12, is a potent inhibitor of an evolutionarily conserved Target of Rapamycin Complex 1 (TORC1), which plays a central role in mediating cellular response to nutrient availability. Given the prominent role of TORC1 in cell growth and proliferation, clinical trials have explored the possibility of using rapamycin as an anticancer agent. Unfortunately, the percentage of patients responding favorably has been low, intensifying the need to find biomarkers able to predict rapamycin sensitivity or resistance. In this study, we elucidate the molecular mechanism underlying partial rapamycin resistance in yeast. Using the yeast deletion collection, we identified 15 deletion strains leading to partial rapamycin resistance. Among these were Cu/Zn-superoxide dismutase Sod1, copper transporter Ctr1, and copper chaperone Lys7, suggesting a role for oxidative stress in rapamycin resistance. Further analysis revealed that all 15 strains exhibit elevated levels of superoxide anions, and we show that elevated levels of reactive oxygen species specifically modify TORC1 such that it is no longer able to fully bind FKBP12:rapamycin. Therefore, elevated oxidative stress modifies TORC1 and prevents its binding to the FKBP12:rapamycin complex, ultimately leading to rapamycin resistance. These results warrant an examination into whether similar reasons explain rapamycin resistance observed in various clinical samples.