Abstract: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 hydrophobi… Show more
“…Significant progress has recently been made towards chemically induced targeted protein degradation using heterobifunctional small molecule ligands (often referred to as degraders or PROTACs for PROteolysis-TArgeting Chimeras) 1–10 . Targeted protein degradation refers to small molecule induced ubiquitination and degradation of disease targets, in which a small molecule simultaneously recruits both a ubiquitin E3 ligase and the target protein to be ubiquitinated (Supplementry Fig.…”
SUMMARY
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.
“…Significant progress has recently been made towards chemically induced targeted protein degradation using heterobifunctional small molecule ligands (often referred to as degraders or PROTACs for PROteolysis-TArgeting Chimeras) 1–10 . Targeted protein degradation refers to small molecule induced ubiquitination and degradation of disease targets, in which a small molecule simultaneously recruits both a ubiquitin E3 ligase and the target protein to be ubiquitinated (Supplementry Fig.…”
SUMMARY
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.
“…Crews and colleagues have also devised a hydrophobic tagging strategy by attaching a recognition ligand to adamantyl and other hydrophobic moieties (e.g., HyT13; Figure 1; 12–17 ). Adamantyl-tagged recognition ligands destabilize their target proteins and recruit Hsp70, inducing ubiquitination and eventual degradation via the proteasome 12, 14 .…”
Targeted protein degradation is a promising strategy for drug design and functional assessment. Several small molecule approaches have been developed that localize target proteins to ubiquitin ligases, inducing ubiquitination and subsequent degradation by the 26S proteasome. We discovered that the degradation of a target protein can also be induced by a recognition ligand linked to tert-butyl carbamate (Boc3)-protected arginine (B3A). Here we show that this process requires the proteasome, but does not involve ubiquitination of the target protein. B3A does not perturb the structure of the target protein; instead a B3A-ligand stabilizes its target protein. B3A ligands stimulate activity of purified 20S proteasome, emonstrating that the tag binds directly to the 20S proteasome. Moreover, purified 20S proteasome is sufficient to degrade target proteins in the presence of their respective B3A-linked recognition ligands. These observations suggest a simple model for B3A-mediated degradation wherein the B3A tag localizes target proteins directly to the 20S proteasome. Thus B3A ligands are the first example of a ubiquitin-free strategy for targeted protein degradation.
“…SARD279 showed more potent inhibition of AR-dependent gene expression (IC 50 = 156 nM) than that for AR degradation, indicating a dual mode of activity through competitive inhibition of AR transactivation and AR depletion. Importantly, in contrast with competitive AR antagonists, eliminating AR protein with SARD279 was found to be antiproliferative in AR-dependent prostate cancer cell lines and also in castration-resistant prostate cell lines, whether resistance to antiandrogens resulted from increased androgen levels or from the F876L AR mutation that converts antagonists into agonists [23]. Figure 7.Selected bifunctional molecules directing target degradation through binding of HSP70 or the 20S proteasome.( A ) Cartoons showing the complexes involved in (i) HSP70-dependent protein degradation mediated by a hydrophobic adamantyl tag (HSP70, heat shock protein 70; Ub, ubiquitin) and (ii) direct recruitment of the 20S proteasome by Boc 3 Arg tags.…”
Section: Hijacking E3 Ligases For Specific Target Degradation Using Bmentioning
Manipulation of the ubiquitin–proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel–Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology.
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