Targeting oncoproteins with a positive selection assay for protein degraders.

Most intracellular proteins lack hydrophobic pockets suitable for altering their function with drug-like small molecules. Recent studies indicate that some undruggable proteins can be targeted by compounds that can degrade them. For example, thalidomide-like drugs (IMiDs) degrade the critical multiple myeloma transcription factors IKZF1 and IKZF3 by recruiting them to the cereblon E3 ubiquitin ligase. Current loss of signal ("down") assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal ("up") assay for degraders. In arrayed chemical screens, we identified novel IMiD-like IKZF1 degraders and Spautin-1, which, unlike the IMiDs, degrades IKZF1 in a cereblon-independent manner. In a pooled CRISPR-Cas9-based screen, we found that CDK2 regulates the abundance of the ASCL1 oncogenic transcription factor. This methodology should facilitate the identification of drugs that directly or indirectly degrade undruggable proteins.

Plasticity in binding confers selectivity in ligand-induced protein degradation.

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 a 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 that are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying interprotein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic interprotein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands.

Translation Termination Factor GSPT1 Is a Phenotypically Relevant Off-Target of Heterobifunctional Phthalimide Degraders.

Protein degradation is an emerging therapeutic strategy with a unique molecular pharmacology that enables the disruption of all functions associated with a target. This is particularly relevant for proteins depending on molecular scaffolding, such as transcription factors or receptor tyrosine kinases (RTKs). To address tractability of multiple RTKs for chemical degradation by the E3 ligase CUL4-RBX1-DDB1-CRBN (CRL4CRBN), we synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752. While both series failed to induce degradation of their consensus targets, individual molecules displayed pronounced efficacy in leukemia cell lines. Orthogonal target identification supported by molecular docking led us to identify the translation termination factor G1 to S phase transition 1 (GSPT1) as a converging off-target, resulting from inadvertent E3 ligase modulation. This research highlights the importance of monitoring degradation events that are independent of the respective targeting ligand as a unique feature of small-molecule degraders.

Diastereoselective synthesis of novel heterocyclic scaffolds through tandem Petasis 3-component/intramolecular Diels-Alder and ROM-RCM reactions.

A high-yielding, stereoselective and extraordinarily complexity-generating Petasis 3-component/intramolecular Diels-Alder reaction has been developed. In combination with ROM-RCM, rapid access to complex sp3-rich heterocyclic scaffolds amenable to subsequent functionalization and library synthesis is provided.

Ligand-Promoted Meta-C-H Arylation of Anilines, Phenols, and Heterocycles.

Here we report the development of a versatile 3-acetylamino-2-hydroxypyridine class of ligands that promote meta-C-H arylation of anilines, heterocyclic aromatic amines, phenols, and 2-benzyl heterocycles using norbornene as a transient mediator. More than 120 examples are presented, demonstrating this ligand scaffold enables a wide substrate and coupling partner scope. Meta-C-H arylation with heterocyclic aryl iodides as coupling partners is also realized for the first time using this ligand. The utility for this transformation for drug discovery is showcased by allowing the meta-C-H arylation of a lenalidomide derivative. The first steps toward a silver-free protocol for this reaction are also demonstrated.

Synthesis of heterocycles through transition-metal-catalyzed isomerization reactions.

Metal-catalyzed isomerization of N- and O-allylic systems is emerging as an effective method to form synthetically useful iminium and oxocarbenium intermediates. In the presence of tethered nucleophiles, several recent examples illuminate this approach as a powerful strategy for the synthesis of structurally complex and diverse heterocycles. In this Concept article, we attempt to cover this area of research through a selection of recent versatile examples.

Build/couple/pair strategy combining the Petasis 3-component reaction with Ru-catalyzed ring-closing metathesis and isomerization.

A "build/couple/pair" pathway for the systematic synthesis of structurally diverse small molecules is presented. The Petasis 3-component reaction was used to synthesize anti-amino alcohols displaying pairwise reactive combinations of alkene moieties. Upon treatment with a ruthenium alkylidene-catalyst, these dienes selectively underwent ring-closing metathesis reactions to form 5- and 7-membered heterocycles and cyclic aminals via a tandem isomerization/N-alkyliminium cyclization sequence.

Solid-phase synthesis of smac peptidomimetics incorporating triazoloprolines and biarylalanines.

Apoptotic induction mechanisms are of crucial importance for the general homeostasis of multicellular organisms. In cancer the apoptotic pathways are downregulated, which, at least partly, is due to an abundance of inhibitors of apoptosis proteins (IAPs) that block the apoptotic cascade by deactivating proteolytic caspases. The Smac protein has an antagonistic effect on IAPs, thus providing structural clues for the synthesis of new pro-apoptotic compounds. Herein, we report a solid-phase approach for the synthesis of Smac-derived tetrapeptide libraries. On the basis of a common (N-Me)AVPF sequence, peptides incorporating triazoloprolines and biarylalanines were synthesized by means of Cu(I)-catalyzed azide-alkyne cycloaddition and Pd-catalyzed Suzuki cross-coupling reactions. Solid-phase procedures were optimized to high efficiency, thus accessing all products in excellent crude purities and yields (both typically above 90%). The peptides were subjected to biological evaluation in a live/dead cellular assay which revealed that structural decorations on the AVPF sequence indeed are highly important for cytotoxicity toward HeLa cells.