Rational Screening for Cooperativity in Small-Molecule Inducers of Protein-Protein Associations.

The hallmark of a molecular glue is its ability to induce cooperative protein-protein interactions, leading to the formation of a ternary complex, despite weaker binding toward one or both individual proteins. Notably, the extent of cooperativity distinguishes molecular glues from bifunctional compounds, which constitute a second class of inducers of protein-protein interactions. However, apart from serendipitous discovery, there have been limited rational screening strategies for the high cooperativity exhibited by molecular glues. Here, we propose a binding-based screen of DNA-barcoded compounds on a target protein in the presence or absence of a presenter protein, using the "presenter ratio", the ratio of ternary enrichment to binary enrichment, as a predictive measure of cooperativity. Through this approach, we identified a range of cooperative, noncooperative, and uncooperative compounds in a single DNA-encoded library screen with bromodomain containing protein (BRD)9 and the VHL-elongin C-elongin B (VCB) complex. Our most cooperative hit compound, 13-7, exhibits micromolar binding affinity to BRD9 but nanomolar affinity for the ternary complex with BRD9 and VCB, with cooperativity comparable to classical molecular glues. This approach may enable the rational discovery of molecular glues for preselected proteins and thus facilitate the transition to a new paradigm of small-molecule therapeutics.

Rational screening for cooperativity in small-molecule inducers of protein-protein associations.

The hallmark of a molecular glue is its ability to induce cooperative protein-protein interactions, leading to the formation of a ternary complex, despite weaker binding towards one or both individual proteins. Notably, the extent of cooperativity distinguishes molecular glues from bifunctional compounds, a second class of inducers of protein-protein interactions. However, apart from serendipitous discovery, there have been limited rational screening strategies for the high cooperativity exhibited by molecular glues. Here, we propose a binding-based screen of DNA-barcoded compounds on a target protein in the presence and absence of a presenter protein, using the "presenter ratio", the ratio of ternary enrichment to binary enrichment, as a predictive measure of cooperativity. Through this approach, we identified a range of cooperative, noncooperative, and uncooperative compounds in a single DNA-encoded library screen with bromodomain (BRD)9 and the VHL-elongin C-elongin B (VCB) complex. Our most cooperative hit compound, 13-7 , exhibits micromolar binding affinity to BRD9 but nanomolar affinity for the ternary complex with BRD9 and VCB, with cooperativity comparable to classical molecular glues. This approach may enable the discovery of molecular glues for pre-selected proteins and thus facilitate the transition to a new paradigm of molecular therapeutics.

Chemical investigations into the biosynthesis of the gymnastatin and dankastatin alkaloids.

Electrophilic natural products have provided fertile ground for understanding how nature inhibits protein function using covalent bond formation. The fungal strain Gymnascella dankaliensis has provided an especially interesting collection of halogenated cytotoxic agents derived from tyrosine which feature an array of reactive functional groups. Herein we explore chemical and potentially biosynthetic relationships between architecturally complex gymnastatin and dankastatin members, finding conditions that favor formation of a given scaffold from a common intermediate. Additionally, we find that multiple natural products can also be formed from aranorosin, a non-halogenated natural product also produced by Gymnascella sp. fungi, using simple chloride salts thus offering an alternative hypothesis for the origins of these compounds in nature. Finally, growth inhibitory activity of multiple members against human triple negative breast cancer cells is reported.

Chemoproteomics-enabled discovery of covalent RNF114-based degraders that mimic natural product function.

The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation-a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.

Bardoxolone conjugation enables targeted protein degradation of BRD4.

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3's has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL.

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific proteins in a proteasome-dependent manner. However, a major limitation of TPD is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent recruiter for the E3 ligase RNF114. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or, in some cases, inactivity. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs, and we demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing selectivity for the degradation of neo-substrate proteins.

The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids1,2. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols3,4. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions2, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death5. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines6, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q10 (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents.

Asymmetric Total Synthesis of Lancifodilactone G Acetate. 1. Diastereoselective Synthesis of CDEFGH Ring System.

The stereoselective construction of the CDEFGH ring system of lancifodilactone G is described. The key steps in this synthesis are (i) ring-closing metathesis for formation of the oxa-bridged eight-membered ring; (ii) an intramolecular Pauson-Khand reaction for construction of the sterically congested F ring; and (iii) sequential cross-metathesis, hydrogenation, and lactonization reactions for installation of the anomerically stabilized bis-spiro ketal fragment of lancifodilactone G.

Biomimetically inspired asymmetric total synthesis of (+)-19-dehydroxyl arisandilactone A.

Complex natural products are a proven and rich source of disease-modulating drugs and of efficient tools for the study of chemical biology and drug discovery. The architectures of complex natural products are generally considered to represent significant barriers to efficient chemical synthesis. Here we describe a concise and efficient asymmetric synthesis of 19-dehydroxyl arisandilactone A-which belongs to a family of architecturally unique, highly oxygenated nortriterpenoids isolated from the medicinal plant Schisandra arisanensis. This synthesis takes place by means of a homo-Michael reaction, a tandem retro-Michael/Michael reaction, and Cu-catalysed intramolecular cyclopropanation as key steps. The proposed mechanisms for the homo-Michael and tandem retro-Michael/Michael reactions are supported by density functional theory (DFT) calculation. The developed chemistry may find application for the synthesis of its other family members of Schisandraceae nortriterpenoids.