Highlights d The dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to Nurr1 d DHI forms a covalent adduct with Nurr1, reacting as the indolequinone with Cys566 d The Nurr1-metabolite structure reveals a previously unreported ligand-binding pocket d DHI stimulates the transcription of Nurr1 target genes underlying dopamine homeostasis

The RAS–RAF pathway is one of the most commonly dysregulated in human cancers1–3. Despite decades of study, understanding of the molecular mechanisms underlying dimerization and activation4 of the kinase RAF remains limited. Recent structures of inactive RAF monomer5 and active RAF dimer5–8 bound to 14-3-39,10 have revealed the mechanisms by which 14-3-3 stabilizes both RAF conformations via specific phosphoserine residues. Prior to RAF dimerization, the protein phosphatase 1 catalytic subunit (PP1C) must dephosphorylate the N-terminal phosphoserine (NTpS) of RAF11 to relieve inhibition by 14-3-3, although PP1C in isolation lacks intrinsic substrate selectivity. SHOC2 is as an essential scaffolding protein that engages both PP1C and RAS to dephosphorylate RAF NTpS11–13, but the structure of SHOC2 and the architecture of the presumptive SHOC2–PP1C–RAS complex remain unknown. Here we present a cryo-electron microscopy structure of the SHOC2–PP1C–MRAS complex to an overall resolution of 3 Å, revealing a tripartite molecular architecture in which a crescent-shaped SHOC2 acts as a cradle and brings together PP1C and MRAS. Our work demonstrates the GTP dependence of multiple RAS isoforms for complex formation, delineates the RAS-isoform preference for complex assembly, and uncovers how the SHOC2 scaffold and RAS collectively drive specificity of PP1C for RAF NTpS. Our data indicate that disease-relevant mutations affect complex assembly, reveal the simultaneous requirement of two RAS molecules for RAF activation, and establish rational avenues for discovery of new classes of inhibitors to target this pathway.

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function.Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3Afragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.

The discovery of covalent inhibitors binding the switch II (SWII) pocket has enabled therapeutic intervention in KRAS G12C driven tumors and represents a milestone in targeting KRAS-driven cancers. However, the transient nature and high energetic barrier required for binding this pocket has been an obstacle in successfully targeting other KRAS mutant oncoproteins. We report the discovery of KRAS Conformation Locking Antibodies for Molecular Probe discovery (CLAMP)s that specifically recognize the unique conformation of KRAS G12C induced 5 by covalent inhibitors. KRAS CLAMPs enable single cell resolution of covalent inhibitor-bound KRAS G12C in cells and in vivo tumor models, providing a biomarker for direct target engagement of KRAS G12C inhibition. KRAS CLAMPs bind multiple KRAS mutants and stabilize an open conformation of the SWII pocket increasing the affinity of weak non-covalent SWII pocket ligands. This work provides new insights into KRAS G12C upon treatment with covalent inhibitors and offers a path towards targeting the SWII pocket in other RAS mutants. 10 3 Main: RAS proteins are small, membrane-bound guanine nucleotide-binding proteins encoded by three genes (HRAS, NRAS and KRAS). RAS proteins act as molecular switches by cycling between active GTP-bound and inactive GDP-bound conformations 1 . The active GTP-bound conformation allows RAS to signal to a diverse set of downstream effectors including RAF, PI3K, and RAL GDS 2-11 and oncogenic mutations in RAS, frequently at position 12, reduce GTP hydrolysis resulting in constitutively active RAS signaling [12][13][14][15] . The picomolar affinity for GTP or GDP, in addition to the lack of obvious pockets for small molecule binding in RAS have hampered drug discovery efforts against oncogenic mutant RAS for several decades.The landmark discovery of KRAS G12C inhibitors that covalently modify the mutant Cys12 residue has provided a novel and promising opportunity for drugging KRAS G12C mutant tumors 16 . Compound 12, ARS-853, ARS-1620, AIM-4, and clinical molecules AMG 510 and MRTX849 bind and stabilize an "open" conformation in the switch II (SWII) region not previously observed in KRAS-GDP or KRAS-GTP [16][17][18][19][20][21][22][23] . The mechanism of action of such SWII pocket covalent binders is through stabilization of this transient pocket via initial binding to the pocket followed by chemical reaction with Cys12 17 . This modification irreversibly locks KRAS G12C in a GDP-bound inactive state by preventing intrinsic or SOS-mediated exchange, causes tumor growth inhibition in pre-clinical models, and is showing promising clinical activity 17,18 . Discovery of these KRAS G12C inhibitors relied heavily on the covalent reactivity with Cys12 to inhibit KRAS G12C protein 24 . Thus, the viability of strategies targeting this pocket in other KRAS mutants lacking this critical mutant cysteine residue remains to be determined.

Conventional efforts relying on high-throughput physical and virtual screening of large compound libraries have failed to yield high-efficiency chemical probes for many of the 48 human nuclear receptors. Here, we investigated whether disulfide-trapping, an approach new to nuclear receptors, would provide effective lead compounds targeting human liver receptor homolog 1 (hLRH-1, NR5A2). Despite the fact that hLRH-1 contains a large ligand binding pocket and binds phospholipids with high affinity, existing synthetic hLRH-1 ligands are of limited utility due to poor solubility, low efficacy or significant off-target effects. Using disulfide-trapping, we identified a lead compound that conjugates with remarkably high-efficiency to a native cysteine residue (Cys346) lining the hydrophobic cavity in the ligand binding domain of hLRH-1. Guided by computational modeling and cellular assays, the lead compound was elaborated into ligands PME8 and PME9 that bind hLRH-1 reversibly (no cysteine reactivity) and increase hLRH-1 activity in cells. When compared with the existing hLRH-1 synthetic agonist RJW100, both PME8 and PME9 showed comparable induction of the LRH-1 dependent target gene CYP24A1 in human HepG2 cells, beginning as early as 3 h after drug treatment. The induction is specific as siRNA-mediated knock-down of hLRH-1 renders both PME8 and PME9 ineffective. These data show that PME8 and PME9 are potent activators of hLRH-1 and suggest that with further development this lead series may yield useful chemical probes for manipulating LRH-1 activity in vivo.