The anti-cancer agent Indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which Indisulam mediates DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-Indisulam-RBM39(RRM2) complex structure to 2.3 Å. DCAF15 has a novel topology which embraces the RBM39(RRM2) domain largely via nonpolar interactions, and Indisulam binds between DCAF15 and RBM39(RRM2) and coordinates additional interactions between the two proteins. Studies with RBM39 point mutants and Indisulam analogs validated the structural model and defined the RBM39 alpha-helical degron motif. The degron is found only in RBM23 and RBM39 and only these proteins were detectably downregulated in Indisulam-treated HCT116 cells. This work further explains how Indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade novel targets.

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

Fragment‐based lead discovery has become a fundamental approach to identify ligands that efficiently interact with disease‐relevant targets. Among the numerous screening techniques, fluorine‐detected NMR has gained popularity owing to its high sensitivity, robustness, and ease of use. To effectively explore chemical space, a universal NMR experiment, a rationally designed fragment library, and a sample composition optimized for a maximal number of compounds and minimal measurement time are required. Here, we introduce a comprehensive method that enabled the efficient assembly of a high‐quality and diverse library containing nearly 4000 fragments and screening for target‐specific binders within days. At the core of the approach is a novel broadband relaxation‐edited NMR experiment that covers the entire chemical shift range of drug‐like 19F motifs in a single measurement. Our approach facilitates the identification of diverse binders and the fast ligandability assessment of new targets.

NMR binding assays are routinely applied in hit finding and validation during early stages of drug discovery, particularly for fragment-based lead generation. To this end, compound libraries are screened by ligand-observed NMR experiments such as STD, T1ρ, and CPMG to identify molecules interacting with a target. The analysis of a high number of complex spectra is performed largely manually and therefore represents a limiting step in hit generation campaigns. Here we report a novel integrated computational procedure that processes and analyzes ligand-observed proton and fluorine NMR binding data in a fully automated fashion. A performance evaluation comparing automated and manual analysis results on (19)F- and (1)H-detected data sets shows that the program delivers robust, high-confidence hit lists in a fraction of the time needed for manual analysis and greatly facilitates visual inspection of the associated NMR spectra. These features enable considerably higher throughput, the assessment of larger libraries, and shorter turn-around times.

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.