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The anticancer 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 the DCAF15–RBM39 interaction, we solved the DCAF15–DDB1–DDA1–indisulam–RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-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 additional targets. The crystal and cryo-electron microscopy structure analysis of the DCAF15–DDB1–DDA1–indisulam–RBM39 complex revealed the detailed mechanism of action of indisulam-induced RBM39 degradation and defined an α-helical degron motif in RBM39.

Target-anchored monovalent degraders are more drug-like than their bivalent counterparts, Proteolysis Targeting Chimeras (PROTACs), while offering greater target specificity control than E3 ligase-anchored monovalent degraders, also known as molecular glues. However, their discovery has typically been serendipitous, and the rules governing their identification remain unclear. This study focuses on the intentional discovery of SMARCA2/A4 monovalent degraders using a library based on SMARCA2/A4 bromodomain-binding ligands. Compound G-6599 emerged as a lead candidate, showing exceptional degradation potency and specificity for SMARCA2/A4. Mechanistic studies reveal that G-6599 operates through the ubiquitin-proteasome pathway and the E3 ligase FBXO22. G-6599 promotes ternary complex formation between SMARCA2 and FBXO22 involving covalent conjugation to a cysteine residue on the latter. Unlike other recently identified FBXO22-dependent degraders, it does not require biotransformation. The selective degradation ability of G-6599 , along with its unique mechanism, highlights the therapeutic potential of target-anchored monovalent degraders. Degraders of SMARCA2/4 have so far relied on bivalent designs. Here, a targeted discovery campaign identified the first monovalent degraders, revealing a highly potent, selective compound that recruits FBXO22 through a covalent mechanism.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRAS G12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRAS G12C covalent modification in vivo and can be used to investigate response heterogeneity to KRAS G12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRAS G12C switch II region (SWII) by stabilizing a specific conformation of KRAS G12C , thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins. Antibodies that lock KRAS mutants in inactive conformations facilitate drug discovery.

In pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine-scanning of the peptide identified three key residues, substitution of each of which results in ~1000 fold loss of binding affinity with a concomitant reduction in antiviral activity. Structural studies demonstrate specific binding of the peptide to the pore of pentameric VP1. Cell-based assays demonstrate nanomolar inhibition (EC50) of BKV infection and suggest that the peptide acts early in the viral entry pathway. Homologous peptide exhibits similar binding to JC polyomavirus VP1 and inhibits infection with similar potency to BKV in a model cell line. Lastly, these studies validate targeting the VP1 pore as a novel strategy for the development of anti-polyomavirus agents.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transient affinity binding interactions are central to life, composing the fundamental elements of biological networks including cell signaling, cell metabolism and gene regulation. Assigning a defined reaction mechanism to affinity binding interactions is critical to our understanding of the associated structure-function relationship, a cornerstone of biophysical characterization. Transient kinetics are currently measured using low throughput methods such as nuclear magnetic resonance, or stop-flow spectrometry-based techniques, which are not practical in many settings. In contrast, label-free biosensors measure reaction kinetics through direct binding, and with higher throughout, impacting life sciences with thousands of publications each year. Here we have developed a methodology enabling label-free biosensors to measure transient kinetic interactions towards providing a higher throughput approach suitable for mechanistic understanding of these processes. The methodology relies on hydrodynamic dispersion modeling of a smooth analyte gradient under conditions that maintain the quasi-steady-state boundary layer assumption. A transient peptide-protein interaction of relevance to drug discovery was analyzed thermodynamically using transition state theory and numerical simulations validated the approach over a wide range of operating conditions. The data establishes the technical feasibility of this approach to transient kinetic analyses supporting further development towards higher throughput applications in life science.

In pursuit of effective therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen amino acid peptide binds to major structural protein VP1 in a new location within the pore with a low nanomolar KD. Alanine scanning of the peptide identified three key residues, substitution of each of which results in ~1000-fold loss of affinity with a concomitant reduction in antiviral activity. NMR spectroscopy and an X-ray structurally-guided model demonstrate specific binding of the peptide to the pore of the VP1 pentamer that constitutes the BKV capsid. Cell-based assays with the peptide demonstrate nanomolar inhibition of BKV infection and suggest that the peptide likely blocks the viral entry pathway between endocytosis and escape from the host cell ER. The peptide motif is highly conserved among the polyomavirus clade, and homologous peptides exhibit similar binding properties for JC polyomavirus and inhibit infection with similar potency to BKV in a model cell line. Substitutions within VP1 or VP2/3 residues involved in VP1-peptide interaction negatively impact viral infectivity, potentially indicating the peptide-binding site within the VP1 pore is relevant for VP1-VP2/3 interactions. The inhibitory potential of the peptide-binding site first reported here may present a novel target for development of new anti-polyomavirus therapies. In summary, we present the first anti-polyomavirus inhibitor that acts via a novel mechanism of action by specifically targeting the pore of VP1.

ABSTRACT The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multidrug-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.

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 Angstroms. 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.