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Abstract: Molecular glues, compounds that bind cooperatively at protein–protein interfaces (PPIs), are revolutionizing chemical biology and drug discovery, allowing the modulation of traditional “undruggable” targets. Here, we focus on a native regulatory PPI between the scaffolding protein 14-3-3 and C-RAF, a key component of the MAPK signaling pathway. Extensive drug discovery efforts have focused on the MAPK pathway due to its central role in oncology and developmental disorders (RASopathies). However, the modulation of its protein complexes is underexplored. C-RAF activity is regulated on multiple levels including dimerization, phosphorylation, and complex formation with 14-3-3, which prevents C-RAF activation by binding to a C-RAF sequence centered on phospho-serine 259. We used a fragment-merging approach to design molecular glues that bound to the composite surface of this 14-3-3/C-RAFpS259 complex. Molecular glues stabilized the inhibitory complex up to 300-fold; their glue-based mechanism of action was confirmed by crystallography and biophysical studies. Selectivity among the other RAF isoforms and other RAF phosphorylation sites was evaluated. The best compounds showed excellent selectivity among a broad panel of 80 14-3-3 clients. Cellular assays demonstrated on-target engagement, enhanced phosphorylation levels of C-RAFpS259, and reduced levels of RAF dimerization and ERK phosphorylation. Overall, this approach enabled chemical biology studies for a C-RAF site that was intrinsically disordered prior to 14-3-3 binding and had not been targeted previously. These molecular glues will be useful chemical probes and starting points for drug discovery efforts to modulate native PPI stabilization in the MAPK pathway with applications in oncology and RASopathies.

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Abstract: DNA polymerase theta (Polθ), which mediates microhomology-mediated end joining (MMEJ) in homologous recombination-deficient (HRD) cancers, has recently emerged as a compelling synthetic lethal target. Combining Polθ inhibition with PARP inhibitors represents a promising strategy to overcome PARP inhibitor resistance. Here, we present the discovery of SY-589, a highly potent (ATPase IC50 = 2.29 nM), selective (selectivity index >1800), and orally bioavailable (F = 107%) Polθ helicase inhibitor, which exhibits robust antitumor efficacy in HRD tumors in vitro (CTG IC50 = 2.71 nM). Notably, SY-589 synergized strongly with the PARP inhibitor Olaparib in vitro (Loewe score >20) and in vivo (TGI = 109%), enhancing antitumor effects while permitting reduced Olaparib dosing. Overall, SY-589 is a promising candidate of Polθ inhibitor and has been positioned as a rational combination partner with PARP inhibitors, aiming to overcome PARP inhibitor resistance and mitigate their dose-limiting toxicities.

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Abstract: Fragment-based inhibitor design is an established and widely used approach in drug discovery pipelines. Despite several examples of drugs originating from this approach, the identification of fragments still suffers from issues with solubility, reactivity, cost and worldwide accessibility. Here, we design a low-cost minimal fragment library (LoCoFrag100) for crystallographic screening, with an average cLogP of 0.03 (median 0.23) and an average of £20/g for each compound, facilitating assembly in any laboratory. Formatted in a 10 × 10 matrix to minimize Tanimoto similarity in the 20 cocktails, we demonstrate its applicability on three structurally distinct enzymes involved in microbial cell wall synthesis. Hit rates range from 1 to 6% among these enzymes, with three fragments suggesting avenues for inhibitor exploration.

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Abstract: RAF activation is essential for MAPK signaling and is mediated by RAS binding and the dephosphorylation of a conserved phosphoserine by the SHOC2–RAS–PP1C complex. MRAS forms a high-affinity SHOC2–MRAS–PP1C (SMP) complex, while canonical RAS isoforms (KRAS, HRAS, NRAS) form analogous but lower-affinity assemblies. Yet, cancers driven by oncogenic KRAS, HRAS, or NRAS remain strongly SHOC2-dependent, suggesting that these weaker complexes contribute to tumorigenesis. To elucidate how canonical RAS proteins form lower-affinity ternary complexes, the cryo-EM structure of the SHOC2–KRAS–PP1C (SKP) complex stabilized by Noonan syndrome mutations is described. The SKP architecture is similar to the SMP complex but forms fewer contacts and buries less surface area due to the absence of MRAS-specific structural features in KRAS that enhance complex stability. RAS inhibitors MRTX1133 and RMC-6236 alter Switch-I/II conformations, thereby blocking SKP assembly more effectively than they disrupt preformed complexes. These RAS inhibitors do not affect SMP formation because they do not bind MRAS. Since MRAS is upregulated in resistance to KRAS inhibition, we characterize a MRAS mutant capable of binding MRTX1133. This MRAS mutant can form an SMP complex, but MRTX1133 blocks its assembly, demonstrating the feasibility of dual SKP and SMP targeting. Overall, our findings define isoform-specific differences in SHOC2–RAS–PP1C complex formation and support a strategy to prevent both SKP and SMP assemblies to overcome resistance in RAS-driven cancers.

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Abstract: Zinc finger antiviral protein (ZAP) is a cytoplasmic protein central to host innate immunity to viral infection. ZAP has no intrinsic catalytic activity but inhibits viral replication by binding to CpG dinucleotides in cytoplasmic viral RNA and recruiting other factors to inhibit protein synthesis and target the RNA for degradation. KHNYN is a ZAP-binding protein required for ZAP-restriction of CpG-rich viral genomes. It contains an extended diKH, PIN nuclease, and CUElike domain, each of which are required for ZAP restriction of viral replication. Here, we report a structural, enzymological, and virological study of KHNYN’s essential PIN nuclease domain. Our crystal structure reveals an extended PIN domain (ex-PIN) containing a conserved N-terminal arm region required for domain stability and an active site tetra-Asp motif, which are both required for antiviral activity. Unlike the weak activity recently reported for the PIN domain, we demonstrate that the KHNYN ex-PIN domain is a highly active Mn2+-dependent single-stranded RNA endonuclease that cleaves with a preference for ApC, ApA, and UpA dinucleotides. These observations extend our view of KHNYN antiviral activity and suggest an unforeseen role for activation by manganese ions in the ZAP–KHNYN antiviral response.