Nuclear receptor-binding SET domain protein 3 (NSD3) is a histone H3K36 methyltransferase implicated in lung squamous cell carcinoma, yet chemical targeting is challenging due to the shallow PWWP reader pocket. Here, we report XSY12, a hydrophobic-tag degrader of the NSD3 PWWP domain that enables cellular NSD3 depletion (DC50 = 2.21 μM; Dmax = 80.2%). AI-guided discovery using a 3D fingerprinting platform (TF3P) identified a new NSD3-PWWP chemotype, which was optimized via deep learning-based molecular generation to the high-affinity ligand SYC2 (KD = 0.07 μM) and subsequently converted into XSY12. XSY12 promotes proteasome-dependent NSD3 depletion accompanied by HSP90-associated signatures, reduces H3K36 methylation, and induces apoptosis and G2/M arrest in NSD3-dependent models. In vivo, XSY12 achieved measurable exposure and significant tumor growth inhibition in an LUSC xenograft model at 100 mg/kg with acceptable tolerability. Collectively, these results provide a practical workflow linking AI-guided ligand discovery to functional degrader development.
Si-Yu Xiu, Zhenyu Jia, Qi Li et al.· Journal of Medicinal Chemist...· 0 citations
ABSTRACT The clinical management of invasive aspergillosis (IA) is limited by the scarcity of antifungal agents and the emergence and spread of drug resistance, which impairs therapeutic efficacy and poses a significant public health threat. Olorofim, a first-in-class clinical antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapeutic option for IA. It shows activity against several filamentous fungi, including Aspergillus spp., both in vitro and in vivo. Olorofim-resistant Aspergillus fumigatus isolates, characterized by various amino acid substitutions (with a hotspot at G119), have been generated under laboratory conditions through olorofim exposure. However, the potential olorofim resistance mechanisms in Aspergillus flavus, the second-most common pathogen causing IA globally, remain entirely unexplored. In this study, we revealed that the residue G118 in A. flavus, conserved in many filamentous fungi, is homologous to G119 in A. fumigatus by DHODH amino acid sequence alignment. Using a CRISPR-based point mutation approach, we further confirmed that substitutions at the DHODH G118 residue confer acquired olorofim resistance in A. flavus. Additionally, we established a Galleria mellonella infection model and observed that these resistant isolates exhibited no obvious fitness cost, and olorofim treatment was ineffective against infections caused by DHODH G118 mutants. Molecular docking models further indicated that substitutions at the G118 locus may introduce steric hindrance, reducing the binding affinity between olorofim and the DHODH active site and thereby driving resistance. IMPORTANCE Invasive aspergillosis, a life-threatening infection in immunocompromised patients, is increasingly difficult to treat due to limited antifungal options and rising resistance. Olorofim, a novel antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapy. While resistance in Aspergillus fumigatus has been linked to DHODH G119 mutations, the mechanisms in the globally prevalent pathogen Aspergillus flavus remained unknown. Using CRISPR-based mutagenesis, we demonstrate that amino acid substitutions at G118 confer olorofim resistance, supported by molecular docking showing reduced drug binding. Importantly, olorofim failed to protect larvae infected with resistant mutants in an infection model. These findings provide essential insights for proactively monitoring and managing olorofim resistance in Aspergillus flavus, aiding clinical and public health responses. This study is registered with ClinicalTrials.gov as NCT05101187 and NCT06969703. Invasive aspergillosis, a life-threatening infection in immunocompromised patients, is increasingly difficult to treat due to limited antifungal options and rising resistance. Olorofim, a novel antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapy. While resistance in Aspergillus fumigatus has been linked to DHODH G119 mutations, the mechanisms in the globally prevalent pathogen Aspergillus flavus remained unknown. Using CRISPR-based mutagenesis, we demonstrate that amino acid substitutions at G118 confer olorofim resistance, supported by molecular docking showing reduced drug binding. Importantly, olorofim failed to protect larvae infected with resistant mutants in an infection model. These findings provide essential insights for proactively monitoring and managing olorofim resistance in Aspergillus flavus, aiding clinical and public health responses. This study is registered with ClinicalTrials.gov as NCT05101187 and NCT06969703.
Hui Xu, Tianyu Liang, Zhongwei Wang et al.· Microbiology spectrum· 0 citations
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