Targeted degradation of PCNA outperforms stoichiometric inhibition to result in programed cell death.

Biodegraders are targeted protein degradation constructs composed of mini-proteins/peptides linked to E3 ligase receptors. We gained deeper insights into their utility by studying Con1-SPOP, a biodegrader against proliferating cell nuclear antigen (PCNA), an oncology target. Con1-SPOP proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut) as it had more potent anti-proliferative effects and uniquely induced DNA damage, cell apoptosis, and necrosis. Proteomics showed that PCNA degradation gave impaired mitotic division and mitochondria dysfunction, effects not seen with the stoichiometric inhibitor. We further showed that doxycycline-induced Con1-SPOP achieved complete tumor growth inhibition in vivo. Intracellular delivery of mRNA encoding Con1-SPOP via lipid nanoparticles (LNPs) depleted endogenous PCNA within hours of application with nanomolar potency. Our results demonstrate the utility of biodegraders as biological tools and highlight target degradation as a more efficacious approach versus stoichiometric inhibition. Once in vivo delivery is optimized, biodegraders may be leveraged as an exciting therapeutic modality.

Repurposing a Histamine Detection Platform for High-Throughput Screening of Histidine Decarboxylase.

Histidine decarboxylase (HDC) is the primary enzyme that catalyzes the conversion of histidine to histamine. HDC contributes to many physiological responses as histamine plays important roles in allergic reaction, neurological response, gastric acid secretion, and cell proliferation and differentiation. Small-molecule modulation of HDC represents a potential therapeutic strategy for a range of histamine-associated diseases, including inflammatory disease, neurological disorders, gastric ulcers, and select cancers. High-throughput screening (HTS) methods for measuring HDC activity are currently limited. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for monitoring HDC activity. The assay is based on competition between HDC-generated histamine and fluorophore-labeled histamine for binding to a Europium cryptate (EuK)-labeled anti-histamine antibody. We demonstrated that the assay is highly sensitive and simple to develop. Assay validation experiments were performed using low-volume 384-well plates and resulted in good statistical parameters. A pilot HTS screen gave a Z' score > 0.5 and a hit rate of 1.1%, and led to the identification of a validated hit series. Overall, the presented assay should facilitate the discovery of therapeutic HDC inhibitors by acting as a novel tool suitable for large-scale HTS and subsequent interrogation of compound structure-activity relationships.