Kahnt, A. | bioRxiv Science

bioRxiv2024

Identification of small molecule enhancers of NK cell tumoricidal activity via a tumor microenvironment-mimicking co-culture assay

The tumor microenvironment (TME) is a pro-cancerous niche harboring immunosuppressive factors that are secreted by cancer cells and the surrounding cancer-supportive tissue, such as kynurenine, prostaglandin E2 and transforming growth factor {beta} (TGF{beta}). These factors dampen the activity of cytotoxic lymphocytes like natural killer (NK) cells, allowing evasion of immune cell-mediated killing. To identify small molecules that counteract the immunosuppressive effect of the TME and restore NK cell-mediated cytotoxicity, we developed a phenotypic co-culture assay of cancer cells and primary lymphocytes suitable for medium-throughput screening. We discovered small molecules that restore NK cell-mediated cytotoxicity through diverse mechanisms. The potent TGF{beta} type I receptor (TGF{beta}R-1) inhibitor, RepSox, stood out as superior to other TGF{beta}R-1 inhibitors due to its ability to abolish the effects of both inhibitory factors used in our setup. This mode of action goes beyond TGF{beta}R-1 inhibition and is related to the simultaneous abrogation of cyclooxygenase 1 (COX1) activity.

immunology↗

bioRxiv2024

STRUCTURE-BASED DESIGN OF PROTACS FOR THE DEGRADATION OF SOLUBLE EPOXIDE HYDROLASE

Soluble epoxide hydrolase (sEH) represents a promising target for inflammation-related diseases as it hydrolyzes highly anti-inflammatory epoxy-fatty acids (EpFAs) to the less active corresponding diols.1 sEH harbours two distinct catalytic domains, the C-terminal hydrolase domain and the N-terminal phosphatase domain which are connected by a proline-rich linker.2 Although potent inhibitors of enzymatic activity are available for both domains, sEH-PROTACs offer the unique ability to simultaneously degrade both domains, mimicking the sEH knockout phenotype associated with beneficial effects as reducing inflammation, attenuating neuroinflammation, and delaying the progression of Alzheimers disease. Herein, we report the structure-based development of a potent sEH-PROTAC as a useful tool compound for the investigation of sEH. In order to facilitate a rapid testing of the synthesized compounds a cell-based sEH degradation assay was developed based on the HiBiT-technology. A structure-activity-relationship (SAR) investigation was performed, based on the crystal structure of previously published sEH inhibitor FL217 where we identified two possible exit vectors. We designed and synthesized a set of 24 PROTACs with varying linkers in a combinatorial manner. Furthermore, co-crystallization of sEH with two selected PROTACs allowed us to explore the binding mode and rationalize the appropriate linker length. After biological and physicochemical investigation, the most suitable PROTAC 23 was identified and applied to degrade sEH in primary human macrophages, marking the successful translation and applicability to non-artificial systems.

biochemistry↗