Discovery of novel 5-phenylpyrazol receptor interacting protein 1(RIP1) kinase inhibitors as anti-necroptosis agents by combining virtual screening and in vitro and in vivo experimental evaluations.

Necroptosis is one of the modes of cell death, and its occurrence and development are associated with the development of numerous diseases. To prevent the progression of necroptosis, it is crucial to inhibit the phosphorylation of three proteins: receptor-interacting protein kinase 1 (RIP1), RIP3, and mixed lineage kinase domain-like protein (MLKL). Through virtual and experimental screening approaches, we have identified 8 small molecular inhibitors with potent antinecroptotic activity and binding affinity to RIP1. Among these compounds, SY-1 demonstrated the most remarkable antinecroptotic activity (EC50 = 105.6 ± 9.6 nM) and binding affinity (RIP1 Kd = 49 nM). It effectively blocked necroptosis and impeded the formation of necrosomes by inhibiting the phosphorylations of the RIP1/RIP3/MLKL pathway triggered by TSZ (TNFα, Smac mimetic and Z-VAD-fmk). Furthermore, SY-1 exhibited a protective effect against tumor necrosis factor (TNF)-induced hypothermia in mice and significantly improved the survival rate (100 %, 30 mg/kg) of mice with systemic inflammatory response syndrome (SIRS) in a dose-dependent manner. Pharmacokinetic parameters of SY-1 were also collected in vitro and in vivo. These results strongly suggest that SY-1 and its derivatives warrant further investigation for their potential therapeutic applications.

Discovery of novel 5-phenyl-1H-pyrrole-2-carboxylic acids as Keap1-Nrf2 inhibitors for acute lung injury treatment.

Oxidative stress is intricately linked to acute lung injury (ALI) and cerebral ischemic/reperfusion (I/R) injury. The Keap1 (Kelch-like ECH-Associating protein 1)-Nrf2 (nuclear factor erythroid 2-related factor 2)-ARE (antioxidant response element) signaling pathway, recognized as a crucial regulatory mechanism in oxidative stress, holds immense potential for the treatment of both diseases. In our laboratory, we initially screened a compound library and identified compound 3, which exhibited a dissociation constant of 5090 nM for Keap1. To enhance its binding affinity, we developed a novel 5-phenyl-1H-pyrrole-2-carboxylic acid Keap1-Nrf2 inhibitor through scaffold hopping from compound 3. Structure-activity relationship studies identified compound 19 as the most potent, with a KD2 of 42.2 nM against Keap1. Furthermore, compound 19 showed significant protection against LPS-induced injury in BEAS-2B cells and promoted Nrf2 nuclear translocation. Subsequently, we investigated its therapeutic effects in mouse models of ALI injury. Compound 19 effectively alleviated symptoms at doses of 15 mg/kg for ALI injury. Additionally, it facilitated Nrf2 translocation to the nucleus, increased Nrf2 levels, and upregulated the expression of HO-1 and NQO1 in affected tissues.

Scaffold hopping derived novel benzoxazepinone receptor-interacting protein kinase 1 (RIP1) inhibitors as anti-necroptosis agents: Anti-inflammatory effect in systemic inflammatory response syndrome (SIRS) and epilepsy.

Necroptosis is a type of regulated cell death known for its pro-inflammatory nature due to the substantial release of cellular contents. The phosphorylation of key proteins, namely RIP1, RIP3, and mixed lineage kinase domain-like protein (MLKL), plays a pivotal role in the processes associated with necroptosis. Consequently, inhibiting the phosphorylation of any of these three key protein kinases could effectively block necroptosis. Utilizing a scaffold hopping strategy, we have successfully designed and synthesized a series of novel RIP1 inhibitors with selective and anti-necrotic properties, using compound o1 as the lead compound. In comparison to o1, SY1 has demonstrated heightened antinecroptosis activity and binding affinity in vitro studies. Moreover, SY1 has exhibited superior efficacy in both in vivo studies, specifically in the context of SIRS, and pharmacokinetic assessments. Furthermore, SY1 has proven effective in significantly suppressing the central inflammatory response induced by epilepsy.