Potent Inhibition of Necroptosis by Simultaneously Targeting Multiple Effectors of the Pathway

Abstract

Necroptosis is an inflammatory form of programmed cell death that has been implicated in various human diseases. Compound 2 is a more potent analogue of the published compound 1 and inhibits necroptosis in human and murine cells at nanomolar concentrations. Several target engagement strategies were employed, including cellular thermal shift assays (CETSA) and diazirine-mediated photoaffinity labeling via a bifunctional photoaffinity probe derived from compound 2. These target engagement studies demonstrate that compound 2 binds to all three necroptotic effector proteins (mixed lineage kinase domain-like protein (MLKL), receptor-interacting serine/threonine protein kinase 1 (RIPK1) and receptor-interacting serine/threonine protein kinase 3 (RIPK3)) at different levels in vitro and in cells. Compound 2 also shows efficacy in vivo in a murine model of systemic inflammatory response syndrome (SIRS).

The Role of the Key Effector of Necroptotic Cell Death, MLKL, in Mouse Models of Disease

“…Unlike catalytically inactive RIPK1 or RIPK3 deficiency in mice, Mlkl −/− mice are not protected against SIRS driven by low dose TNFα [20] or A20 deficiency [20]. When wild-type mice are pre-treated with compound 2, a potent inhibitor of necroptosis that binds to RIPK1, RIPK3 and MLKL, hypothermia is delayed [81]. Together these findings suggest protection against SIRS is necroptosis-independent and occurs upstream of MLKL.…”

There’s more to death than life: Noncatalytic functions in kinase and pseudokinase signaling

“…Together, these act as cues for conformational transitions that disable the suppressor function of the pseudokinase domain and promote the killer function of the N-terminal 4HB domain. While ATP binding does not appear to serve a physiological function in dictating MLKL activation, it does raise the question whether the ATP-binding site could be targeted pharmacologically to block conformational transitions and prevent errant MLKL activation ( 54 , 58 ).…”

“…Such a role was proposed for the MLKL pseudokinase, whose conformational interconversion is promoted by phosphorylation. A small molecule series identified by DSF is known to bind MLKL’s pseudoactive site, although deducing the precise contribution of ligation to pathway inhibition is confounded by the compounds showing affinity for the upstream kinases in the pathway RIPK1 and RIPK3 ( 54 , 58 ). Similar to JAK2 pseudokinase and kinase domain ligands ( 103 ), this illustrates the challenges of pharmacologically targeting pseudokinases selectively, considering their ancestral origins commonly reside in gene or domain duplication events.…”

The regulation of necroptosis by post-translational modifications

“…[55,56]). Consequently, errant necroptosis has been implicated in the pathology of inflammatory syndromes [57], atherosclerosis [58], cardiac [59] and kidney [60] ischemia/reperfusion injury, sepsis [61][62][63], inflammatory bowel disease [64], neurodegenerative diseases [65], and cancer [66], although some of these attributions remain the matter of debate [62,[67][68][69][70][71][72]. In addition, damage caused by some bacterial infections have been proposed to be attributable to excessive necroptosis and inflammation [73,74].…”

“…It is foreseeable that targeting MLKL could be advantageous because of MLKL's specific role in executing necroptosis, although the only selective agent reported to date, NSA, is a covalent modifier of MLKL and is thus not suitable for clinical development [28]. Recent studies suggest that small molecules that target RIPK1, RIPK3, and MLKL simultaneously may prove an effective strategy in targeting necroptotic diseases pharmacologically [63].…”