Effects of Necrostatin-1, an Inhibitor of Necroptosis, and its Inactive Analogue Nec-1i on Basal Cardiovascular Function

Abstract

Inhibitionof receptor-interacting serine/threonine-protein kinase 1 (RIP1) by necrostatin-1 (Nec-1) alleviates cardiac injury due to prevention of necroptotic cell death. Its inactive analogue necrostatin-1i (Nec-1i), lacking RIP1 activity, serves as a suitable control. It is unknown if these agents influence the heart function in the absence of damaging stimuli. For this purpose, we measured intraarterial blood pressure (systolic -sBP and diastolic -dBP) and ECG parameters after a bolus administration of Nec-1 and Nec-1i in rats during 30 min. Nec-1, unlike Nec-1i, increased sBP and dBP, as well as heart rate reaching the peak at 20 min. The P wave duration tended to be decreased and the duration of the PR interval was shortened by Nec-1 indicating faster conduction of the impulses through atria to the ventricles.The drugs did not influence the QTc interval duration and no episode of ventricular arrhythmia was observed. In summary, Nec-1 temporarily modulates blood pressure and electrical function of the healthy heart. These effects of Nec-1 are likely due to its off-target action or RIP1 has an important role in the regulation of cardiovascular function independently of its action on the necroptotic pathway.

Determination of Novel Highly Effective Necrostatin Nec-1s in Rat Plasma by High Performance Liquid Chromatography Hyphenated with Quadrupole-Time-of-Flight Mass Spectrometry

“…When considering the concentration levels found in the pharmacokinetic study of Nec-1 (see the Introduction section) [ 13 ], the developed HPLC-Q-TOF method seems to be enough sensitive (see Section 2.2 Method validation) and, by that, easily applicable also for similar pharmacological and clinical studies of Nec-1s (even if Nec-1s exhibits 2-times higher inhibition activity than Nec-1). More specifically, the method could be useful for discovery of relationships and correlations between plasma concentrations of Nec-1s and its selected pharmacological effects (e.g., influence of the contractile and electrical activity of the heart as described in [ 11 ], or stabilization of pre-existing aneurisms [ 12 ]). Such knowledge could lead to further therapy optimization.…”

“…However, its relatively low kinase selectivity evidenced by the concomitant inhibition of indoleamine 2,3-dioxygenase (IDO), a potent immunomodulatory enzyme [ 9 ], may significantly influence the outcomes of such studies, mainly those associated with inflammatory response [ 10 ]. In addition, we have indicated that Nec-1, may also influence protein kinases regulating excitation-contraction coupling (ECC) of the heart and thereby its contractile and electrical activity [ 11 ]. An inactive analogue, Nec-1i (Nec-1i, N-demethylated thiohydantoin analogue of Nec-1), lacking the capability to inhibit RIP1 and probably also other protein kinases without any effects on ECC [ 11 ], can serve as a suitable control.…”

“…In addition, we have indicated that Nec-1, may also influence protein kinases regulating excitation-contraction coupling (ECC) of the heart and thereby its contractile and electrical activity [ 11 ]. An inactive analogue, Nec-1i (Nec-1i, N-demethylated thiohydantoin analogue of Nec-1), lacking the capability to inhibit RIP1 and probably also other protein kinases without any effects on ECC [ 11 ], can serve as a suitable control. However, this compound was also found to be an IDO inhibitor [ 10 ].…”

Evidence of necroptosis in hearts subjected to various forms of ischemic insults

“…Its inactive variant Nec-1i lacks the RIP1-targeting effects, however, it retains the ability to inhibit IDO (Degterev et al 2013;Takahashi et al 2012). In addition, we have recently reported that Nec-1 likely exerts off-target pharmacodynamic effects modulating basal heart function (Szobi et al 2016). In fact, after a bolus application, Nec-1 increased systolic and diastolic blood pressure as well as heart rate.…”

Cardioprotection of ischaemic preconditioning is associated with inhibition of translocation of MLKL within the plasma membrane

“…Indeed, pThr287‐CaMKIIδ expression was the greatest in the PC+N group. Intriguingly, in the absence of I/R damage (Figure E), Nec‐1s perfusion increased CaMKIIδ phosphorylation at Thr287 suggesting that RIP1 kinase may modulate the activity of this protein kinase even under baseline conditions …”