Introduction: Nitrate ester bearing 6-piperazinyl-purine analogues (denoted MK drugs) are cardioprotective in infarction animal models and act as inhibitors of Janus kinase (JAK) and Rho-associated kinase (ROCK) [1-3]. Despite the presence of nitrate ester moiety, the MK drugs do not release nitric oxide (NO).

Methods: We utilized organic chemistry platforms to design a dinitrate ester derivative denoted MK177, cell-free and cellular assays to elucidate antithrom-botic and anti-ischemic mechanisms. Furthermore, we also used tissue models to analyze vasodilation, and animal models to evaluate drug activities in vivo.

Results: In anesthetized pigs, intravenous infusion of MK177 produced“nitroglycerin-like”effects on vital parameters. Analysis of exhaled air confirmed release of NO. MK177 caused concentration-dependent relaxation of iliac arteries (87±6.8 % relaxation of precontracted arteries, mean value ±SD, n=5) and this effect was mediated by activation of the NO/cyclic GMP signaling pathway. It is noteworthy that other mononitrate or non-nitrate MKs did not cause NO-induced vasodilation. In cellular model systems, MK177 evoked antithrombotic effects by targeting ROCK in a NO-independent manner. Specifically, MK177 inhibited platelet aggregation induced by collagen (72±12.6 % inhibition of aggregation, mean value±SD, n=7). Western blot analyses confirmed that MK177 reduced ROCK-dependent phosphorylation of myosin phosphatase sub-unit (MYPT-1) in platelets. Finally, kinase screening assay revealed that MK177 concentration-dependently inhibited ROCK and JAK (Kd values around 5μM).

Conclusion: We have developed a bifunctional drug molecule, MK177, that acts by NO-dependent and NO-independent mechanisms. MK 177 induced car-diovascular NO effects in vivo and relaxed vessels in vitro. MK177 also prevented blood platelet activation via NO-independent ROCK inhibition. The bifunctional nature of MK177 can be of significance in future management of thrombotic and ischemic disease. Collectively, the novel cardio-protective and bifunctional drug MK177 has promising therapeutic potential.

References:

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2. Kardeby C, Paramel GV, Pournara D, Fotopoulou T, Sirsjö A, Koufaki M, Fransén K, Grenegård M. A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition. Eur J Pharmacol. 2019;15(857):172428-172434. https://doi.org/10.1016/j.ejphar.2019.172428

3. Paramel GV, Lindkvist M, Idosa BA, Sebina LS, Kardeby C, Fotopoulou T, Pournara D, Kritsi E, Ifanti E, Zervou M, Koufaki M, Grenegård M, Fransén K. Novel purine analogues regulate IL-1βrelease via inhibition of JAK activity in human aortic smooth muscle cells. Eur J Pharmacol. 2022;15(929):175128-175135. https://doi.org/10.1016/j.ejphar.2022.175128