*Article* **Discovery of a Necroptosis Inhibitor Improving Dopaminergic Neuronal Loss after MPTP Exposure in Mice**

**Sara R. Oliveira, Pedro A. Dionísio, Maria M. Gaspar, Maria B. T. Ferreira, Catarina A. B. Rodrigues, Rita G. Pereira, Mónica S. Estevão, Maria J. Perry, Rui Moreira, Carlos A. M. Afonso, Joana D. Amaral and Cecília M. P. Rodrigues \***

> Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal; sararoliveira@ff.ulisboa.pt (S.R.O.); pedelandionisio@gmail.com (P.A.D.); mgaspar@ff.ulisboa.pt (M.M.G.); mariabtferreira@gmail.com (M.B.T.F.); cataxana@gmail.com (C.A.B.R.); ragpereira@gmail.com (R.G.P.); monica.estevao@gmail.com (M.S.E.); mjprocha@ff.ulisboa.pt (M.J.P.); rmoreira@ff.ulisboa.pt (R.M.); carlosafonso@ff.ulisboa.pt (C.A.M.A.); jamaral@ff.ulisboa.pt (J.D.A.) **\*** Correspondence: cmprodrigues@ff.ulisboa.pt; Tel.: +351-217946490

**Abstract:** Parkinson's disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SN). Necroptosis is a caspase-independent form of regulated cell death mediated by the concerted action of receptor-interacting protein 3 (RIP3) and the pseudokinase mixed lineage domain-like protein (MLKL). It is also usually dependent on RIP1 kinase activity, influenced by further cellular clues. Importantly, necroptosis appears to be strongly linked to several neurodegenerative diseases, including PD. Here, we aimed at identifying novel chemical inhibitors of necroptosis in a PD-mimicking model, by conducting a two-step screening. Firstly, we phenotypically screened a library of 31 small molecules using a cellular model of necroptosis and, thereafter, the hit compound effect was validated in vivo in a sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) PD-related mouse model. From the initial compounds, we identified one hit—Oxa12—that strongly inhibited necroptosis induced by the pan-caspase inhibitor zVAD-fmk in the BV2 murine microglia cell line. More importantly, mice exposed to MPTP and further treated with Oxa12 showed protection against MPTP-induced dopaminergic neuronal loss in the SN and striatum. In conclusion, we identified Oxa12 as a hit compound that represents a new chemotype to tackle necroptosis. Oxa12 displays in vivo effects, making this compound a drug candidate for further optimization to attenuate PD pathogenesis.

**Keywords:** MPTP; necroptosis; neurodegeneration; Parkinson's disease; small molecules
