Neurotherapeutics https://doi.org/10.1007/s13311-019-00717-4 ORIGINAL ARTICLE Axonal and Myelin Neuroprotection by the Peptoid BN201 in Brain Inflammation Pablo Villoslada 1 & Gemma Vila 1 & Valeria Colafrancesco 1 & Beatriz Moreno 1 & Begoña Fernandez-Diez 1 & Raquel Vazquez 1 & Inna Pertsovskaya 1 & Irati Zubizarreta 1 & Irene Pulido-Valdeolivas 1 & Joaquin Messeguer 2 & Gloria Vendrell-Navarro 2 & Jose Maria Frade 3 & Noelia López-Sánchez 3 & Meritxell Teixido 4 & Ernest Giralt 4 & Mar Masso 5 & Jason C Dugas 6 & Dmitri Leonoudakis 6 & Karen D. Lariosa-Willingham 6 & Lawrence Steinman 7 & Angel Messeguer 2 # The American Society for Experimental NeuroTherapeutics, Inc. 2019 Abstract The development of neuroprotective therapies is a sought-after goal. By screening combinatorial chemical libraries using in vitro assays, we identified the small molecule BN201 that promotes the survival of cultured neural cells when subjected to oxidative stress or when deprived of trophic factors. Moreover, BN201 promotes neuronal differentiation, the differentiation of precursor cells to mature oligodendrocytes in vitro , and the myelination of new axons. BN201 modulates several kinases participating in the insulin growth factor 1 pathway including serum –glucocorticoid kinase and midkine, inducing the phosphorylation of NDRG1 and the translocation of the transcription factor Foxo3 to the cytoplasm. In vivo , BN201 prevents axonal and neuronal loss, and it promotes remyelination in models of multiple sclerosis, chemically induced demyelination, and glaucoma. In summary, we provide a new promising strategy to promote neuroaxonal survival and remyelination, potentially preventing disability in brain diseases. Key Words Neuroprotection . neuroinflammation . neurodegenerative diseases . multiple sclerosis . glaucoma Introduction Brain diseases represent a significant clinical challenge due to Electronic supplementary material The online version of this article the known sensitivity of the central nervous system (CNS) to (https://doi.org/10.1007/s13311-019-00717-4 ) contains supplementary damage and its limited capacity for repair and regeneration. material, which is available to authorized users. Brain injury and brain diseases are notorious for significant long-term disability [ 1, 2]. After insult, damage to neurons and * Pablo Villoslada axons may induce several forms of neuropathology including [email protected] necrosis, apoptosis, autophagia, necroptosis, and pyroptosis. Further, brain injury may disrupt the connections between 1 Center for Neuroimmunology, Institut d ’Investigacions Biomediques neurons, as what occurs following axon degeneration (anter- August Pi Sunyer, Casanova 145, Centre Cellex 3A, ograde or Wallerian degeneration, retrograde degeneration or 08036 Barcelona, Spain dying-back, or even transynaptic degeneration), synaptic loss, 2 Institute for Advanced Chemistry of Catalonia, Consejo Superior de or dendritic pruning [ 3–5]. Although some of these processes Investigaciones Cientificas, Barcelona 08034, Spain take place early after insult, others may be delayed for weeks, 3 Instituto Cajal, Consejo Superior de Investigaciones Cientificas, months, or even years. For these reasons, the discovery of Madrid 28002, Spain agents that promote neuroprotection is a health priority. 4 Institute for Research in Biomedicine, Barcelona 08028, Spain In multiple sclerosis (MS), the autoimmune attack and 5 Bionure Farma SL, Barcelona 94025, Spain chronic inflammation produces a massive destruction of mye- 6 Myelin Repair Foundation, Saratoga, NY 94085, USA lin, with loss of oligodendrocytes and their precursors in the 7 Stanford University, Palo Alto, CA 94305, USA long term, as well as relative axonal loss. Long-term disability P. Villoslada et. al. is mainly dependent on axonal loss, which is the result of the PC12 cells were maintained in Dulbecco ’s modified acute inflammatory damage and degeneration of chronically Eagle ’s medium (DMEM) supplemented with 2.5% fetal bo- demyelinated axons. Because current immunotherapy de- vine serum (FBS), 15% horse serum (HS), and penicillin/ creases the frequency of autoimmune attacks but chronic in- streptomycin (P/S). The SH-SY5Y cell line was maintained flammation persists, neuroprotection is being pursued as com- in 50% Ham ’s F12 medium and 50% Earle ’s minimal essen- bination therapy to prevent high levels of disability [ 6]. On the tial medium, supplemented with 10% FBS, 2 mM L-gluta- other hand, glaucoma is a neurodegenerative disease of the mine, and 1% P/S. The NSC-34 cell line was cultivated in visual pathway, with high intraocular pressure being 1 of the DMEM with 10% FBS and 1% P/S. The rat Schwannoma most common predisposing factors and the only 1 targeted with RN22 cell line was cultured in DMEM with 10% FBS and approved therapies. However, in most cases, even though treat- P/S. All the cell cultures were maintained at 37 °C in 5% CO 2 ed with ocular pressure-lowering drugs, the disease keeps and they were grown in 60 and 100 mm tissue culture dishes progressing, and for this reason, neuroprotection is defined as (Beckton Dickinson, Franklin Lakes, NJ). a high priority for preventing blindness due to this condition [ 7]. In order to develop new therapies for prevention of brain PC12 Differentiation Assay damage [ 1–6], we screened combinatorial libraries of small chemicals in order to identify compounds that favor the sur- PC12 cell differentiation and survival was measured by plat- vival or differentiation of neuronal precursors and the survival ing cells onto collagen-coated 24-well plates and adding NGF of mature and immature neurons in the presence of stressors (100 ng/ml, [ 11 ]) or the small chemicals to the cultures at (including oxidative stress or trophic factor deprivation). We different concentrations (2-20-100 ng/ml and 2-20- also assessed the capacity of such compounds to promote the 50 μg/ml) (see additional details in Fig. S1 ). The number of survival and differentiation of myelin-forming cells. The differentiated cells with neurite processes greater than 2 cell screening included in vitro cell assays and in vivo models of bodies in length were counted after 5 days of treatment, neuroinflammation and neurodegeneration. We describe the counting 100 cells in 3 randomly selected fields in each well small chemical BN201 that displays an array of neuroprotec- (at least 300 cells were assessed at random in each experi- tive effects in neurons and myelin-forming cells, and is being ment) [ 11 ]. developed at present as a neuroprotective therapy for MS and optic neuritis (NCT03630497). In Vitro Oxidative Stress Survival Assays RN22 cells were plated in 24-well plates (20,000 cells/well) in Material and Methods DMEM alone and after allowing the cells to adhere for 3 days, and copper sulfate (CuSO 4, 150 μM) was added in the pres- Chemical Libraries ence or absence of NGF (100 ng/ml) or BN201 (1-10-50 ng/ml or 1-10 μg/ml [ 12 ]. After 24 h, cell viability was studied by Two different chemical libraries developed at the Institute of determining the amount of MTT (Sigma, St Louis, MI, USA) Medical Advanced Chemistry of Catalonia, CSIC, Barcelona, that was reduced to insoluble purple formazan. After remov- Spain (Prof Angel Messeguer), were tested: first, N- ing the medium, the water-insoluble formazan was solubilized alkylglycine trimers (peptoids), and second, cyclic in DMSO (Sigma) and the dissolved material was measured tetralkylammonium salts. The cyclic tetraalkylammonium on a spectrophotometer at a wavelength of 570 nm, salts library was composed of 66 master compounds (44 com- subtracting the background at 650 nm. pounds contained a 6-membered ring, whereas 22 contained a Human SH-SY5Y neuroblastoma cells were first differen- 7-membered ring) [ 8]. The peptoid (oligomers of N- tiated to a neuronal phenotype with retinoic acid (10 μM) for alkylglycine units) library consisted of controlled mixtures 6 days and they were then pretreated for 3 days with BN201 at constructed under the positional scanning format, and it was different doses (0.03, 0.1, 0.5, 1, 3, 5, 10, 20, and 100 μM) in composed by 5120 compounds [ 9, 10 ]. The lyophilized com- fresh medium, with or without K252a (200 nM). MPP + pounds were dissolved in H 2O with 5% DMSO for the in vitro (100 μM) or H 2O2 (100 μM) was then added after 30 min assays and were tested in controlled mixtures at 1 to 5 mg/ml. and the number of surviving cells was determined by quanti- fying the MTT staining 48 h later as described above [ 13 , 14 ]. Cell Lines Trophic Factor Deprivation In Vitro Assay All cell lines were obtained from the American Tissue Culture Collection (Manassas, VA, USA) and were tested and con- NSC-34 cells were seeded in 24-well poly-lysine-coated trolled for a number of expansion cycles. All in vitro experi- plates (30,000 cells/well) and preincubated for 24 h in ments were repeated twice. DMEM plus 10% FBS with various doses of BN201 (0.2, Axonal and Myelin Neuroprotection by the Peptoid BN201 in Brain Inflammation 0.1, 2, 20, and 50 μg/ml), or with granulocyte-colony stimu- − mean RLU of vehicle control) / (mean RLU of EC80 con- lating factor (G-CSF) (2 μg/ml) or brain-derived trophic factor trol − mean RLU of vehicle control)). (BDNF) (20 ng/ml) as positive controls [ 15 ]. The medium was The CNS receptor assay is based in the binding to a radio- then removed and replaced with fresh DMEM without FBS, actively labeled CNS receptor. Compound binding was calcu- and after 48 h, cell viability was assayed by the MTT assay. lated as percentage inhibition of the binding of a radioactively labeled ligand specific for each target. Results showing an inhibition or stimulation higher than 50% are considered to In Vitro Remyelination Assays represent significant effects of the test compounds. The CETSA assay was conducted by Pelago Biosciences Purified retinal ganglion cells (4000/well) from P7 rats were Inc. (Solna, Sweden) in the SH-S5Y5 cell line. cultured in 96-well plates for 10 days in culture media in order to produce newly generated axons.