Polypharmacology is a new trend in amyotrophic lateral sclerosis (ALS) therapy and an effective way of addressing a multifactorial etiology involving excitotoxicity, mitochondrial dysfunction, oxidative stress, and microglial activation. Inspired by a reported clinical trial, we converted a riluzole (1)-rasagiline (2) combination into single-molecule multi-target-directed ligands. By a ligand-based approach, the highly structurally integrated hybrids 3-8 were designed and synthesized. Through a target- and phenotypic-based screening pipeline, we identified hit compound 6. It showed monoamine oxidase A (MAO-A) inhibitory activity (IC50 = 6.9 μM) rationalized by in silico studies as well as in vitro brain permeability. By using neuronal and non-neuronal cell models, including ALS-patient-derived cells, we disclosed for 6 a neuroprotective/neuroinflammatory profile similar to that of the parent compounds and their combination. Furthermore, the unexpected MAO inhibitory activity of 1 (IC50 = 8.7 μM) might add a piece to the puzzle of its anti-ALS molecular profile.

Biomedical Research Center University Hospital Hradec Kralove Sokolska 581 500 05 Hradec Králové Czech Republic

Center for Neuroscience and Cell Biology University of Coimbra 3004 504 Coimbra Portugal

CIQUP Department of Chemistry and Biochemistry Faculty of Sciences University of Porto 4169 007 Porto Portugal

Department of Chemical and Pharmaceutical Sciences University of Trieste 34127 Trieste Italy

Department of Chemistry in Pharmaceutical Sciences Organic and Medicinal Chemistry Unit Faculty of Pharmacy Universidad Complutense 28040 Madrid Spain

Department of Pharmaceutical and Pharmacological Sciences University of Padova 35131 Padova Italy

Department of Pharmacy and Biotechnology Alma Mater Studiorum University of Bologna 40126 Bologna Italy

Dipartimento di Scienze della Salute Università Magna Græcia di Catanzaro Campus S Venuta Viale Europa 88100 Catanzaro Italy

Institute for Interdisciplinary Research University of Coimbra Casa Costa Alemão Pólo 2 Rua D Francisco de Lemos 3030 789 Coimbra Portugal

Mitotag Lda Biocant Park 3060 197 Cantanhede Portugal

Net4Science Academic Spin Off Università Magna Græcia di Catanzaro Campus S Venuta Viale Europa 88100 Catanzaro Italy

Research Unit for Sport and Physical Activity University of Coimbra 3040 248 Coimbra Portugal

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Kiernan M. C.; Vucic S.; Cheah B. C.; Turner M. R.; Eisen A.; Hardiman O.; Burrell J. R.; Zoing M. C. Amyotrophic Lateral Sclerosis. Lancet 2011, 377 (9769), 942–955. 10.1016/S0140-6736(10)61156-7. PubMed DOI

Martinez A.; Palomo Ruiz M. D.; Perez D. I.; Gil C. Drugs In Clinical Development For The Treatment Of Amyotrophic Lateral Sclerosis. Expert Opin. Invest. Drugs 2017, 26 (4), 403–414. 10.1080/13543784.2017.1302426. PubMed DOI

Mullard A. ALS Antisense Drug Falters In Phase III. Nat. Rev. Drug Discovery 2021, 20 (12), 883–885. 10.1038/d41573-021-00181-w. PubMed DOI

Martín-Cámara O.; Arribas M.; Wells G.; Morales-Tenorio M.; Martín-Requero Á.; Porras G.; Martínez A.; Giorgi G.; López-Alvarado P.; Lastres-Becker I.; et al. Multitarget Hybrid Fasudil Derivatives As A New Approach To The Potential Treatment Of Amyotrophic Lateral Sclerosis. J. Med. Chem. 2022, 65 (3), 1867–1882. 10.1021/acs.jmedchem.1c01255. PubMed DOI PMC

Cavalli A.; Bolognesi M. L.; Minarini A.; Rosini M.; Tumiatti V.; Recanatini M.; Melchiorre C. Multi-Target-Directed Ligands To Combat Neurodegenerative Diseases. J. Med. Chem. 2008, 51 (3), 347–372. 10.1021/jm7009364. PubMed DOI

Dhasmana S.; Dhasmana A.; Narula A. S.; Jaggi M.; Yallapu M. M.; Chauhan S. C. The Panoramic View Of Amyotrophic Lateral Sclerosis: A Fatal Intricate Neurological Disorder. Life Sci. 2022, 288, 120156.10.1016/j.lfs.2021.120156. PubMed DOI

Waibel S.; Reuter A.; Malessa S.; Blaugrund E.; Ludolph A. C. Rasagiline Alone And In Combination With Riluzole Prolongs Survival In An ALS Mouse Model. J. Neurol. 2004, 251 (9), 1080–1084. 10.1007/s00415-004-0481-5. PubMed DOI

Ludolph A. C.; Schuster J.; Dorst J.; Dupuis L.; Dreyhaupt J.; Weishaupt J. H.; Kassubek J.; Weiland U.; Petri S.; Meyer T.; et al. Safety And Efficacy Of Rasagiline As An Add-On Therapy To Riluzole In Patients With Amyotrophic Lateral Sclerosis: A Randomised, Double-Blind, Parallel-Group, Placebo-Controlled, Phase 2 Trial. Lancet Neurol. 2018, 17 (8), 681–688. 10.1016/S1474-4422(18)30176-5. PubMed DOI

Maruyama W.; Youdim M. B.; Naoi M. Antiapoptotic Properties Of Rasagiline, N-Propargylamine-1(R)-Aminoindan, And Its Optical (S)-Isomer, TV1022. Ann. N.Y. Acad. Sci. 2001, 939, 320–329. 10.1111/j.1749-6632.2001.tb03641.x. PubMed DOI

Dong X. X.; Wang Y.; Qin Z. H. Molecular Mechanisms Of Excitotoxicity And Their Relevance To Pathogenesis Of Neurodegenerative Diseases. Acta Pharmacol. Sin. 2009, 30 (4), 379–387. 10.1038/aps.2009.24. PubMed DOI PMC

Sala G.; Arosio A.; Conti E.; Beretta S.; Lunetta C.; Riva N.; Ferrarese C.; Tremolizzo L. Riluzole Selective Antioxidant Effects In Cell Models Expressing Amyotrophic Lateral Sclerosis Endophenotypes. Clin. Psychopharmacol. Neurosci. 2019, 17 (3), 438–442. 10.9758/cpn.2019.17.3.438. PubMed DOI PMC

Prati F.; Cavalli A.; Bolognesi M. Navigating The Chemical Space Of Multitarget-Directed Ligands: From Hybrids To Fragments In Alzheimer’s Disease. Molecules 2016, 21 (4), 466.10.3390/molecules21040466. PubMed DOI PMC

Sweeney J. B.; Rattray M.; Pugh V.; Powell L. A. Riluzole-Triazole Hybrids As Novel Chemical Probes For Neuroprotection In Amyotrophic Lateral Sclerosis. ACS Med. Chem. Lett. 2018, 9 (6), 552–556. 10.1021/acsmedchemlett.8b00103. PubMed DOI PMC

Mignani S.; Majoral J. P.; Desaphy J. F.; Lentini G. From Riluzole To Dexpramipexole Via Substituted-Benzothiazole Derivatives For Amyotrophic Lateral Sclerosis Disease Treatment: Case Studies. Molecules 2020, 25 (15), 332010.3390/molecules25153320. PubMed DOI PMC

Bar-Am O.; Amit T.; Youdim M. B.; Weinreb O. Neuroprotective And Neurorestorative Potential Of Propargylamine Derivatives In Ageing: Focus On Mitochondrial Targets. J. Neural Transm. 2016, 123 (2), 125–135. 10.1007/s00702-015-1395-3. PubMed DOI

Wager T. T.; Hou X.; Verhoest P. R.; Villalobos A. Moving Beyond Rules: The Development Of A Central Nervous System Multiparameter Optimization (CNS MPO) Approach To Enable Alignment Of Druglike Properties. ACS Chem. Neurosci. 2010, 1 (6), 435–449. 10.1021/cn100008c. PubMed DOI PMC

Jordan A. D.; Luo C.; Reitz A. B. Efficient Conversion Of Substituted Aryl Thioureas To 2-Aminobenzothiazoles Using Benzyltrimethylammonium Tribromide. J. Org. Chem. 2003, 68 (22), 8693–8696. 10.1021/jo0349431. PubMed DOI

Pansarasa O.; Bordoni M.; Dufruca L.; Diamanti L.; Sproviero D.; Trotti R.; Bernuzzi S.; La Salvia S.; Gagliardi S.; Ceroni M.; Cereda C. Lymphoblastoid Cell Lines As A Model To Understand Amyotrophic Lateral Sclerosis Disease Mechanisms. Dis. Models Mech. 2018, 11 (3), dmm03162510.1242/dmm.031625. PubMed DOI PMC

Di L.; Kerns E. H.; Fan K.; Mcconnell O. J.; Carter G. T. High Throughput Artificial Membrane Permeability Assay For Blood-Brain Barrier. Eur. J. Med. Chem. 2003, 38 (3), 223–232. 10.1016/S0223-5234(03)00012-6. PubMed DOI

Statland J. M.; Moore D.; Wang Y.; Walsh M.; Mozaffar T.; Elman L.; Nations S. P.; Mitsumoto H.; Fernandes J. A.; Saperstein D.; Hayat G.; Herbelin L.; Karam C.; Katz J.; Wilkins H. M.; Agbas A.; Swerdlow R. H.; Santella R. M.; Dimachkie M. M.; Barohn R. J. Rasagiline For Amyotrophic Lateral Sclerosis: A Randomized, Controlled Trial. Muscle Nerve 2019, 59 (2), 201–207. 10.1002/mus.26335. PubMed DOI PMC

Mandel S.; Weinreb O.; Amit T.; Youdim M. B. Mechanism Of Neuroprotective Action Of The Anti-Parkinson Drug Rasagiline And Its Derivatives. Brain Res. Rev. 2005, 48 (2), 379–387. 10.1016/j.brainresrev.2004.12.027. PubMed DOI

Binda C.; Hubálek F.; Li M.; Herzig Y.; Sterling J.; Edmondson D. E.; Mattevi A. Crystal Structures Of Monoamine Oxidase B In Complex With Four Inhibitors Of The N-Propargylaminoindan Class. J. Med. Chem. 2004, 47 (7), 1767–1774. 10.1021/jm031087c. PubMed DOI

Salado I. G.; Redondo M.; Bello M. L.; Perez C.; Liachko N. F.; Kraemer B. C.; Miguel L.; Lecourtois M.; Gil C.; Martinez A.; Perez D. I. Protein Kinase CK-1 Inhibitors As New Potential Drugs For Amyotrophic Lateral Sclerosis. J. Med. Chem. 2014, 57 (6), 2755–2772. 10.1021/jm500065f. PubMed DOI PMC

Martínez-González L.; Rodríguez-Cueto C.; Cabezudo D.; Bartolomé F.; Andrés-Benito P.; Ferrer I.; Gil C.; Martín-Requero Á.; Fernández-Ruiz J.; Martínez A.; de Lago E. Motor Neuron Preservation And Decrease Of In Vivo TDP-43 Phosphorylation By Protein CK-1δ Kinase Inhibitor Treatment. Sci. Rep. 2020, 10 (1), 4449.10.1038/s41598-020-61265-y. PubMed DOI PMC

Bissaro M.; Federico S.; Salmaso V.; Sturlese M.; Spalluto G.; Moro S. Targeting Protein Kinase CK1δ With Riluzole: Could It Be One Of The Possible Missing Bricks To Interpret Its Effect In The Treatment Of ALS From A Molecular Point Of View?. ChemMedChem 2018, 13 (24), 2601–2605. 10.1002/cmdc.201800632. PubMed DOI

Allen S. P.; Duffy L. M.; Shaw P. J.; Grierson A. J. Altered Age-Related Changes In Bioenergetic Properties And Mitochondrial Morphology In Fibroblasts From Sporadic Amyotrophic Lateral Sclerosis Patients. Neurobiol. Aging 2015, 36 (10), 2893–2903. 10.1016/j.neurobiolaging.2015.07.013. PubMed DOI

Lastres-Becker I.; Porras G.; Arribas-Blazquez M.; Maestro I.; Borrego-Hernandez D.; Boya P.; Cerdan S.; Garcia-Redondo A.; Martinez A.; Martin-Requero A. Molecular Alterations In Sporadic And SOD1-ALS Immortalized Lymphocytes: Towards A Personalized Therapy. Int. J. Mol. Sci. 2021, 22 (6), 300710.3390/ijms22063007. PubMed DOI PMC

Cunha-Oliveira T.; Silva D. F.; Segura L.; Baldeiras I.; Marques R.; Rosenstock T.; Oliveira P. J.; Silva F. S. G. Redox Profiles Of Amyotrophic Lateral Sclerosis Lymphoblasts With Or Without Known SOD1 Mutations. Eur. J. Clin. Invest. 2022, 10.1111/eci.13798. PubMed DOI

Silva F. S. G.; Starostina I. G.; Ivanova V. V.; Rizvanov A. A.; Oliveira P. J.; Pereira S. P. Determination Of Metabolic Viability And Cell Mass Using A Tandem Resazurin/Sulforhodamine B Assay. Curr. Protoc. Toxicol. 2016, 68, 2.24.1–22.24.15. 10.1002/cptx.1. PubMed DOI

Yi H.; Maruyama W.; Akao Y.; Takahashi T.; Iwasa K.; Youdim M. B.; Naoi M. N-Propargylamine Protects SH-SY5Y Cells From Apoptosis Induced By An Endogenous Neurotoxin, N-Methyl-(R)-Salsolinol, Through Stabilization Of Mitochondrial Membrane And Induction Of Anti-Apoptotic Bcl-2. J. Neural Transm. 2006, 113 (1), 21–32. 10.1007/s00702-005-0299-z. PubMed DOI

Tatton W.; Chalmers-Redman R.; Tatton N. Neuroprotection By Deprenyl And Other Propargylamines: Glyceraldehyde-3-Phosphate Dehydrogenase Rather Than Monoamine Oxidase B. J. Neural Transm. 2003, 110 (5), 509–515. 10.1007/s00702-002-0827-z. PubMed DOI

Koh J. Y.; Kim D. K.; Hwang J. Y.; Kim Y. H.; Seo J. H. Antioxidative And Proapoptotic Effects Of Riluzole On Cultured Cortical Neurons. J. Neurochem. 1999, 72 (2), 716–723. 10.1046/j.1471-4159.1999.0720716.x. PubMed DOI

Anzini M.; Chelini A.; Mancini A.; Cappelli A.; Frosini M.; Ricci L.; Valoti M.; Magistretti J.; Castelli L.; Giordani A.; Makovec F.; Vomero S. Synthesis And Biological Evaluation Of Amidine, Guanidine, And Thiourea Derivatives Of 2-Amino(6-Trifluoromethoxy)Benzothiazole As Neuroprotective Agents Potentially Useful In Brain Diseases. J. Med. Chem. 2010, 53 (2), 734–744. 10.1021/jm901375r. PubMed DOI

Criddle D. N.; Gillies S.; Baumgartner-Wilson H. K.; Jaffar M.; Chinje E. C.; Passmore S.; Chvanov M.; Barrow S.; Gerasimenko O. V.; Tepikin A. V.; Sutton R.; Petersen O. H. Menadione-Induced Reactive Oxygen Species Generation Via Redox Cycling Promotes Apoptosis Of Murine Pancreatic Acinar Cells. J. Biol. Chem. 2006, 281 (52), 40485–40492. 10.1074/jbc.M607704200. PubMed DOI

Sala G.; Arosio A.; Conti E.; Beretta S.; Lunetta C.; Riva N.; Ferrarese C.; Tremolizzo L. Riluzole Selective Antioxidant Effects in Cell Models Expressing Amyotrophic Lateral Sclerosis Endophenotypes. Clin. Psychopharmacol. Neurosci. 2019, 17 (3), 438–442. 10.9758/cpn.2019.17.3.438. PubMed DOI PMC

Dessi F.; Benari Y.; Charriautmarlangue C. Riluzole Prevents Anoxic Injury In Cultured Cerebellar Granule Neurons. Eur. J. Pharmacol. 1993, 250 (2), 325–328. 10.1016/0014-2999(93)90398-2. PubMed DOI

Bonneh-Barkay D.; Ziv N.; Finberg J. Characterization Of The Neuroprotective Activity Of Rasagiline In Cerebellar Granule Cells. Neuropharmacology 2005, 48 (3), 406–416. 10.1016/j.neuropharm.2004.10.016. PubMed DOI

Liu J.; Wang F. Role of Neuroinflammation in Amyotrophic Lateral Sclerosis: Cellular Mechanisms and Therapeutic Implications. Front. Immunol. 2017, 8, 100510.3389/fimmu.2017.01005. PubMed DOI PMC

Henkel J.; Beers D.; Zhao W.; Appel S. Microglia In ALS: The Good, The Bad, And The Resting. J. NeuroImmune Pharmacol. 2009, 4 (4), 389–398. 10.1007/s11481-009-9171-5. PubMed DOI

Trudler D.; Weinreb O.; Mandel S. A.; Youdim M. B.; Frenkel D. DJ-1 Deficiency Triggers Microglia Sensitivity To Dopamine Toward A Pro-Inflammatory Phenotype That Is Attenuated By Rasagiline. J. Neurochem. 2014, 129 (3), 434–447. 10.1111/jnc.12633. PubMed DOI

Hassanzadeh K.; Roshangar L.; Habibi-asl B.; Farajnia S.; Izadpanah E.; Nemati M.; Arasteh M.; Mohammadi S. Riluzole Prevents Morphine-Induced Apoptosis In Rat Cerebral Cortex. Pharmacol. Rep. 2011, 63 (3), 697–707. 10.1016/S1734-1140(11)70581-3. PubMed DOI