Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome.

Marco Borgogno,Alessandra Turci,Corinne Portioli,Jacopo Manigrasso,Annalisa Savardi,Laura Cancedda,Marco De Vivo,Andrea Armirotti,Andrea Contestabile,Giuliana Ottonello,Sine Mandrup Bertozzi

doi:10.1021/acs.jmedchem.1c00603

Abstract

Intracellular chloride concentration [Cl–]i is defective in several neurological disorders. In neurons, [Cl–]i is mainly regulated by the action of the Na+–K+–Cl– importer NKCC1 and the K+–Cl– exporter KCC2. Recently, we have reported the discovery of ARN23746 as the lead candidate of a novel class of selective inhibitors of NKCC1. Importantly, ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. Here, we describe the discovery and extensive characterization of this chemical class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivatives. In particular, we present compound 40 (ARN24092) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compounds for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio.

Highlights

In recent years, a large body of constantly increasing experimental evidence has indicated modulation of intracellular chloride concentration [Cl−]i as a valuable therapeutic strategy for a number of neurological conditions, including Down syndrome (DS).1−3 [Cl−]i is mainly regulated in neurons by the sodium (Na+)−potassium (K+)−chloride (Cl−) importer NKCC1 and the K+−Cl− exporter KCC2
We have recently reported the discovery of 3(N,N-dimethylsulfamoyl)-4-((8,8,8-trifluorooctyl)amino)benzoic acid, compound 1 (ARN23746 in Figure 1), as a potent and selective NKCC1 inhibitor, with in vivo efficacy in mouse models of DS and autism, potentially in other neurodevelopmental disorders characterized by impaired [Cl−]i
We refined the first bumetanide’s pharmacophore by superimposing it with structures of other unspecific NKCC1 inhibitors. We used this model as a search filter for the virtual screening of our institution’s chemical library and other chemical libraries from commercial vendors (∼135,000 compounds, in total). This computational effort identified a total of 253 compounds that we tested at two concentrations (10 and 100 μM) in a Cl− influx assay in HEK293 cells lines transfected with NKCC1.5 Among these 253 compounds, we identified the two structurally related 2-amino-5-nitro-benzenesulfonamide derivatives 3 (ARN22393) and 4 (ARN22394), diversified for the substituent on the amino group (Table 1)

Summary

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*sı Supporting Information nNAeBau+Sr−ToKlRo+gA−icCCaTll−:diiImsnoptrrdoaerctreesl.rluINlnaKrnCecCuhrl1oornainsd,de[tCchole−n]Kci e+in−stCrmalta−iionenlxypo[rCertgleu−r]laiKteCidsCbd2ye. fRethcetecievanecttlyiion, nwseoefvheatrhvaeel reported the discovery of ARN23746 as the lead candidate of a novel class of selective inhibitors of NKCC1. FRethcetecievanecttlyiion, nwseoefvheatrhvaeel reported the discovery of ARN23746 as the lead candidate of a novel class of selective inhibitors of NKCC1. ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. We describe the discovery and extensive characterization of this chemical class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivatives. We present compound 40 (ARN24092) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compounds for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio

■ INTRODUCTION

■ CONCLUDING REMARKS AND FUTURE WORK

Author Contributions

■ ACKNOWLEDGMENTS

■ REFERENCES