Sephin1

Sephin1 is a selective inhibitor of stress-induced PPP1R15A and targets disease associated with accumulation of misfolded protein. [1]
PPP1R15A is a regulator subunit of protein phosphatase 1 and regulates stress-induced eIF2α (α subunit of eukaryotic translation initiation factor 2). It brings PP1 (serine/threonine-protein phosphatase) to dephosphorylate eIF2α. Phosphorylation of eIF2α reduces protein synthesis and prevents the accumulation of misfolded protein in ER (endoplasmic reticulum). Thus inhibiting PPP1R15A prolongs the phosphorylation of eIF2α, and benefits the treatment for protein misfolding disease. [1]
In cells treated with 5μm Sephin1 for 6 hours, Sephin1 specifically disrupted the PPP1R15A-PP1c complex without affecting PPP115B-PP1c complex. As a consequence, Sephin1 prolonged eIF2a phosphorylation in HeLa cells after stress (*2.5 μg/ml tunicamycin treatment) and delayed translational recovery. Sephin1 recused cell from cytotoxic ER stress* in wild-type cell but not the PPP1R15A mutant cell. [1]
In mice administrated orally with 1-5mg/kg Sephin1 for given time, Sephin1 exhibited no adverse effects on rotarod performances, total body weight gain or memory. Treating MPZ (Deletion of serine 63 of myelin protein zero) mutant mice with 1mg/kg orally twice a day, prevented the molecular, morphological and motor defects. Oral treatment of 5 mg/kg of Sephin1 once a day, prevented motor deficits, motor neuron loss and the molecular defects in SOD1 (Mutant and misfolding-prone superoxide dismutase 1) mutant mice. [1]
Reference:
1: Das I, Krzyzosiak A, Schneider K, Wrabetz L, D'Antonio M, Barry N, Sigurdardottir A, Bertolotti A. Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit. Science. 2015 Apr 10; 348(6231):239-42.

Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit.[Pubmed:25859045]

Science. 2015 Apr 10;348(6231):239-42.

Protein phosphorylation regulates virtually all biological processes. Although protein kinases are popular drug targets, targeting protein phosphatases remains a challenge. Here, we describe Sephin1 (selective inhibitor of a holophosphatase), a small molecule that safely and selectively inhibited a regulatory subunit of protein phosphatase 1 in vivo. Sephin1 selectively bound and inhibited the stress-induced PPP1R15A, but not the related and constitutive PPP1R15B, to prolong the benefit of an adaptive phospho-signaling pathway, protecting cells from otherwise lethal protein misfolding stress. In vivo, Sephin1 safely prevented the motor, morphological, and molecular defects of two otherwise unrelated protein-misfolding diseases in mice, Charcot-Marie-Tooth 1B, and amyotrophic lateral sclerosis. Thus, regulatory subunits of phosphatases are drug targets, a property exploited here to safely prevent two protein misfolding diseases.

PPP1R15A-mediated dephosphorylation of eIF2alpha is unaffected by Sephin1 or Guanabenz.[Pubmed:28447936]

Elife. 2017 Apr 27;6.

Dephosphorylation of translation initiation factor 2 (eIF2alpha) terminates signalling in the mammalian integrated stress response (ISR) and has emerged as a promising target for modifying the course of protein misfolding diseases. The [(o-chlorobenzylidene)amino]guanidines (Guanabenz and Sephin1) have been proposed to exert protective effects against misfolding by interfering with eIF2alpha-P dephosphorylation through selective disruption of a PP1-PPP1R15A holophosphatase complex. Surprisingly, they proved inert in vitro affecting neither stability of the PP1-PPP1R15A complex nor substrate-specific dephosphorylation. Furthermore, eIF2alpha-P dephosphorylation, assessed by a kinase shut-off experiment, progressed normally in Sephin1-treated cells. Consistent with its role in defending proteostasis, Sephin1 attenuated the IRE1 branch of the endoplasmic reticulum unfolded protein response. However, repression was noted in both wildtype and Ppp1r15a deleted cells and in cells rendered ISR-deficient by CRISPR editing of the Eif2s1 locus to encode a non-phosphorylatable eIF2alpha (eIF2alpha(S51A)). These findings challenge the view that [(o-chlorobenzylidene)amino]guanidines restore proteostasis by interfering with eIF2alpha-P dephosphorylation.

Decoding the selectivity of eIF2alpha holophosphatases and PPP1R15A inhibitors.[Pubmed:28759048]

Nat Struct Mol Biol. 2017 Sep;24(9):708-716.

The reversible phosphorylation of proteins controls most cellular functions. Protein kinases have been popular drug targets, unlike phosphatases, which remain a drug discovery challenge. Guanabenz and Sephin1 are selective inhibitors of the phosphatase regulatory subunit PPP1R15A (R15A) that prolong the benefit of eIF2alpha phosphorylation, thereby protecting cells from proteostatic defects. In mice, Sephin1 prevents two neurodegenerative diseases, Charcot-Marie-Tooth 1B (CMT-1B) and SOD1-mediated amyotrophic lateral sclerosis (ALS). However, the molecular basis for R15A inhibition is unknown. Here we reconstituted human recombinant eIF2alpha holophosphatases, R15A-PP1 and R15B-PP1, whose activity depends on both the catalytic subunit PP1 (protein phosphatase 1) and either R15A or R15B. This system enabled the functional characterization of these holophosphatases and revealed that Guanabenz and Sephin1 induced a selective conformational change in R15A, detected by resistance to limited proteolysis. This altered the recruitment of eIF2alpha, preventing its dephosphorylation. This work demonstrates that regulatory subunits of phosphatases are valid drug targets and provides the molecular rationale to expand this concept to other phosphatases.