Accumulation of misfolded α-synuclein protein in intracellular inclusion bodies of dopaminergic neurons underlies the pathogenesis of Synucleinopathies, which include Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB). Therefore, preventing the accumulation of misfolded α-synuclein in dopaminergic neurons or their clearance could in principle offer intervention strategies against Synucleinopathies, which currently remain untreatable. In this study, we employ the Affinity-directed PROtein Missile (AdPROM) system consisting of the substrate receptor of the CUL2-E3 ligase complex VHL and a nanobody selectively recognising the human α-synuclein protein and demonstrate targeted degradation of endogenous α-synuclein from human cell lines with remarkable selectivity. We further demonstrate that targeted degradation of α-synuclein impedes the pre-formed fibril (PFF)-induced aggregation of α-synuclein in primary neurons derived from rats expressing human α-synuclein. This approach also represents the first demonstration of nanobody-guided proteasomal degradation of some reported clinically relevant α-synuclein variants.
Bill Carton, Géraldine Gelders, Gajanan Sathe et al.· npj Parkinson's Disease· 0 citations
Proteins lacking defined ligandable pockets remain challenging drug targets. Here, we develop a molecular glue-based PROTAC (MGPROTAC) approach that chemically conjugates a molecular glue stabilizer to a VHL-recruiting ligand to capture and ubiquitinate the 14-3-3/Estrogen receptor α (ERα) complex. Our designed MGPROTACs engage a composite interface between 14-3-3 and the disordered F-domain of ERα, promoting cooperative complex formation and targeted ubiquitination. Biophysical characterization revealed distinct linker-dependent cooperativities across the MGPROTAC series, which influenced both cellular permeability and ubiquitination efficiency. Cryo-EM of the most cooperative MGPROTAC uncovered de novo VHL–14-3-3ζ contacts, while molecular dynamics simulations rationalize the stabilizing interactions underlying cooperativity. Strikingly, fine-tuning linker design enables selective ubiquitination of distinct complex subunits. These findings establish a structural and mechanistic framework for integrating molecular glue and PROTAC principles, expanding the scope of drug discovery to previously intractable protein complexes. Proteins lacking defined ligandable pockets are challenging drug targets. Here the authors combine molecular glue-stabilizer and PROTAC strategies to enable ubiquitination of the 14-3-3/estrogen receptor α complex, revealing cooperative linker-dependent activity and a structural basis for targeted E3 ligase recruitment.
Carlo J. A. Verhoef, Charlotte Crowe, M. Nakasone et al.· Nature Communications· 0 citations