Structural dynamics of RAF1-HSP90-CDC37 and HSP90 complexes reveal asymmetric client interactions and key structural elements.

RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 µs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1's unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.

Cinpanemab in Early Parkinson Disease: Evaluation of Biomarker Results From the Phase 2 SPARK Clinical Trial.

BACKGROUND AND OBJECTIVES: Sensitive, reliable, and scalable biomarkers are needed to accelerate the development of therapies for Parkinson disease (PD). In this study, we evaluate the biomarkers of early PD diagnosis, disease progression, and treatment effect collected in the SPARK. METHODS: Cinpanemab is a human-derived monoclonal antibody binding preferentially to aggregated forms of extracellular α-synuclein. SPARK was a randomized, double-blind, placebo-controlled, phase 2 multicenter trial evaluating 3 cinpanemab doses administered intravenously every 4 weeks for 52 weeks with an active treatment dose-blind extension period for up to 112 weeks. SPARK enrolled 357 participants diagnosed with PD within 3 years, aged 40-80 years, ≤2.5 on the modified Hoehn and Yahr scale, and with evidence of striatal dopaminergic deficit. The primary outcome was change from baseline in the Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale total score. Secondary and exploratory biomarker outcomes evaluated change from baseline at week 52 relative to placebo. Dopamine transporter SPECT and MRI were used to quantify changes in the nigrostriatal dopamine pathway and regional atrophy. CSF and plasma samples were used to assess change in total α-synuclein levels, α-synuclein seeding, and neurofilament light chain levels. SPARK was conducted from January 2018 to April 2021 and terminated due to lack of efficacy. RESULTS: Approximately 3.8% (15/398) of SPECT-imaged participants did not have evidence of dopaminergic deficit and were screen-failed. Binary classification of α-synuclein seeding designated 93% (110/118) of the enrolled CSF subgroup as positive for α-synuclein seeds at baseline. Clinical disease progression was observed, with no statistically significant difference in cinpanemab groups compared with that in placebo. Ninety-nine percent of participants with positive α-synuclein seeding remained positive through week 52. No statistically significant changes from baseline were observed between treatment groups and placebo across biomarker measures. Broadly, there was minimal annual change with high interindividual variability across biomarkers-with striatal binding ratios of the ipsilateral putamen showing the greatest mean change/SD over time. DISCUSSION: Biomarker results indicated enrollment of the intended population with early PD, but there was no significant correlation with disease progression or clear evidence of a cinpanemab treatment effect on biomarker measures. Suitable biomarkers for evaluating disease severity and progression in early PD trials are still needed. TRIAL REGISTRATION INFORMATION: NCT03318523 (clinicaltrials.gov/ct2/show/NCT03318523); Submitted October 24, 2017; First patient enrolled January 2018.