α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson’s disease (PD). Despite the protein’s pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14 N/ 15 N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson’s disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson’s disease patients in a phase 2 trial, has had in animal models of PD.
MYC is a transcription factor involved in fundamental cellular functions such as proliferation, apoptosis, differentiation, metabolism and, as lately discovered, immune response. [1,2] It is deregulated in a wide variety of aggressive human cancers and its overexpression is one of the most common events associated with tumorigenesis, making it one of the most attractive yet challenging targets in oncology.[3] c-MYC protein structure is mostly disordered in its monomeric state, and assumes a helix-loop-helix fold upon binding to its protein partner MAX. The absence of clefts or hydrophobic pockets (typical of enzymes) and the large protein-protein interaction surface formed in the c-MYC/MAX complex represent a challenge for the design of small molecule inhibitors. Moreover, the intra-nuclear localization of c-MYC renders the use of antibodies impossible. Hence, it is not surprising that conventional drug development has failed and no therapeutic has yet progressed into clinical trials.[4] Here we describe IDP-121, a stapled peptide specifically designed to target c-MYC protein. A library based on c, l, n-MYC and MAX druggable regions was designed, with a bias towards disruption of c-MYC (residual) intra-protein interactions. The primary goal in the initial screen was to identify compounds able to block c-MYC's fold-like state that allows binding to MAX. A functional (cellular) assay was used for our screening program to ensure cell and nuclear penetration. Stapling technology was implemented to overcome the lack of stability and cell permeability common to unmodified peptides, whilst retaining c-MYC specificity and a large surface coverage. The lead compound, IDP-121, is stable to proteolysis and is rapidly internalized into cells, distributing efficiently into the nucleus (see Abstract 3). The interaction with c-MYC was characterized by HSQC-NMR, Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR). IDP-121 binds specifically to c-MYC, with a Kd of 400 nM, an affinity ten-fold higher than c-MYC/MAX interaction, allowing IDP-121 to displace MAX from the binding site. SAR studies were conducted to tailor the elements essential for its biological function. Isolated protein complex (ELISA), and pull-down and FRET experiments confirmed disruption of the c-MYC/MAX complex by IDP-121 (see Abstracts 2). The drug-like properties and efficacy in hematological and solid tumors of IDP-121 have been demonstrated in vivo (see Abstracts 2 and 3). In addition, IDP-121 has progressed through GLP toxicology studies without evidence of major systemic toxicity, allowing for the first viable Myc-targeted therapy to enter Phase 1 clinical trials in 2021. 1.Casey S.C. et al. Science 2016; 352 (6282): 227–231 2.Casey S.C. et al. Blood 2018; 131 (18): 2007-20153.Dang CV. Cell. 2012;149(1):22-35.4. Whitfield J. R., Front. Cell Dev. Biol. 2017 Citation Format: Carmen Calvis, Andreas Beier, Michael Feichtinger, Theresa Höfurthner, Miguel Moreno, Ramon Messeguer, Robert Konrat, Santiago Esteban, Laura Nevola. IDP-121, a first in class staple peptide targeting c-MYC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2471.
NMR spectroscopy has matured into a powerful tool to characterize interactions between biological molecules at atomic resolution, most importantly even under near to native (physiological) conditions. The field of in-cell NMR aims to study proteins and nucleic acids inside living cells. However, cells interrogate their environment and are continuously modulated by external stimuli. Cell signaling processes are often initialized by membrane receptors on the cell surface; therefore, characterizing their interactions at atomic resolution by NMR, hereafter referred as on-cell NMR, can provide valuable mechanistic information. This review aims to summarize recent on-cell NMR tools that give information about the binding site and the affinity of membrane receptors to their ligands together with potential applications to in vivo drug screening systems.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.