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Peptide-Based PROTAC: The Predator of Pathological Proteins

2020; Elsevier BV; Volume: 27; Issue: 6 Linguagem: Inglês

10.1016/j.chembiol.2020.06.002

2451-9456

Yan Zi Au, Tingjian Wang, Logan H. Sigua, Jun Qi,

Ubiquitin and proteasome pathways

Qu et al., 2020Qu J. Ren X. Xue F. He Y. Zhang R. Zheng Y. Huang H. Wang W. Zhang J. Specific Knockdown of α-Synuclein by Peptide-Directed Proteasome Degradation Rescued Its Associated Neurotoxicity.Cell Chem. Biol. 2020; (this issue): 751-762Google Scholar demonstrate a peptide-induced targeted degradation of the alpha-synuclein protein, a hallmark of Parkinson's disease. Using a modular three-component design, the target-protein-specific, cell-permeable peptide disposed of alpha-synuclein via the ubiquitin-proteasome pathway rather than the standard autophagy-lysosome pathway. Qu et al., 2020Qu J. Ren X. Xue F. He Y. Zhang R. Zheng Y. Huang H. Wang W. Zhang J. Specific Knockdown of α-Synuclein by Peptide-Directed Proteasome Degradation Rescued Its Associated Neurotoxicity.Cell Chem. Biol. 2020; (this issue): 751-762Google Scholar demonstrate a peptide-induced targeted degradation of the alpha-synuclein protein, a hallmark of Parkinson's disease. Using a modular three-component design, the target-protein-specific, cell-permeable peptide disposed of alpha-synuclein via the ubiquitin-proteasome pathway rather than the standard autophagy-lysosome pathway. Treatments of neurodegenerative diseases have been a challenge, in part due to the lack of "druggable" targets. Although diverse in clinical manifestation, neurodegenerative diseases share one common feature, which is the presence of protein aggregates (Gan et al., 2018Gan L. Cookson M.R. Petrucelli L. La Spada A.R. Converging pathways in neurodegeneration, from genetics to mechanisms.Nat. Neurosci. 2018; 21: 1300-1309Crossref PubMed Scopus (121) Google Scholar). Among these proteins, aggregation of alpha-synuclein (α-syn) protein is a characteristic of synucleinopathies, including Parkinson's disease (PD) (Stefanis, 2012Stefanis L. α-Synuclein in Parkinson's disease.Cold Spring Harb. Perspect. Med. 2012; 2: a009399Crossref PubMed Scopus (475) Google Scholar). α-syn is correlated to PD at both genetic and protein levels. Familial PD has been linked to mutations or amplification in the SNCA gene, while aggregates of α-syn protein are the main component of Lewy bodies, the pathological hallmark of PD (Stefanis, 2012Stefanis L. α-Synuclein in Parkinson's disease.Cold Spring Harb. Perspect. Med. 2012; 2: a009399Crossref PubMed Scopus (475) Google Scholar). With mounting genetic and molecular evidence suggesting a causative role of α-syn in the pathogenesis of PD, it is not surprising that α-syn is a candidate therapeutic target for this disease. Additionally, aggregation of α-syn damages lysosomal function, which hampers its clearance from the cell via the lysosomal pathway (Gan et al., 2018Gan L. Cookson M.R. Petrucelli L. La Spada A.R. Converging pathways in neurodegeneration, from genetics to mechanisms.Nat. Neurosci. 2018; 21: 1300-1309Crossref PubMed Scopus (121) Google Scholar). Thus, a degradation strategy involving an alternative proteolysis system could be a potential therapeutic strategy to eliminate the α-syn protein. In this study, Qu et al. utilized the proteasome system to degrade α-syn to bypass the compromised autosome-lysosome pathway in PD by designing a peptide-based proteolysis targeting chimeras (PROTAC) (Qu et al., 2020Qu J. Ren X. Xue F. He Y. Zhang R. Zheng Y. Huang H. Wang W. Zhang J. Specific Knockdown of α-Synuclein by Peptide-Directed Proteasome Degradation Rescued Its Associated Neurotoxicity.Cell Chem. Biol. 2020; (this issue): 751-762Google Scholar). PROTAC is a protein depletion approach that utilizes small molecules or peptides to artificially bridge a protein of interest with E3 ligase, which promotes the addition of ubiquitin to the target protein, thereby flagging it for subsequent proteasomal degradation. To date, more than 600 members of the E3 ubiquitin ligases have been reported, and some of them have been employed in PROTAC, namely cereblon (CRBN), Von Hippel-Lindau (VHL), DCAF, and cellular inhibitor of apoptosis (cIAP) (Burslem and Crews, 2020Burslem G.M. Crews C.M. Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery.Cell. 2020; 181: 102-114Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). With the development of PROTAC and its event-driven mechanism of action, some proteins that were previously deemed undruggable using occupancy-driven inhibitor can now be selectively degraded based on putative protein-protein interactions. Compared to small-molecule target protein binders, the use of peptides has been limited due to the lack of a strong binder as well as the cell permeability issue of the peptide-based strategy. To develop a cell-permeable PROTAC for α-syn, Qu et al. designed a synthetic trifunctional peptide, referred to as CPD-PBD-PTM peptide hereafter, with a cell-penetrating moiety (CPD), α-syn binding domain (PBD), and proteasome targeting motif (PTM) to achieve α-syn-specific protein degradation in SK-N-SH neuroblastoma cell lines as well as primary neurons (Figure 1). Prior studies on the α-syn protein family identified a crucial region in the beta-synuclein protein that interacts with α-syn both selectively and potently (Shaltiel-Karyo et al., 2010Shaltiel-Karyo R. Frenkel-Pinter M. Egoz-Matia N. Frydman-Marom A. Shalev D.E. Segal D. Gazit E. Inhibiting α-synuclein oligomerization by stable cell-penetrating β-synuclein fragments recovers phenotype of Parkinson's disease model flies.PLoS One. 2010; 5: e13863Crossref PubMed Scopus (68) Google Scholar), which was adapted by Qu et al. in the CPD-PBD-PTM peptide PROTAC to specifically recognize the intracellular α-syn. To bridge α-syn in close proximity with components of the proteasomal machinery, the authors added a short minimal element, RRRG-motif, as the PTM portion of the CPD-PBD-PTM peptide that is recognizable by E3 ligases in the ubiquitin-proteasome pathways (Bonger et al., 2011Bonger K.M. Chen L.C. Liu C.W. Wandless T.J. Small-molecule displacement of a cryptic degron causes conditional protein degradation.Nat. Chem. Biol. 2011; 7: 531-537Crossref PubMed Scopus (114) Google Scholar). Penetration of the CPD-PBD-PTM peptide through the cell membrane is achieved using a short stretch of 11 arginine-rich basic amino acid sequence derived from the trans-activator of transcription (TAT) protein of the human immunodeficient virus (HIV). Apart from peptides, this specific TAT-derived segment is also capable of delivering nucleic acids and small particles, as well as proteins across the cell membranes (Brooks et al., 2005Brooks H. Lebleu B. Vivès E. Tat peptide-mediated cellular delivery: back to basics.Adv. Drug Deliv. Rev. 2005; 57: 559-577Crossref PubMed Scopus (545) Google Scholar). Importantly, this TAT peptide is sufficient to carry peptide cargos across the blood-brain barrier in rat models (Fan et al., 2014Fan X. Jin W.Y. Lu J. Wang J. Wang Y.T. Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation.Nat. Neurosci. 2014; 17: 471-480Crossref PubMed Scopus (69) Google Scholar). Together, this 25-amino-acid-long exogenous CPD-PBD-PTM peptide molecule is sufficient to induce time- and concentration-dependent downregulation of α-syn proteins in cellular models. Indeed, α-syn proteins showed significant depletion in SK-N-SH cells, while levels of other neurodegeneration-associated proteins such as Tau, amyloid precursor protein, and DJ-1 remained unchanged. Although the RRRG-motifs are known to govern the stability of endogenous and exogenous proteins, it is not intuitive which specific members of the E3 ligase family are responsible. Proteomics analysis revealed that the RRRG-motif located on the C-terminal of the CPD-PBD-PTM peptide interacts with TRIP12, STUB1, and UHRF1 proteins, and these E3 ligases are likely to promote the ubiquitination and subsequent degradation of α-syn proteins in SK-N-SH cells. Overexpression of α-syn in cellular systems increases in mitochondrial reactive oxygen species (ROS), which in turn compromises the normal function of mitochondria (Stefanis, 2012Stefanis L. α-Synuclein in Parkinson's disease.Cold Spring Harb. Perspect. Med. 2012; 2: a009399Crossref PubMed Scopus (475) Google Scholar). Importantly, using the CPD-PBD-PTM peptide, Qu and collogues were able to reverse this phenotype in primary neurons, SK-N-SH, and HEK293T cell lines overexpressed with α-syn. Moreover, the CPD-PBD-PTM peptide can rescue the reduction of cellular viability and the increased cytotoxicity caused by α-syn overexpression, suggesting that the peptide has a beneficial effect by reducing the α-syn burden. Other neurodegenerative-associated proteins have been targeted for degradation using similar approaches. By changing the moieties of the multi-functional peptides, one can direct the protein of interests to be removed via different disposable pathways, namely lysosomal- and proteasomal-mediated pathways. Two studies targeted the Tau protein, a hallmark of Alzheimer's disease (AD), using short peptide-based PROTAC (Chu et al., 2016Chu T.T. Gao N. Li Q.Q. Chen P.G. Yang X.F. Chen Y.X. Zhao Y.F. Li Y.M. Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation.Cell Chem. Biol. 2016; 23: 453-461Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, Lu et al., 2018Lu M. Liu T. Jiao Q. Ji J. Tao M. Liu Y. You Q. Jiang Z. Discovery of a Keap1-dependent peptide PROTAC to knockdown Tau by ubiquitination-proteasome degradation pathway.Eur. J. Med. Chem. 2018; 146: 251-259Crossref Scopus (83) Google Scholar). These studies employed the Kelch-like ECH-associated protein-1 (Keap1) component of the E3 ligase complex or VHL E3 ligase to induce proteasomal-dependent Tau protein downregulation. These Tau-targeting peptides consist of an additional 4-amino-acid linker, separating the Tau-binding motif and the ligase binding domain, and the cell-penetrating domain is located in the C-terminal domain as opposed to the N-terminal domain used by Qu and colleagues. In another study, Fan et al. demonstrated that α-syn protein can be disposed of via the lysosomal degradation pathway using the cell-penetrating motif and α-syn binding domain adopted by Qu et al., but replacing the proteasome binding domain with chaperone-mediated autophagy (CMA)-targeting motif. In addition to Tau and α-syn, peptides that mediate the endosome-lysosome degradation can also be applied to target larger proteins such as PSD-95 (95 kDa) and DAPK1 (160 kDa) (Fan et al., 2014Fan X. Jin W.Y. Lu J. Wang J. Wang Y.T. Rapid and reversible knockdown of endogenous proteins by peptide-directed lysosomal degradation.Nat. Neurosci. 2014; 17: 471-480Crossref PubMed Scopus (69) Google Scholar). Collectively, these studies expand the avenues of using peptides to degrade proteins of interest both (1) in a research setting for the targets that do not have de novo chemical compounds and (2) for potential use in therapeutics. The major advantage of peptide-based PROTAC is the potential to expand the range of targetable proteins by utilizing amino acid sequences from known protein-protein interaction partners. The design of the three-component peptide including cell permeability motif can broaden the scope of target protein degradation in vitro. Using transfection or transduction, researchers could introduce the peptide-based PROTAC in the form of cDNA to a given cellular model to test for on- and off-target degradation before investing in peptide development. Target-specific PROTAC reduces the barrier of assessing the knockout of protein of interest in primary or patient-derived cells compared to techniques such as CRISPR-Cas9, which requires the expression of functional and active Cas9 within the cellular system. Just like translation of any potential therapeutics, applications of peptide-based PROTAC in treating disease is challenging. More efforts are required to ensure the biosafety and the stability of peptides-based treatments and, in the case of neurodegenerative diseases, to address the formidable challenge of transporting therapeutics across the blood-brain barrier to reach the intended cells. This work was supported in part by grants from the National Cancer Institute (NCI) CA233601 , U54HD093540-03 , CA222218 , CA176745 , CA142106 , and P50-CA100707-15 (to J.Q.), Lymphoma Leukemia Society TRP award (to J. Q.), the Alex's Lemonade Stand Foundation (J.Q.). Figure created using BioRender (https://biorender.com/). J.Q. is a scientific co-founder and consultant for Epiphanes and a shareholder of Zentalis. Specific Knockdown of α-Synuclein by Peptide-Directed Proteasome Degradation Rescued Its Associated NeurotoxicityQu et al.Cell Chemical BiologyApril 30, 2020In Briefα-Syn is an intrinsically disordered protein that is associated with the core pathology in PD. Qu et al. designed a peptide, composed of a cell-penetrating domain, an α-syn protein-binding domain, and a proteasome-targeting motif, to induce α-syn degradation. Partial reduction of α-syn induced by the peptide prevents its associated cytotoxicity. Full-Text PDF Open Archive