Induced protein degradation: an emerging drug discovery paradigm
TL;DR: Induced protein degradation has the potential to reduce systemic drug exposure, the ability to counteract increased target protein expression that often accompanies inhibition of protein function and the potential ability to target proteins that are not currently therapeutically tractable, such as transcription factors, scaffolding and regulatory proteins.
Abstract: Small-molecule drug discovery has traditionally focused on occupancy of a binding site that directly affects protein function, and this approach typically precludes targeting proteins that lack such amenable sites. Furthermore, high systemic drug exposures may be needed to maintain sufficient target inhibition in vivo, increasing the risk of undesirable off-target effects. Induced protein degradation is an alternative approach that is event-driven: upon drug binding, the target protein is tagged for elimination. Emerging technologies based on proteolysis-targeting chimaeras (PROTACs) that exploit cellular quality control machinery to selectively degrade target proteins are attracting considerable attention in the pharmaceutical industry owing to the advantages they could offer over traditional small-molecule strategies. These advantages include the potential to reduce systemic drug exposure, the ability to counteract increased target protein expression that often accompanies inhibition of protein function and the potential ability to target proteins that are not currently therapeutically tractable, such as transcription factors, scaffolding and regulatory proteins.
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TL;DR: The present work describes the development of proteolysis targeting chimeras (PROTACs) of HDAC8 based on substituted benzhydroxamic acids previously reported as potent and selectiveHDAC8 inhibitors that show anti-neuroblastoma activity in cells.
Abstract: In addition to involvement in epigenetic gene regulation, histone deacetylases (HDACs) regulate multiple cellular processes through mediating the activity of non-histone protein substrates. The knockdown of HDAC8 isozyme is associated with the inhibition of cell proliferation and apoptosis enhancement in several cancer cell lines. As shown in several studies, HDAC8 can be considered a potential target in the treatment of cancer forms such as childhood neuroblastoma. The present work describes the development of proteolysis targeting chimeras (PROTACs) of HDAC8 based on substituted benzhydroxamic acids previously reported as potent and selective HDAC8 inhibitors. Within this study, we investigated the HDAC8-degrading profiles of the synthesized PROTACs and their effect on the proliferation of neuroblastoma cells. The combination of in vitro screening and cellular testing demonstrated selective HDAC8 PROTACs that show anti-neuroblastoma activity in cells.
9 citations
TL;DR: A structure‐guided biophysical approach is described to probe the protein–protein interaction (PPI) between the Cullin‐2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel‐Lindau complex (CRL2VHL) using peptides.
Abstract: Cullin RING E3 ubiquitin ligases (CRLs) are large dynamic multi-subunit complexes that control the fate of many proteins in cells. CRLs constitute attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. Here we describe a structure-guided biophysical approach to probe the protein-protein interaction (PPI) between the Cullin-2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel-Lindau complex (CRL2VHL) using peptides. Two peptides were shown to bind at the targeted binding site on Elongin C, named the "EloC site", with micromolar dissociation constants, providing a starting point for future optimization. Our results suggest ligandability of the EloC binding site to short linear peptides, unveiling the opportunity and challenges to develop small molecules that have the potential to target selectively the Cul2-adaptor PPI within CRLs.
9 citations
TL;DR: The first application of hydrophobic tagging is applied to induce degradation of CDK9-cyclin T1 heterodimer, which is required to keep productive transcription of oncogenes in cancers and inhibited AR and Myc-driven oncogenic transcriptional programs.
Abstract: Aberrant hyperactivation of cyclins results in carcinogenesis and therapy resistance in cancers. Direct degradation of the specific cyclin or cyclin-dependent kinase (CDK)-cyclin complex by small-molecule degraders remains a great challenge. Here, we applied the first application of hydrophobic tagging to induce degradation of CDK9-cyclin T1 heterodimer, which is required to keep productive transcription of oncogenes in cancers. LL-K9-3 was identified as a potent small-molecule degrader of CDK9-cyclin T1. Quantitative and time-resolved proteome profiling exhibited LL-K9-3 induced selective and synchronous degradation of CDK9 and cyclin T1. The expressions of androgen receptor (AR) and cMyc were reduced by LL-K9-3 in 22RV1 cells. LL-K9-3 exhibited enhanced anti-proliferative and pro-apoptotic effects compared with its parental CDK9 inhibitor SNS032 and suppressed downstream signaling of CDK9 and AR more effectively than SNS032. Moreover, LL-K9-3 inhibited AR and Myc-driven oncogenic transcriptional programs and exerted stronger inhibitory effects on several intrinsic target genes of AR than the monomeric CDK9 PROTAC (Thal-SNS032).
9 citations
TL;DR: Wang et al. as mentioned in this paper reported the design and synthesis of novel PROTAC molecules based on an oseltamivir scaffold to combat severe annual influenza outbreaks, which showed good anti-H1N1 activity and efficient influenza neuraminidase (NA) degradation activity.
Abstract: Annual and sporadic influenza outbreaks pose a great threat to human health and the economy worldwide. Moreover, the frequent mutation of influenza viruses caused by antigen drift complicates the application of antiviral therapeutics. As such, there is an urgent need for novel antiviral agents to tackle the problem of insufficient efficacy of licensed drugs. Inspired by the success of the newly emerged PROTACs (PROteolysis TArgeting Chimeras) strategy, we report herein the design and synthesis of novel PROTAC molecules based on an oseltamivir scaffold to combat severe annual influenza outbreaks. Among these, several compounds showed good anti-H1N1 activity and efficient influenza neuraminidase (NA) degradation activity. The best compound, 8e, effectively induced influenza NA degradation in a dose-dependent manner and relied on the ubiquitin–proteasome pathway. Moreover, Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain (H1N1, H274Y). A molecular docking study demonstrated that Compound 8e had good hydrogen-bonding and hydrophobic interactions with both the active sites of NA and Von Hippel-Lindau (VHL) proteins, which could effectively drive the favorable interaction of these two proteins. Thus, as the first report of a successful anti-influenza PROTAC, this proof of concept will greatly widen the application range of the PROTAC technique to antiviral drug discovery.
9 citations
TL;DR: In this article , the most active PROTAC molecule U3i has a high affinity for PRC2 complex (KD = 16.19 nM) and show good inhibitory effects on MDA-MB-231 (IC50 = 0.57 μM).
Abstract: EZH2 is usually overexpressed in TNBC and other tumors, which has a great influence on the occurrence, development and prognosis of tumors. However, current EZH2 inhibitors, including Tazemetostat and GSK126, affect the methyl catalytic capacity of EZH2 and have little effect on the tumorigenic activity of EZH2 itself, resulting in poor efficacy against most solid tumors. Herein, we designed and optimized proteolytic targeting chimeras (PROTACs) precision targeting EZH2. The most active PROTAC molecule U3i has a high affinity for PRC2 complex (KD = 16.19 nM) and show good inhibitory effects on MDA-MB-231 (IC50 = 0.57 μM) and MDA-MB-468 (IC50 = 0.38 μM) cells. Compared with that of the GSK126, the growth inhibitory activities of U3i against these two TNBC cells increased by approximately 20- and 30-fold. Further studies showed that U3i can degrade PRC2 complex in TNBC cells, induce apoptosis, and cause little damage to normal cells. Therefore, U3i is a potential anticancer molecule for TNBC treatment.
9 citations
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TL;DR: Experimental and computational approaches to estimate solubility and permeability in discovery and development settings are described in this article, where the rule of 5 is used to predict poor absorption or permeability when there are more than 5 H-bond donors, 10 Hbond acceptors, and the calculated Log P (CLogP) is greater than 5 (or MlogP > 415).
14,026 citations
TL;DR: This study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
12,865 citations
TL;DR: The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage as discussed by the authors.
Abstract: Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
12,265 citations
01 Feb 2013
TL;DR: Two different type II CRISPR/Cas systems are engineered and it is demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
Abstract: Genome Editing Clustered regularly interspaced short palindromic repeats (CRISPR) function as part of an adaptive immune system in a range of prokaryotes: Invading phage and plasmid DNA is targeted for cleavage by complementary CRISPR RNAs (crRNAs) bound to a CRISPR-associated endonuclease (see the Perspective by van der Oost). Cong et al. (p. 819, published online 3 January) and Mali et al. (p. 823, published online 3 January) adapted this defense system to function as a genome editing tool in eukaryotic cells. A bacterial genome defense system is adapted to function as a genome-editing tool in mammalian cells. [Also see Perspective by van der Oost] Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
10,746 citations
TL;DR: The results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents and the generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of ‘personalized’ therapeutic regimens.
Abstract: The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of 'personalized' therapeutic regimens.
6,417 citations