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: This review endeavors to exam the potential role of UPS in p-tau aggregation, and how pathogenic tau may inflict other abnormalities such as amyloid beta accumulation in Alzheimer's disease.
12 citations
TL;DR: This work reviews some of the main PROTACs that have been reported recently and discusses their potential therapeutic benefits over classical enzyme inhibition, and future research is expected to focus on the delivery and bioavailability of PROTacs due to their high molecular weight.
Abstract: Proteolysis-targeting chimeras (PROTACs) are an emerging tool for therapeutic intervention by reducing or eliminating disease-causing proteins. PROTACs are bifunctional molecules that consist of a target protein ligand, a linker and an E3 ligase ligand, which mediate the polyubiquitination of the target protein, ultimately leading to the target protein degradation by the ubiquitin–proteasome pathway. We review some of the main PROTACs that have been reported recently and discuss their potential therapeutic benefits over classical enzyme inhibition. Future research is expected to focus on the delivery and bioavailability of PROTACs due to their high molecular weight (700–1000 Da).
12 citations
25 Feb 2021
TL;DR: In this article, small molecules that induce degradation could represent an attractive, simple option to overcoming the limitations of both traditional modulators and PROTACs, and a general screening approach can be successful in identifying useful start points for developing small-molecule degraders.
Abstract: Targeted protein degradation (TPD) is a recent strategy, utilizing the cell's proteostasis machinery to deplete specific proteins. This represents a paradigm shift in early drug discovery, away from occupancy-driven to event-driven mechanisms.Recent efforts have focused on the development of proteolysis-targeting chimeras (PROTACs). These heterobifunctional molecules combine a target-specific binding moiety linked to an E3 ligase ligand and trigger selective ubiquitination of the target protein, marking it for proteasomal degradation. While these molecules can be highly efficacious, they generally have unfavorable physicochemical properties due to their large size.In contrast, smaller molecules that induce degradation could represent an attractive, simple option to overcoming the limitations of both traditional modulators and PROTACs. These molecules may have a range of mechanisms: recruitment of an E3 ligase (molecular glues), introduction of hydrophobic areas, or inducing local unfolding, each of which triggers degradation.We recently completed a high-throughput screen of 111,000 compounds in a cellular HiBiT assay in an effort to identify such molecules. Preliminary analysis indicates that we have been able to identify alternative small-molecule degraders. We highlight methods for triage, characterization, selectivity, and mode of action. In summary, we believe that these types of small-molecule degraders, which may possibly have more acceptable physicochemical properties than the inherently larger heterobifunctional molecules, are an exciting approach for inducing TPD, and we illustrate that a general screening approach can be successful in identifying useful start points for developing such molecules.
12 citations
TL;DR: In this article, a method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a single target-of-interest was presented.
Abstract: Peptide secondary metabolites are common in nature and have diverse pharmacologically-relevant functions, from antibiotics to cross-kingdom signaling. Here, we present a method to design large libraries of modified peptides in Escherichia coli and screen them in vivo to identify those that bind to a single target-of-interest. Constrained peptide scaffolds were produced using modified enzymes gleaned from microbial RiPP (ribosomally synthesized and post-translationally modified peptide) pathways and diversified to build large libraries. The binding of a RiPP to a protein target leads to the intein-catalyzed release of an RNA polymerase σ factor, which drives the expression of selectable markers. As a proof-of-concept, a selection was performed for binding to the SARS-CoV-2 Spike receptor binding domain. A 1625 Da constrained peptide (AMK-1057) was found that binds with similar affinity (990 ± 5 nM) as an ACE2-derived peptide. This demonstrates a generalizable method to identify constrained peptides that adhere to a single protein target, as a step towards “molecular glues” for therapeutics and diagnostics. Peptide secondary metabolites have a diverse range of functions. Here the authors present a method to design and screen a large library of modified peptides in E. coli against a target of interest.
12 citations
TL;DR: A quantitative assay for the discovery and characterization of E3 ligase ligands that is based on the thermophoretic behavior of a custom reporter ligand is described, which can be employed for the high-throughput screening of compound libraries for a given ligase but also for hit validation.
Abstract: Repurposing E3 ubiquitin ligases for targeted protein degradation via customized molecular glues or proteolysis-targeting chimeras (PROTACs) is an increasingly important therapeutic modality. Currently, a major limitation in the design of suitable molecular glues and PROTACs is our fragmentary understanding of E3 ligases and their ligand space. We here describe a quantitative assay for the discovery and characterization of E3 ligase ligands that is based on the thermophoretic behavior of a custom reporter ligand. Thereby, it is orthogonal to commonly employed fluorescence-based assays and less affected by the optical properties of test compounds. It can be employed for the high-throughput screening of compound libraries for a given ligase but also for hit validation, which we demonstrate with the identification of unexpected well-binders and non-binders, yielding new insights into the ligand space of cereblon (CRBN).
12 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