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 potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets is demonstrated and DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL.
Abstract: B-cell lymphoma extra large (BCL-XL) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis-targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL. The first BCL-XL-degrading PROTAC achieves safer and more potent antitumor activity than dual BCL-XL and BCL-2 inhibitor navitoclax because of reduced dose-limiting platelet toxicity and high target specificity.
289 citations
TL;DR: It is shown that varying the linker between warhead and E3 ligand and the orientation of the E3ligase allow tuning PROTAC selectivity toward different p38 isoforms, resulting in degradation of p38α or p38δ.
Abstract: PROteolysis-TArgeting Chimeras (PROTACs) are hetero-bifunctional molecules that recruit an E3 ubiquitin ligase to a given substrate protein resulting in its targeted degradation. Many potent PROTACs with specificity for dissimilar targets have been developed; however, the factors governing degradation selectivity within closely-related protein families remain elusive. Here, we generate isoform-selective PROTACs for the p38 MAPK family using a single warhead (foretinib) and recruited E3 ligase (von Hippel-Lindau). Based on their distinct linker attachments and lengths, these two PROTACs differentially recruit VHL, resulting in degradation of p38α or p38δ. We characterize the role of ternary complex formation in driving selectivity, showing that it is necessary, but insufficient, for PROTAC-induced substrate ubiquitination. Lastly, we explore the p38δ:PROTAC:VHL complex to explain the different selectivity profiles of these PROTACs. Our work attributes the selective degradation of two closely-related proteins using the same warhead and E3 ligase to heretofore underappreciated aspects of the ternary complex model. PROTACs enable targeted protein degradation by recruiting an E3 ligase to a specific substrate but the determinants of selectivity are not fully understood. Here, the authors show that varying the linker between warhead and E3 ligand and the orientation of the E3 ligase allow tuning PROTAC selectivity toward different p38 isoforms.
279 citations
University of New Mexico1, University of Copenhagen2, European Bioinformatics Institute3, University of North Carolina at Chapel Hill4, National Institutes of Health5, Icahn School of Medicine at Mount Sinai6, Baylor College of Medicine7, University of California, San Francisco8, University of Zurich9, AstraZeneca10, GlaxoSmithKline11, University of Miami12, Yale University13, Google14
TL;DR: How the systematic collection and processing of a wide array of genomic, proteomic, chemical and disease-related resource data by the IDG Knowledge Management Center have enabled the development of evidence-based criteria for tracking the target development level (TDL) of human proteins is discussed.
Abstract: A large proportion of biomedical research and the development of therapeutics is focused on a small fraction of the human genome. In a strategic effort to map the knowledge gaps around proteins encoded by the human genome and to promote the exploration of currently understudied, but potentially druggable, proteins, the US National Institutes of Health launched the Illuminating the Druggable Genome (IDG) initiative in 2014. In this article, we discuss how the systematic collection and processing of a wide array of genomic, proteomic, chemical and disease-related resource data by the IDG Knowledge Management Center have enabled the development of evidence-based criteria for tracking the target development level (TDL) of human proteins, which indicates a substantial knowledge deficit for approximately one out of three proteins in the human proteome. We then present spotlights on the TDL categories as well as key drug target classes, including G protein-coupled receptors, protein kinases and ion channels, which illustrate the nature of the unexplored opportunities for biomedical research and therapeutic development.
274 citations
TL;DR: The current state of BET inhibitor biology is reviewed, the next wave of bromodomain inhibitors with clinical potential in oncology and non-oncology indications are discussed, and the lessons learned from BET inhibitor programmes should affect efforts to develop drugs that target non-BET bRomodomains and other epigenetic readers.
Abstract: Less than a decade ago, it was shown that bromodomains, acetyl lysine 'reader' modules found in proteins with varied functions, were highly tractable small-molecule targets. This is an unusual property for protein-protein or protein-peptide interaction domains, and it prompted a wave of chemical probe discovery to understand the biological potential of new agents that targeted bromodomains. The original examples, inhibitors of the bromodomain and extra-terminal (BET) class of bromodomains, showed enticing anti-inflammatory and anticancer activities, and several compounds have since advanced to human clinical trials. Here, we review the current state of BET inhibitor biology in relation to clinical development, and we discuss the next wave of bromodomain inhibitors with clinical potential in oncology and non-oncology indications. The lessons learned from BET inhibitor programmes should affect efforts to develop drugs that target non-BET bromodomains and other epigenetic readers.
269 citations
TL;DR: The evolution of the approach to target validation, hit and lead optimization, pharmacokinetic/pharmacodynamic modelling and drug safety testing, which have helped improve the quality of candidate drug nomination, as well as the development of the right culture.
Abstract: In 2011, AstraZeneca embarked on a major revision of its research and development (R&D) strategy with the aim of improving R&D productivity, which was below industry averages in 2005-2010. A cornerstone of the revised strategy was to focus decision-making on five technical determinants (the right target, right tissue, right safety, right patient and right commercial potential). In this article, we describe the progress made using this '5R framework' in the hope that our experience could be useful to other companies tackling R&D productivity issues. We focus on the evolution of our approach to target validation, hit and lead optimization, pharmacokinetic/pharmacodynamic modelling and drug safety testing, which have helped improve the quality of candidate drug nomination, as well as the development of the right culture, where 'truth seeking' is encouraged by more rigorous and quantitative decision-making. We also discuss where the approach has failed and the lessons learned. Overall, the continued evolution and application of the 5R framework are beginning to have an impact, with success rates from candidate drug nomination to phase III completion improving from 4% in 2005-2010 to 19% in 2012-2016.
261 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