Publications by authors named "Rohan E J Beckwith"

12 Publications

  • Page 1 of 1

A Novel Luminescence-Based High-Throughput Approach for Cellular Resolution of Protein Ubiquitination Using Tandem Ubiquitin Binding Entities (TUBEs).

SLAS Discov 2020 04 30;25(4):350-360. Epub 2020 Jan 30.

Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, Cambridge, MA, USA.

Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.
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http://dx.doi.org/10.1177/2472555219901261DOI Listing
April 2020

CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.

Nat Chem Biol 2020 01 9;16(1):50-59. Epub 2019 Dec 9.

Novartis Institutes for BioMedical Research, Cambridge, MA, USA.

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing's sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing's sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.
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http://dx.doi.org/10.1038/s41589-019-0424-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116157PMC
January 2020

Reactive carbon species tamed for synthesis.

Nature 2018 02;554(7690):36-38

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http://dx.doi.org/10.1038/d41586-018-01308-7DOI Listing
February 2018

Reactive carbon species tamed for synthesis.

Nature 2018 Feb;554(7690):36-38

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http://dx.doi.org/10.1038/d41586-018-01308-7DOI Listing
February 2018

Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

Nature 2016 Mar;531(7596):598-603

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland.

The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.
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http://dx.doi.org/10.1038/nature17416DOI Listing
March 2016

Late-stage C-H functionalization of complex alkaloids and drug molecules via intermolecular rhodium-carbenoid insertion.

Nat Commun 2015 Jan 12;6:5943. Epub 2015 Jan 12.

Department of Global Discovery Chemistry, Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

Alkaloids constitute a large family of natural products possessing diverse biological properties. Their unique and complex structures have inspired numerous innovations in synthetic chemistry. In the realm of late-stage C-H functionalization, alkaloids remain a significant challenge due to the presence of the basic amine and a variety of other functional groups. Herein we report the first examples of dirhodium(II)-catalysed intermolecular C-H insertion into complex natural products containing nucleophilic tertiary amines to generate a C-C bond. The application to a diverse range of alkaloids and drug molecules demonstrates remarkable chemoselectivity and predictable regioselectivity. The capacity for late-stage diversification is highlighted in the catalyst-controlled selective functionalizations of the alkaloid brucine. The remarkable selectivity observed, particularly for site-specific C-H insertion at N-methyl functionalities, offers utility in a range of applications where efficient installation of synthetic handles on complex alkaloids is desired.
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http://dx.doi.org/10.1038/ncomms6943DOI Listing
January 2015

Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide.

Nature 2014 Aug 16;512(7512):49-53. Epub 2014 Jul 16.

1] Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland [2] University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland.

In the 1950s, the drug thalidomide, administered as a sedative to pregnant women, led to the birth of thousands of children with multiple defects. Despite the teratogenicity of thalidomide and its derivatives lenalidomide and pomalidomide, these immunomodulatory drugs (IMiDs) recently emerged as effective treatments for multiple myeloma and 5q-deletion-associated dysplasia. IMiDs target the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4(CRBN)) and promote the ubiquitination of the IKAROS family transcription factors IKZF1 and IKZF3 by CRL4(CRBN). Here we present crystal structures of the DDB1-CRBN complex bound to thalidomide, lenalidomide and pomalidomide. The structure establishes that CRBN is a substrate receptor within CRL4(CRBN) and enantioselectively binds IMiDs. Using an unbiased screen, we identified the homeobox transcription factor MEIS2 as an endogenous substrate of CRL4(CRBN). Our studies suggest that IMiDs block endogenous substrates (MEIS2) from binding to CRL4(CRBN) while the ligase complex is recruiting IKZF1 or IKZF3 for degradation. This dual activity implies that small molecules can modulate an E3 ubiquitin ligase and thereby upregulate or downregulate the ubiquitination of proteins.
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http://dx.doi.org/10.1038/nature13527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4423819PMC
August 2014

Plate-based diversity selection based on empirical HTS data to enhance the number of hits and their chemical diversity.

J Biomol Screen 2009 Jul 16;14(6):690-9. Epub 2009 Jun 16.

Lead Discovery Informatics, Center for Proteomic Chemistry Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA.

Typically, screening collections of pharmaceutical companies contain more than a million compounds today. However, for certain high-throughput screening (HTS) campaigns, constraints posed by the assay throughput and/or the reagent costs make it impractical to screen the entire deck. Therefore, it is desirable to effectively screen subsets of the collection based on a hypothesis or a diversity selection. How to select compound subsets is a subject of ongoing debate. The authors present an approach based on extended connectivity fingerprints to carry out diversity selection on a per plate basis (instead of a per compound basis). HTS data from 35 Novartis screens spanning 5 target classes were investigated to assess the performance of this approach. The analysis shows that selecting a fingerprint-diverse subset of 250K compounds, representing 20% of the screening deck, would have achieved significantly higher hit rates for 86% of the screens. This measure also outperforms the Murcko scaffold-based plate selection described previously, where only 49% of the screens showed similar improvements. Strikingly, the 2-fold improvement in average hit rates observed for 3 of 5 target classes in the data set indicates a target bias of the plate (and thus compound) selection method. Even though the diverse subset selection lacks any target hypothesis, its application shows significantly better results for some targets-namely, G-protein-coupled receptors, proteases, and protein-protein interactions-but not for kinase and pathway screens. The synthetic origin of the compounds in the diverse subset appears to influence the screening hit rates. Natural products were the most diverse compound class, with significantly higher hit rates compared to the compounds from the traditional synthetic and combinatorial libraries. These results offer empirical guidelines for plate-based diversity selection to enhance hit rates, based on target class and the library type being screened.
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http://dx.doi.org/10.1177/1087057109335678DOI Listing
July 2009

Catalytic asymmetric reactions for organic synthesis: the combined C-H activation/siloxy-cope rearrangement.

J Org Chem 2004 Dec;69(26):9241-7

Department of Chemistry, University at Buffalo, State University of New York, New York 14260-3000, USA.

Tetrakis(N-[4-dodecylbenzenesulfonyl]-(L)-prolinate) dirhodium [Rh(2)(S-DOSP)(4)]-catalyzed decomposition of vinyldiazoacetates in the presence of allyl silyl ethers results in the formation of the direct C-H insertion product and the product derived from a combined C-H activation/siloxy-Cope rearrangement. Both products are formed with very high diastereoselectivity (>94% de) and high enantioselectvity (78-93% ee). Under thermal or microwave conditions, the direct C-H insertion product undergoes a siloxy-Cope rearrangement in a stereoselective manner.
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http://dx.doi.org/10.1021/jo048429mDOI Listing
December 2004

Catalytic enantioselective C-H activation by means of metal-carbenoid-induced C-H insertion.

Chem Rev 2003 Aug;103(8):2861-904

Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260-3000, USA.

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http://dx.doi.org/10.1021/cr0200217DOI Listing
August 2003

New strategic reactions for organic synthesis: catalytic asymmetric C-H activation alpha to oxygen as a surrogate to the aldol reaction.

J Org Chem 2003 Aug;68(16):6126-32

Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000, USA.

The C-H activation of silyl ethers by means of rhodium carbenoid-induced C-H insertion represents a very direct method for the stereoselective synthesis of silyl-protected beta-hydroxy esters. The reaction can proceed with very high regio-, diastereo-, and enantioselectivity and represents a surrogate to the aldol reaction. The reaction is catalyzed by the rhodium prolinate complex Rh(2)(S-DOSP)(4). A critical requirement for the high chemoselectivity is the use of donor/acceptor-substituted carbenoids such as those derived from methyl aryldiazoacetates. A range of silyl ethers may be used such as allyl silyl ethers, tetraalkoxysilanes, and even simple trimethylsilyl alkyl ethers. In general, C-H activation preferentially occurs at methylene sites, as the reactivity is controlled by a delicate balance between steric and electronic effects.
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http://dx.doi.org/10.1021/jo034533cDOI Listing
August 2003