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Arrowsmith, C.

Publications and source records attributed to Arrowsmith, C..

At least 19 recordsLinked to original sources

An Atlas of Short Linear Motif-Mediated Human Protein-Protein Interactions

Short linear motifs (SLiMs) within intrinsically disordered protein regions mediate transient interactions crucial for cell physiology1. However, the global interaction landscape of human SLiMs remains largely uncharted. Here we present the Atlas of SLiM-mediated Human protein-protein Interactions (ASHI), which maps more than 20,000 interactions by screening over 800 human protein domains against a library of one million peptides tiling the human disordered proteome. ASHI expands the SLiM interactome, uncovers novel binding modes for known peptide-binding domains, and reveals unexpected peptide-binding activities in enzymes, chaperones, RNA-binding proteins, and modification-reader domains. Furthermore, intrinsically disordered regions emerge as densely encoded interaction platforms where interaction specificity is governed by diverse mechanisms, including key motif determinants, flanking residues, competition, and multivalency. These data provide an unprecedented foundation for modeling dynamic interaction networks, interpreting disease-associated variants, and decoding the dark proteome.

biochemistry↗

Direct-to-Biology Enables Rapid Identification of Potent FBXO22 Degraders

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules that bring a ubiquitin E3 ligase into proximity of a target protein to polyubiquitinate and degrade the target. PROTACs act catalytically, offering distinct advantages over conventional inhibitors and are the subject of intense study. The development of PROTACs involves extensive optimization of the chemical moiety linking two different protein-binding chemotypes, often requiring the synthesis, purification and testing of hundreds of PROTAC candidates. We used this approach to rapidly explore the landscape of targeted degradation of four different targets in parallel, combining and comparing a recently reported FBXO22-recruiting chemical warhead with warheads for the commonly used CRBN and VHL E3 ligases. Using a limited number of compounds (175 compounds in total) we observed no FBXO22-dependent degradation of these four targets. However, our libraries generated potent FBXO22 homo-PROTACs inducing self-degradation, as well as CRBN- and VHL-mediated degraders of FBXO22.

cell biology↗

Identification and Characterization of a Small Molecule Ligand for the Huntingtin-HAP40 Complex

Huntingtons disease (HD) is caused by a CAG repeat expansion mutation, giving rise to a polyglutamine expansion in the huntingtin (HTT). However, the explicit molecular functions of HTT and opportunities for direct pharmacological modulation remain incompletely understood. Here, we report the discovery of a small molecule ligand for the full-length HTT protein in complex with its partner, HAP40. Using affinity selection mass spectrometry (AS-MS), we identified a stereoselective binder, whose binding was characterized by surface plasmon resonance, hydrogen-deuterium exchange mass spectrometry, and cryo-electron microscopy at 2.3 [A] resolution. The ligand binds HTT-HAP40 in vitro with single-digit micromolar affinity and one-to-one stoichiometry at a druggable interface previously predicted computationally. In silico studies predicted and experimental analyses confirmed the (R)-enantiomer as the eutomer and initial structure activity relationship was established experimentally. This work details a structurally-validated chemical scaffold and highlights a ligandable pocket which could enable development of chemical probes for probing HTT biology, as well as therapeutics such as degraders and imaging agents for HD.

pharmacology and toxicology↗

Development of a dual chemical probe for the USP16 and HDAC6 zinc-finger ubiquitin-binding domain

Ubiquitin-specific peptidase 16 (USP16) is a deubiquitinase that specifically cleaves ubiquitin from histone H2A, and modulates gene expression, cell cycle regulation, and various other cellular processes. The USP16 zinc-finger ubiquitin-binding domain (UBD) binds the free C-terminal end of both ubiquitin and ISG15, two major signaling proteins that mediate many biological pathways. Because the precise function of USP16-UBD and its interactions remains unclear, a small molecule antagonist targeting the USP16-UBD could enable cellular studies to elucidate its biological role. Here we report SGC-UBD1031 (15), a chemical probe targeting USP16-UBD with similar in vitro binding profiles to HDAC6-UBD and selectivity over nine other UBDs. In cellular assays, 15 disrupts the interaction between the C-terminus of ISG15 and USP1-UBD, as well as the interaction between ISG15 and HDAC6 UBD, at a concentration of 1 M. The corresponding enantiomer SGC-UBD1031N (16), does not interfere with these interactions, even at concentrations as high as 30 M, and thus serves as a negative control. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/684218v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@50def6org.highwire.dtl.DTLVardef@15dedc4org.highwire.dtl.DTLVardef@1a50169org.highwire.dtl.DTLVardef@1ffbb4b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

Cathepsin K as a Key SARS-CoV-2 Cell Entry Protease and Dual-Inhibition Target

SARS-CoV-2 relies on host proteases to prime its spike protein for cell entry through either the endosomal or plasma membrane pathway. Although cysteine cathepsins are known to mediate the endosomal route, the identity of the dominant enzyme has remained unclear. Here, we identify human Cathepsin K (hCatK), a lysosomal cysteine protease, as a previously unrecognized yet functionally important mediator of spike activation. While human Cathepsin L (hCatL) has long been regarded as the principal endosomal protease for spike processing, inhibition of hCatK with the selective inhibitor Odanacatib suppressed viral infection in endothelial cells as effectively as the broad-spectrum cysteine protease inhibitor E-64d, implicating hCatK as a key driver of spike processing during the endosomal viral entry. Comprehensive enzymatic profiling demonstrated that hCatK exhibits 24- to 63-fold higher catalytic efficiency toward the Furin-cleavage site (FCS) sequence than hCatL and displays a distinct substrate-recognition pattern at the Omicron FCS relative to the Wuhan variant. We further demonstrate that hCatK is an off-target of Nirmatrelvir, a clinically approved 3CL-Mpro inhibitor, with a sub-micromolar potency (IC50 = 0.6 {+/-} 0.1 {micro}M). A 1.9 [A] crystal structure of the hCatK-Nirmatrelvir complex delineates the molecular basis of inhibitor binding and supports the rational design of dual-acting antivirals. Collectively, these findings redefine the landscape of host proteases involved in SARS-CoV-2 spike activation and establish hCatK as a previously overlooked but strategic target for antiviral intervention.

biochemistry↗

Glioblastoma stem cells show transcriptionally correlated spatial organization

Glioblastoma (GBM) is an aggressive brain cancer with a poor survival rate. Despite hundreds of clinical trials, there is no effective targeted therapy. Glioblastoma stem cells (GSCs) are an important GBM model system. In culture, these cells form spatial structures that share morphological aspects with their source tumors. We collected 17,000 phase contrast images of 15 patient-derived GSC lines growing to confluence. We find that GSCs grow in characteristic multicellular patterns depending on their transcriptional state. Interpretable computer vision algorithms identified specific image features that predict transcriptional state across multiple cell confluency levels. This relationship will be useful in developing GSC screens where image features can be used to identify how GSC biology changes in response to perturbations simply by imaging cultured cells on plates.

cancer biology↗

Heli-SMACC: Helicase-targeting SMAll Molecule Compound Collection

Helicases have emerged as promising targets for the development of antiviral drugs; however, the family remains largely undrugged. To support the focused development of viral helicase inhibitors we identified, collected, and integrated all chemogenomics data for all available helicases from the ChEMBL database. After thoroughly curating and enriching the data with relevant annotations we have created a derivative database of helicase inhibitors which we dubbed Heli-SMACC (Helicase-targeting SMAll Molecule Compound Collection). The current version of Heli-SMACC contains 20,432 bioactivity entries for viral, human, and bacterial helicases. We have selected 30 compounds with promising viral helicase activity and tested them in a SARS-CoV-2 NSP13 ATPase assay. Twelve compounds demonstrated ATPase inhibition and a consistent dose-response curve. The Heli-SMACC database may serve as a reference for virologists and medicinal chemists working on the development of novel helicase inhibitors. Heli-SMACC is publicly available at https://smacc.mml.unc.edu. HighlightsO_LIWe created a curated Helicase-Targeting SMAll Molecule Compound Collection (Heli-SMACC). C_LIO_LIHeli-SMACC covers 29 human, viral, and bacterial helicases. C_LIO_LITwelve of thirty selected compounds demonstrated inhibitory activity in a SARS-CoV-2 NSP13 ATPase Assay. C_LIO_LIHeli-SMACC is freely available online at https://smacc.mml.unc.edu. C_LI TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/602122v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@8e098forg.highwire.dtl.DTLVardef@115cb2borg.highwire.dtl.DTLVardef@1cd9da3org.highwire.dtl.DTLVardef@2870a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

PRMT5 is required for full-length HTT expression by repressing multiple proximal intronic polyadenylation sites

Expansion of the CAG trinucleotide repeat tract in exon 1 of the Huntingtin (HTT) gene above a threshold of [~]36 repeats causes Huntingtons disease (HD) through the expression of a polyglutamine-expanded form of the HTT protein. This mutation triggers wide-ranging cellular and biochemical pathologies leading to cognitive, motor, and psychiatric symptoms in HD patients. As accurate splicing is required to produce the full-length HTT protein of [~]348 kDa, targeting HTT splicing with small molecule drugs is a compelling approach to lower HTT protein levels to treat HD, and splice modulators are being tested in the clinic. Here, we identify PRMT5 as a novel regulator of HTT mRNA splicing and alternative polyadenylation. PRMT5 inhibition disrupts the splicing of HTT introns 9 and 10, leading to activation of multiple proximal intronic polyadenylation sites within these introns and promoting premature termination, cleavage and polyadenylation (PCPA) of the HTT mRNA, thus lowering total HTT protein levels. We also detected increasing levels of these truncated, intron-containing HTT transcripts across a series of neuronal differentiation samples which correlated with lower PRMT5 expression. Notably, PRMT5 inhibition in glioblastoma (GBM) stem cells potently induced neuronal differentiation. We posit that PRMT5-mediated regulation of intronic polyadenylation, premature termination and cleavage of the HTT mRNA modulates HTT expression and plays an important role during embryonic development and neuronal differentiation.

neuroscience↗

Epigenetic vulnerabilities of leukemia harboring inactivating EZH2 mutations

Epigenetic regulators such as the polycomb repressive complex 2 (PRC2) play a critical role in both normal development and carcinogenesis. Mutations and functional dysregulation of PRC2 complex components such as EZH2 are implicated in various forms of cancer and associated with poor prognosis. This study investigated the epigenetic vulnerabilities of acute myeloid leukemia (AML) and myelodysplastic/myeloproliferative disorders (MDS/MPN) by performing a chemical probe screen in patient cells. Paradoxically, we observed increased sensitivity to EZH2 and EED inhibitors in AML and MDS/MPN patient cells harboring EZH2 mutations. Expression analysis indicated that EZH2 inhibition elicited upregulation of pathways responsible for cell death and growth arrest, specifically in patient cells with mutant EZH2. The identified EZH2 mutations had drastically reduced catalytic activity, resulting in lower cellular H3K27me3 levels and were associated with decreased EZH2 and PRC2 component EED protein levels. Overall, this study provides an important understanding of the role of EZH2 dysregulation in blood cancers and may indicate disease etiology for these poor prognosis AML and MDS/MPN cases.

cancer biology↗

Chemical Tools for the Gid4 Subunit of the Human E3 Ligase C-terminal to LisH (CTLH) Degradation Complex

We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.

biophysics↗

Recruitment of FBXO22 for Targeted Degradation of NSD2

Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.

biochemistry↗

Probing the mechanism of Cbl-b inhibition by a small-molecule inhibitor

Cbl-b is a RING-type E3 ubiquitin ligase that is expressed in several immune cell lineages, where it negatively regulates the activity of immune cells. Cbl-b has specifically been identified as an attractive target for cancer immunotherapy due to its role in promoting an immunosuppressive tumor environment, and Nx-1607, is in phase I clinical trials for advanced solid tumor malignancies. Using a suite of biophysical and cellular assays, we confirmed potent binding of C7683 (an analogue of Nx-1607) to the full-length Cbl-b and its N-terminal fragment containing the TKBD-LHR-RING domains. To further elucidate its mechanism of inhibition, we determined the co-crystal structure of Cbl-b with C7683, revealing compound interaction with both the TKBD and LHR, but not the ring domain. Here, we provide structural insights into a novel mechanism of Cbl-b inhibition by a small-molecule inhibitor that locks the protein in an inactive conformation by acting as an intramolecular glue.

immunology↗

RAS and PP2A activities converge on epigenetic gene regulation

RAS-mediated human cell transformation requires inhibition of the tumor suppressor Protein Phosphatase 2A (PP2A). Both RAS and PP2A mediate their effects by phosphoregulation, but phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, based on mass spectrometry phosphoproteome data we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168 and TP53BP1. Mechanistically, we validate co-regulation of NuRD chromatin repressor complex by RAS and PP2A. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter de-repression and activation of oncogenic transcription. Notably, transcriptional de-repression by PP2A inhibition was associated with increased euchromatin and decrease in global DNA methylation. Further, targeting of RAS- and PP2A-regulated epigenetic proteins decreased viability of KRAS-mutant human lung cancer cells. Collectively the results indicate that epigenetic protein complexes involved in oncogenic gene expression constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Further, the results provide a rich source for future understanding of phosphorylation as a previously unappreciated layer of regulation of epigenetic gene regulation in cancer, and in other RAS/PP2A-regulated cellular processes.

cancer biology↗

cGAS recruitment to micronuclei is dictated by pre-existing nuclear chromatin status.

Micronuclei (MN) are aberrant cytosolic compartments containing broken genomic fragments or whole lagging chromosomes. MN envelopes irreversibly rupture, allowing the viral receptor cGAS to localize to MN and initiate an inflammatory signalling cascade. Here, we demonstrate that MN envelope rupture is not sufficient for cGAS localization. Unlike MN that arise following ionizing radiation (IR), ruptured MN generated from acute transcription stressors DRB or siSRSF1 are refractory to cGAS localization. Recruitment of cGAS to MN is blocked by inhibiting the histone methyltransferase DOT1L prior to IR exposure, demonstrating that cGAS recruitment to MN is dictated by nuclear chromatin organization at the time of DNA damage. Loss of cGAS+ MN, caused either by acute transcription stressors or by preventing DOT1L-deposited histone methylation, corresponded to significantly decreased cGAS-dependent inflammatory signalling. These results implicate nuclear chromatin organization in micronuclear composition and activity, influencing the ability of damage-induced MN to retain cytosolic proteins upon rupture.

cell biology↗

Structure Activity Relationship of USP5 Allosteric Inhibitors

USP5 is a deubiquitinase that has been implicated in a range of diseases, including cancer, but no USP5-targeting chemical probe has been reported to date. Here, we present the progression of a chemical series that occupies the C-terminal ubiquitin-binding site of a poorly characterized zinc-finger ubiquitin binding domain (ZnF-UBD) of USP5 and allosterically inhibits the catalytic activity of the enzyme. Systematic exploration of the structure-activity relationship, complemented with crystallographic characterization of the ZnF-UBD bound to multiple ligands, led to the identification of 64, which binds to the USP5 ZnF-UBD with a KD of 2.8 {micro}M. 64 is selective over the structurally similar ZnF-UBD domain of HDAC6 and inhibits USP5 catalytic activity in vitro with an IC50 of 26 {micro}M. This study provides a chemical and structural framework for the discovery of a chemical probe to delineate USP5 function in cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/444542v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@3fc7bcorg.highwire.dtl.DTLVardef@15233e0org.highwire.dtl.DTLVardef@1cbf34corg.highwire.dtl.DTLVardef@d237d0_HPS_FORMAT_FIGEXP M_FIG Table of Contents Graphic C_FIG

biochemistry↗

Design, Synthesis and Evaluation of WD-repeat containing protein 5 (WDR5) degraders

Histone H3K4 methylation serves as post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferases (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat containing protein 5 (WDR5) that has also been associated with controlling long non-coding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMTs complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic function. This study presents the design, synthesis and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.

cancer biology↗

Rational Design and Synthesis of Selective PRMT4 Inhibitors: a New Chemotype for Development of Cancer Therapeutics

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates arginine residues of histone H3 and non-histone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and potentially therapeutics. While several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Here, we report the structure-based design of a new class of alanine containing 3-arylindoles as potent and selective PRMT4 inhibitors and describe key structure activity relationships for this class of compounds.

pharmacology and toxicology↗

A semi-automated intestinal organoid screening method demonstrates epigenetic control of epithelial maturation

Intestinal organoids are an excellent model to study epithelial biology. Yet, the selection of analytical tools to accurately quantify heterogeneous organoid cultures remains limited. Here, we developed a semi-automated organoid screening method, which we applied to a library of highly specific chemical probes to identify epigenetic regulators of intestinal epithelial biology. The role of epigenetic modifiers in adult stem cell systems, such as the intestinal epithelium, is still undefined. Based on this resource data, we identified several targets that affected epithelial cell differentiation, including HDACs, EP300/CREBBP, LSD1, and type I PRMTs, which were verified by complementary methods. For example, we show that inhibiting type I PRMTs, which leads enhanced epithelial differentiation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are powerful tools to study intestinal epithelial biology and may have therapeutic potential.

cell biology↗