Search

Amplification of chromosomal material derived from 12q13-15 is common in human cancer and believed to result in overexpression of multiple collaborating oncogenes. To define the oncogenes involved, we overexpressed genes recurrently amplified in human liposarcoma using a zebrafish model of the disease. We found several genes whose overexpression collaborated with AKT in sarcomagenesis, including the tRNA methyltransferase METTL1. This was surprising, because AKT phosphorylates METTL1 to inactivate its enzymatic activity. Indeed, phosphomimetic S27D or catalytically dead alleles phenocopied the oncogenic activity of wild-type METTL1. We found that METTL1 binds the multi-tRNA synthetase complex, which contains many of the cellular aminoacyl-tRNA synthetases and promotes tRNA aminoacylation, polysome formation, and protein synthesis independent of its methyltransferase activity. METTL1-amplified liposarcomas were hypersensitive to actinomycin D, a clinical inhibitor of ribosome biogenesis. We propose that METTL1 overexpression promotes sarcomagenesis by stimulating tRNA aminoacylation, protein synthesis, and tumor cell growth independent of its methyltransferase activity., Competing Interests: Declaration of interests T.A. is a consultant for Bayer, AstraZeneca, and Astellas Pharma and receives research funding from Epizyme, EMD Serono, Karyopharm Therapeutics, SpringWorks Therapeutics, Astellas Pharma, Deciphera, and Pharma Mar. J.A.M. is a founder, equity holder, and advisor to Entact Bio, serves on the scientific advisory board (SAB) of 908 Devices, and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks, TUO Therapeutics, and Takeda. J.R.P. is a consultant for ProFound Therapeutics. R.I.G. is a co-founder and advisory board member of Redona Therapeutics, and scientific advisor to Alida Biosciences. A.G. is an advisory board member of Attivare Therapeutics., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4 DCAF16 ligase to the second bromodomain of BRD4 (BRD4 BD2 ). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4 BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16 Cys58 . This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4 BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology., Competing Interests: Competing interests: B.L.E. has received research funding from Celgene, Deerfield, Novartis and Calico Life Sciences and consulting fees from AbbVie. He is a member of the scientific advisory board (SAB) for and a shareholder of Neomorph, Inc., TenSixteen Bio, Skyhawk Therapeutics and Exo Therapeutics. E.S.F. is a founder, SAB member and equity holder of Civetta Therapeutics, Lighthorse Therapeutics, Proximity Therapeutics and Neomorph, Inc. (board of directors). He is an equity holder in and SAB member for Avilar Therapeutics and Photys Therapeutics and a consultant to Novartis, Sanofi, EcoR1 Capital and Deerfield. The Fischer laboratory receives or has received research funding from Deerfield, Novartis, Ajax, Interline and Astellas. N.S.G. is a founder, SAB member and equity holder in Syros, C4, Allorion, Lighthorse, Voronoi, Inception, Matchpoint, CobroVentures, GlaxoSmithKline, Larkspur (board member), Shenandoah (board member) and Soltego (board member). The Gray laboratory receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, Springworks, Interline and Sanofi. M.S. has received research funding from Calico Life Sciences. K.A.D. receives or has received consulting fees from Kronos Bio and Neomorph, Inc. J.Q. is an equity holder of Epiphanes and Talus Bioscience and receives or has received research funding from Novartis. J.A.M. is a founder and equity holder of and advisor to Entact Bio, serves on the SAB of 908 Devices and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks, TUO Therapeutics and Bruker. Y.-D.L. is currently employed by Leerink Partners. M.W.M. is currently employed by Novartis Venture Fund. K.P. is currently employed by AbbVie. R.J.L. is currently employed by Flagship Pioneering. B.J.G. is currently employed by Blueprint Medicines. I.Y. is currently employed by Matchpoint Therapeutics. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple Python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC 50 < 5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jarrod A Marto, Ph.D. reports a relationship with Entact Bio that includes: equity or stocks. Jarrod A Marto, Ph.D. reports a relationship with 908 Devices that includes: board membership. Jarrod A Marto, Ph.D. reports a relationship with Vertex Pharmaceuticals Incorporated, Astra Zeneca, Taiho, Springworks, TUO Therapeutics, Takeda that includes: funding grants. Sara J. Buhrlage reports a relationship with Entact Bio that includes: equity or stocks. Sara J. Buhrlage reports a relationship with Novartis Institutes for Biomedical Research, AbbVie, Kinogen, TUO Therapeutics, and Takeda that includes: funding grants. Sirano Dhe-Paganon reports a relationship with NIBR, Deerfield, Taiho, and TUO Therapeutics that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The eukaryotic RNA polymerase II (Pol II) multi-protein complex transcribes mRNA and coordinates several steps of co-transcriptional mRNA processing and chromatin modification. The largest Pol II subunit, Rpb1, has a C-terminal domain (CTD) comprising dozens of repeated heptad sequences (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), each containing five phospho-accepting amino acids. The CTD heptads are dynamically phosphorylated, creating specific patterns correlated with steps of transcription initiation, elongation, and termination. This CTD phosphorylation 'code' choreographs dynamic recruitment of important co-regulatory proteins during gene transcription. Genetic tools were used to engineer protease cleavage sites across the CTD (msCTD), creating tryptic peptides with unique sequences amenable to mass spectrometry analysis. However, phosphorylation isoforms within each msCTD sequence are difficult to resolve by standard reversed phase chromatography typically used for LC-MS/MS applications. Here, we use a panel of synthetic CTD phosphopeptides to explore the potential of hydrophilic interaction and electrostatic repulsion hydrophilic interaction (HILIC and ERLIC) chromatography as alternatives to reversed phase separation for CTD phosphopeptide analysis. Our results demonstrate that ERLIC provides improved performance for separation of singly- and doubly-phosphorylated CTD peptides for sequence analysis by LC-MS/MS. Analysis of native yeast msCTD confirms that phosphorylation on Ser5 and Ser2 represents the major endogenous phosphoisoforms. We expect this methodology will be especially useful in the investigation of pathways where multiple protein phosphorylation events converge in close proximity.

Advances in mass spectrometry (MS) have enabled high-throughput analysis of proteomes in biological systems. The state-of-the-art MS data analysis relies on database search algorithms to quantify proteins by identifying peptide-spectrum matches (PSMs), which convert mass spectra to peptide sequences. Different database search algorithms use distinct search strategies and thus may identify unique PSMs. However, no existing approaches can aggregate all user-specified database search algorithms with a guaranteed increase in the number of identified peptides and a control on the false discovery rate (FDR). To fill in this gap, we proposed a statistical framework, Aggregation of Peptide Identification Results (APIR), that is universally compatible with all database search algorithms. Notably, under an FDR threshold, APIR is guaranteed to identify at least as many, if not more, peptides as individual database search algorithms do. Evaluation of APIR on a complex proteomics standard dataset showed that APIR outpowers individual database search algorithms and empirically controls the FDR. Real data studies showed that APIR can identify disease-related proteins and post-translational modifications missed by some individual database search algorithms. The APIR framework is easily extendable to aggregating discoveries made by multiple algorithms in other high-throughput biomedical data analysis, e.g., differential gene expression analysis on RNA sequencing data. The APIR R package is available at https://github.com/yiling0210/APIR., (© The Author(s) 2024. Published by Oxford University Press and Science Press on behalf of the Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation and Genetics Society of China.)

A target identification platform derived from the bioorthogonal activation of reactive species is described. We explore the reactivity of halogenated enamine N -oxides and report that the previously undisclosed α,γ-halogenated enamine N -oxides can be reduced biooorthogonally by diboron reagents to produce highly electrophilic α,β-unsaturated haloiminium ions suitable for labeling a range of amino acid residues on proteins in a 1,2- or 1,4-fashion. Affinity labeling reagents bearing this motif enable ligand-directed protein modification and afford highly sensitive and selective target identification in unbiased chemoproteomics experiments. Target identification is supported in both cell lysate and live cells., Competing Interests: J.K. has received sponsored research funding from Taiho previously. J.A.M. is a founder, equity holder, and advisor to Entact Bio, serves on the SAB of 908 Devices, and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks, and TUO Therapeutics. J.K. and S.S. are inventors on a provisional U.S. patent application based in part on this work.

A systematic strategy to develop dual-warhead inhibitors is introduced to circumvent the limitations of conventional covalent inhibitors such as vulnerability to mutations of the corresponding nucleophilic residue. Currently, all FDA-approved covalent small molecules feature one electrophile, leaving open a facile route to acquired resistance. We conducted a systematic analysis of human proteins in the protein data bank to reveal ∼400 unique targets amendable to dual covalent inhibitors, which we term "molecular bidents". We demonstrated this strategy by targeting two kinases: MKK7 and EGFR. The designed compounds, ZNL-8162 and ZNL-0056, are ATP-competitive inhibitors that form two covalent bonds with cysteines and retain potency against single cysteine mutants. Therefore, molecular bidents represent a new pharmacological modality with the potential for improved selectivity, potency, and drug resistance profile., Competing Interests: The authors declare the following competing financial interest(s): N.S.G. is a founder, science advisory board member (SAB), and equity holder in Syros, C4, Allorion, Lighthorse, Voronoi, Inception, Matchpoint, CobroVentures, GSK, Shenandoah (board member), Larkspur (board member), and Soltego (board member). The Gray laboratory receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, Springworks, Interline, and Sanofi. T.Z. is a scientific founder, equity holder, and consultant of Matchpoint, equity holder of Shenandoah. P.A.J. has received consulting fees from AbbVie, Accutar Biotech, Allorion Therapeutics, AstraZeneca, Bayer, Biocartis, Boehringer Ingelheim, Chugai Pharmaceutical Co., Daiichi Sankyo, Duality, Eisai, Eli Lilly, Frontier Medicines, Hongyun Biotechnology, Merus, Mirati Therapeutics, Monte Rosa, Novartis, Nuvalent, Pfizer, Roche/Genentech, Scorpion Therapeutics, SFJ Pharmaceuticals, Silicon Therapeutics, Syndax, Takeda Oncology, Transcenta, and Voronoi; sponsored research support from AstraZeneca, Boehringer Ingelheim, Daiichi Sankyo, Eli Lilly, Puma Technology, Revolution Medicines, and Takeda Oncology; post-marketing royalties from a DFCI owned patent on EGFR mutation licensed to Lab Corp; and has stock in Gatekeeper Pharmaceuticals. J.A.M. is a founder, equity holder, and advisor to Entact Bio, serves on the SAB of 908 Devices, and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks, and TUO Therapeutics. J.C. is a co-founder for Matchpoint Therapeutics. J.C. is a scientific co-founder M3 Bioinformatics & Technology Inc., and consultant and equity holder for Soltego and Allorion. M.J.E. has received sponsored research support from Novartis, Takeda, Springworks, and Sanofi and consulting income from Novartis. All other authors declare no competing interests., (© 2024 The Authors. Published by American Chemical Society.)

The pyrazolopyrimidine (PP) heterocycle is a versatile and widely deployed core scaffold for the development of kinase inhibitors. Typically, a 4-amino-substituted pyrazolopyrimidine binds in the ATP-binding pocket in a conformation analogous to the 6-aminopurine of ATP. Here, we report the discovery of ZNL0325 which exhibits a flipped binding mode where the C3 position is oriented toward the ribose binding pocket. ZNL0325 and its analogues feature an acrylamide side chain at the C3 position which is capable of forming a covalent bond with multiple kinases that possess a cysteine at the αD-1 position including BTK, EGFR, BLK, and JAK3. These findings suggest that the ability to form a covalent bond can override the preferred noncovalent binding conformation of the heterocyclic core and provides an opportunity to create structurally distinct covalent kinase inhibitors.

Human cytomegalovirus (HCMV) is an important pathogen for which new antiviral drugs are needed. HCMV, like other herpesviruses, encodes a nuclear egress complex (NEC) composed of two subunits, UL50 and UL53, whose interaction is crucial for viral replication. To explore whether small molecules can exert selective antiviral activity by inhibiting NEC subunit interactions, we established a homogeneous time-resolved fluorescence (HTRF) assay of these interactions and used it to screen >200,000 compound-containing wells. Two compounds, designated GK1 and GK2, which selectively inhibited this interaction in the HTRF assay with GK1 also active in a co-immunoprecipitation assay, exhibited more potent anti-HCMV activity than cytotoxicity or activity against another herpesvirus. At doses that substantially reduced HCMV plaque formation, GK1 and GK2 had little or no effect on the expression of viral proteins and reduced the co-localization of UL53 with UL50 at the nuclear rim in a subset of cells. GK1 and GK2 contain an acrylamide moiety predicted to covalently interact with cysteines, and an analog without this potential lacked activity. Mass spectrometric analysis showed binding of GK2 to multiple cysteines on UL50 and UL53. Nevertheless, substitution of cysteine 214 of UL53 with serine (C214S) ablated detectable inhibitory activity of GK1 and GK2 in vitro, and the C214S substitution engineered into HCMV conferred resistance to GK1, the more potent of the two inhibitors. Thus, GK1 exerts selective antiviral activity by targeting the NEC. Docking studies suggest that the acrylamide tethers one end of GK1 or GK2 to C214 within a pocket of UL53, permitting the other end of the molecule to sterically hinder UL50 to prevent NEC formation. Our results prove the concept that targeting the NEC with small molecules can selectively block HCMV replication. Such compounds could serve as a foundation for development of anti-HCMV drugs and as chemical tools for studying HCMV., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: C.G. is a cofounder of Quantum Therapeutics Inc., a company that uses computational methods for drug development, and a cofounder and consultant to Virtual Discovery Inc., which is a fee-for-service company for computational drug discovery. S.D. is a consultant to Virtual Discovery Inc., which is a fee-for-service company for computational drug discovery, and a voluntary member of the Non-Governmental Research Organization Biologic. J.A.M. is a founder, equity holder, and advisor to Entact Bio, serves on the Scientific Advisory Board of 908 Devices, and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks and TUO Therapeutics. D.M.C. has received research funding from Biota, which has been subsumed into what is now Vaxart, and an unrestricted gift from SmithKlineBeecham, which has been subsumed into what is now GSK., (Copyright: © 2023 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Site-specific modification of amino acid residues in protein binding pockets using sulfonyl exchange chemistry expands the druggable proteome by enabling the development of covalent modulators that target residues beyond cysteine. Sulfonyl fluoride and triazole electrophiles were incorporated previously into the cereblon (CRBN) molecular glue degrader EM12, to covalently engage His353 within the CRBN sensor loop, but these probes had poor human plasma stability. Attenuation of intrinsic reactivity through the development of sulfonyl pyrazoles, imidazoles, and nucleobases enhanced plasma stability, and several compounds retained efficient labeling of His353. For example, sulfonyl imidazole EM12-SO 2 Im covalently blocked the CRBN binding site and possessed excellent metabolic stability in human plasma, liver microsomes, and hepatocytes. These results highlight the potential suitability of sulfonyl imidazole and related sulfur(VI)-diazole exchange (SuDEx) warheads for covalent drug development and further exemplify the therapeutic promise of site-specific histidine targeting., Competing Interests: The authors declare the following competing financial interest(s): The CPD receives research funding from Deerfield., (© 2023 American Chemical Society.)

Despite the considerable efficacy observed when targeting a dispensable lineage antigen, such as CD19 in B cell acute lymphoblastic leukaemia 1,2 , the broader applicability of adoptive immunotherapies is hampered by the absence of tumour-restricted antigens 3-5 . Acute myeloid leukaemia immunotherapies target genes expressed by haematopoietic stem/progenitor cells (HSPCs) or differentiated myeloid cells, resulting in intolerable on-target/off-tumour toxicity. Here we show that epitope engineering of donor HSPCs used for bone marrow transplantation endows haematopoietic lineages with selective resistance to chimeric antigen receptor (CAR) T cells or monoclonal antibodies, without affecting protein function or regulation. This strategy enables the targeting of genes that are essential for leukaemia survival regardless of shared expression on HSPCs, reducing the risk of tumour immune escape. By performing epitope mapping and library screenings, we identified amino acid changes that abrogate the binding of therapeutic monoclonal antibodies targeting FLT3, CD123 and KIT, and optimized a base-editing approach to introduce them into CD34 + HSPCs, which retain long-term engraftment and multilineage differentiation ability. After CAR T cell treatment, we confirmed resistance of epitope-edited haematopoiesis and concomitant eradication of patient-derived acute myeloid leukaemia xenografts. Furthermore, we show that multiplex epitope engineering of HSPCs is feasible and enables more effective immunotherapies against multiple targets without incurring overlapping off-tumour toxicities. We envision that this approach will provide opportunities to treat relapsed/refractory acute myeloid leukaemia and enable safer non-genotoxic conditioning., (© 2023. The Author(s).)

Many cereblon (CRBN) ligands have been used to develop proteolysis targeting chimeras (PROTACs), but all are reversible binders of the E3 ubiquitin ligase. We recently described the use of sulfonyl exchange chemistry to design binders that covalently engage histidine 353 in CRBN for the first time. Here we show that covalent CRBN ligands can be used to develop efficient PROTAC degraders. We demonstrate that the fluorosulfate PROTAC FS-ARV-825 covalently labels CRBN in vitro , and in cells the BRD4 degrader is insensitive to wash-out and competition by potent reversible CRBN ligands, reflecting enhanced pharmacodynamics. We anticipate that covalent CRBN-based PROTACs will enhance degradation efficiencies, thus expanding the scope of addressable targets using the heterobifunctional degrader modality., Competing Interests: LHJ serves on the SAB for, and holds equity in, Interline Therapeutics, Umbra Therapeutics, Rapafusyn Pharmaceuticals and Ananke Therapeutics. He also holds equity in Jnana Therapeutics and consults for Matchpoint Therapeutics. The Center for Protein Degradation at DFCI receives research funding from Deerfield. E. S. F. is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Lighthorse Therapeutics, Proximity Therapeutics, and Neomorph, Inc. (board of directors). E. S. F. is an equity holder and SAB member for Avilar Therapeutics and Photys Therapeutics and a consultant to Novartis, Sanofi, EcoR1 Capital, and Deerfield. The Fischer lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline, Voronoi, and Astellas. KAD is a consultant for Kronos Bio and Neomorph Inc. J. A. M. is a founder, equity holder, and advisor to Entact Bio, serves on the SAB of 908 Devices, and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks and TUO Therapeutics., (This journal is © The Royal Society of Chemistry.)

As originally described some 40 years ago, protein ubiquitination was thought to serve primarily as a static mark for protein degradation. In the ensuing years, it has become clear that 'ubiquitination' is a structurally diverse and dynamic post-translational modification and is intricately involved in a myriad of signaling pathways in all eukaryote cells. And like other key pathways in the functional proteome, ubiquitin signaling is often disrupted, sometimes severely so, in human pathophysiology. As a result of its central role in normal physiology and human disease, the ubiquitination field is now represented across the full landscape of biomedical research from fundamental structural and biochemical studies to translational and clinical research. In recent years, mass spectrometry has emerged as a powerful technology for the detection and characterization of protein ubiquitination. Herein we detail qualitative and quantitative proteomic methods using a compare/contrast approach to highlight their strengths and weaknesses., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jarrod A Marto reports financial support was provided by Vertex Pharmaceuticals Incorporated. Jarrod A Marto reports financial support was provided by AstraZeneca Pharmaceuticals LP. Jarrod A Marto reports financial support was provided by Taiho Oncology Inc. Jarrod A Marto reports financial support was provided by SpringWorks Therapeutics Inc. Jarrod A Marto reports financial support was provided by TUO Therapeutics. Sara J Buhrlage reports financial support was provided by AbbVie Inc. Sara J Buhrlage reports financial support was provided by TUO Therapeutics. Sara J Buhrlage reports equipment, drugs, or supplies was provided by AbbVie Inc. Sara J Buhrlage reports equipment, drugs, or supplies was provided by Novartis Institutes for BioMedical Research Inc., (Copyright © 2023. Published by Elsevier B.V.)

Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03-176 (compound 22 ) as a selective, cysteine-covalent TEAD inhibitor. MYF-03-176 (compound 22 ) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1-JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher K inact compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.K I compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics 1-3 . Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs 4-6 . The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans -labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4 DCAF16 ligase to the second bromodomain of BRD4 (BRD4 BD2 ). BRD4 BD2 , in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4 BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16 Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4 BD2 revealed that the loop conformation around BRD4 His437 , rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology., Competing Interests: Competing Interests B.L.E. has received research funding from Celgene, Deerfield, Novartis, and Calico and consulting fees from GRAIL. He is a member of the scientific advisory board and shareholder for Neomorph Inc., TenSixteen Bio, Skyhawk Therapeutics, and Exo Therapeutics. E.S.F is a founder, scientific advisory board (SAB) member, and equity holder of Civetta Therapeutics, Lighthorse Therapeutics, Proximity Therapeutics, and Neomorph, Inc. (board of directors). He is an equity holder and SAB member for Avilar Therapeutics and Photys Therapeutics and a consultant to Novartis, Sanofi, EcoR1 Capital, and Deerfield. The Fischer lab receives or has received research funding from Deerfield, Novartis, Ajax, Interline and Astellas. N.S.G. is a founder, science advisory board member (SAB) and equity holder in Syros, C4, Allorion, Lighthorse, Voronoi, Inception, Matchpoint, CobroVentures, GSK, Larkspur (board member), Shenandoah (board member), and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Jansen, Kinogen, Arbella, Deerfield, Springworks, Interline and Sanofi. M.S. has received research funding from Calico Life Sciences LLC. K.A.D is a consultant to Kronos Bio and Neomorph Inc. J.Q. is an equity holder of Epiphanes, Talus Bioscience, and receives or has received research funding from Novartis. J.A.M. is a founder, equity holder, and advisor to Entact Bio, serves on the SAB of 908 Devices, and receives or has received sponsored research funding from Vertex, AstraZeneca, Taiho, Springworks and TUO Therapeutics. K.P. is currently employed by Abbvie. B.J.G. is currently employed by Blueprint Medicines.

Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space., (© 2023. The Author(s).)

The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration., Competing Interests: MF, YG, YL is one of the inventors on TEAD inhibitor patents (WO2020081572A1), WL, NK, HS, SF, PG, EG, JL, KS, MK, WJ, JJ, ZH, JT, AB, MR, MR, JR, JM, JM, SD No competing interests declared, JC is a consultant to Soltego, Jengu, Allorion, EoCys, and equity holder for Soltego, Allorion, EoCys, and M3 bioinformatics & technology Inc, TZ is a scientific funder, equity holder and consultant in Matchpoint. T.Z. is one of the inventors on TEAD inhibitor patents (WO2020081572A1), NG is a founder, science advisory board (SAB) member and equity holder in Syros, Jengu, C4, B2S, Allorion, Inception, GSK, Larkspur (board member), Soltego (board member) and Matchpoint. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Interline, Springworks and Sanofi. TEAD inhibitors developed in this manuscript are licensed to a start-up (Lighthorse) where Gray has a financial interest. N.S.G. is one of the inventors on TEAD inhibitor patents (WO2020081572A1), (© 2022, Fan, Lu et al.)

The fate of pyruvate is a defining feature in many cell types. One major fate is mitochondrial entry via the mitochondrial pyruvate carrier (MPC). We found that diffuse large B cell lymphomas (DLBCLs) consume mitochondrial pyruvate via glutamate-pyruvate transaminase 2 to enable α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid cycle in DLBCLs. As a result, MPC inhibition led to decreased glutaminolysis in DLBCLs, opposite to previous observations in other cell types. We also found that MPC inhibition or genetic depletion decreased DLBCL proliferation in an extracellular matrix (ECM)-like environment and xenografts, but not in a suspension environment. Moreover, the metabolic profile of DLBCL cells in ECM is markedly different from cells in a suspension environment. Thus, we conclude that the synergistic consumption and assimilation of glutamine and pyruvate enables DLBCL proliferation in an extracellular environment-dependent manner.

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.

Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of the list of targetable amino acid residues is required to augment the synthetic bology toolkit of site-selective protein modifications. This work describes the first rational targeting of a specific histidine residue in a protein binding site using sulfonyl exchange chemistry. Structure-based drug design was used to incorporate sulfonyl fluoride and triazole reactive groups into the isoindolinone thalidomide congener EM12 to yield potent covalent inhibitors of the cereblon E3 ubiquitin ligase complex through engagement of His353. Conversely, the fluorosulfate derivative EM12-FS labels His353, but degrades a novel neosubstrate, the protein N-terminal glutamine amidohydrolase NTAQ1, which is involved in the N-end rule pathway and DNA damage response. Targeted protein degradation using cereblon ligands has become an important new drug discovery modality and the chemical probes and covalent labeling strategy described here will broadly impact this exciting area of therapeutic research., Competing Interests: L. H. J. serves on the scientific advisory boards for, and holds equity in, Interline Therapeutics and Rapafusyn Pharmaceuticals, consults for Umbra Therapeutics, Ananke Therapeutics and Matchpoint Therapeutics, and holds equity in Jnana Therapeutics. The Center for Protein Degradation at DFCI receives research funding from Deerfield. E. S. F. is a founder, member of the SAB, and equity holder of Civetta Therapeutics, Jengu Therapeutics, Proximity Therapeutics, and Neomorph Inc, SAB member and equity holder in Avilar Therapeutics and Photys Therapeutics, and a consultant to Astellas, Sanofi, Novartis, Deerfield and EcoR1 capital. The Fischer laboratory receives or has received research funding from Novartis, Deerfield, Ajax, Interline, and Astellas. N. S. G. is a founder, an SAB, and an equity holder in Syros, Jengu, Inception, C4, B2S, Voronoi, Matchpoint, GSK, CobroVentures, Larkspur (board member), and Soltego (board member). The Gray laboratory receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Arbella, Epiphanes, Deerfield, and Sanofi. J. C. is a consultant for Soltego, Jengu, Allorion and Matchpoint Therapeutics, and holds equity in Soltego, Allorion, Matchpoint and M3 Bioinformatics & Technology Inc. K. A. D. is a consultant for Kronos Bio and Neomorph Inc. J. A. M. serves on the SAB of 908 Devices and receives sponsored research support from Vertex, AstraZeneca, Taiho, and TUO., (This journal is © The Royal Society of Chemistry.)

Henning et al. (2022) report development of a novel class of agents, bivalent deubiquitinase (DUB)-targeting chimeras (DUBTACs), that can selectively stabilize protein targets. These findings encourage further pursuit of targeted protein stabilization as a new paradigm in chemical biology and drug discovery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

We describe a complete open-source hardware/software solution for high performance thermostatted peptide fraction collection to support mass spectrometry experiments with complex proteomes. The instrument is easy to assemble using parts readily available through retail channels at a fraction of the cost compared to typical commercial systems. Control software is written in Python allowing for rapid customization. We demonstrate several useful applications, including the automated deposition of LC separated peptides for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) as well as collection and concatenation of peptide fractions from nanoflow HPLC separations., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C terminus through the central pore of the hexameric double ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48., Competing Interests: Declaration of interests J.A.M. serves on the scientific advisory board of 908 Devices and receives sponsored research support from AstraZeneca and Vertex. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mutations in the Janus Kinase 2 (JAK2) gene resulting in constitutive kinase activation represent the most common genetic event in myeloproliferative neoplasms (MPN), a group of diseases involving overproduction of one or more kinds of blood cells, including red cells, white cells, and platelets. JAK2 kinase inhibitors, such as ruxolitinib, provide clinical benefit, but inhibition of wild-type (wt) JAK2 limits their clinical utility due to toxicity to normal cells, and small molecule inhibition of mutated JAK2 kinase activity can lead to drug resistance. Here, we present a strategy to target mutated JAK2 for degradation, using the cell's intracellular degradation machinery, while sparing non-mutated JAK2. We employed a chemical genetics screen, followed by extensive selectivity profiling and genetic studies, to identify the deubiquitinase (DUB), JOSD1, as a novel regulator of mutant JAK2. JOSD1 interacts with and stabilizes JAK2-V617F, and inactivation of the DUB leads to JAK2-V617F protein degradation by increasing its ubiquitination levels, thereby shortening its protein half-life. Moreover, targeting of JOSD1 leads to the death of JAK2-V617F-positive primary acute myeloid leukemia (AML) cells. These studies provide a novel therapeutic approach to achieving selective targeting of mutated JAK2 signaling in MPN., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes., Competing Interests: Declaration of interests J.A.M. serves on the SAB of 908 Devices and receives sponsored research funding from Vertex and AstraZeneca. S.J.B. serves on the SAB of Adenoid Cystic Carcinoma Foundation. The authors have submitted patent applications related to compounds in this manuscript., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

Development of inhibitors targeting CDK12/13 is of increasing interest as a potential therapy for cancers as these compounds inhibit transcription of DNA damage response (DDR) genes. We previously described THZ531, a covalent inhibitor with selectivity for CDK12/13. In order to elucidate structure-activity relationship (SAR), we have undertaken a medicinal chemistry campaign and established a focused library of THZ531 analogs. Among these analogs, BSJ-01-175 demonstrates exquisite selectivity, potent inhibition of RNA polymerase II phosphorylation, and downregulation of CDK12-targeted genes in cancer cells. A 3.0 Å co-crystal structure with CDK12/CycK provides a structural rational for selective targeting of Cys1039 located in a C-terminal extension from the kinase domain. With moderate pharmacokinetic properties, BSJ-01-175 exhibits efficacy against an Ewing sarcoma tumor growth in a patient-derived xenograft (PDX) mouse model following 10 mg/kg once a day, intraperitoneal administration. Taken together, BSJ-01-175 represents the first selective CDK12/13 covalent inhibitor with in vivo efficacy reported to date., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N.S.G. is a founder, science advisory board member (SAB) and equity holder in Gatekeeper, Syros, Petra, C4, B2S, Aduro and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield. Ephiphanes and Sanofi. N.S.G., T. Z. and B. J. are named inventors on patent applications covering compounds described in this manuscript., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Covalent drugs constitute cornerstones of modern medicine. The past decade has witnessed growing enthusiasm for development of covalent inhibitors, fueled by clinical successes as well as advances in analytical techniques associated with the drug discovery pipeline. Among these, mass spectrometry-based chemoproteomic methods stand out due to their broad applicability from focused analysis of electrophile-containing compounds to surveying proteome-wide inhibitor targets. Here, we review applications of both foundational and cutting-edge chemoproteomic techniques across target identification, hit discovery, and lead characterization/optimization in covalent drug discovery. We focus on the practical aspects necessary for the general drug discovery scientist to design, interpret, and evaluate chemoproteomic experiments. We also present three case studies on clinical stage molecules to further showcase the real world significance and future opportunities of these methodologies.

Ligand-directed bioconjugation strategies have been used for selective protein labeling in live cells or tissue samples in applications such as live-cell imaging. Here we hypothesized that a similar strategy could be used for targeted protein degradation. To test this possibility, we developed a series of CDK2-targeting N -acyl- N -alkylsulfonamide (NASA)-containing acylation probes. The probes featured three components: a CDK2 homing ligand, a CRL4 CRBN E3 ligase recruiting ligand, and a NASA functionality. We determined that upon target binding, NASA-mediated reaction resulted in selective functionalization of Lys89 on purified or native CDK2. However, we were unable to observe CDK2 degradation, which is in contrast to the efficient degradation achieved by the use of a structurally similar reversible bivalent degrader. Our analysis suggests that the lack of degradation is due to the failure to form a productive CDK2:CRBN complex. Therefore, although this work demonstrates that NASA chemistry can be used for protein labeling, whether this strategy could enable efficient protein degradation remains an open question., Competing Interests: The authors declare the following competing financial interest(s): N.S.G. is a founder, science advisory board (SAB) member, and equity holder in Syros, Jengu, Inception, C4, B2S, Larkspur (board member), and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Arbella, Epiphanes, Deerfield, and Sanofi. J.A.M. serves on the SAB of 908 Devices and has received sponsored research support from AstraZeneca and Vertex. E.S.F. is a founder, SAB member, and equity holder in Civetta Therapeutics, Jengu Therapeutics (board member), and Neomorph Inc., an equity holder in C4 Therapeutics, and a consultant to Novartis, Sanofi, EcoR1 Capital, and Deerfield. The Fischer lab receives or has received research funding from Astellas, Novartis, Voronoi, Ajax, and Deerfield. S.D.-P. receives or has received research funding from the Linde Family Foundation, Taiho Pharmaceuticals, and the Doris Duke Charitable Foundation., (© 2021 American Chemical Society.)

R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders., (© 2020 Fukuda et al.)

MKK4/7 are kinases that phosphorylate JNKs and regulate the MAPK signaling pathway. Their overexpression has been associated with tumorigenesis and aggressiveness in cancers such as breast, prostate, non-small cell lung, and pediatric leukemia, making them a potential target for inhibitor development. Here, we report the discovery, development, and validation of a dual MKK4/7 inhibitor, BSJ-04-122, that covalently targets a conserved cysteine located before the DFG motif and displays excellent kinome selectivity. BSJ-04-122 exhibits potent cellular target engagement and induces robust target-specific downstream effects. The combination of the dual MKK4/7 inhibitor with a selective, covalent JNK inhibitor demonstrated an enhanced antiproliferative activity against triple-negative breast cancer cells. Taken together, the results show that BSJ-04-122 represents a pharmacological probe for MKK4/7 and credential covalent targeting as a way to explore the therapeutic potential of these kinases., Competing Interests: Declaration of Interests N.S.G. is a founder of, science advisor at, and equity holder in C4, Syros, Petra, Soltego, B2S, Aduro, and Gatekeeper Pharmaceuticals. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, Epiphanes, and Sanofi. The other authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Long non-coding RNAs (lncRNAs) are emerging regulators of pathophysiological processes including atherosclerosis. Using RNA-seq profiling of the intima of lesions, here we identify a macrophage-specific lncRNA MAARS (Macrophage-Associated Atherosclerosis lncRNA Sequence). Aortic intima expression of MAARS increases by 270-fold with atherosclerotic progression and decreases with regression by 60%. MAARS knockdown reduces atherosclerotic lesion formation by 52% in LDLR -/- mice, largely independent of effects on lipid profile and inflammation, but rather by decreasing macrophage apoptosis and increasing efferocytosis in the vessel wall. MAARS interacts with HuR/ELAVL1, an RNA-binding protein and important regulator of apoptosis. Overexpression and knockdown studies verified MAARS as a critical regulator of macrophage apoptosis and efferocytosis in vitro, in an HuR-dependent manner. Mechanistically, MAARS knockdown alters HuR cytosolic shuttling, regulating HuR targets such as p53, p27, Caspase-9, and BCL2. These findings establish a mechanism by which a macrophage-specific lncRNA interacting with HuR regulates apoptosis, with implications for a broad range of vascular disease states.

Interleukin 1 (IL-1) receptor-associated kinases (IRAKs) are serine/threonine kinases that play critical roles in initiating the innate immune response against foreign pathogens. Additionally, dysregulation of IRAK1 signaling plays a role in neoplastic disorders. For example, IRAK1 was shown to be important for survival and proliferation in many B-cell lymphomas, including Waldenström's macroglobulinemia (WM) and ABC subtype Diffused Large B-cell Lymphoma (DLBCL) cells. Here, we report the discovery of a highly potent and selective covalent inhibitor of IRAK1, JH-X-119-01. Intact protein MS labeling studies confirmed that JH-X-119-01 irreversibly labels IRAK1 at C302. This compound exhibited cytotoxic activity at single digit micromolar concentrations in a panel of WM, DLBCL, and lymphoma cell lines expressing MYD88. Cotreatment of JH-X-119-01 with the BTK inhibitor ibrutinib resulted in synergistic killing effects in these systems. Taken together, JH-X-119-01 represents a highly selective probe of IRAK1 for further development., Competing Interests: The authors declare the following competing financial interest(s): N.S.G. is a founder, science advisory board member (SAB), and equity holder in Gatekeeper, Syros, Petra, C4, B2S, Aduro, Inception, Allorion, Jengu and Soltego (board member). The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi. S.P.T. lab receives or has received funding from Pharmacyclics/Abbvie, Janssen Pharmaceuticals, Beigene, and Bristol Myers Squibb. J.A.M. serves on the SAB of 908 Devices. The Marto lab receives sponsored research support from Vertex and Astra-Zeneca. S.B. is a scientific advisory board member of Adenoid Cystic Carcinoma Foundation. H.W. is a co-founder, SAB member, and equity holder of Ventus Therapeutics., (© 2020 American Chemical Society.)

Notch signaling regulates squamous cell proliferation and differentiation and is frequently disrupted in squamous cell carcinomas, in which Notch is tumor suppressive. Here, we show that conditional activation of Notch in squamous cells activates a context-specific gene expression program through lineage-specific regulatory elements. Among direct Notch target genes are multiple DNA damage response genes, including IER5 , which we show is required for Notch-induced differentiation of squamous carcinoma cells and TERT-immortalized keratinocytes. IER5 is epistatic to PPP2R2A , a gene that encodes the PP2A B55α subunit, which we show interacts with IER5 in cells and in purified systems. Thus, Notch and DNA-damage response pathways converge in squamous cells on common genes that promote differentiation, which may serve to eliminate damaged cells from the proliferative pool. We further propose that crosstalk involving Notch and PP2A enables tuning and integration of Notch signaling with other pathways that regulate squamous differentiation., Competing Interests: LP, ML, TT, JR, CL, WL, CJ, LS, AS, JM, GA No competing interests declared, SB SCB is on the SAB for Erasca, Inc, receives sponsored research funding from Novartis and Erasca, Inc, and is a consultant for IFM therapeutics and Ayala Pharmaceuticals. JA JCA is a consultant for Ayala Pharmaceuticals and for Cellestia, Inc, There is no conflict of interest with the work described in this manuscript., (© 2020, Pan et al.)

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.

Genetic depletion of cyclin-dependent kinase 12 (CDK12) or selective inhibition of an analog-sensitive CDK12 reduces DNA damage repair gene expression, but selective inhibition of endogenous CDK12 is difficult. Here, we report the development of MFH290, a novel cysteine (Cys)-directed covalent inhibitor of CDK12/13. MFH290 forms a covalent bond with Cys-1039 of CDK12, exhibits excellent kinome selectivity, inhibits the phosphorylation of serine-2 in the C-terminal domain (CTD) of RNA-polymerase II (Pol II), and reduces the expression of key DNA damage repair genes. Importantly, these effects were demonstrated to be CDK12-dependent as mutation of Cys-1039 rendered the kinase refractory to MFH290 and restored Pol II CTD phosphorylation and DNA damage repair gene expression. Consistent with its effect on DNA damage repair gene expression, MFH290 augments the antiproliferative effect of the PARP inhibitor olaparib.

The PI5P4Ks have been demonstrated to be important for cancer cell proliferation and other diseases. However, the therapeutic potential of targeting these kinases is understudied due to a lack of potent, specific small molecules available. Here, we present the discovery and characterization of a pan-PI5P4K inhibitor, THZ-P1-2, that covalently targets cysteines on a disordered loop in PI5P4Kα/β/γ. THZ-P1-2 demonstrates cellular on-target engagement with limited off-targets across the kinome. AML/ALL cell lines were sensitive to THZ-P1-2, consistent with PI5P4K's reported role in leukemogenesis. THZ-P1-2 causes autophagosome clearance defects and upregulation in TFEB nuclear localization and target genes, disrupting autophagy in a covalent-dependent manner and phenocopying the effects of PI5P4K genetic deletion. Our studies demonstrate that PI5P4Ks are tractable targets, with THZ-P1-2 as a useful tool to further interrogate the therapeutic potential of PI5P4K inhibition and inform drug discovery campaigns for these lipid kinases in cancer metabolism and other autophagy-dependent disorders., Competing Interests: Declaration of Interests J.A.M. is a member of the scientific advisory board (SAB) of 908 Devices. L.C.C. is a founder and member of the Board of Directors of Agios Pharmaceuticals and is a founder and receives research support from Petra Pharmaceuticals. These companies are developing novel therapies for cancer. N.S.G. is a founder, SAB member, and equity holder in Gatekeeper, Syros, Petra, C4, B2S, and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi. N.S.G., T.Z., and N.P.K. are inventors on a patent application covering chemical matter in this publication owned by the Dana-Farber Cancer Institute., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Due to their role in many important signaling pathways, phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive targets for the development of experimental therapeutics for cancer, metabolic, and immunological disorders. Recent efforts to develop small molecule inhibitors for these lipid kinases resulted in compounds with low- to sub-micromolar potencies. Here, we report the identification of CVM-05-002 using a high-throughput screen of PI5P4Kα against our in-house kinase inhibitor library. CVM-05-002 is a potent and selective inhibitor of PI5P4Ks, and a 1.7 Å X-ray structure reveals its binding interactions in the ATP-binding pocket. Further investigation of the structure-activity relationship led to the development of compound 13 , replacing the rhodanine-like moiety present in CVM-05-002 with an indole, a potent pan-PI5P4K inhibitor with excellent kinome-wide selectivity. Finally, we employed isothermal cellular thermal shift assays (CETSAs) to demonstrate the effective cellular target engagement of PI5P4Kα and -β by the inhibitors in HEK 293T cells.

Chronic inflammation is linked to diverse disease processes, but the intrinsic mechanisms that determine cellular sensitivity to inflammation are incompletely understood. Here, we show the contribution of glucose metabolism to inflammation-induced changes in the survival of pancreatic islet β-cells. Using metabolomic, biochemical and functional analyses, we investigate the protective versus non-protective effects of glucose in the presence of pro-inflammatory cytokines. When protective, glucose metabolism augments anaplerotic input into the TCA cycle via pyruvate carboxylase (PC) activity, leading to increased aspartate levels. This metabolic mechanism supports the argininosuccinate shunt, which fuels ureagenesis from arginine and conversely diminishes arginine utilization for production of nitric oxide (NO), a chief mediator of inflammatory cytotoxicity. Activation of the PC-urea cycle axis is sufficient to suppress NO synthesis and shield cells from death in the context of inflammation and other stress paradigms. Overall, these studies uncover a previously unappreciated link between glucose metabolism and arginine-utilizing pathways via PC-directed ureagenesis as a protective mechanism.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor frequently deregulated in lymphoid malignancies. BCL6 engages with number of corepressors, and these protein-protein interactions are being explored as a strategy for drug development. Here, we report the development of an irreversible BCL6 inhibitor TMX-2164 that uses a sulfonyl fluoride to covalently react with the hydroxyl group of Tyrosine 58 located in the lateral groove. TMX-2164 exhibits significantly improved inhibitory activity compared to that of its reversible parental compound and displays sustained target engagement and antiproliferative activity in cells. TMX-2164 therefore represents an example of a tyrosine-directed covalent inhibitor of BCL6 which demonstrates advantages relative to reversible targeting., Competing Interests: The authors declare the following competing financial interest(s): Eric S. Fischer is a founder, science advisory board (SAB) member, and equity holder in Civetta Therapeutics and an equity holder and SAB member in C4 Therapeutics. The Fischer lab receives or has received research funding from Astellas, Novartis, Voronoi, and Deerfield. Jarrod A. Marto serves on the SAB of 908 Devices and has received sponsored research support from AstraZeneca and Vertex. Nathanael S. Gray is a founder, SAB member, and equity holder in Gatekeeper, Syros, Petra, C4, B2S, Aduro, and Soltego. The Gray lab receives or has received research funding from Novartis, Takeda, Astellas, Taiho, Janssen, Kinogen, Voronoi, Her2llc, Deerfield, and Sanofi., (Copyright © 2020 American Chemical Society.)

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.

Bile salt hydrolase (BSH) enzymes are widely expressed by human gut bacteria and catalyze the gateway reaction leading to secondary bile acid formation. Bile acids regulate key metabolic and immune processes by binding to host receptors. There is an unmet need for a potent tool to inhibit BSHs across all gut bacteria to study the effects of bile acids on host physiology. Here, we report the development of a covalent pan-inhibitor of gut bacterial BSHs. From a rationally designed candidate library, we identified a lead compound bearing an alpha-fluoromethyl ketone warhead that modifies BSH at the catalytic cysteine residue. This inhibitor abolished BSH activity in conventional mouse feces. Mice gavaged with a single dose of this compound displayed decreased BSH activity and decreased deconjugated bile acid levels in feces. Our studies demonstrate the potential of a covalent BSH inhibitor to modulate bile acid composition in vivo.

SRC is a major regulator of many signaling pathways and contributes to cancer development. However, development of a selective SRC inhibitor has been challenging, and FDA-approved SRC inhibitors, dasatinib and bosutinib, are multitargeted kinase inhibitors. Here, we describe our efforts to develop a selective SRC covalent inhibitor by targeting cysteine 277 on the P-loop of SRC. Using a promiscuous covalent kinase inhibitor (CKI) SM1-71 as a starting point, we developed covalent inhibitor 15a , which discriminates SRC from other covalent targets of SM1-71 including TAK1 and FGFR1. As an irreversible covalent inhibitor, compound 15a exhibited sustained inhibition of SRC signaling both in vitro and in vivo . Moreover, 15a exhibited potent antiproliferative effects in nonsmall cell lung cancer cell lines harboring SRC activation, thus providing evidence that this approach may be promising for further drug development efforts.

Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile lncRNAs derived specifically from the aortic intima of Ldlr mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 ( -/- mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 ( SNHG12 ) is highly expressed in the vascular endothelium and decreases during lesion progression. SNHG12 knockdown accelerated atherosclerotic lesion formation by 2.4-fold in Ldlr -/- mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of SNHG12 reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD SNHG12 interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of SNHG12 reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD + expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.SNHG12 knockdown. SNHG12 expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Eradicating tumor dormancy that develops following epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) treatment of EGFR-mutant non-small cell lung cancer, is an attractive therapeutic strategy but the mechanisms governing this process are poorly understood. Blockade of ERK1/2 reactivation following EGFR TKI treatment by combined EGFR/MEK inhibition uncovers cells that survive by entering a senescence-like dormant state characterized by high YAP/TEAD activity. YAP/TEAD engage the epithelial-to-mesenchymal transition transcription factor SLUG to directly repress pro-apoptotic BMF, limiting drug-induced apoptosis. Pharmacological co-inhibition of YAP and TEAD, or genetic deletion of YAP1, all deplete dormant cells by enhancing EGFR/MEK inhibition-induced apoptosis. Enhancing the initial efficacy of targeted therapies could ultimately lead to prolonged treatment responses in cancer patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)