Publications by authors named "Markus Schirle"

54 Publications

Chemoproteomics-enabled discovery of covalent RNF114-based degraders that mimic natural product function.

Cell Chem Biol 2021 Jan 22. Epub 2021 Jan 22.

Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Cambridge, MA 02139, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Nutritional Sciences and Toxicology, Univerity of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, Berkeley, CA 94720, USA. Electronic address:

The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation-a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.
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http://dx.doi.org/10.1016/j.chembiol.2021.01.005DOI Listing
January 2021

BET bromodomain inhibitors regulate keratinocyte plasticity.

Nat Chem Biol 2021 Mar 18;17(3):280-290. Epub 2021 Jan 18.

Novartis Institutes for BioMedical Research, Basel, Switzerland.

Although most acute skin wounds heal rapidly, non-healing skin ulcers represent an increasing and substantial unmet medical need that urgently requires effective therapeutics. Keratinocytes resurface wounds to re-establish the epidermal barrier by transitioning to an activated, migratory state, but this ability is lost in dysfunctional chronic wounds. Small-molecule regulators of keratinocyte plasticity with the potential to reverse keratinocyte malfunction in situ could offer a novel therapeutic approach in skin wound healing. Utilizing high-throughput phenotypic screening of primary keratinocytes, we identify such small molecules, including bromodomain and extra-terminal domain (BET) protein family inhibitors (BETi). BETi induce a sustained activated, migratory state in keratinocytes in vitro, increase activation markers in human epidermis ex vivo and enhance skin wound healing in vivo. Our findings suggest potential clinical utility of BETi in promoting keratinocyte re-epithelialization of skin wounds. Importantly, this novel property of BETi is exclusively observed after transient low-dose exposure, revealing new potential for this compound class.
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http://dx.doi.org/10.1038/s41589-020-00716-zDOI Listing
March 2021

Bardoxolone conjugation enables targeted protein degradation of BRD4.

Sci Rep 2020 09 23;10(1):15543. Epub 2020 Sep 23.

Department of Chemistry, University of California, Berkeley, CA, 94720, USA.

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3's has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.
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http://dx.doi.org/10.1038/s41598-020-72491-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511954PMC
September 2020

Discovery of a Functional Covalent Ligand Targeting an Intrinsically Disordered Cysteine within MYC.

Cell Chem Biol 2021 Jan 22;28(1):4-13.e17. Epub 2020 Sep 22.

Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, Berkeley, CA 94720, USA. Electronic address:

MYC is a major oncogenic transcriptional driver of most human cancers that has remained intractable to direct targeting because much of MYC is intrinsically disordered. Here, we have performed a cysteine-reactive covalent ligand screen to identify compounds that could disrupt the binding of MYC to its DNA consensus sequence in vitro and also impair MYC transcriptional activity in situ in cells. We have identified a covalent ligand, EN4, that targets cysteine 171 of MYC within a predicted intrinsically disordered region of the protein. We show that EN4 directly targets MYC in cells, reduces MYC and MAX thermal stability, inhibits MYC transcriptional activity, downregulates multiple MYC transcriptional targets, and impairs tumorigenesis. We also show initial structure-activity relationships of EN4 and identify compounds that show improved potency. Overall, we identify a unique ligandable site within an intrinsically disordered region of MYC that leads to inhibition of MYC transcriptional activity.
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http://dx.doi.org/10.1016/j.chembiol.2020.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854864PMC
January 2021

Manumycin polyketides act as molecular glues between UBR7 and P53.

Nat Chem Biol 2020 11 22;16(11):1189-1198. Epub 2020 Jun 22.

Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.

Molecular glues are an intriguing therapeutic modality that harness small molecules to induce interactions between proteins that typically do not interact. However, such molecules are rare and have been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention. We postulated that natural products bearing one or more electrophilic sites may be an unexplored source of new molecular glues, potentially acting through multicovalent attachment. Using chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells, and engage in molecular glue interactions with the neosubstrate tumor-suppressor TP53, leading to p53 transcriptional activation and cell death. Our results reveal an anticancer mechanism of this natural product family, and highlight the potential for combining chemoproteomics and multicovalent natural products for the discovery of new molecular glues.
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http://dx.doi.org/10.1038/s41589-020-0557-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572527PMC
November 2020

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL.

ACS Chem Biol 2020 07 25;15(7):1788-1794. Epub 2020 Jun 25.

Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, United States.

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific proteins in a proteasome-dependent manner. However, a major limitation of TPD is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent recruiter for the E3 ligase RNF114. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or, in some cases, inactivity. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs, and we demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing selectivity for the degradation of neo-substrate proteins.
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http://dx.doi.org/10.1021/acschembio.0c00348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891886PMC
July 2020

Characterizing Drug-Target Interactions: Shifting towards the Clinic.

Authors:
Markus Schirle

Trends Pharmacol Sci 2020 05 16;41(5):295-297. Epub 2020 Mar 16.

Novartis Institutes for BioMedical Research, Inc., 181 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

Recently, Perrin et al. reported the application of thermal proteome profiling (TPP), a cellular thermal shift assay with an unbiased proteomics readout to complex tissue samples from model organisms and patient-derived whole blood. This study demonstrates for the first time that TPP enables organ-specific drug target engagement and identification studies during the later stages of drug discovery and even in a clinical setting.
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http://dx.doi.org/10.1016/j.tips.2020.03.001DOI Listing
May 2020

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

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

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

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

A small-molecule inhibitor of C5 complement protein.

Nat Chem Biol 2019 07 17;15(7):666-668. Epub 2019 Jun 17.

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

The complement pathway is an important part of the immune system, and uncontrolled activation is implicated in many diseases. The human complement component 5 protein (C5) is a validated drug target within the complement pathway, as an anti-C5 antibody (Soliris) is an approved therapy for paroxysmal nocturnal hemoglobinuria. Here, we report the identification, optimization and mechanism of action for the first small-molecule inhibitor of C5 complement protein.
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http://dx.doi.org/10.1038/s41589-019-0303-9DOI Listing
July 2019

Harnessing the anti-cancer natural product nimbolide for targeted protein degradation.

Nat Chem Biol 2019 07 17;15(7):747-755. Epub 2019 Jun 17.

Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.
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http://dx.doi.org/10.1038/s41589-019-0304-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592714PMC
July 2019

Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications.

ACS Chem Biol 2019 11 13;14(11):2430-2440. Epub 2019 May 13.

Department of Molecular and Cell Biology , University of California, Berkeley , Berkeley , California 94720 , United States.

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.
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http://dx.doi.org/10.1021/acschembio.8b01083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422721PMC
November 2019

Discovery of a ZIP7 inhibitor from a Notch pathway screen.

Nat Chem Biol 2019 02 14;15(2):179-188. Epub 2019 Jan 14.

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

The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
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http://dx.doi.org/10.1038/s41589-018-0200-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251565PMC
February 2019

An Activity-Based Probe Targeting Non-Catalytic, Highly Conserved Amino Acid Residues within Bromodomains.

Angew Chem Int Ed Engl 2019 01 27;58(4):1007-1012. Epub 2018 Dec 27.

Structural Genomic Consortium (SGC), University of Oxford, Oxford, OX3 7DQ, UK.

Bromodomain-containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl-lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule-based approach to covalently modify recombinant and endogenous bromodomain-containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in-gel visualization and pull-down of the desired bromodomains.
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http://dx.doi.org/10.1002/anie.201807825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492141PMC
January 2019

Previously Uncharacterized Vacuolar-type ATPase Binding Site Discovered from Structurally Similar Compounds with Distinct Mechanisms of Action.

ACS Chem Biol 2019 01 18;14(1):20-26. Epub 2018 Dec 18.

Novartis Institutes for BioMedical Research , 250 Massachusetts Avenue , Cambridge , Massachusetts 02139 , United States.

Using a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiency screen strongly supported this finding. Competitive photoaffinity labeling experiments demonstrated that the ATP6V0A2 subunit of the v-ATPase complex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsible for proton translocation. While other mechanisms of v-ATPase regulation have been described, such as dissociation of the complex or inhibition by natural products including bafilomycin A1 and concanamycin, this work provides detailed insight into a distinct binding pocket within the v-ATPase complex.
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http://dx.doi.org/10.1021/acschembio.8b00656DOI Listing
January 2019

A Photoaffinity Labeling-Based Chemoproteomics Strategy for Unbiased Target Deconvolution of Small Molecule Drug Candidates.

Methods Mol Biol 2017 ;1647:1-18

Novartis Institutes for Biomedical Research, 181 Massachusetts Avenue, Cambridge, MA, 02139, USA.

The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.
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http://dx.doi.org/10.1007/978-1-4939-7201-2_1DOI Listing
April 2018

Direct Interaction of Chivosazole F with Actin Elicits Cell Responses Similar to Latrunculin A but Distinct from Chondramide.

ACS Chem Biol 2017 09 15;12(9):2264-2269. Epub 2017 Aug 15.

Novartis Institutes for BioMedical Research , Novartis Pharma AG, Forum 1 Novartis Campus, CH-4056 Basel, Switzerland.

The microbial metabolite Chivosazole F has been described to affect the cytoskeleton and to inhibit actin polymerization in vitro. Applying orthogonal genomic and proteomics approaches, we now show for the first time that Chivosazole F exerts its effect by directly interacting with actin and demonstrate the cellular impact of Chivosazole F in an unbiased, genome-wide context in yeast and in mammalian cells. Furthermore, mutation-based resistance mapping identifies two SNPs located in the putative Chivosazole F binding site of actin. Comparing chemogenomic profiles and responses to the Chivosazole F-resistant SNPs shows a partially conserved mechanism of action for Chivosazole F and Latrunculin A, but clear divergence from Chondramide. In addition, C14orf80 is an evolutionarily highly conserved ORF, lacking any functional annotation. As editing of C14orf80 leads to Chivosazole F hyper-resistance, we propose a function for this gene product in counteracting perturbation of actin filaments.
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http://dx.doi.org/10.1021/acschembio.7b00385DOI Listing
September 2017

Examining the influence of specificity ligands and ATP-competitive ligands on the overall effectiveness of bivalent kinase inhibitors.

Proteome Sci 2016 17;15:17. Epub 2017 Jul 17.

Novartis Institutes for BioMedical Research, Cambridge, MA 02139 USA.

Background: Identifying selective kinase inhibitors remains a major challenge. The design of bivalent inhibitors provides a rational strategy for accessing potent and selective inhibitors. While bivalent kinase inhibitors have been successfully designed, no comprehensive assessment of affinity and selectivity for a series of bivalent inhibitors has been performed. Here, we present an evaluation of the structure activity relationship for bivalent kinase inhibitors targeting ABL1.

Methods: Various SNAPtag constructs bearing different specificity ligands were expressed in vitro. Bivalent inhibitor formation was accomplished by synthesizing individual ATP-competitive kinase inhibitors containing a SNAPtag targeting moiety, enabling the spontaneous self-assembly of the bivalent inhibitor. Assembled bivalent inhibitors were incubated with K562 lysates, and then subjected to affinity enrichment using various ATP-competitive inhibitors immobilized to sepharose beads. Resulting eluents were analyzed using Tandem Mass Tag (TMT) labeling and two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Relative binding affinity of the bivalent inhibitor was determined by calculating the concentration at which 50% of a given kinase remained bound to the affinity matrix.

Results: The profiling of three parental ATP-competitive inhibitors and nine SNAPtag conjugates led to the identification of 349 kinase proteins. In all cases, the bivalent inhibitors exhibited enhanced binding affinity and selectivity for ABL1 when compared to the parental compound conjugated to SNAPtag alone. While the rank order of binding affinity could be predicted by considering the binding affinities of the individual specificity ligands, the resulting affinity of the assembled bivalent inhibitor was not predictable. The results from this study suggest that as the potency of the ATP-competitive ligand increases, the contribution of the specificity ligand towards the overall binding affinity of the bivalent inhibitor decreases. However, the affinity of the specificity components in its interaction with the target is essential for achieving selectivity.

Conclusion: Through comprehensive chemical proteomic profiling, this work provides the first insight into the influence of ATP-competitive and specificity ligands binding to their intended target on a proteome-wide scale. The resulting data suggest a subtle interplay between the ATP-competitive and specificity ligands that cannot be accounted for by considering the specificity or affinity of the individual components alone.
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http://dx.doi.org/10.1186/s12953-017-0125-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513037PMC
July 2017

Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells.

Sci Rep 2017 02 16;7:42728. Epub 2017 Feb 16.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Forum 1 Novartis Campus, CH-4056 Basel, Switzerland.

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action.
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http://dx.doi.org/10.1038/srep42728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311948PMC
February 2017

Tankyrase Inhibitor Sensitizes Lung Cancer Cells to Endothelial Growth Factor Receptor (EGFR) Inhibition via Stabilizing Angiomotins and Inhibiting YAP Signaling.

J Biol Chem 2016 07 26;291(29):15256-66. Epub 2016 May 26.

From the Department of Developmental and Molecular Pathways, Novartis Institute of Biomedical Research, Cambridge, Massachusetts 02139 and

YAP signaling pathway plays critical roles in tissue homeostasis, and aberrant activation of YAP signaling has been implicated in cancers. To identify tractable targets of YAP pathway, we have performed a pathway-based pooled CRISPR screen and identified tankyrase and its associated E3 ligase RNF146 as positive regulators of YAP signaling. Genetic ablation or pharmacological inhibition of tankyrase prominently suppresses YAP activity and YAP target gene expression. Using a proteomic approach, we have identified angiomotin family proteins, which are known negative regulators of YAP signaling, as novel tankyrase substrates. Inhibition of tankyrase or depletion of RNF146 stabilizes angiomotins. Angiomotins physically interact with tankyrase through a highly conserved motif at their N terminus, and mutation of this motif leads to their stabilization. Tankyrase inhibitor-induced stabilization of angiomotins reduces YAP nuclear translocation and decreases downstream YAP signaling. We have further shown that knock-out of YAP sensitizes non-small cell lung cancer to EGFR inhibitor Erlotinib. Tankyrase inhibitor, but not porcupine inhibitor, which blocks Wnt secretion, enhances growth inhibitory activity of Erlotinib. This activity is mediated by YAP inhibition and not Wnt/β-catenin inhibition. Our data suggest that tankyrase inhibition could serve as a novel strategy to suppress YAP signaling for combinatorial targeted therapy.
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http://dx.doi.org/10.1074/jbc.M116.722967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946938PMC
July 2016

ERG signaling in prostate cancer is driven through PRMT5-dependent methylation of the Androgen Receptor.

Elife 2016 05 16;5. Epub 2016 May 16.

Department of Oncology, Novartis Institutes for BioMedical Research, Cambridge, United States.

The TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR's ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.
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http://dx.doi.org/10.7554/eLife.13964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909395PMC
May 2016

Conversion of a Single Polypharmacological Agent into Selective Bivalent Inhibitors of Intracellular Kinase Activity.

ACS Chem Biol 2016 Jan 6;11(1):121-31. Epub 2015 Nov 6.

Novartis Institutes for Biomedical Research , Cambridge, Massachusetts 02139, United States.

Loss-of-function studies are valuable for elucidating kinase function and the validation of new drug targets. While genetic techniques, such as RNAi and genetic knockouts, are highly specific and easy to implement, in many cases post-translational perturbation of kinase activity, specifically pharmacological inhibition, is preferable. However, due to the high degree of structural similarity between kinase active sites and the large size of the kinome, identification of pharmacological agents that are sufficiently selective to probe the function of a specific kinase of interest is challenging, and there is currently no systematic method for accomplishing this goal. Here, we present a modular chemical genetic strategy that uses antibody mimetics as highly selective targeting components of bivalent kinase inhibitors. We demonstrate that it is possible to confer high kinase selectivity to a promiscuous ATP-competitive inhibitor by tethering it to an antibody mimetic fused to the self-labeling protein SNAPtag. With this approach, a potent bivalent inhibitor of the tyrosine kinase Abl was generated. Profiling in complex cell lysates, with competition-based quantitative chemical proteomics, revealed that this bivalent inhibitor possesses greatly enhanced selectivity for its target, BCR-Abl, in K562 cells. Importantly, we show that both components of the bivalent inhibitor can be assembled in K562 cells to block the ability of BCR-Abl to phosphorylate a direct cellular substrate. Finally, we demonstrate the generality of using antibody mimetics as components of bivalent inhibitors by generating a reagent that is selective for the activated state of the serine/threonine kinase ERK2.
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http://dx.doi.org/10.1021/acschembio.5b00847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4943861PMC
January 2016

Gift from Nature: Cyclomarin A Kills Mycobacteria and Malaria Parasites by Distinct Modes of Action.

Chembiochem 2015 Nov 16;16(17):2433-6. Epub 2015 Oct 16.

Novartis Institutes for BioMedical Research, Novartis Campus, 4056, Basel, Switzerland.

Malaria continues to be one of the most devastating human diseases despite many efforts to limit its spread by prevention of infection or by pharmaceutical treatment of patients. We have conducted a screen for antiplasmodial compounds by using a natural product library. Here we report on cyclomarin A as a potent growth inhibitor of Plasmodium falciparum and the identification of its molecular target, diadenosine triphosphate hydrolase (PfAp3Aase), by chemical proteomics. Using a biochemical assay, we could show that cyclomarin A is a specific inhibitor of the plasmodial enzyme but not of the closest human homologue hFHIT. Co-crystallisation experiments demonstrate a unique binding mode of the inhibitor. One molecule of cyclomarin A binds a dimeric PfAp3Aase and prevents the formation of the enzyme⋅substrate complex. These results validate PfAp3Aase as a new drug target for the treatment of malaria. We have previously elucidated the structurally unrelated regulatory subunit ClpC1 of the ClpP protease as the molecular target of cyclomarin A in Mycobacterium tuberculosis. Thus, cyclomarin A is a rare example of a natural product with two distinct and specific modes of action.
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http://dx.doi.org/10.1002/cbic.201500472DOI Listing
November 2015

Identifying compound efficacy targets in phenotypic drug discovery.

Drug Discov Today 2016 Jan 10;21(1):82-89. Epub 2015 Aug 10.

Developmental & Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA. Electronic address:

The identification of the efficacy target(s) for hits from phenotypic compound screens remains a key step to progress compounds into drug development. In addition to efficacy targets, the characterization of epistatic proteins influencing compound activity often facilitates the elucidation of the underlying mechanism of action; and, further, early determination of off-targets that cause potentially unwanted secondary phenotypes helps in assessing potential liabilities. This short review discusses the most important technologies currently available for characterizing the direct and indirect target space of bioactive compounds following phenotypic screening. We present a comprehensive strategy employing complementary approaches to balance individual technology strengths and weaknesses.
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http://dx.doi.org/10.1016/j.drudis.2015.08.001DOI Listing
January 2016

Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

Angew Chem Int Ed Engl 2015 Aug 15;54(35):10149-54. Epub 2015 Jul 15.

Novartis Institutes for BioMedical Research, Novartis Campus, 4056 Basel (Switzerland).

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1.
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http://dx.doi.org/10.1002/anie.201505069DOI Listing
August 2015

Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo.

Nat Cell Biol 2014 Nov 19;16(11):1069-79. Epub 2014 Oct 19.

Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.
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http://dx.doi.org/10.1038/ncb3053DOI Listing
November 2014

Quantitative Lys-ϵ-Gly-Gly (diGly) proteomics coupled with inducible RNAi reveals ubiquitin-mediated proteolysis of DNA damage-inducible transcript 4 (DDIT4) by the E3 ligase HUWE1.

J Biol Chem 2014 Oct 21;289(42):28942-55. Epub 2014 Aug 21.

From the Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139,

Targeted degradation of proteins through the ubiquitin-proteasome system (UPS) via the activities of E3 ubiquitin ligases regulates diverse cellular processes, and misregulation of these enzymes contributes to the pathogenesis of human diseases. One of the challenges facing the UPS field is to delineate the complete cohort of substrates for a particular E3 ligase. Advances in mass spectrometry and the development of antibodies recognizing the Lys-ϵ-Gly-Gly (diGly) remnant from ubiquitinated proteins following trypsinolysis have provided a tool to address this question. We implemented an inducible loss of function approach in combination with quantitative diGly proteomics to find novel substrates of HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase), an E3 ligase implicated in cancer and intellectual disabilities. diGly proteomics results led to the identification of DNA damage-inducible transcript 4 (DDIT4) as a putative HUWE1 substrate. Cell-based assays demonstrated that HUWE1 interacts with and regulates ubiquitination and stability of DDIT4. Together these data suggest a model in which HUWE1 mediates DDIT4 proteasomal degradation. Our results demonstrate proof of concept that inducible knockdown of an E3 ligase in combination with diGly proteomics provides a potentially advantageous method for identifying novel E3 substrates that may help to identify candidates for therapeutic modulation in the UPS.
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http://dx.doi.org/10.1074/jbc.M114.573352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200252PMC
October 2014

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

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

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

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

Target identification for a Hedgehog pathway inhibitor reveals the receptor GPR39.

Nat Chem Biol 2014 May 16;10(5):343-9. Epub 2014 Mar 16.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.

Hedgehog (Hh) signaling determines cell fate during development and can drive tumorigenesis. We performed a screen for new compounds that can impinge on Hh signaling downstream of Smoothened (Smo). A series of cyclohexyl-methyl aminopyrimidine chemotype compounds ('CMAPs') were identified that could block pathway signaling in a Smo-independent manner. In addition to inhibiting Hh signaling, the compounds generated inositol phosphates through an unknown GPCR. Correlation of GPCR mRNA expression levels with compound activity across cell lines suggested the target to be the orphan receptor GPR39. RNA interference or cDNA overexpression of GPR39 demonstrated that the receptor is necessary for compound activity. We propose a model in which CMAPs activate GPR39, which signals to the Gli transcription factors and blocks signaling. In addition to the discovery of GPR39 as a new target that impinges on Hh signaling, we report on small-molecule modulators of the receptor that will enable in vitro interrogation of GPR39 signaling in different cellular contexts.
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http://dx.doi.org/10.1038/nchembio.1481DOI Listing
May 2014

Mining the bacterial unknown proteome: identification and characterization of a novel family of highly conserved protective antigens in Staphylococcus aureus.

Biochem J 2013 Nov;455(3):273-84

*Research Department, Novartis Vaccines and Diagnostics, 53100 Siena, Italy.

In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.
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http://dx.doi.org/10.1042/BJ20130540DOI Listing
November 2013