Direct Interaction of Chivosazole F with Actin Elicits Cell Responses

Subscriber access provided by WILFRID LAURIER UNIV

Letter

Direct interaction of Chivosazole F with actin elicits cell responses similar to Latrunculin A but distinct from Chondramide Ireos Filipuzzi, Jason Ray Thomas, Verena Pries, David Estoppey, Michael Salcius, Christian Studer, Markus Schirle, and Dominic Hoepfner ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00385 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Direct interaction of Chivosazole F with actin elicits cell responses similar to Latrunculin A but distinct from Chondramide

Ireos Filipuzzi1, Jason Ray Thomas 2, Verena Pries1, David Estoppey1, Michael Salcius2, Christian Studer1, Markus Schirle2*, Dominic Hoepfner1*

1

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Forum 1 Novartis Campus,

CH-4056 Basel, Switzerland 2

Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, MA

02139, USA * Corresponding authors: [email protected], [email protected]

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract 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 chemo-genomic 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 evolutionary 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.


Page 2 of 17

Page 3 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Most eukaryotic cells contain a complex protein network known as the cytoskeleton 1. It consists of three distinct components -microfilaments, intermediate filaments and microtubules- and exerts not only structural and mechanical tasks like establishing cell shape and rigidity, but is also actively implicated in many essential processes such as cell motility, cell division, vesicle-mediated transport, muscle contraction and many more 1. Microfilaments are composed of linear polymers (F-actin) of globular actin (G-actin), one of the most highly evolutionary conserved proteins, differing by only 20% in species as diverse as humans and algae 2. Actin is also the most abundant protein in eukaryotic cells, accounting for about 10% of total protein mass 3 and possesses the ability to polymerize into filaments which then are further complexed in higher-ordered structures by binding a variety of structural and regulatory proteins 4

. Furthermore, mutations in actin and actin-binding proteins have been reported to lead to diseases

including neurodegeneration 5 and cancer 6. It is therefore not surprising that studying actin-mediated processes and profiling actin modulators as potential therapeutics are of central interest to a large scientific and bio-medical community. Selective and potent modulators are a prerequisite to study and dissect the various functions of actin and microfilaments and several compounds have been identified that mediate their effects by binding to actin directly. Jasplakinolide 7 and Latrunculin A 8 have proven their usefulness in interrogating actinrelated processes, but these reagents have to be tediously isolated from marine sponges or synthesized in elaborated reactions 9. Other reagents, like Cytochalasin D, have only limited applicability in fungi 9 and Phalloidin suffers from the lack of penetrating living cells 10. For Chivosazole F, a macrolide isolated from the myxobacterium Sorangium cellulosum, potent anti-proliferative activity has been reported against filamentous fungi, yeasts, insect cells and also a range of mammalian cells 11. As a microbial metabolite, a main advantage of Chivosazole F is that it can be isolated from microbial cultures, thus avoiding dependencies on marine sources or chemical synthesis. While Chivosazole F was found to disrupt the cytoskeletal dynamics in cultured cells and to inhibit actin polymerization in vitro 12, the molecular efficacy target of this macrolide has so far not been described. The goals of this study were i) to confirm direct interaction of Chivosazole F with actin and ii) to assess the effects of actin perturbation at a genomic scale for the first time. Applying a combination of genomic and proteomics strategies for target identification and mode of action elucidation, we show for the first time that Chivosazole F exerts its effect by physically interacting with actin and resistance mapping identifies two SNPs located in the putative Chivosazole F binding site. Furthermore we confirm previous findings that the mechanism of action of Chivosazole F is similar to the one of Latrunculin A but clearly differs from Chondramide. Chemo-genomic profiling in in mammalian cells then allows us to visualize the ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

impact of Chivosazole F on actin in an unbiased, genome-wide context and additionally to propose a first genetic link for an uncharacterized ORF to actin.

Results and Discussion Chemoproteomics identifies actin and actin-binding proteins complexed to Chivosazole F Approaches combining small molecule affinity chromatography and mass spectrometry (MS)-based proteomics have been successfully applied for identifying protein targets of bioactive compounds 13. However, these approaches require the derivatization of the compound of interest for immobilization to a matrix. In particular for complex natural products like Chivosazole F this can be challenging. We thus turned to a random display approach using photo-crosslinking 14 15. N-Hydroxysuccinimidyl-activated sepharose beads were modified to present a photo-reactive diazirine group and mixed with Chivosazole F. UV irradiation of the mixture leads to random immobilization of the target compound on beads via carbene insertion or addition (Fig. 1A). In order to account for the fact that the effective coupling concentration is hard to predict, we generated two batches of matrix using two different concentrations of photocrosslinker (2 and 10 µmol/ml (Fig.1B)). The prepared Chivosazole F-matrices were incubated with HEK293T lysates that had been pre-incubated with 100 μM free Chivosazole F or with DMSO, respectively. Bead-bound proteins were then eluted and subjected to LC-MS/MS-based protein identification and quantitation using isobaric labeling tags. Amongst the 3443 proteins with 2 or more quantifiable peptides (Supplemental Data Table 1), several proteins showed robust, >2 fold competition by 100 μM free Chivosazole F with both matrices (Fig. 1C). Notably, these include two isoforms of actin itself (ACTB and ACTC1), while all other candidates are known actin-binding proteins (DBN1, LIMA1, TPM1, TPM3, MYL6 and MYL12B). Several weaker hits that showed robust competition with only one of the matrices are also known actin interactors (NEXN, MYH9, MYH10 and PFN1). While this dataset is in line with direct modulation of the actin cytoskeleton, and presents several key candidates, it does not yet provide the resolution required for unambiguous deconvolution of the direct physical target(s) of Chivosazole F. Size-exclusion chromatography validates actin as target of Chivosazole F As actin was a prominent, but not the only hit in the pull-down experiment, we wanted to see if Chivosazole F binds directly to actin by using SEC-TID, an in vitro approach combining size-exclusion chromatography with MS-based compound quantitation 16. Two additional proteins involved in regulation of the actin cytoskeleton, the Arp2/3 complex member ARPC2 as well as the actin capping protein CAPG were included as controls. Chivosazole F was incubated with purified actin, ARPC2 or CAPG


Page 4 of 17

Page 5 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and the protein-bound compound fraction was separated from unbound compound by centrifugationbased size-exclusion chromatography. The flow-through fraction containing protein-compound complexes was subjected to SDS-based denaturation followed by MS-based quantitation of released compound. As shown in Fig. 1D, Chivosazole F formed a complex with actin but neither with ARPC2 nor with CAPG, thus demonstrating that actin is a direct interactor of Chivosazole F. Genome wide mutagenesis identifies Chivosazole F-resistance conferring mutations in actin Random mutagenesis followed by selection and analysis of resistant clones not only allows the identification and/or validation of cellular targets, but also has the potential to give insights into the mechanism of protein-drug interaction and localization of the exact binding site 17, 18. Exploiting the high evolutionary conservation of the actin network 2 and the pan-species activity of Chivosazole F 11 we mutagenized a culture of Saccharomyces cerevisiae cells in an unbiased fashion, followed by selection on lethal concentrations of Chivosazole F. After three days, resistant colonies were isolated, genomic DNA prepared, ACT1 PCR-amplified and sequenced. Analysis identified two distinct point-mutations in ACT1, R183K and R335K. These mutations were engineered into a wild type strain by homologous recombination, eliminating potential effects from second-site mutations, and subjected to validation (Fig. 2). The resulting strains had wild-type like growth kinetics and a morphologically normal actin cytoskeleton. Spotting serial cell dilutions on Chivosazole F-containing plates demonstrated that both point-mutations were able to induce hyper resistance, compared to a wild type control (Fig. 2A). Resistance-testing in liquid cultures revealed that ACT1 R183K shifted the IC50 three-fold and ACT1 R335K two-fold, respectively (Fig. 2B), confirming the resistance observed on solid medium. Moderate alleviation of the toxicity by the tested mutations was expected as the Arginine to Lysine transition result in minor structural modifications only. As strong evolutionary conservation of actin suggests little flexibility in the protein sequence it is unlikely to find functional mutations that induce more pronounced changes. Interestingly, ACT1 R183K and ACT1 R335K behaved differently, when we tested against other actin toxins like the depolymerizer Latrunculin A or the stabilizer Chondramide 9. As ACT1 R335K led to hyper resistance but R183K led to hyper sensitivity to Latrunculin A (Fig. 2C), we hypothesize that the binding site for Chivosazole F and Latrunculin A partially overlap. Whereas R335K induces resistance against both drugs by modulating a common area of drug binding, R183K does this only for Chivosazole F, but increases penetrance for Latrunculin A, by modulating a locus lateral of the binding site. Both mutants, however, lead to hyper sensitivity to Chondramide (Fig. 2D). This is expected as filament stabilizers like Chondramide interfere with actin at the barbed end and thus resistance by impairment of binding is ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

unlikely. Filament destabilizers like Chivosazole F and Latrunculin A however interact with the ATPbinding cleft in immediate vicinity of the tested mutations 9. 2-way dose-response assays using combinations of either Latrunculin A or Chondramide with Chivosazole F were performed to determine potential synergistic or antagonistic effects (Supplemental Fig 2). The data recapitulated the individual resistance/hypersenstitivity phenotpyes depicted in the Fig. 2 but Latrunculin A and Chivosazole F combinations showed no obvious synergistic effects. This adds further support for partially overlapping binding sites in the ATP-binding cleft. Combinations of Chondramide and Chivosazole F however showed clear antagonism, recapitulating their antagonsitic mechanism of actin and distinct binding sites. Chemogenomic profiling in yeast reveals the impact of Chivosazole F on the actin network Conserved binding of Chivosazole F and Latrunculin A to the ATP-binding site would be expected to exert similar cellular effects but to be distinct to those of Chondramide. To test the effect in a cellular context, we subjected these three actin toxins to genome-wide haploinsufficiency profiling (HIP) and homozygous profiling (HOP) in S. cerevisiae 19. Heterozygous deletions (HIP) indicate pathways directly affected by the compound, whereas homozygous deletions (HOP) indicate synthetic lethality and compensating pathways to those directly affected by the compound. Testing of all three compounds resulted in profiles rich in hits related to actin biology like the ARP2/3 and the CTT complex (Fig. 3A, B; Supplemental Data Table 1). As published, the heterozygous ACT1/act1 deletion strain did not score hypersensitivity due to compensatory feedback mechanisms and a resulting lack of haploinsufficiency 20. HIP profile correlation analysis identified many common hits linked to actin folding shared by Chivosazole F with an overall correlation coefficient of 0.34 (Fig. 3C). The profiles of Chivosazole F and Chondramide displayed unique sets of hits and an overall correlation coefficient of 0.12, reflecting the differential depolymerization/stabilization mechanisms. Based on our analysis, Chivosazole F and Latrunculin A are not only proposed to share a partially conserved binding site but also exert similar cellular effects. Thus, microbially produced Chivosazole F constitutes an attractive research reagent alternative to the costly Latrunculin A from marine sources. Inhibitor-sensitized CRISPR screen reveals the impact of Chivosazole F on the mammalian actin network Roles of the different cytoskeletal elements in fungi and mammalian cells are distinct and intermediate filaments absent in fungi. In yeast, vesicular and organellar transport is accomplished exclusively by myosin motors and the actin cytoskeleton whereas in mammalian cells this function is mainly dependent on dynein, kinesin motors and microtubules 1. To obtain a genetic fingerprint on the modulation of the actin cytoskeleton by Chivosazole F, we deployed a recently published, genome-wide CRISPR/Cas9-based ACS Paragon Plus Environment

Page 6 of 17

Page 7 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


chemogenomic profiling strategy that recapitulates the key features of the yeast HIP HOP experiment in mammalian cells 21 22. The obtained data was plotted to identify genes that when edited confer hypersensitivity or resistance (Fig. 3E, F; Supplemental Data Table 3). Strongest hypersensitivity was observed for alpha 4 actinin (ACTN4), the actin remodeling protein (FLII) and myotrophin (MTPN). Intermediate hits were gamma 1 actin (ACTG1), tropomodulin 3 (TMOD3), the diaphanous related formin 3 (DIAPH3) the FLII interacting protein 2 (LRRFIP2), exportin 6 (XPO6) and mesogenin 1 (MSGN1). Weaker scores were also found for beta actin (ACTB), myosin light chain 6 (MYL6), dynactin subunit 6 (DCTN6) and the subunits 2 and 4 of the ARP2/3 complex (Fig. 3E). Compound resistance was observed upon CRISPR editing of coronin 1C (CORO1C) which spatially targets Arp2/3 complex and cofilin to opposite ends of actin networks. In addition resistance was also found for the actin capping proteins CAPZB, CAPZA1 and CD2AP stabilizing filament growth 23. These data clearly show that reducing levels of various negative regulators of the catabolic filament pathway can partially compensate the effects of the filament destabilized Chivosazole F in a cellular environment. As the uncharacterized, but evolutionary conserved ORF C14orf80 also scores significantly, we hypothesize an actin-stabilizing function for this gene product. It would be intriguing to profile and compare to Latrunculin A, however, the method requires several liters of tissue culture medium and thus Latrunculin A compound amounts are prohibitive. Thus to the best of our knowledge this is the first unbiased, genome-wide chemogenomic profile of an actin toxin in mammalian cells.

Acknowledgments We would like to thank the Novartis Natural Products Unit for compound isolation and purification of Chivosazole F, T. Jaeg for experimental support, J. Nagel for mass spectrometry, R. Riedl and T. Aust for HIP HOP profiling and P. Furet for structural work. Supporting Information The supporting information is available, free of charge at http://pubs.acs.org. This includes the validation of HIP and HOP hits (Supplemental Fig. 1), 2D dose-response testing of Chivosazole F, Latrunculin A and Chondramide (Supplemental Fig. 2), protein identification and quantitation of the chemoproteomics approach (Supplemental Data Table 1), the genome-wide MADL and Z-score values for HIP and HOP hits (Supplemental Data Table 2) and the genome-wide scores for the CRISPR profiling of Chivosazole F (Supplemental Data Table 3).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Founding The authors declare the following competing financial interest(s): All authors of the manuscript are employees of the Novartis Institutes for BioMedical Research and may own stock in the company. Material and Methods Chivosazole F Chivosazole F was isolated from Sorangium cellulosum and purified as published 11. Purity was assessed by analytical LC/MS/MS and found to be >90%. Compound was stored at 4°C in the dark. Solutions were prepared in 90% DMSO as 10 mM stocks, stored at 4°C and used within 3 months. Synthesis of diazirine-derivatized resin for immobilization of Chivosazole F and chemoproteomics For synthesis of diazirine-bearing photocrosslinker resin, 5 ml packed resin volume of EAH sepharose 4B (GE Healthcare) was washed 3 times with 10 mL anhydrous DMSO (Sigma-Aldrich). For the synthesis of resin with a coupling density of 2 µmol/ml, the EAH sepharose resin was resuspended in 5 ml anhydrous DMSO and 10 µmol LC-SDA (Pierce Biotechnology) and 75 µL triethylamine (Sigma-Aldrich) were added. The reaction mixture was vortexed, spun at 100 x g for 2 min and a 50 µl aliquot of the supernatant was saved for LC-MS analysis. After overnight incubation at room temp with end-over-end agitation, the reaction mixture was spun at 100 x g for 2 min and a 50 µl aliquot of the supernatant was saved for LCMS analysis. Completion of coupling was inferred by loss of starting material following LC-MS analysis. 250 µl of 2-(2-aminoethoxy)ethanol (Sigma-Aldrich) was added to the reaction mixture, vortexed, and allowed to incubate overnight. Finally, the diazirine-derivatized resin was washed 3 times with 10 ml anhydrous DMSO. For the synthesis of diazirine-derivatized resin at a coupling density of 10 µmol/ml, the above protocol was followed except that 50 µmol of LC-SDA was added. Chivosazole F was immobilized on the diazirine-derivatized resin by photo-crosslinking as described previously 15, except that the compound was solubilized in ethanol. Generation of cell lysates from flash frozen HEK293T cell pellets, compound competition and affinity enrichments as well as proteomics sample preparation, quantitative mass spectrometry and data analysis were done as described previously 24. iTRAQ 114 and 115 were used for samples pre-treated with 100 µM Chivosazole F and DMSO, respectively, and subjected to enrichment on the Chivosazole F affinity matrix with 2 µmol/ml coupling density; iTRAQ 116 and 117 were used for the corresponding samples using the matrix with 10 µmol/ml coupling density.


Page 8 of 17

Page 9 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In vitro binding assay Size-exclusion chromatography for target identification (SEC-TID) was performed as described 16 with the exception that commercially available proteins were used in the buffers as supplied by the vendors in the following concentrations: Human platelet-derived non-muscle actin (APHL095, Cytoskeleton, Inc; 14.5 μM), rabbit skeletal muscle actin (AKL095, Cytoskeleton, Inc; 14.5 μM), recombinant human CAPG (NBP1-30303, Novus Biologicals, LLC; 13 μM), recombinant human ARPC2 (AB140555, Abcam; 3.3 μM). Chivosazole F was tested at 10 μM in replicate. Genome wide mutagenesis The isolation and characterization of Chivosazole F-resistant mutants was performed as described 18 with the exception that ACT1 was amplified by PCR from genomic DNA of the mutants using the oligonucleotides 5'-GAT GGT GTT ACT CAC GTC-3' and 5'-GAT TCT GTG CGA TAG CGC GTA GAA AAG GGA GAG AC-3', purified and sequenced directly. The minimal inhibitory concentration (MIC) for Chivosazole F was found to be 1 µM for the used strain BY4743Δ8 25 on SD agar. Engineering of the resistance-conferring mutations Genomic DNA of the wild type and mutant yeasts was isolated, the ACT1 gene amplified by PCR using the oligonucleotides 5'-GAT GGT GTT ACT CAC GTC-3' and 5'-GAT TCT GTG CGA TAG CGC GTA GAA AAG GGA GAG AC-3' and gel purified. The LEU2 gene was generated by PCR from the vector BYIntLEU 26 using the oligonucleotides 5'-GTC TCT CCC TTT TCT ACG CGC TAT CGC ACA GAA TC-3' and 5’ CCT ATG AAC ATA TTC CAT TTT G-3' and gel purified. The ACT1-LEU2 cassettes were generated by PCR using 10 ng of each purified product produced above as PCR template and 5'-CAT ATG ATA CAC GGT CCA ATG GAT AAA CAT TTT TTA TCA ACA CTA TGC CTA TGA ACA TAT TCC ATT TTG-3' and 5'-GAT GGT GTT ACT CAC GTC-3' as oligonucleotides. The gel purified cassettes were then transformed into wild type yeast, transformants selected on SD-LEU and then tested for resistance on SD-LEU supplemented with 1 µM Chivosazole F. Correct integration into the ACT1 locus and integrity of the ACT1 gene of the engineered resistant (mutants) and sensitive (wild type) clones was verified by sequencing. Validation of resistant mutants Colony formation and liquid growth assays were performed as described 27. Chemogenomic profiling in yeast HIP and HOP was exactly performed as previously described 19. Chivosazole F was tested at 12 µM, Latrunculin A at 0.7 µM and Chondramide at 90 µM.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 17

Inhibitor-sensitized CRISPR screen The screen was performed as previously described 21. Chivosazole F was profiled at 90 nM.

Figure Legends Fig. 1: Chivosazole F chemoproteomics and in vitro binding assay data indicate binding to actin in cell lysate and in vitro A: Schematic of the generation of a random display Chivosazole F affinity matrix using photocrosslinker beads B: Schematic of the quantitative chemoproteomics workflow C: Unbiased, competition-based chemoproteomics identifies actin-containing protein complexes as interactors of Chivosazole F in HEK293T lysate. Scatter plot showing competition of binding to affinity matrix by 100 µM Chivosazole F for two replicate experiments at a photocrosslinker density of 2 µmol/ml and 10 µmol/ml, respectively. Data are plotted as Log10 fold change over DMSO control for 3443 human proteins with 2 or more quantified peptides. Dashed lines denote 50% competition. D: Chivosazole F shows direct binding in vitro to actin from human platelets as well as rabbit skeletal muscle as assessed by SEC-TID 16. No binding is observed for two additional proteins involved in regulation of the actin cytoskeleton, ARPC2 and CAPG. Mass spectrometry-derived peak areas for Chivosazole are plotted for replicate analyses. Fig. 2: Confirmation and characterization of Chivosazole F-resistant mutants A: Saturated ACT1 wild type (WT), as well as engineered ACT1 R183K (R183K) and ACT1 R335K (R335K) cultures were adjusted to 10 OD600 and 3µl of serial 1:3 dilutions spotted on YPD agar supplemented with indicated concentrations of Chivosazole F. Growth was scored after three days incubation at 30°C. B: Liquid growth assays were performed in quadruplicate biological replicates as described 19 and mean IC50 values calculated by logistic regression. IC50 WT: 0.14 µM, IC50 R183K 0.38 µM and IC50R335K: 0.27 µM. C: Cross-reactivity of Chivosazole F-resistant mutants to Latrunculin A was determined as above. IC50 WT: 0.07 µM, IC50 R183K: 0.11 µM and IC50R335K: 0.04 µM. D: Cross-reactivity of Chivosazole F-resistant mutants to Chondramide were determined as above. IC50 WT: 16.7 µM, IC50 R183K: 11.9 µM and IC50R335K: 15.1 µM. Fig. 3: Chemogenomic profiling of Chivosazole F A: HIP and B: HOP profiles of Chivosazole F tested in two independent biological replicates at 12 µM are displayed. Strain sensitivity of deletions is plotted against statistical significance (z-score) as previously described 19. Squares indicate essential, circles non-essential genes of the S. cerevisiae genome. The


Page 11 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


complete HIP and HOP hit lists for Chivosazole F, Latrunculin A and Chondramide are given in Suppl. Data Table. 1 Alignments of deletion sensitivities of two independent biological replicates of C: Latrunculin A at 0.7 µM and D: Chondramide at 90 µM to Chivosazole F at 12 µM are displayed and r2 values given. Hits scoring against both drugs are highlighted. Squares indicate essential, circles non-essential genes. The depletion E: and the enrichment F: of individual guide RNAs, compared to a no drug control, from an inhibitor-sensitized CRISPR screen of Chivosazole F at 90 nM in HCT116 cells is shown. The RSA p-value, a gene-level measure for conserved depletion (RSA down) or enrichment (RSP up) of its respective guides is plotted against Q, a gene-level effect size corresponding to the RSA p-value for depletion (Q1) or enrichment (Q4) 21. Supplemental Fig. 1: Validation of HIP and HOP hits Liquid growth assays were performed in duplicates (HO control in quadruplicates) as described 19 and dose-response curves generated by logistic regression. Supplemental Fig. 2: 2D dose-response testing of Chivosazole F, Latrunculin A and Chondramide. Liquid growth assays were performed in triplicate as described 19 and % growth, as measured by OD600, is displayed by a color gradient. Latrunculin A was tested in a dose-range of 400 - 0.4 nM, Chivosazole F of 4000 - 4.4 nM, and Chondramide of 40 - 0.04 μM, respectively. As outlined in the schematic representation at the bottom, antagonistic, neutral or synergistic effects are expected to result in indicated shape of growth distribution. Supplemental Data Table 1: Chemoproteomics data Protein identification and quantitation source data for Fig. 1C. Supplemental Data Table 2: MADL and Z-scores for Chivosazole F, Latrunculin A and Chondramide Genome-wide MADL and Z-score values for HIP and HOP profile hits. Supplemental Data Table 3: Compound/CRISPR profiling data for Chivosazole F Genome-wide scores for Chivosazole F profiling experiment in HCT116 cells at 50 and 90 nM.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 17

References 1. Alberts B, J. A., Lewis J, Raff M, Roberts K Walter P. (2002) The Cytoskeleton, In Mol. Biol. Cell, p Chapter 16, Garland Science, New York. 2. Gunning, P. W., Ghoshdastider, U., Whitaker, S., Popp, D., and Robinson, R. C. (2015) The evolution of compositionally and functionally distinct actin filaments, J. Cell Sci. 128, 2009-2019. 3. Kiuchi, T., Nagai, T., Ohashi, K., and Mizuno, K. (2011) Measurements of spatiotemporal changes in Gactin concentration reveal its effect on stimulus-induced actin assembly and lamellipodium extension, J. Cell Biol. 193, 365-380. 4. Dominguez R, H. K. (2011) Actin Structure and Function, Annu. Rev. Biophys. 40, 169-186. 5. Eira, J., Silva, C. S., Sousa, M. M., and Liz, M. A. (2016) The cytoskeleton as a novel therapeutic target for old neurodegenerative disorders, Prog. Neurobiol. 141, 61-82. 6. Davidson, A. J., and Wood, W. (2016) Unravelling the Actin Cytoskeleton: A New Competitive Edge?, Trends Cell Biol. 26, 569-576. 7. Holzinger, A. (2010) Jasplakinolide: An Actin-Specific Reagent that Promotes Actin Polymerization, Methods Mol Biol 586, 71-87. 8. Yarmola, E. G., Somasundaram, T., Boring, T. A., Spector, I., and Bubb, M. R. (2000) Actin-latrunculin A structure and function:differential modulation of actin-binding protein function by latrunculin A, J. Biol. Chem. 275, 28120-28127. 9. Allingham, J. S., Klenchin, V. A., and Rayment, I. (2006) Actin-targeting natural products: structures, properties and mechanisms of action, Cell. Mol. Life Sci. 63, 2119-2134. 10. Melak, M., Plessner, M., and Grosse, R. (2017) Actin visualization at a glance, J. Cell Sci. 130, 525-530. 11. Irschik, H., Jansen, R., Gerth, K., Höfle, G., and Reichenbach, H. (1995) Chivosazol A, a New Inhibitor of Eukaryotic Organisms Isolated from Myxobacteria, J. Antibiot. 48, 5. 12. Diestel R, I. H., Jansen R, Khalil MW, Reichenbach H and Sasse F. (2009) Chivosazoles A and F, Cytostatic Macrolides from Myxobacteria, Interfere with Actin, ChemBioChem 10, 2900-2903. 13. Schmitt, E. K., Hoepfner, D., and Krastel, P. (2016) Natural products as probes in pharmaceutical research, J. Ind. Microbiol. Biotechnol. 43, 249-260. 14. Kanoh, N., Honda, K., Simizu, S., Muroi, M., and Osada, H. (2005) Photo-Cross-Linked Small-Molecule Affinity Matrix for Facilitating Forward and Reverse Chemical Genetics, Angew. Chem., Int. Ed. 44, 3559-3562. 15. Takayama, H., Moriya, T., and Kanoh, N. (2012) Preparation of Photo-Cross-Linked Small Molecule Affinity Matrices for Affinity Selection of Protein Targets for Biologically Active Small Molecules, Methods Mol Biol. 800, 75-83. 16. Salcius M, B. A., Hao Q, Li S, Tutter A, Raphael J, Jahnke W, Rondeau JM, Bourgier E, Tallarico J, Michaud GA. (2014) SEC-TID: A Label-Free Method for Small-Molecule Target Identification, J. Biomol. Screening 19, 11. 17. Filipuzzi I, C. S., Perruccio F, Knapp B, Fu Y, Studer C, Pries V, Riedl R, Helliwell S B, Petrovic K T, Movva NR, Sanglard D, Tao J and Hoepfner D. (2016) High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines, PLoS Genet. 12, e1006374. 18. Shimada K, F. I., Stahl M, Helliwell S B, Studer C, Hoepfner D, Seeber A, Loewith R, Movva N R and Gasser S M. (2013) TORC2 Signaling Pathway Guarantees Genome Stability in the Face of DNA Strand Breaks, Mol. Cell. 51, 829-839. 19. Hoepfner D, H. S. B., Sadlish H, Schuierer S, Filipuzzi I, Brachat S, Bhullar B, Plikat U, Abraham Y, Altorfer M, Aust T, Baeriswyl L, Cerino 2, Chang L, Estoppey D, Eichenberger J, Frederiksen M, Hartmann N, Hohendahl A, Knapp B, Krastel P, Melin N, Nigsch F, Oakeley EJ, Petitjean V, Petersen F, Riedl R, Schmitt EK, Staedtler F, Studer C, Tallarico JA, Wetzel S, Fishman MC, Porter JA, Movva NR. (2014) High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions, Microbiol. Res. 169, 107-120. ACS Paragon Plus Environment

Page 13 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20. Mulholland, J., Wesp, A., Riezman, H., and Botstein, D. (1997) Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle, Molecular Biology of the Cell 8, 14811499. 21. Estoppey D, H. J., Guy CT, Harrington E, Thomas JR, Schirle M, Cuttat R, Waldt A, Gerrits B, Yang Z, Schuierer S, Pan X, Xie K, Carbone W, Knehr J, Lindeman A, Russ C, Frias E, Hoffman GR, Varadarajan M, Ramadan N, Reece-Hoyes JS, Wang Q, Chen X, McAllister G, Roma G, Bouwmeester T and Hoepfner D. (2017) Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells, Sci. Rep. 7, 42728. 22. Estoppey D, L. C., Janoschke M, Lee BH, Wan KF, Dong H, Mathys P, Filipuzzi I, Schuhmann T, Riedl R, Aust T, Galuba O, McAllister G, Russ C, Spiess M, Bouwmeester T, Bonamy GMC and Hoepfner D. (2017) The Natural Product Cavinafungin Selectively Interferes with Zika and Dengue Virus Replication by Inhibition of the Host Signal Peptidase, Cell Reports 19, 451-460. 23. Xu, j. C., jf; Pollard, TD;. (1998) Effect of capping protein, CapZ, on the length of actin filaments and mechanical properties of actin filament networks, Cell Motil. Cytoskeleton 42, 9. 24. Krastel P, R. S., Schirle M, Ross NT, Perruccio F, Aspesi P Jr, Aust T, Buntin K, Estoppey D, Liechty B, Mapa F, Memmert K, Miller H, Pan X, Riedl R, Thibaut C, Thomas J, Wagner T, Weber E, Xie X, Schmitt EK and Hoepfner D. (2015) Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties, Angew. Chem., Int. Ed. 54, 10149-10154. 25. Hoepfner D, M. C., Lim CS, Studer C, Riedl R, Aust T, McCormack SL, Plouffe DM, Meister S, Schuierer S, Plikat U, Hartmann N, Staedtler F, Cotesta S, Schmitt EK, Petersen F, Supek F, Glynne RJ, Tallarico JA, Porter JA, Fishman MC, Bodenreider C, Diagana TT, Movva NR and Winzeler EA. (2012) Selective and Specific Inhibition of the Plasmodium falciparum Lysyl-tRNA Synthetase by the Fungal Secondary Metabolite Cladosporin, Cell Host Microbe 11, 654-663. 26. Pierce, S. E., Davis, R. W., Nislow, C., and Giaever, G. (2007) Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures, Nat. Protocols 2, 29582974. 27. Pries V, C. S., Riedl R, Aust T, Schuierer S, Tao J, Filipuzzi I and Hoepfner D. (2016) Advantages and Challenges of Phenotypic Screens: The Identification of Two Novel Antifungal Geranylgeranyltransferase I Inhibitors, J. Biomol. Screening 21, 306-315.



A

O O

OH N

H

O

OH OH O

Chivosazole F

OH O

N N

H N

N H

O

Chivosazole F

O

H N

N H

UV (365 nm)

O

HEK293T lysates

B

DMSO

100 μM Chivosazole F

2 µmol/ml

DMSO

100 μM Chivosazole F

Chivosazole F O N H

10 µmol/ml

H N O

Chivosazole F O

H N

N H

O

iTRAQ- based Quantitative Proteomics

C

D 0.4

400

0.2

-0.4

100

PFN1

-0.2

0

0.2

Log10 (100mM Chivosazole F/DMSO) (Coupling density: 2mmol/mL)


CAPG (human, recombinant)

-0.6

200

ARPC2 (human, recombinant)

ELAC1 TPM1 -0.4 TPM3 LIMA1 DBN1 ACTC1 -0.6 MYL6 MYL12B ACTB

300

Actin (human, platelet)

-0.2

PPP1R12A RAI14 MYH10 NEXN MYH9

Actin (rabbit, skeletal muscle)

0

Intensity (AU)

Log10 (100mM Chivosazole F/DMSO) (Coupling density: 10mmol/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 17

A

ChivoF Actin

B

WT

0

μM R183K R335K

0.8 0.6 0.4

WT

0.04

C Growth (OD600)

0.5 μM R183K

ACT1 WT ACT1 R183K ACT1 R335K

0.2

R335K WT 1.0 μM R183K R335K

R335K WT 2.0 μM R183K R335K

D

4

0.8 0.6 0.4


WT 1.5 μM R183K

0.1 0.4 1 Chivosazole F (μM)

1

0.2

0.01 0.04 0.1 Latrunculin A (μM)

Growth (OD600)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Growth (OD600)

Page 15 of 17

0.4

0.8 0.6 0.4


0.2 4


10 Chondramide (μM)

40


0 sensitivity

B

10

-10

ARC19 ARC15 ARP3

CCT6 CCT4 TCP1 CCT5 CCT2 CCT8 CCT3

-30 SEC4 -14

-10

C

AKR1 -6 -2 z-score

-20

-20 -30

10

NPL60

ARPC2 ARPC4 DCTN6

-5 LRRFIP2 XPO6 MSGN1 DIAPH3 POFUT1 ACTG1 TMOD3 -10

MTPN

FLII

-10

GSP1 PDR5 HXT12 -30

-20 -10 0 sensitivity Chivosazole F

0

ACTB

GOLGA6

0

-20

F

-5 CD2AP

-10

C14orf80 CAPZA1 CAPZB

-15

ACTN4

CORO1C

-20 0 Q1

10

2

r2=0.12

RSA up

MYL6

-6 -2 z-score

-10

CCT8 GSP1

RPL10 CCT2 PDR5 SEC4 HXT12 -30 -30 -20 -10 0 sensitivity Chivosazole F 0

-10

D

CCT3

E

PTR2

-14

r2=0.34

AKR1

YFR016C

-40

2

0 -10

BUD6 BNI1

-10

sensitivity Chondramide

sensitivity Latrunculin A

10

BNR1

0

-20

-40

10

ACT1 sensitivity

A

RSA down

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 17

-10

0 Q4


10

Page 17 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for table of contents 82x41mm (300 x 300 DPI)


Direct Interaction of Chivosazole F with Actin Elicits Cell Responses

Recommend Documents