Cutting Edge Chemical Biology: Report from the 2016 International

In Focus pubs.acs.org/acschemicalbiology

Cutting Edge Chemical Biology: Report from the 2016 International Symposium on Chemical Biology, January 13−15, Geneva, Switzerland Alexander Adibekian*,† and Pierre Stallforth*,‡ †

School of Chemistry and Biochemistry, NCCR Chemical Biology, University of Geneva, 30 quai Ernest-Ansermet, Geneva, Switzerland ‡ Junior Research Group Chemistry of Microbial Communication, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute, Beutenbergstrasse 11a, 07745 Jena, Germany

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biology and disease using chemical tools as well as state-of-theart “omics” technologies. Recent work from Cravatt and coworkers unraveled the biochemical mechanism of the neurodegenerative disease PHARC via an untargeted metabolomics approach. This disorder is caused by mutations in serine hydrolase ABHD12 that serves as a principal lysophosphatidylserine (Lyso-PS) lipase in the mammalian brain, leading to accumulation of Lyso-PS lipids, which then promote microglial and neurobehavioral abnormalities.1 Interestingly, a selective small molecule inhibitor of hydrolase ABHD16A, discovered in the same laboratory, is able to reverse elevated production of Lyso-PS in lymphoblasts from human PHARC (ABHD12-null) subjects, thus providing pharmacological evidence that ABHD16A is a major enzyme responsible for generating lysoPSs in vivo.2 Concluding, Benjamin Cravatt provided a general overview of the past and currently ongoing research efforts in his lab in proteomic profiling and ligand discovery. Kevan Shokat from the Department of Cellular and Molecular Pharmacology, University of California San Francisco provided an overview of his lab’s efforts around small molecule inhibitors of NTPases, especially those that target the mutated, oncogenic forms of these enzymes. His group has, for example, developed a new generation of mTOR inhibitors that are able to overcome cancer-related resistance mutations in this protein. Kevan Shokat’s lab has also developed allosteric inhibitors of the mutated small GTPase K-Ras.3 Somatic mutations in K-Ras represent very common activating lesions in cancer and respond poorly to standard cancer therapies. These novel allosteric inhibitors are electrophilic small molecules that bind to a cysteine that only occurs on the oncogenic mutant form of K-Ras (G12C). Upon binding to K-Ras, these molecules reverse the nucleotide preference of K-Ras(G12C) toward GDP over GTP and impair binding to effectors B-Raf and C-Raf. These inhibitors provide an excellent starting point for development of a new generation of anticancer therapeutics. Jeffrey Glenn from the Stanford School of Medicine, who works in the field of molecular virology, presented a strategy to combat Hepatitis δ Virus (HDV) infections. Interestingly, the virus requires proteins from the hepatitis B virus for its own assembly and thus can only propagate in the presence of HBV. Hepatitis D is considered the worst form of hepatitis due to its fast progression to liver cirrhosis, as well as the increased

he 2016 International Symposium on Chemical Biology was organized by the Swiss National Centre of Competence in Research (NCCR) Chemical Biology. The NCCR Chemical Biology is located at the University of Geneva (UniGE; Director: Prof. Howard Riezman) and the Ecole Polytechnique Fédérale de Lausanne (EPFL; Co-director: Prof. Kai Johnsson). The symposium was held on January 13−15, 2016 at the Campus Biotech (Figure 1), a new center for

Figure 1. Front view of the Campus Biotech facility in Geneva, Switzerland (Photo credit: Daniel Abegg).

biotechnology and life science research that was formed through a unique partnership between the University of Geneva and EPFL and is located within walking distance from the city center of Geneva. This exciting scientific venue attracted more than 240 attendees from around the world. With the aim of providing a sound overview of key challenges in cutting edge chemical biology, as well as fostering an open dialogue with networking opportunities, the organizing committee selected 15 plenary lectures with diverse scope under the broad subject of chemical biology. The scientific program was rounded up by four poster sessions with over 60 posters. The symposium started on Wednesday afternoon with Benjamin Cravatt from the Department of Chemical Physiology at The Scripps Research Institute, whose laboratory is specialized in mapping biochemical pathways in human © 2016 American Chemical Society

Published: April 15, 2016 816

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further diversity through synthesis.9 An alternative to BIOS, which Herbert Waldmann introduced, is achieved by deconstructing natural products into fragments and recombining these fragments into biologically active composites. A number of illustrative examples demonstrated how the abovementioned concepts are applied and implemented in his lab. Alanna Schepartz from the Department of Chemistry, Yale University presented how her lab approaches the fundamental question of signal transduction, i.e., the transfer of information through the cell membrane. In particular, she addressed the question of how the identity of a receptor ligand can alter downstream molecular processes. The molecular prerequisites for kinase activity of the Epidermal Growth Factor (EGF)Receptor in the respective signal transduction pathway were investigated. Importantly, EGF-Receptor mutations in the kinase domain are implicated in up to a third of non-smallcell lung cancer cases. Alanna Schepartz highlighted that mutations in the kinase domain can lead to structural changes in the cytoplasmic juxtamembrane segment (JM), a segment that links the kinase domain with the extracellular and transmembrane regions.11 Additionally, a detailed picture of the processes that occur upon binding of the different ligands (epidermal growth factors) of the receptor and the concomitant structural changes in the JM domain were elucidated in great detail.10 Ulrike Eggert from the Department of Chemistry and Randall Division of Cell and Molecular Biophysics, King’s College London provided an overview of the chemical and cell biology approaches developed in her lab to study the mechanism of cytokinesis. Cytokinesis is the last step in the cell cycle and is inherently difficult to study because it is a highly complex and dynamic process. Ulrike Eggert’s lab found that prazosin, a well-studied antihypertensive drug, also inhibits endocytic sorting via an off-target interaction with the dopamine receptor D3 (DRD3).12 Prazosin also inhibits cytokinesis, possibly through stabilizing the interaction between DRD3 and the coatomer COPI. In the second part of her talk, Ulrike Eggert presented a systematic approach for mapping lipidomic changes during the cell cycle.13 LC-MS-based lipidomics profiling identified 11 structurally distinct lipids accumulating in dividing cells. In parallel, an RNAi knockdown of lipid biosynthetic enzymes identified 23 enzymes required for cell division. This work sheds light on the so far uncovered relationship between the cellular lipidome and a fundamental cellular process such as cell division. Nathanael Gray from the Dana-Farber Cancer Institute and Harvard Medical School gave a lecture on the ever-growing field of small-molecule kinase inhibitors, including the compounds recently discovered in his own laboratory. Dysregulated kinase activities are very common in human cancer, and more than 20 kinase inhibitors have already been approved by the U.S. Food and Drug Administration for cancer treatment. For example, Imatinib, an ATP-competitive inhibitor of the Bcr-Abl oncoprotein, has revolutionized the treatment of chronic myelogenous leukemia (CML), but the disease eventually develops resistance to imatinib through expression of a drug insensitive Bcr-Abl mutant. To overcome this resistance, Nathanael Gray’s lab has identified allosteric Bcr-Abl inhibitors that target the myristate binding site of this protein.14 One promising new strategy for combating cancer involves targeting of the transcriptional machinery (or the machineryassociated enzymes) with small molecules. The Gray laboratory discovered compound THZ1 as a first-in-class, covalent,

incidence of patients developing liver cancer. Therapeutic options are limited; the standard treatment, Interferon-α, is only effective in about a quarter of the patients. Jeffrey Glenn described a strategy that targets the host prenylation machinery, which farnesylates the large delta antigen,4 a critical step that is required for virus replication. The farnesyltransferase inhibitor Lonafarnib (either alone or in combination with other antivirals such as Ritonavir and/or IF-α) showed excellent results both in a mouse model as well as in clinical phase II studies.5 Drastic reductions in viral loads were observed, when compared to the current standard of treatment, Interferon-α. Christian Eggeling from the MRC Human Immunology Unit and Wolfson Imaging Center Oxford, Weatherall Institute of Molecular Medicine University of Oxford gave some insights into the advances and challenges of fluorescence microscopy of living cells. In his talk, he focused on two of his main research topics, particularly highlighting the need, and their endeavor, to achieve high spatial resolution and high sensitivity, with a specific focus on demands put on chemistry (e.g., labeling). First, he discussed their advances in imaging T-cells, thereby understanding the organization of various molecules on the cell surface. This insight is crucial to grasping the cellular response mechanisms of the human immune system. Second, he focused on microscopic methods to analyze the lipid plasma membrane organization. In particular, the use of stimulated emission depletion microscopy in combination with fluorescence correlation microscopy (STED-FCS) has enabled the visualization, and thus analysis, of the dynamic nature of lipid rafts.6 Adam Cohen from the departments of Chemistry and Chemical Biology, and Physics, Harvard University discussed his lab’s endeavor to study the activity of a large number of neurons while minimizing the perturbation of the system. Using their genetically encoded fluorescent probes of membrane voltage, it is possible to image neurons with high spatial and temporal resolution.7 Recent work uses this technique to understand neurological diseases by the use of human induced pluripotent stem cells that can be differentiated into neurons. Introducing his membrane voltage probes into neurons derived from both healthy individuals and those bearing neurological diseases enables him to visualize differences in the firing patterns of neurons. Thus, differences in activity can be linked to a particular neurological disease. In his talk, it became apparent that this approach is not limited to neuronal networks and can be utilized to alter other cells. For instance, HEK cells can be artificially equipped with ion channels.8 Addition of the florescent membrane potential probe to these cells allows for high throughput screening of pain-altering drugs. This was tested on NaV1.7, an ion channel specific for transmitting pain signals, which was screened for inhibitors. Herbert Waldmann from the Department of Chemical Biology at the Max-Planck-Institute for Molecular Physiology gave an overview of his lab’s approach to identify and design small, natural product-like molecules that modulate the dynamics of biological processes. A common theme of this conference was, as stressed by the speaker, the importance of phenotypic screens in the development of both new drugs and chemical probes. Another important question, also addressed by the speaker, is how to reduce the complexity of natural products without losing biological activity. Biology-orientated synthesis (BIOS) uses core structures obtained from natural products as scaffolds of compound collections to access diversity around a biologically active structure. This approach harnesses the diversity obtained through evolution and creates 817

DOI: 10.1021/acschembio.6b00267 ACS Chem. Biol. 2016, 11, 816−820

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ACS Chemical Biology

product tunicamycin.21 Its toxicity has precluded the use of this natural product as an antibiotic; therefore an understanding of the molecular basis of pharmacological activity and toxicity is necessary to access tunicamycin analogs with higher potency and less toxicity. In the last part of his talk, he described a novel combinatorial approach of his lab to combat bacterial infectious diseases.22 Dirk Trauner from the Department of Chemistry at the Ludwig-Maximilians-Universität shared his latest developments in the field of photopharmacology. This highly dynamic research field is based on light-induced control of biological function via the use of synthetic photoswitches. A combination of biological insight and creative chemistry allows for the design of photoswitchable probes that alter numerous biological processes.23 Since azobenzenes are ideally suited for the construction of photoswitches, the concept of azosteres is an important starting point in the design of photoswitches. Azosteres are structural motifs in ligands or drugs that can be mimicked by azobenzene, thus enabling development of a photoswitchable version of the respective compound. This approach was used, for instance, in the synthesis of photostatin, a photoswitch used to probe microtubule dynamics.24 On the other hand, long-alkyl-chain-containing molecules (e.g., fatty acids) can be made photoswitchable.25 A photoswitchable version for capsaicin was made, and the amount of active and inactive isomer can be titrated by tuning the wavelength. Mark Bunnage from Pfizer and the University of Oxford gave an account on the rise of chemical biology in drug discovery from a “pharma” perspective. In particular, he discussed the importance of well-designed and well-characterized chemical probes for biological targets;26 a newly created community-driven platform27 should enable the sharing of crucial information about the properties, scopes, and limitations of these tools. Yet another challenge which Mark Bunnage discussed was that of choosing the “right” target, a factor ultimately linked to the success of a drug. Echoing the sentiment of many of the previous speakers, he discussed how the most challenging part of finding the right target is often target validation,28 stressing the importance of phenotypic screens. Finally, he showed some of his team’s efforts on targeting bromodomains.29 Daniel Müller from the Department of Biosystems Science and Engineering, ETH Basel presented his lab’s approach toward an understanding and quantitative characterization of fundamental cellular processes such as mitosis. In collaboration with Anthony Hyman and co-workers, Daniel Müller’s lab has found that cells generate an outward rounding force, which increases upon entering mitosis. This force is the result of an osmotic pressure, which is balanced by the contracting actomyosin cortex. Thus, cells are able to control their volume and shape by regulating the actomyosin-cortex-dependent surface tension and the osmotic gradient.30 More recently, it was found that, upon mitotic progression, myosin II increasingly accumulates at the cell cortex to increase tension and that there is a correlation between the amount of myosin at the cortex, cortex tension, and intracellular pressure. Finetuning of the myosin II concentration at the mitotic cell cortex and thereby of the mitotic cell pressure is taken by both p21activated kinases and the Rho kinase.31 The truly exceptional quality of all talks was reflected throughout lively question and answer sessions. Yet, gathering an international panel of highly recognized speakers in the field of chemical biology would not have been possible without

cysteine-reactive inhibitor of cyclin-dependent kinase 7 (CDK7).15 For instance, THZ1 showed potent activity in Tcell acute lymphoblastic leukemia (T-ALL cells) and may eventually prove useful for the treatment of many other cancer forms. David Liu from the Department of Chemistry and Chemical Biology, Harvard University shared the most recent results from his laboratory in drug discovery and protein evolution. Liu’s lab has pioneered the field of DNA-encoded small molecule libraries as a means for rapid and efficient discovery of drug candidates. Most recently, this technology has been applied to identify a physiologically active inhibitor of insulindegrading enzyme (IDE) that binds to a cavity distant from the catalytic site. It has been long speculated that inhibiting IDE, and therefore stopping endogenous insulin degradation, might help to treat type-2 diabetes. Pharmacological inhibition of IDE in mice using the newly discovered inhibitor provides compelling evidence that IDE not only regulates insulin levels but also controls the endogenous levels of glucagon and amylin.16 Using phage-assisted continuous evolution (PACE), David Liu’s lab evolves disease-relevant proteins toward improved variants. Remarkably, this technology is implementable in evolving many different classes of proteins. For example, PACE was recently applied to obtain hepatitis C virus (HCV) protease variants with up to 30-fold drug resistance in only 1 to 3 days.17 Thus, protease PACE can help in identifying mutated variants of proteins resistant to clinically relevant drugs and, thus, illustrate the possible vulnerabilities of these drugs. Priscilla Yang from the Department of Microbiology and Immunobiology, Harvard Medical School, whose laboratory is specialized in studying host−virus interactions, provided insights into new strategies for the development of inhibitors of dengue virus (DV) and hepatitis C virus (HCV) infections. Priscilla Yang’s lab has found that the well-characterized kinase inhibitors AZD0530 and dasatinib potently block the DV infection in cell culture. Using RNAi-mediated depletion and overexpression of known kinase targets of these compounds, they identified that Fyn kinase is involved in dengue virus 2 replication and, thus, is likely the major causative factor in the antiviral activity of the aforementioned kinase inhibitors.18 Furthermore, Yang and co-workers made use of LC-MS-based lipid metabolite profiling in an HCV cell culture infection model to identify steady-state accumulation of desmosterol, a direct precursor of cholesterol.19 Consequently, pharmacological inhibition or RNAi depletion of the Δ-7-sterol reductase (DHCR7), an enzyme responsible for the last step in the desmosterol biosynthesis, inhibits HCV; moreover, this antiviral effect is rescued upon the addition of exogenous desmosterol but not other sterols. Thus, desmosterol may have a unique function and HCV replication, and the enzymes responsible for desmosterol biosynthesis could represent novel pharmacological targets for a new generation of antivirals. Ben Davis from the Department of Chemistry at the University of Oxford focused on three aspects of his current research. First, he introduced a savvy method that allows selective labeling of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB).20 This method was used both as a diagnostic tool as well as a chemical probe to understand the infection modes of TB. The method harnesses the microbe’s lipidating enzyme antigen 85, which catalyzes lipidation of 1,1trehalose. In the second part of his talk, Ben Davis discussed his group’s ongoing efforts on elucidating the biosynthesis of the natural 818

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Gray, N. S. (2006) Allosteric inhibitors of Bcr-abl−dependent cell proliferation. Nat. Chem. Biol. 2, 95−102. (15) Kwiatkowski, N., Zhang, T., Rahl, P. B., Abraham, B. J., Reddy, J., Ficarro, S. B., Dastur, A., Amzallag, A., Ramaswamy, S., Tesar, B., Jenkins, C. E., Hannett, N. M., McMillin, D., Sanda, T., Sim, T., Kim, N. D., Look, T., Mitsiades, C. S., Weng, A. P., Brown, J. R., Benes, C. H., Marto, J. A., Young, R. A., and Gray, N. S. (2015) Targeting transcription regulation in cancer with a covalent CDK7 inhibitor. Nature 511, 616−620. (16) Maianti, J. P., McFedries, A., Foda, Z. H., Kleiner, R. E., Du, X. Q., Leissring, M. A., Tang, W.-J., Charron, M. J., Seeliger, M. A., Saghatelian, A., and Liu, D. R. (2015) Anti-diabetic activity of insulindegrading enzyme inhibitors mediated by multiple hormones. Nature 511, 94−98. (17) Dickinson, B. C., Packer, M. S., Badran, A. H., and Liu, D. R. (2014) A system for the continuous directed evolution of proteases rapdly reveals drug-resistance mutations. Nat. Commun. 5, 1−8. (18) de Wispelaere, M., LaCroix, A. J., and Yang, P. L. (2013) The Small Molecules AZD0530 and Dasatinib Inhibit Dengue Virus RNA Replication via Fyn Kinase. J. Virol. 87, 7367−7381. (19) Rodgers, M. A., Villareal, V. A., Schaefer, E. A., Peng, L. F., Corey, K. E., Chung, R. T., and Yang, P. L. (2012) Lipid Metabolite Profiling Identifies Desmosterol Metabolism as a New Antiviral Target for Hepatitis C Virus. J. Am. Chem. Soc. 134, 6896−6899. (20) Backus, K. M., Boshoff, H. L., Barry, C. S., Boutureira, O., Patel, M. K., D’Hooge, F. C. O., Lee, S. S., Via, L. E., Tahlan, K., Barry, C. E., III, and Davis, B. G. (2011) Uptake of unnatural trehalose analogs as a reporter for Mycobacterium tuberculosis. Nat. Chem. Biol. 7, 228−235. (21) Wyszynski, F. J., Lee, S. S., Yabe, T., Wang, H., GomezEscribano, J. P., Bibb, M. J., Lee, S. J., Davies, G. J., and Davis, B. G. (2012) Biosynthesis of the tunicamycin antibioticsproceeds via unique exo-glycal intermediates. Nat. Chem. 4, 539−546. (22) Kong, L., Vijayakrishnan, B., Kowarik, M., Park, J., Zakharova, A. N., Neiwert, L., Faridmoayer, A., and Davis, B. G. (2016) An antibacterial vaccination strategy based on a glycoconjugate containing the core lipopolysaccharide tetrasaccharide Hep. Nat. Chem. 8, 242. (23) Broichhagen, J., Frank, J. A., and Trauner, D. (2015) A Roadmap to Success in Photopharmacology. Acc. Chem. Res. 48, 1947−1960. (24) Borowiak, M., Nahaboo, W., Reynders, M., Nekolla, K., Jalinot, P., Hasserodt, J., Rehberg, M., Delattre, M., Zahler, S., Vollmar, A., Trauner, D., and Thorn-Seshold, O. (2015) Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 162, 403−411. (25) Frank, J. A., Moroni, M., Moshourab, R., Sumser, M., Lewin, G. R., and Trauner, D. (2015) Photoswitchable fatty acids enable optical control of TRPV1. Nat. Commun. 6, 1−11. (26) Arrowsmith, C. H., Audia, J. E., Austin, C., Baell, J., Bennett, J., Blagg, J., Bountra, C., Brennan, P. E., Brown, P. J., Bunnage, M. E., Buser-Doepner, C., Campbell, R. M., Carter, A. J., Cohen, P., Copeland, R. A., Cravatt, B., Dahlin, J. L., Dhanak, D., Edwards, A. M., Frederiksen, M., Frye, S. V., Gray, N., Grimshaw, C. E., Hepworth, D., Howe, T., Huber, K. V. M, Jin, J., Knapp, S., Kotz, J. D., Kruger, R. G., Lowe, D., Mader, M. M., Marsden, B., Mueller-Fahrnow, A., Müller, S., O’Hagan, R. C., Overington, J. P., Owen, D. R., Rosenberg, S. H., Ross, R., Roth, B., Schapira, M., Schreiber, S. L., Shoichet, B., Sundström, M., Superti-Furga, G., Taunton, J., Toledo-Sherman, L., Walpole, C., Walters, M. A., Willson, T. M., Workman, P., Young, R. N., and Zuercher, W. J. (2015) The promise and peril of chemical probes. Nat. Chem. Biol. 11, 536−541. (27) Chemical Probes. www.chemicalprobes.org. (28) Bunnage, M. E., Gilbert, A. M., Jones, L. H., and Hett, E. C. (2015) Know your target, know your molecule. Nat. Chem. Biol. 11, 368−372. (29) Picaud, S., Da Costa, D., Thanasopoulou, A., Filippakopoulos, P., Fish, P. V., Philpott, M., Fedorov, O., Brennan, P., Bunnage, M. E., Owen, D. R., Bradner, J. E., Taniere, P., O’Sullivan, B., Muller, S., Schwaller, J., Stankovic, T., and Knapp, S. (2013) PFI-1, a Highly

sponsors. In particular, we would like to mention BASF, F. Hoffmann-La Roche, Novartis Pharma AG, and Syngenta Crop Protection AG who generously supported this event.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected].

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ACKNOWLEDGMENTS The authors thank Phaedra Simitsek, the Organizing Committee, and the NCCR Chemical Biology for this spectacular and successful meeting.

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REFERENCES

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DOI: 10.1021/acschembio.6b00267 ACS Chem. Biol. 2016, 11, 816−820