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/0/)CBD8( Page 1: Welcome Page 2: Agenda for Thursday, November 10th: ICBS Trainee Symposium Page 4: Agenda for Friday, November 11th Page 9: Agenda for Friday, November 12th Page 16: Agenda for Friday, November 13th Page 18: Sponsors & Conference Organization Page 19: Poster Abstracts (in alphabetical order)

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Welcome to ICBS 2021: Looking towards the next decade of chemical biology To commemorate the 10th anniversary of the International Chemical Biology Society (ICBS), the 2021 annual meeting centers on the theme of looking towards the next decade of chemical biology and will both reflect on the accomplishments of the last decade as well as look towards new technologies and discoveries that will shape the field in the decades ahead.

As innovative chemistry, novel technologies, and conceptual advances in chemical biology are transforming the landscape of the life sciences and health industry, it is timely for chemical biologists from both academic and industry perspectives to highlight new discoveries and breakthroughs, discuss opportunities and challenges, and identify emerging concepts and synergy.

To maximize active interactions, ICBS2021 will feature thought leaders for keynote and plenary lectures, early career Rising Stars whose science will impact the future, focused sessions on specialized topics, a student- and postdoc-oriented Young Chemical Biologist Forum, and podium flash presentations.

Sponsors and exhibitors will be an integral parts of the conference in support of the global chemical biology community. Poster and podium presentation awards will be provided to support and recognize outstanding international trainees. Your input is welcome. We are looking forward to hosting this exciting event, a decade in the making!

Your ICBS2021 conference co-chairs,

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Wednesday, November 10th: Pre-Conference Trainee Symposium 10:00 – 11:45 AM EST: Trainee Presentation Session #1 Sponsored by Royal Society of Chemistry Journals Main Theatre Opening Remarks: Kun Qian, Emory University, ICBS-Emory, US Chair: Aylin Binici, MPI-Dortmund, ICBS-Dortmund, Germany Speakers:

• Brodie Bailey, Walter and Eliza Hall Institute of Medical Research, Australia The Development of a Novel Antimalarial Class with Slow to Moderate Erythrocytic Stage Activity

• Guoliang Zhu, ECUST, China Molecular networking- and Caenorhabditis elegans assay-assisted characterization of anti-infective acylcoprogen siderophores

• Catia Pierotti, Walter and Eliza Hall Institute of Medical Research, Australia Small molecule inhibitor of necroptosis targets multiple protein effectors to potently block cell death

• Laura Depta, Drug Discovery Hub-Dortmund, Germany Development and evaluation of a cellular model system to dissect isoform-selectivity of Akt inhibitors

• Thomas Whitmarsh-Everiss, Technical University of Denmark, Denmark Identification of inhibitors of cholesterol transport proteins through the synthesis of a diverse, sterol-inspired compound collection

• Ilana Kotliar, Rockefeller University, US Multiplexed analysis of the secretin-like GPCR-RAMP interactome by suspension bead array

12:00 – 1:00 PM EST: Career Talk + Panel Discussion Main Theatre Moderator: Shannon Miller, Harvard University & the Broad Institute, ICBS-Boston, US Panelists:

• Guillaume Garivet, Lab Team Leader in Chemical & Process Development, BASF, Germany

• Jiafeng Geng, Associate Director of Research Projects/Patent Agent, Emory University, US

• Adam Kositzke, Research Associate, Bioanalytic Department at Covance (Labcorp Drug Development), US

• Brooke Bullock Lao, Principal Scientist, Pfizer, US • Kali Miller, Research Safety Specialist, Stanford University, US

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1:00 – 2:00 PM EST: Trainee Presentation Session #2 Sponsored by Royal Society of Chemistry Journals Main Theatre Chair: Dacheng Fan, Emory University, ICBS-Emory, US Speakers:

• Joomyung V. Jun, Massachusetts Institute of Technology, US A Chemoselective Protein Modification for Traceless Cytosolic Entry

• Anneliese M. Gest, UC Berkeley, US Development of an Optical Method to Measure Membrane Potential in Neurons

• Zhi Lin, Harvard University, US A chemical proteomics platform reveals eIF3i as a direct target of lenalidomide

• Brendan Dwyer, Scripps Research Institute – Florida, US Chemoproteomic-enabled target identification of the curcusone natural products reveals new chemotherapeutic and targeted protein degradation strategies

2:10 – 3:00 PM EST: Keynote Lecture by Jason Gestwicki Protein-protein interactions at the C-termini Main Theatre Introduction: Chad Altobelli, UC San Francisco, ICBS-UCSF, US Speaker: Jason Gestwicki, UC San Francisco

Bio: Dr. Gestwicki completed undergraduate studies at SUNY Fredonia in 1997 and earned his PhD from University of Wisconsin in 2002. He then performed postdoctoral studies at Stanford University prior to starting his independent group at University of Michigan in 2005. In 2013, his group relocated to the University of California, San Francisco (UCSF), joining the Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Diseases (IND). At UCSF, he serves as the Associate Director of Academic Affairs in the IND and as Director of the Chemistry & Chemical Biology (CCB) Ph.D. program. Dr. Gestwicki is also an Associate Editor at ACS Chemical Biology and co-founder of multiple biotechnology companies. He has published 190+ manuscripts and trained 40+ graduate students and postdoctoral fellows. Abstract: To maintain protein homeostasis (proteostasis), each cell must carefully balance the decision to retain or degrade a protein. This decision is not always made correctly, as apparent in neurodegenerative diseases, where misfolded proteins accumulate in diseases neurons. Thus, there is interest in better understanding how cells maintain proteostasis, with the goal of finding ways that balance can be therapeutically restored. For each protein, the decision to “degrade” is typically driven by direct, physical protein-protein interactions (PPIs). For example, chaperone binding is used to discern whether the protein is unfolded. Likewise, PPIs between the protein, chaperones and the ubiquitin conjugation machinery are often used to drive turnover. Our goal is to understand and perturb these PPIs using a chemical biology strategy. Here, we focus on an interesting and relatively under explored category of PPIs: those that occur at N- and C-termini. The chemical environment at the ends of a polypeptide is often unique (e.g. pKa values, dynamics) and these regions can be a hotspot for post-translational modifications (PTMs). Thus, new strategies might be required to chemically manipulate terminal PPIs. Here, we will discuss progress on developing methods towards that goal, including high throughput methods for studying terminal PPIs.

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Thursday, November 11th: Main Conference 9:45 – 11:00 AM EST: Welcome, Opening Remarks + Opening Session Global advances in chemical biology Main Theatre Chair: Cheryl Arrowsmith, University of Toronto, Canada Speakers:

• Bahne Stechmann, EU-OPENSCREEN, Germany • Stephan Schürer, University of Miami, US • Yan Zhang, National Natural Science Foundation of China • Bridget Wagner, Broad Institute, US • Cheryl Arrowsmith, University of Toronto, Canada

11:00 – 11:45 AM EST: Opening Keynote Lecture by Peter Schultz Playing with the molecules of life Main Theatre Introduction: Shuibing Chen, Weill Cornell School of Medicine, US Speaker: Peter Schultz, CEO of Scripps Research, US

Bio: Peter Schultz has made pioneering contributions at the interface between chemistry and biology, notably the exploitation of molecular diversity in the synthesis of new medicines and materials, and the rational expansion of the genetic code. Schultz has demonstrated that traditional chemical tools used together with modern cellular and molecular biology methods can enable chemists to manipulate cellular machinery in amazing new ways. For example, his work has allowed us to add new building blocks to the genetic code, removing a billion-year constraint on living organisms. Schultz has also harnessed molecular diversity to create new catalysts, medicines and materials—first in reprogramming the immune system to make enzyme-like catalysts, and more recently developing and applying the use of large combinatorial libraries to find new materials and innovative drugs for aging, cancer, and infectious disease. Coauthor of over 600 scientific publications, Schultz has trained over 300 coworker and is a founder of nine companies that have pioneered the application of molecular diversity technologies to address multiple challenges in human health and materials science. He is a Professor of Chemistry at Scripps Research as well as its President and CEO. Abstract: Our research program combines the tools and principles of chemistry with the molecules and processes of living cells to synthesize new molecules and molecular assemblies with novel physical, chemical and biological functions. By studying the structure and function of the resulting molecules, new insights can be gained into the mechanisms of complex biological and chemical systems. Examples of this synergistic chemical/biological approach to synthesis will be discussed including (1) the addition of amino acids with novel biological, chemical and physical properties to the genetic codes of prokaryotic and eukaryotic organisms, (2) recapitulating the evolution of mitochondria in a synthetic eukaryotic system, (3) characterizing organisms with chimeric RNA-DNA genomes, and (4) and the identification of small molecules that control stem cell fate in vivo.

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11:45 AM – 12:45 PM EST: Concurrent Session #1 Bioengineering commodities of the future Theatre A Chairs:

• Bauke Albada, Wageningen University, Netherlands • Abhishek Chatterjee, Boston College, US

Speakers: • Michelle Chang, UC Berkeley, US

Hybrid biocatalysis for engineered olefin production from glucose • Pamela Peralta-Yahya, Georgia Institute of Technology, US

Bioconversion of CO2 to value-added chemicals • Michael Jewett, Northwestern University, US

Bioengineering beyond cells: repurposing ribosomes for novel polymers • Angela Steinauer, ETH Zurich, Switzerland

A bacterial protein cage goes viral 11:45 AM – 12:45 PM EST: Concurrent Session #2 Versatile tools for the chemistry-biology interface Theatre B Chairs:

• Peng Chen, Peking University, China • Jaisankar Parasuraman, CSIR-Indian Institute of Chemical Biology, India

Speakers: • Andrea Rentmeister, University of Muenster, Germany

Enzymatic photocaging of nucleic acids: A strategy for controlling methyltransferase target sites by light

• Guifang Jia, Peking University, China Epitranscriptome engineering boosts crop productivity

• Anneliese Gest, UC Berkeley, US Optical determination of neuronal membrane potential using fluorescence lifetime imaging microscopy

• Hankum Park, Seoul National University, Korea Early endosome proteomics and its application to spatial snapshots of APP intramembrane processing

• Himadri Sekhar Sarkar, CSIR-Indian Institute of Chemical Biology, India Development of a new chemical biology tool enabling reversible optical control of protein labeling

12:45 – 1:45 PM EST: Industrial contributions to advancing chemical biology Main Theatre Chair: Douglas Auld, Novartis, US Speakers:

• Paul Brennan, University of Oxford, UK Chemical Probes in Target Discovery

• Jeremy Jenkins, Novartis, US Creation of a sustainable MoA library through augmented intelligence

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• Frances Rodriguez-Rivera, Merck, US Small molecule photocatalysis enables drug target identification via energy transfer

1:45 – 2:45 PM EST: Concurrent Session #3 Antimicrobial discovery and resistance: Tackling an upcoming health crisis Theatre A Chairs:

• Claudia Jessen-Trefzer, University of Freiburg, Germany • Stephan Hacker, Leiden University

Speakers: • Anna Hirsch, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS),

Germany Discovery of Submicromolar Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa using Rational Design

• David Hilko, Griffith University, Australia In search of better nucleotide chemical probes trhough improved synthetic chemistry

• Yaojun Tong, Shanghai Jiao Tong University, China CRISPR meets actinomycetes, a new era of natural products discovery is coming

• Snehlata Saini, IISER - Bhopal, India Inhibition of bacterial signal recognition particle system by antisense PNA: A novel antibacterial strategy

• Chris Meier, University of Hamburg, Germany Development of broad spectrum antivirals against emerging infections targeting a host cell enzyme

1:45 – 2:45 PM EST: Concurrent Session #4 Drug screening during a new pandemic: Meeting the balance between drug repurposing and novel viral biology Theatre B Chairs:

• Arnab Chatterjee, Scripps Research Institute, US • Shuibing Chen, Cornell University, US

Speakers: • Sumit Chanda, Sanford Burnham Prebys, US

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals • Matthew Friedman, University of Maryland School of Medicine, Center for Pathogen

Research, US Therapeutic Development for SARS-CoV-2

• Johan Neyts, KU Leuven, Belgium Towards potent drugs for the treatment and prophylaxis of SARS-CoV2 infections

• Samuel Constant, Epithelix, Switzerland 3D human airway epithelial models to study SARS-CoV-2 pathogenesis

• Sara Cherry, University of Pennsylvania, US COVID-19: Antiviral discovery pipeline

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2:45 – 3:30 PM EST: Keynote Lecture by Deb Hung Chemical biological investigations of infection Main Theatre Introduction: Laura Kiessling, Massachusetts Institute of Technology, US Speaker: Deb Hung, Harvard University, US

Bio: Deborah Hung is a physician-scientist combining chemical and genomic approaches to define host-pathogen interactions and to reveal the critical pressure points of infectious disease. By deploying small organic molecules on a genome-wide scale to both perturb and understand bacterial infection, she hopes to identify new therapeutic prospects for a variety of devastating pathogens, including Vibrio cholerae, Pseudomonas aeruginosa and Mycobacterium tuberculosis. Deborah received her Ph.D. in chemistry from Harvard University, where she worked in Stuart Schreiber’s laboratory to characterize the chemical and biological properties of discodermolide, a small molecule isolated from marine sponges that stabilizes microtubules. More recently, she pursued her postdoctoral research in the laboratory of John Mekalanos at Harvard Medical School, using a high-throughput chemical screen to identify a small molecule that inhibits two major virulence factors of Vibrio cholerae, a gram-negative bacterium that causes an acute intestinal diarrhea. When given orally, the inhibitor can protect mice from the effects of V. cholerae infection. Although cholera outbreaks are relatively rare in the United States, the disease is epidemic in many non-industrialized countries where water sanitation is poor. Deborah received her medical degree from Harvard Medical School and completed a residency in internal medicine and fellowships in infectious disease and critical care medicine at Brigham and Women’s Hospital and Massachusetts General Hospital. Currently, she holds positions as an infectious disease physician at Brigham and Women’s Hospital and Massachusetts General Hospital and an attending critical care physician in the Medical Intensive Care Unit at Brigham and Women’s Hospital.

3:30 – 4:00 PM EST: ICBS Business Meeting (OPEN TO ALL) Main Theatre Chairs:

• Haian Fu, Chair, ICBS Board of Directors • Jonathan Baell, ICBS President • Zaneta Nikolovska-Coleska, ICBS President Elect

4:00 – 6:00 PM: Networking + Professional Development Networking Lounge

• 4 – 5: Live Poster Sessions, Sponsored by ChemBridge • 4 – 5: Meet the Editors: Cell Chemical Biology • 5 – 6: Meet the Editors: Royal Society of Chemistry Chemical Biology, Organic &

Biomolecular Chemistry, and Medicinal Chemistry • 5 – 6: Meet the Editors: ACS Medicinal Chemistry, Chemical Biology, and Biochemistry • 4 – 6: Collaborative Drug Discovery (CDD): Exhibitor Booth + Live Demonstrations • 4 – 6: Networking Social

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8:00 – 9:45 PM EST: Breakthrough Chemical Biology in the Asia/Oceania Region Main Theatre Opening Remarks: Jonathan Baell, Monash University, Australia Chairs:

• Yan-Mei Li, Tsinghua University, China • Mikiko Sodeoka, RIKEN, Japan

Speakers: • Feng Shao, National Institute of Biological Sciences, China

Pyroptosis in antibacterial & antitumor immunity • Rebecca Ritchie, Monash University, Australia

Probing biased formyl peptide receptor agonism with small molecules to limit myocardial ischaemic injury

• Hyun-Woo Rhee, Seoul National University, Korea In vivo mitochondrial matrix proteome profiling reveals an antioxidant NADPH oxidoreductase in the muscle tissues

• Junko Ohkanda, Shinshu University, Japan Fusicoccin: A chemical modulator for 14-3-3 interactions

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Friday, November 12th

10:00 – 10:45 AM EST: Keynote Lecture by Dame Carol Robinson New roles for mass spectrometry in chemical biology Main Theatre Introduction: Sally-Ann Poulsen, Griffith University, Australia Speaker: Dame Carol Robinson, University of Oxford, UK

Bio: Carol holds the Oxford Chair of Dr. Lee’s Professor of Chemistry and is the first Director of the Kavli Institute for Nanoscience Discovery at the University of Oxford. She is recognised for using mass spectrometry to further research into the 3D structure of proteins and their complexes. Her current interest is in uncovering the synergy of lipid and drug binding. With this information she is exploring new ways to characterise receptor-signalling complexes. Carol’s graduate education was completed whilst working full-time in industry. She was subsequently admitted to the University of Cambridge where she completed her PhD in two years. Following an eight-year career break to begin raising her three children, she returned to research at Oxford. In 2001 she became the first female Professor of Chemistry at the University of Cambridge, returning to Oxford in 2009 to take up the Chair of Dr. Lee’s Professor of Chemistry. Her research has attracted numerous international awards and prizes. Her distinctions include International Honorary Member of the American Academy of Arts and Sciences (2021), Foreign Associate of the US National Academy of Sciences (2017), Dame Commander of the Order of the British Empire (DBE) in 2013 for her contributions to Science and Industry and she is also a former President of the UK Royal Society of Chemistry.

Abstract: Scientists have used mass spectrometry for more than a century to separate and detect gaseous ions of small molecules leading to historic applications in elucidating the structure of natural products. With the advent of electrospray came the ability to study intact proteins and subsequently proteins in their folded states with non-covalent interactions preserved. Recent breakthroughs allow this technology to be applied to both soluble and membrane protein complexes and allow us to probe small molecule binding to multiprotein targets. For membrane proteins assemblies, released directly into the gas phase of the mass spectrometer from micelles or lipid-based solubilisation vehicles, we are able to capture small molecule binding. I will illustrate these capabilities with a range of receptors and transporters including GPCRs and ABC transporters. In my lecture I will also demonstrate how we can exploit new mass spectrometry approaches to understand protein interactions from lipidic environments. These experiments are allowing us to focus on the numerous roles played by lipids in governing interactions and in stabilising different conformations of membrane proteins. Understanding the interplay between lipids, proteins and drugs is not only a fascinating line of research but will, I believe, provide vital information for next generation therapeutics.

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10:45 – 11:45 AM EST: Concurrent Session #5 Chemical biology approaches to unravel cell death pathways Theatre A Chairs:

• Kamyar Hadian, Helmholtz-Muenchen, Germany • Zaneta Nikolovska-Coleska, University of Michigan, US

Speakers: • Brent Stockwell, Columbia University, US

Ferroptosis: mechanisms and therapeutic applications • Stephen Tait, University of Glasgow, UK

Targeting caspase activity to engage anti-tumour immunity • Yilong Zou, Westlake University, China

Targeting lipid metabolism and ferroptosis in cancer • Catia Pierotti, The Walter and Eliza Hall Institute of Medical Research (WEHI),

Australia Small molecule inhibitor of necroptosis targets multiple protein effectors to potently block cell death

• A. Nikolai von Krusenstiern, Columbia University, US Lipid peroxidation in the endoplasmic reticulum drives ferroptosis

10:45 – 11:45 AM EST: Concurrent Session #6 The power of phenotypic screening to uncover new cellular targets Theatre B Chair: Bridget Wagner, Broad Institute, US Speakers:

• Jason Sello, UC San Francisco, US Using small molecules to to restore drug susceptibility in pathogenic Candida species

• Ellen Berg, CSO, Eurofins, US Phenotypic screening and the future of drug discovery

• Jeremy Baskin, Cornell University, US Click Chemistry-Enabled CRISPR Screening Reveals GSK3 as a Regulator of Phospholipase D Signaling

• Douglas Selinger, CEO, Plex Research, Inc., US Plex: A novel form of artificial intelligence (AI) for the identification of compound targets and mechanism of action

• Laura Doherty, Harvard Medical School & Broad Institute, US Integrating multi-omics phenotypic screens reveals function and therapeutic potential of deubiquitinating enzymes

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11:45 AM – 12:30 PM EST: ICBS Global Lectureship Award & Presentation Main Theatre Chair: Jin Zhang, UC San Diego, US 2021 ICBS Global Lectureship Awardee: Yimon Aye, Ecole Polytechnique Federale de Lausanne – EPFL What we do in the dark: Illuminating signaling functions of electrophile-sensing proteins that evade conventional target ID

Bio: Yimon Aye read chemistry at Oxford University (2000-2004), UK, and received her PhD in organic chemistry in 2009 from Harvard University, USA, with Prof. David Evans. Taking a sharp switch in research discipline she joined Prof. JoAnne Stubbe’s laboratory at MIT, USA, as a Damon Runyon cancer research postdoctoral fellow to study anticancer drugs’ mode-of-action. Since mid-2012, she has been leading her independent laboratory, formerly at Cornell University, USA, and presently at Swiss Federal Institute of Technology Lausanne (EPFL), Switzerland. Science in the Aye lab (https://leago.epfl.ch) strives to understand various non-canonical cellular signaling processes. Her lab is most well-known for investigating electrophile signaling, a nuanced communication mode whereby on-target engagement between specific reactive metabolites and target proteins, orchestrates functional biological responses at cellular and organismal levels. Their parallel contributions include research into nucleotide signaling pathways regulated by ligand-driven changes in protein–protein associations of importance in genome regulation. Abstract: Defining the precise biological impacts of localized reactive electrophilic metabolites and related small-molecule drugs has proven to be highly challenging. Traditional approaches to study such non-enzymatic modifications had relied on in vitro work and/or swamping cells with excess electrophiles. However, these conditions often do not recapitulate localized signaling behaviors, and teasing apart on- vs. off-target responses, against issues such as toxicity, metabolic conversion, etc., remains complex. Latest technologies thus pursued target profiling; yet, a major question mark remained as to how important modifications of these protein-hits are, in terms of measured phenotypic or pharmacological output. My laboratory proposed that for a protein to sense and respond to an electrophile often manifesting short half-life in cells, the protein should have elevated reactivity for that electrophile. We thus devised a strategy, later termed T-REX, to present in close proximity such proteins with a native, unfettered electrophile, and assay (1) to what extent the protein reacts with this freely-diffusible electrophile; and (2) should some degree of labeling occur, to what extent such substoichiometric modification is sufficient to elicit a functional signaling ouput. Over the past 9 years, T-REX, and a related development, G-REX electrophile-responsivity profiling in living systems, have enabled us to identify bona fide first responders that interact with native signaling electrophiles under close to endogenous redox signaling conditions (i.e., “kcat/Km”-like). Our data show that these first responders lie at nexuses between electrophile- and canonical-signaling pathways. Thus, these proteins translate information encoded by electrophiles to, for instance, phosphate or ubiquitin to reroute signaling pathway flux often conserved across evolution, even at the organismal level. We have also shown recently that the electrophile-sensing characteristics that are unique to native electrophiles can be transferred to custom-designed inhibitors.

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12:30 – 1:30 PM EST: Concurrent Session #7 Carbohydrate chemistry: From new methodologies to applications in chemical glycobiology Theatre A Chairs:

• Carmen Galan, University of Bristol, UK • Boris Vauzeilles, CNRS-ICSN, France

Speakers: • Sabine Flitsch, University of Manchester, UK

Applications of biocatalysis in glycoconjugate synthesis • Christophe Biot, Universite de Lille, France

Such a longing: shining sugars • Cristina De Castro, Università degli Studi di Napoli Federico II, Italy

Viral glycosylation according to a giant (virus) perspective • Koichi Fukase, Osaka University, Japan

Structural and synthetic studies on lipopolysacharides and lipid A from gut-associated lymphoid-tissue-resident Alcaligenes faecalis, and their application as safe mucosal immunoadjuvants

12:30 – 1:30 PM EST: Concurrent Session #8 Chemical proteomics in health and disease Theatre B Chairs:

• Ray Moellering, University of Chicago, US • Christina M. Woo, Harvard University, US

Speakers • Christopher Parker, Scripps Research Institute, US

Proteome-wide ligand and target discovery in cells • Peng Zou, Peking University, China

Spatiotemporally resolved subcellular protein post-translational modifications profiling • Tomonori Tamura, Kyoto University, Japan

Ligand-directed NASA chemistry for rapid protein labeling and covalent inhibition

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1:30 – 2:15 PM: Keynote Lecture by Dan Nomura Reimagining druggability using chemoproteomic platforms Main Theatre Introduction: Huw Davies, Emory University, US Speaker: Dan Nomura, UC Berkeley, US

Bio: Dan Nomura is a Professor of Chemical Biology in the Departments of Chemistry, Molecular and Cell Biology, and Nutritional Sciences and Toxicology at the University of California, Berkeley and an Adjunct Professor in the Department of Pharmaceutical Chemistry at UCSF. Since 2017, he has also been the Director of the Novartis-Berkeley Center for Proteomics and Chemistry Technologies focused on using chemoproteomic platforms to tackle the undruggable proteome. He is also Co-Founder and Head of the Scientific Advisory Board of Frontier Medicines. He earned his B.A. in Molecular and Cell Biology and Ph.D. in Molecular Toxicology at UC Berkeley with Professor John Casida and was a postdoctoral fellow at Scripps Research with Professor Ben Cravatt before returning to Berkeley as a faculty member in 2011. Among his honors are selection as a Searle Scholar, American Cancer Society Research Scholar Award, the Department of Defense Breakthroughs Award, Eicosanoid Research Foundation Young Investigator Award, and the Mark Foundation for Cancer Research ASPIRE award. Abstract: The Nomura Research Group is focused on reimagining druggability using chemoproteomic platforms to develop transformative medicines. One of the greatest challenges that we face in discovering new disease therapies is that most proteins are considered “undruggable,” in that most proteins do not possess known binding pockets or “ligandable hotspots” that small-molecules can bind to modulate protein function. Our research group addresses this challenge by advancing and applying chemoproteomic platforms to discover and pharmacologically target unique and novel ligandable hotspots for disease therapy. We currently have three major research directions. Our first major focus is on developing and applying chemoproteomics-enabled covalent ligand discovery approaches to rapidly discover small-molecule therapeutic leads that target unique and novel ligandable hotspots for undruggable protein targets and pathways. Our second research area focuses on using chemoproteomic platforms to expand the scope of targeted protein degradation technologies. Our third research area focuses on using chemoproteomics-enabled covalent ligand discovery platforms to develop new induced proximity-based therapeutic modalities. Collectively, our lab is focused on developing next-generation transformative medicines through pioneering innovative chemical technologies to overcome challenges in drug discovery. 2:15 – 3:15 PM EST: Concurrent Session #9 Next generation drug targets for cancer therapy In partnership with the European Federation for Medicinal Chemistry (EFMC) Theatre A Chair: Rui Moreira, Universidade de Lisboa, Portugal Speakers:

• Yimon Aye, Ecole Polytechnique Federale de Lausanne (EPFL), Switzerland Leveraging precision reactive metabolite signaling toward precision lead discovery

• Tiago Rodrigues, Universidade de Lisboa, Portugal From machine learned drug-target correlations to translation

• Stephan Hacker, Leiden University, Netherlands Covalent Inhibitors for the Proteome-wide Identification of New Druggable Targets for Antibiotics

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2:15 – 3:15 PM EST: Concurrent Session #10 Hijacking and targeting the ubiquitin system Theatre B Chairs:

• Stefan Knapp, University of Frankfurt, Germany • Xiangbing Qi, National Institute of Biological Sciences, China

Speakers: • Monique Mulder, Leiden University, Netherlands

Probing into the ubiquitin machinery • Ting Han, National Institute of Biological Sciences, China

Molecular glue discovery: Serendipity or Inevitability? • Georg Winter, CeMM - Austrian Academy of Science, Austria

Chemical genetics to identify and characterize small-molecule degraders • Xiulei Mo, Emory University, US

Discovery of hypomorph mutation-directed small molecule protein-protein interaction inducers/molecular glues

3:15 – 3:45 PM EST: Reflecting on the first decade of ICBS Main Theatre Introduction: Haian Fu, Emory University, US Speaker: Christopher Austin, Flagship Pioneering, US

Bio: Christopher Austin is a CEO-Partner at Flagship Pioneering in Cambridge, MA. In that role, he serves as CEO of one of Flagship’s franchise companies and advises on the operation and creation of other Flagship entities. Before joining Flagship in 2021, Dr. Austin served for almost a decade as the founding director of the National Center for Advancing Translational Sciences at the NIH, where he formulated the strategic vision and scientific directions of the new center, and led its efforts in developing, demonstrating, and disseminating scientific and operational advances across the spectrum of translational science to get more treatments to more patients more quickly, from target validation to preclinical therapeutic development to clinical trials to community health implementation. Before NCATS, Austin founded and directed a number of scientific and technology initiatives at the National Human Genome Research Institute at NIH to derive biological insights and therapeutic potential from the human genome. Austin came to NIH in 2002 from Merck, where his work focused on genome-based discovery of novel targets and drugs, with a particular focus on common complex neuropsychiatric diseases. He received his A.B. in biology from Princeton and M.D. from Harvard Medical School, did clinical training in internal medicine and neurology at Massachusetts General Hospital, and completed a research fellowship in genetics at Harvard.

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4:00 – 6:00 PM: Networking + Professional Development Networking Lounge

• 4 – 5: Meet the Editors: Royal Society of Chemistry Chemical Biology, Organic & Biomolecular Chemistry, and Medicinal Chemistry

• 4 – 5: Meet the Editors: Cell Chemical Biology • 5 – 6: Live Poster Sessions, Sponsored by ChemBridge • 5 – 6: Meet the Editors: ACS Medicinal Chemistry, Chemical Biology and Biochemistry • 4 – 6: Collaborative Drug Discovery (CDD): Exhibitor Booth + Live Demonstrations • 4 – 6: Networking Social

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Saturday, November 13th 10:00 – 10:45 AM EST: Keynote Lecture by Dan Yang Small molecule-based probes for molecular imaging, diagnostics, and medicine Main Theatre Introduction: Hiroyuki Osada, RIKEN, Japan Speaker: Dan Yang, Westlake University, China

Bio: Dan Yang received her B.Sc. degree in chemistry from Fudan University in 1985. Through the U.S.-China Chemistry Graduate Program, she obtained an M.A. from Columbia in 1988 under the direction of Professor Ronald Breslow. She then joined Professor Daniel Kahne’s group at Princeton and earned her Ph.D. in 1991. In the same year, she won a postdoctoral fellowship award from the Cancer Research Institute in New York to support her two-year research in Professor Stuart Schreiber’s group at Harvard. She started her independent career on organic chemistry and chemical biology at the University of Hong Kong in 1993 and was later promoted to a chair professor of chemistry and the Morningside Professor in Chemical Biology. In August 2021, Prof. Yang joined Westlake University as a chair professor in both School of Life Sciences and School of Science. Abstract: In this lecture, our recent progress on using small molecule-based molecular probes for cell biology will be presented in the following aspects: (1) developing fluorescent and chemiluminescent probes for highly selective and sensitive detection of individual reactive oxygen species in cells tissues and animals; (2) developing highly selective fluorescent probes for cell imaging of lipids, enzymes, proteins, nucleic acids and chromatins. Those molecular probes are powerful toolbox for investigating intracellular redox signaling pathways, cell growth and death processes and can be applied to diagnostics, high-throughput screening and drug discovery. 10:45 – 11:45 AM EST: Rising Stars Session Sponsored by the American Chemical Society (ACS) family of Journals Main Theatre Introduction: Haian Fu (ICBS), Emory University, US Chairs:

• Peng Chen (ACS), Peking University, China • Laura Kiessling (ACS), Massachusetts Institute of Technology, US

2021 ICBS Young Chemical Biologist Award Winners: • Leslie Aldrich, University of Illinois at Chicago, US

Chasing Challenging Targets: Development of selective inhibitors of the autophagy pathway to impact cancer therapy

• Jeremy Baskin, Cornell University, US Chemical Tools that IMPACT Lipid Signaling

• Nir London, Weizmann Institute of Science, Israel Covalent Ligand Directed Release Chemistry

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11:45 AM – 1:00 PM EST: Future Forum Looking towards the next decade of chemical biology Main Theatre Moderators:

• Milka Kostic, Dana Farber Cancer Institute, US • Will Pomerantz, University of Minnesota, US

Panelists: • Gonçalo Bernardes, Professor, University of Cambridge, UK and Group Leader,

Instituto de Medicina Molecular, Lisbon, Portugal • Jay Bradner, President of the Novartis Institute for Biomedical Research (NIBR), US • Masatoshi Hagiwara, Professor and Chair, Kyoto University, Japan • Laura Kiessling, Professor, Massachusetts Institute of Technology, US • Yamuna Krishnan, Professor, University of Chicago, US • Zaneta Nikolovska-Coleska, President Elect of the ICBS, Professor, University of

Michigan, US 1:00 – 1:10 PM EST: Closing Remarks Main Theatre

• Zaneta Nikolovska-Coleska, President Elect of the ICBS, University of Michigan, US

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Thank you to our sponsors!

Conference Organization & Leadership

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Identification and characterization of small-molecule inhibitors targeting

Pregnancy-Associated Plasma Protein-A

Ejaz Ahmad1, Ahmed S. A. Mady1, Yuting Yang1, Nurul Ansari1, Vidhya Premkumar1,

Ahmed Mostafa1, Richard Miller1,2, David Lombard1,2,3, Zaneta Nikolovska-Coleska1,3

1Department of Pathology, University of Michigan, Ann Arbor, MI 48109 USA 2University of Michigan Geriatrics Center, Ann Arbor, MI 48109 USA 3Rogel Cancer Center, University of Michigan Medical School, Ann Arbor, MI 48109 USA ahmadej@umich.edu; ejazaaa@gmail.com

Pregnancy-associated plasma protein-A (PAPP-A) is an extracellular circulating and membrane-associated pappalysin metalloprotease from metzincin superfamily. PAPP-A is a key regulator of IGF bioavailability by specific cleavage of insulin like growth factor binding proteins (IGFBPs), leading to increase concentration of free IGF and initiating IGF signaling pathway. PAPP-A knockout (KO) mice have extended lifespan by ~20-40% and they are protected from age-associated pathologies, including cardiomyopathy, atherosclerosis, nephropathy, neurodegeneration and cancers. These findings are validating PAPP-A as an attractive target for pharmacological interventions for age-associated pathologies.

Despite the attractiveness of PAPP-A as an anti-aging target, no small molecule PAPP-A inhibitors have yet been described. Applying a high throughput screening (HTS) strategy several classes of PAPP-A inhibitors were identified and validated. To measure PAPP-A’s proteolytic activity and discover inhibitors, we developed and optimized homogeneous 384-well plate format fluorescence quenching-based biochemical assay (Z’ factor of 0.83) using peptide substrate based on the IGFBP-4. Compounds with demonstrated dose-dependent inhibition were clustered for diversity and analyzed by integrating number of filters to provide small-molecules with drug-like properties. Selected compounds were purchased for further validation by integrating several orthogonal biochemical and functional assays, developed to support hit confirmation. Identified hit compounds inhibited PAPP-A with IC50 values ranging from 0.2 to 150 µM in fluorescence quenching-assay. These results were further confirmed with secondary immunoblot assay and probing for cleaved products, demonstrating that compounds can inhibit PAPP-A-mediated cleavage of full-length IGFBPs proteins, including IGFBP-4 and IGFBP-5. Performing classical steady-state mechanism of action studies, as well as testing the IC50 dependence on the concentration of the substrate, it was determined that the validated hits displayed uncompetitive mechanisms of PAPP-A inhibition. Initial functional characterization was focused on evaluation of the ability of PAPP-A inhibitors to modulate PAPP-A dependent phenotypes in cells, in particular reducing IGF1R phosphorylation. To assess whether identified hits could block IGF signaling, we measured phosphorylation of IGF1R in HEK293 cells upon stimulation with IGF1 in the presence of IGFBP-5 and PAPP-A. As expected, validated compounds blocked IGF1 signaling in HEK293 cells in a dose-dependent fashion, as measured by phosphorylation of IGF1R.

Using high throughput screening, we identified and validated several classes of small-molecule PAPP-A inhibitors. Currently we are performing structure-activity relationship studies with the most promising compounds towards developing chemical tools for pharmacological in vitro and in vivo PAPP-A inhibition and studying its functional role in aging-associated diseases.

Abstract for Poster Presentation

Monitoring of O-GlcNacylated Proteins in Living Cells

Through the Use of Non Canonical Amino Acids

Christer ABOU ANNY1, Isabelle HUVENT1, Corentin SPRIET1, Régis FAURE3, Sébastien NOUAILLE3, Stéphanie Olivier-Van Stichelen2, Christophe BIOT1, Tony LEFEBVRE1

1 CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionelle, Lille, France. 2 Department of Biochemistry, Medical College of Wisconsin (MCW), Milwaukee, Wisconsin, 53226, USA. 3 Toulouse Biotechnology Institute-Bio & Chemical Engineering (TBI), INSA, Toulouse, France. *Contact: christer.abouanny@univ-lille.fr The O-GlcNAcylation is a post-translational modification (PTM) of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) which is regulated by a unique couple of enzymes. O-GlcNAc transferase (OGT) transfers the GlcNAc residue from UDP-GlcNAc, the final product of the hexosamine biosynthetic pathway (HBP), whereas O-GlcNAcase (OGA) removes it. Visualizing the O-GlcNAcylation on a given protein in living cells is therefore essential to understand its functions in a complex biological system as a whole. Thus, we propose a novel approach to generate trackable PTM-specific proteins in space and time to study their behaviors in various physio-pathological contexts. Thanks to the advent of synthetic biology, this strategy is a powerful and promising technology to build tailored proteins and it will be achieved by incorporating an unnatural amino acid known as phenylselenocysteine (by genetic code expansion) into a specific site of the target protein. In this approach, we will use the β-catenin and OTX-2 as proteins models and track their O-GlcNAcylation and phosphorylation statuses in living cells. This design would allow the study of O-GlcNAcylation at residues Serine 23 and Threonine 41 of β-catenin and phosphorylation at Threonine 41. Serine 23 and Threonine 41 were shown to actively participate in β-catenin susceptibility to the proteasome. This innovative approach will make it possible to track the protein carrying non-hydrolyzable modifications, S-GlcNAc or C-phosphonate (instead of the highly labile O-GlcNAc and O-phosphate moieties). Each protein form will exhibit a specific fluorescence and will be tracked in living cells in response to various chemicals or nutrients. This approach combining protein engineering and chemobiology will provide valuable knowledge on the behavior of proteins in various cellular contexts and will open the field to a wide range of studies in many scientific fields.

Chemical endocannabinoid-derived probes identified a potential regulating role for peroxiredoxins and small GTPAses

Ian de Busa,b, Michiel Balversb, Renger Witkampb, Han Zuilhofa, Bauke Albada*a

a Laboratory of Organic Chemistry, b Laboratory of Human Nutrition and Health Wageningen University, Stippeneng 4, 6708 WE, the Netherlands.

* bauke.albada@wur.nl The poly-unsaturated fatty acid (PUFA)-derivative docosahexaenoyl ethanolamide (DHEA) possess immunoregulatory and anti-inflammatory effects. Although previous studies revealed some of their molecular targets, much of their receptor interactions and metabolism remains unknown. Chemical bi-functional probes containing an UV-activatable diazirine and a terminal alkyne were used to selectively target and identify novel protein targets. We synthesized bi-functional endocannabinoid probes to selectively identify new protein interaction targets of DHEA in LPS-stimulated RAW264.7 macrophages. This proteomic methodology revealed interactions between DHEA and COX-2, PRDXs, and small GTPases, which was supported by confocal fluorescent microscopy studies. Previous work by us demonstrated that COX-2 is indeed able to metabolize DHEA, resulting in the formation of 13-HDHEA and 16-HDHEA. To further understand the immune modulating potential of this newly discovered DHEA metabolic route, we analyzed the effects of 13-HDHEA and 16-HDHEA on the synthesis of immune markers in incubated LPS-stimulated RAW264.7 macrophages. A combination of ELISAs, mass spectrometry, and transcriptomic analysis revealed that 13-HDHEA and 16-HDHEA have immune regulating properties which were less pronounced when compared to their parent DHEA. In conclusion, this study revealed novel targets and metabolites of DHEA, and directions for future studies are defined to understand the full immunological potential of these interactions.

The Development of a Novel Antimalarial Class with Slow to Moderate Erythrocytic Stage Activity

Brodie Bailey1,2*, Dr Brad Sleebs1, Prof. Alan Cowman1, Dr William Nguyen1, and Dr Paul Jackson3

1 Department of Chemical Biology, Walter and Eliza Hall Institute of Medical Sciences, VIC, Australia, 2 Department of Medical Biology, University of Melbourne, VIC, Australia,

3Janssen Pharmaceuticals, California, USA.

* = presenting author

Malaria has long been heralded a “preventable and curable disease.” However, parasite resistance against all currently available antimalarials, including the first-line treatment Artemisinin combination therapy (ACT), threatens our efforts to control the disease. An urgent need has arisen towards the development of antimalarials with novel mechanisms of action.

In collaboration with Janssen Pharmaceuticals and Medicines for Malaria Venture, a high-throughput screen was undertaken against the asexual blood stage of Plasmodium falciparum, identifying several novel antimalarial classes. One of these series is the focus of the present studies and is mediated by an unknown mechanism of action. Medicinal chemistry optimisation has generated potent nanomolar inhibitors which have been used to characterise the series’ activity in parasites phenotypically.1 The series was identified to act with a slow to moderate rate of kill and are equipotent in P. falciparum multidrug resistant strains. Mechanistic studies using a range of a chemoproteomics and chemogenomic methods are currently underway in the hope of identifying a novel P. falciparum therapeutic target. 1. Bailey, B. L.; Nguyen, W.; Ngo, A.; Goodman, C. D.; Gancheva, M. R.; Favuzza, P.; Sanz, L. M.; Gamo, F.-J.; Lowes, K. N.; McFadden, G. I.; Wilson, D. W.; Laleu, B.; Brand, S.; Jackson, P. F.; Cowman, A. F.; Sleebs, B. E. Optimisation of 2-(N-phenyl carboxamide) triazolopyrimidine antimalarials with moderate to slow acting erythrocytic stage activity. Bioorganic Chemistry 2021, 105244.

Development of Selective Class I Protein Arginine Methyltransferase Inhibitors Through Fragment-based Drug Design Approach

Debomita Bhattacharya,†Alice Li,‡Barnali Paul,†MasoudVedadi,‡Arindam Talukdar†*

†Department of Organic and Medicinal Chemistry, CSIR-IICB, 4, Raja S.C. Mullick Road, Kolkata - 700032, India ‡Structural Genomics Consortium, Canada, MaRS South Tower, College Street Toronto, ON M5G 1L7

Email id: debomita1992@gmail.com

Methyltransferases areenzymes regulating epigenetic traits of multicellular organisms bycatalyzing the methylationof specific lysine and arginine residues which controls chromatin compaction, cellular differentiation and repress or activate transcription. Two classes of enzyme, protein arginine methyltransferase (PRMTs) and protein lysine methyltransferase (PKMTs) catalyze the transfer of methyl group from the cofactor S-adenosyl- L-methione (SAM) to substrate peptides.Some aberrant methylation by methyltransferases may cause diseases like cancer, diabetes, muscular disorders, neurodegenerative diseases, inflammatory disorders etc. Unlike genetic mutation, epigenetic mutations are reversible in nature.Thus one novel and promising therapeutic approach consists of targeting theseaberrantly expressedepigenetic proteins. Several potent small molecule fragment inhibitors of Class I PRMTs are present in the literature containing flexible alkyl amino side chain, thought to be a possible arginine mimic (Fig 1).1These fragments were found to be pan inhibitors of Class I PRMT, as these PRMTs share moderate to high similarity in sequence. Herein, we report rational design developed potent Class I PRMT inhibitors with good potency and selectivity by applying fragment-based drug design approach.

Structure-based ligand optimization was performed by strategic incorporation of fragment hits on the drug-like quinazoline core and subsequently fragment growth was done in desired orientation.Initially, to understand appropriate orientation for fragment growth we overlapped the co-crystal structures of both PRMT1 (PDB-6NT2) 2and PRMT6 (PDB- 5EGS)1 which revealed the existence of a hydrophobic shelf comprised of a number of hydrophobic residues such as tyrosine, valine and leucine. In our design, we assumed that the quinazoline core might occupy the hydrophobic pocket and gain π- π stacking interaction along with other conventional interactions with the surrounding residues. A clear SAR was established and the lead compounds displayed inhibition in nano molar range against Class I PRMTs. These lead inhibitors displayed ~ 100 fold selectivity when tested against a panel of 31 human methyltransferases comprising of lysine methyltransferases (PKMTs), DNA and RNA methyltransferases (DNMTs/ RNMT’s), histone lysine demethylase (KMT’s) and methyllysine and methylarginine reader proteins. References (1)J. Med. Chem. 2016, 59, 1176–1183

(2) Cancer Cell. 2019, 36, 100-114

Design in the dark – illuminating the druggability of 53BP1 for BRCA-1 breast cancer

Beatrice Chiew1,2, Menachem Gunzberg2, Caroline Foley3, Stephen J. Headey4, Stephen V. Frye3, Lindsey J. Ingerman3, Bradley C. Doak2 and Martin J. Scanlon2

1School of Environmental and Life sciences, The University of Newcastle, Callaghan, NSW, Australia

2 Monash FBDD Platform (MFP), Monash University, Parkville, Victoria, Australia

3Eshelman school of pharmacy, University of North Carolina, Chapel Hill, North Carolina, USA

4 School of Science, RMIT university, Melbourne, Victoria, Australia

Email: Beatrice.chiew@newcastle.edu.au

Breast cancer is the most common form of cancer affecting women. An estimated 2 million people were diagnosed with breast cancer in 2020 – averaging 6 000 diagnoses every day. Those who carry mutations to the BRCA-1 breast cancer susceptibility gene are 6-times more likely to develop breast cancer due to a breakdown of DNA double strand break repair pathways.

53BP1 is a large reader protein which mediates DNA damage repair pathway choice. The knockout of 53BP1 in a BRCA-1 mutant background has been shown to restore normal DNA end joining pathways in mice and abolish tumour development. This appears to be linked to 53BP1's ability to recognize a key histone binding partner to localize to sites of DNA double strand break. This interplay between 53BP1 and the BRCA-1 breast cancer susceptibility gene provides an avenue to prophylactically treat BRCA-1 breast cancer. However, high quality 53BP1 chemical probes are required to validate its therapeutic potential or druggability.

Fragment-Based drug discovery (FBDD) is an established means of drug discovery that is particularly useful in developing chemical probes for protein-protein interaction interfaces. A key concession of FBDD is that initial hits tend to display weak binding to protein targets, and that structural data is heavily relied upon to develop these hits to higher affinity. Here, we present a means to develop chemical probes for the 53BP1 using a Fragment-Based workflow without need of structural data; termed the "Rapid Elaboration of Fragments into Leads" (REFiL) workflow. This workflow utilizes microscale parallel synthesis and Off-rate screening by Surface Plasmon Resonance to expedite the production of lead-like compounds to establish the druggability of a protein of interest. This workflow has enabled the development of 4 small molecule 53BP1 binders that are equipotent to the histone binding partner.

Designing of Imidazo[1,2-a]Pyridine based TLR7 and TLR9 Antagonist

Nirmal Das,†# Swarnali Roy,† Purbita Bandopadhyay,‡ # Dipyaman Ganguly,‡ *#

Arindam Talukdar†*#

†Department of Organic and Medicinal Chemistry, CSIR-IICB, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata - 700032, India

‡ Department of Cancer Biology & Inflammatory Disorder; CSIR-IICB, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata - 700032, India

Email id: atalukdar@iicb.res.in #Academy of Scientific and Innovative Research, Ghaziabad-201002, India

Aberrant expression of innate immune system leads to progression of autoimmune disorder and other metabolic diseases. TLR7 and TLR9 play a driving role in recognition of endogenous immune complexes that contains self-nucleic acids. Thus targeting TLR7/9 protein with intellectual design of antagonist can be of huge clinical importance. In this present work, we have focused on imidazo[1,2-a]pyridine core which is a ‘drug prejudice’1 scaffold and till date is not modified in the field of antagonist designing targeting hTLR7/9 based on imidazo[1,2-a]pyridine core. Here we elucidate an extensive SAR study on this core to gain insight about how step by step modifications can lead towards achieving potent TLR7/9 antagonist. We are able to attain potent hTLR7 and hTLR9 antagonism with an IC50 value <500 nM and <50 nM respectively. The active compounds showed excellent in vitro human and mice plasma stability along with moderate Caco-2 permeability. This establishes that imidazo[1,2-a]pyridine core can be exploited as dual hTLR7 an hTLR9 antagonist in clinical context.

Figure 1. Our designed new class of potent hTLR7 and hTLR9 antagonist.

The main objective of the study was to develop a potent hTLR7 and hTLR9 antagonist with improved pharmacokinetics (PK) and pharmacodynamics (PD) data. We propose discovery of novel imidazo[1,2-a]pyridine core as dual TLR7/9 inhibitor through systematic optimization at C2, C3 and C7 position. Our design is loosely inspirited by previously reported compound 12 and 22. The SAR study revealed that specific and strategic substitution on imidazo[1,2-a]pyridine core at C-2, C-3, and C-7 are essential to get the optimum potency along with good PK and PD data. The design contain strategically incorporated weakly basic amine group keeping in mind that endosomal pH is acidic and hTLR3/7/8/9 are endosomal TLRs. The SAR showed the aromatic group at C-2 position have a very crucial role to impart potency. On addition of heterocyclic aliphatic chain at C-3 position, compounds become more potent but PK of these compounds were poor. At C-7 position we placed N-substituted piperazine ring to make the molecule basic in nature. SAR showed that lone pair of N atom of piperazine ring is very important to get potency.

Thus our newly designed imidazo[1,2-a]pyridine derivatives being very potent as well as stable in human and mice plasma along with moderate Caco-2 permeable could pave the way for the development as an effective drug for autoimmune disorder.

References (1) Current Topics in Medicinal Chemistry. 2017; 17(2). (2) J. Med. Chem. 2019, 62 (15), 7015−7031.

ICBS Abstract HyPep: A Sequence Homology Search Algorithm for Neuropeptide Identification Vu, N.; Cao, W.; Yen, H.; Fields, L*; Li, L. *Contact information: lawashburn@wisc.edu Despite immense efforts to develop software capable of determining the most probable peptide sequence from a biological sample using an established database, there still exists very little computational options designed specifically for matching de novo sequenced peptides with peptide database sequences. Characterization of neuropeptides has different challenges compared to traditional peptide queries, as the model organism of choice, blue crabs (C. sapidus), has a largely unsequenced genome. To mitigate these challenges, we developed a software, HyPep, which uses database matching to identify de novo sequences that match known neuropeptide sequences. First, a database of known neuropeptide sequences was curated from using transcriptome mining and empirical mass spectrometry-based de novo sequencing, which serves as a template to compare unknown sequences to. Following this, de novo sequencing of raw mass spectral data is conducted in a third-party software search, such as PEAKS. De novo sequences identified from this step are passed through the HyPep sequence homology search. In the sequence homology search, de novo sequencing results are compared with the database to assign matches, and a degree of homology score is calculated. Here, the user has the ability to modify the tolerance of this score, by defining a sliding window size, which corresponds to the number of varying sites between the database and experiment that are tolerated. This strategy enables the discovery of novel neuropeptide isoforms that belong to a neuropeptide family. We have found that the unique HyPep algorithm is competitive with other sequence homology search algorithms, with the distinct advantage that HyPep is open source. Given this, our goal is to further optimize HyPep specifically for neuropeptides, a diverse class of signaling molecules that are challenging to characterize due to lack of suitable computational tools. By designing an open-source software specifically for neuropeptides, we now have a more substantial toolset to answer important questions related to neuropeptides with greater ease and accuracy.

Directed evolution of orthogonal RNA–RBP pairs through library-vs-library in vitro selection Keisuke Fukunaga, Yohei Yokobayashi Nucleic Acid Chemistry and Engineering Unit, Okinawa Institute of Science and Technology

Graduate University (OIST), Onna, Okinawa 904-0495, Japan

keisuke.fukunaga@oist.jp (KF)

ABSTRACT RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation

and RNA processing in cells. They are also useful for various applications in cell biology and

synthetic biology. However, re-engineering novel and orthogonal RNA-RBP pairs from natural

components remains challenging while such synthetic RNA-RBP pairs could significantly expand

the RNA-RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro

selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by

EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 1012 unique RNA sequences

and 4.0 × 108 unique phage-displayed L7Ae-scaffold (LS) proteins, we selected RNA-RBP

complexes through a two-step affinity purification process. After six rounds of library-vs-library

selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS).

Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and

orthogonal RNA-RBP pairs that exhibit picomolar affinity and >4000-fold selectivity. REFERENCE 1. Fukunaga, K. and Yokobayashi, Y. (2021) Nucleic Acids Res., in press, doi:

10.1093/nar/gkab527.

Multiplexed analysis of the secretin-like GPCR-RAMP interactome by suspension bead array

Authors: Ilana B. Kotliar1,2, Emily Lorenzen1, Tea Dodig-Crnkovic3, Elisa Pin3, Emilie Ceraudo1, Roger Vaughan4, Mathias Uhlèn3,5, Thomas Huber1, Jochen M. Schwenk3, Thomas P. Sakmar1,6

Affiliation: 1Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, 1230 York Ave., New York, NY 10065, USA. 2Tri-Institutional PhD Program in Chemical Biology, New York, NY 10065, USA. 3Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, 171 65 Solna, Sweden. 4Center for Clinical and Translational Science, The Rockefeller University, 1230 York Ave., New York, NY 10065, USA. 5AlbaNova University Center, School Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, 106 91 Stockholm, Sweden. 6Department of Neurobiology, Care Sciences and Society, Section for Neurogeriatrics, Karolinska Institutet, 171 64 Solna, Sweden.

Email address of presenting author: ikotliar@rockefeller.edu

Abstract:

Receptor activity-modifying proteins (RAMPs) have been shown to modulate the functions of several different G protein-coupled receptors (GPCRs), but the potential for direct interactions among the three known RAMPs and hundreds of GPCRs has not been measured. Focusing on the secretin-like family of GPCRs, we engineered 23 epitope-tagged GPCRs and three epitope-tagged RAMPs. We used these constructs in a multiplexed suspension bead array (SBA) immunoassay that we developed for detection of GPCR-RAMP complexes. The SBA assay results elucidate the complete interactome for secretin-like GPCRs with RAMPs and validate polyclonal Human Protein Atlas antibodies against 19 of the receptors tested. A subset of GPCR-RAMP interactions that were detected using the SBA assay were verified in cell membranes using the semi-quantitative in situ proximity ligation assay (PLA). The PLA indicates a range of interactions that is consistent with the results of the SBA assay. In total, we found GPCR-RAMP interactions for the majority of the 23 GPCRs tested, suggesting that GPCR-RAMP interactions are more common than previously appreciated. Our SBA approach to proteomics analysis of membrane protein interactions can be scaled up to elucidate the entire GPCR-RAMP interactome. Since RAMPs may regulate GPCR pharmacology and biology, characterization of GPCR-RAMP interactions will provide essential information to advance GPCR-targeted drug development.

References

1. Lorenzen E, Dodig-Crnkovic T, Kotliar IB, Pin E, Ceraudo E, Vaughan RD, Uhlen M, Huber T, Schwenk JM, Sakmar TP (2019) Multiplexed Analysis of the Secretin-like GPCR-RAMP Interactome. Sci Adv 5(9):eaaw2778.

Title: A systems pharmacology approach identifies differential network targeting of ROS1 inhibitors and lorlatinib polypharmacology Authors: Yi Liao1, Xueli Li1, Bin Fang1, Victoria Izumi1, Eric A. Welsh1, Eric B. Haura1,John M. Koomen1, Robert C. Doebele2, Uwe Rix1 Authors’ affiliations: 1H. Lee Moffitt Cancer Center & Research Institute, United States of America 2University of Colorado Denver -Anschutz Medical Campus, United States of America Contact: yi.liao@moffitt.org

BACKGROUND: ROS1-fusions have been identified as potent oncogenic drivers in non-small cell lung cancer (NSCLC). However, most patients develop resistance to ROS1 inhibition by acquiring point mutations or through compensatory cellular signaling. Therefore, multiple second- and third- generation ROS1-directed tyrosine kinase inhibitors (TKIs) are currently being developed. However, their relative efficacy and target selectivity in ROS1-rearranged NSCLC cells are incompletely understood. METHODS: ROS1 TKIs were tested across a panel of patient-derived ROS1-rearranged NSCLC cell lines using 2D and 3D viability assays and in vitro kinase assays. Changes in p-ROS1 were detected by Western blotting. Coupleable probes of four ROS1 inhibitors were chemically synthesized and validated by in vitro kinase assays. Quantitative chemical proteomics, global, and tyrosine phosphoproteomics were performed by using liquid chromatography coupled with tandem mass spectrometry. Kinase targets were integrated with global and tyrosine phosphoproteomic data using protein-protein interaction network analysis to reveal a network-wide difference of the four ROS1 inhibitors. siRNAs, PYK2is, and SRC TKIs were applied for functional validation of identified targets. RESULTS: ROS1-rearranged cell lines showed a broad spectrum of ROS1 TKI sensitivity. Lorlatinib not only exhibited the highest potency for inhibition of TPM3-ROS1 kinase activity and ROS1 autophosphorylation but displayed disproportionately stronger activity at reducing cell viability than other ROS1 TKIs. Quantitative competitive chemical proteomics confirmed the cognate target potencies and also identified off-targets across four ROS1 TKIs. Integration of identified kinase targets with global and tyrosine phosphoproteomic data using protein-protein interaction network analysis revealed common and differential signaling effects of the four ROS1 inhibitors, with lorlatinib acting more on focal adhesion signaling than other ROS1 inhibitors. Functional validation using in vitro kinase assays, pharmacological inhibitors, and RNA interference revealed a polypharmacology mechanism of lorlatinib by targeting PYK2 in addition to ROS1. Subsequent rational combination of different ROS1 inhibitors with focal adhesion inhibitors such as PYK2 or SRC TKIs displayed strong synergy. Our results demonstrate that the observed cellular potency of lorlatinib is the result of polypharmacology involving ROS1 (primary) and PYK2 (secondary) inhibition. CONCLUSION: All ROS1-targeted TKIs effectively inhibited p-ROS1 but were differentially potent for the inhibition of cell viability or TPM3-ROS1 kinase activity. A comprehensive chemical systems biology approach not only revealed a polypharmacology mechanism of lorlatinib by dual targeting of ROS1 and PYK2 but also identified effective drug combinations of lorlatinib with SRC inhibitors.

Targeting DNA abasic sites by an aminoquinoxaline compound induces cytotoxicity and DNA damage in combination with anticancer drug chlorambucil in human colorectal carcinoma cells

Chandra Sova Mandi,† Tridib Mahata,† Dipendu Patra,†, ‡ Jeet Chakraborty,† Achyut Bora,†, ‡ Ritesh

Pal,†, ‡ and Sanjay Dutta*, †, ‡

† Organic and Medicinal Chemistry Division, CSIR- Indian Institute of Chemical Biology

4, Raja S. C. Mullick Road, Kolkata- 700032, West Bengal, India. ‡ Academy of Scientific and Innovative Research (AcSIR), Ghaziabad- 201002, India.

*Corresponding author E-mail: chandra791.sm@gmail.com (Chandra Sova Mandi, Presenting Author)

Abstract

In living cells, DNA repair has to tackle many different types of lesions. Probably the most common is

the abasic sites (AP sites) generated via intrinsic and extrinsic agents. In particular, the base excision

repair (BER) pathway repair the modified base through glycosylase that hydrolyses the glycosidic

bond, resulting in the AP site. Subsequently, the AP site is removed by AP lyase/endonucleases and

the damaged nucleotide is removed. If unrepaired, AP sites are lethal for the cells. Some cancer cells

contain high levels of glutathione such as colon cancer cells that affect chemotherapeutic potentials

due to acquired resistance due to glutathione. To inhibit AP sites repair appears like an exquisite

strategy to potentiate the action of DNA alkylating drugs. Hence, AP sites are a favourable target for

cancer therapy. Here, we report a monoquinoxaline amine (1e) and investigated its recognition

towards AP-DNA compared to the native DNA, that can bind and cleave abasic sites in AP-DNA. At

100 nM concentration, 1e selectively cleaves at abasic sites in DNA in vitro. 1e also binds to the THF

analog of the abasic site in nanomolar to submicromolar range depending on the nucleotide sequence

opposite to the abasic site and also induces a structural change in abasic DNA. Further, we

demonstrated in vitro the reduction of the nitroquinoxaline derivative (1d) into an amine (1e) in the

presence of thiol, glutathione. The cellular toxicity of 1d and 1e in combination with chlorambucil was

evaluated and showed a response at a lower concentration, induced DNA damage and apoptosis in

HCT-116 cells. Consequently, the combination showed a potential strategy for targeted therapy, and

the outcome of this study provides a definitive approach that will help to optimize the therapeutic

applications for targeting abasic sites in cancer cells.

Discovery of hypomorph mutation-directed small molecule protein-protein interaction inducers/molecular glues Xiulei Mo1, Cong Tang1, Qiankun Niu1, Alafate Wahafu1, Xuan Yang1, Min Qui1, Andrey A. Ivanov1,2, Yuhong Du1,2, and Haian Fu1,2,3 1 Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA 30322, USA 2 Emory Chemical Biology Discovery Center, Emory University School of Medicine, Atlanta, GA 30322, USA 3 Department of Hematology and Medical Oncology and Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA Presenting author: Xiulei Mo, xmo@emory.edu Abstract

Tumor suppressor genes represent a major class of oncogenic “drivers” and offer robust window for therapeutic intervention. However, direct targeting loss-of-function tumor suppressor genes remains challenging because that majority of tumor suppressors do not have enzymatic activity and exert their normal function through protein-protein interaction (PPI). Noteworthy, a unique class of tumor suppressor mutations are missense mutations encoding single amino acid substitutions that lead to hypomorph PPI (hypoPPI). These tumor suppressor mutations are defined as “loss-of-interaction” mutation. We aim to directly target this undrugged “loss-of-interaction” tumor suppressor space through discovery of small molecule hypoPPI inducers/molecular glues to restore their anticancer functions. SMAD4 is such a tumor suppressor with “loss-of-interaction” mutations in many cancers that disrupt its normal PPI with SMAD3 downstream of TGFb signaling. Using SMAD4 as a proof-of-concept study, we explored a variant-directed chemical biology approach to reverse the lost function of tumor suppressors using SMAD4R361H/SMAD3 hypomorph interaction as an example. From a chemogenomic library screen, we have identified Ro-31-8220, a bisindolylmaleimide derivative, as a SMAD4R361H/SMAD3 interaction inducer. Ro-31-8220 reactivated the dormant SMAD4R361H-mediated transcriptional activity and restored TGFb-induced tumor suppression activity in SMAD4 mutant cancer cells. Thus, demonstration of Ro-31-8220 as a SMAD4R361H/SMAD3 interaction inducer illustrates a general strategy to reverse the lost function of tumor suppressors with hypomorph mutations and supports a systematic approach to develop small molecule PPI molecular glues for biological insights. Such hypomorph mutation-directed small molecule PPI molecular glues may be developed as potential therapeutic agents to selectively restore the tumor suppression function of mutated proteins for cancer therapy, allowing the leverage of the vast undrugged mutated tumor suppressor target space.

Development of proximity labeling via histidine oxidation by singlet oxygen and Fc-selective functionalization of antibody

○Keita Nakane1), Tatsuya Niwa2), Michihiko Tsushima3), Hideki Taguchi2), Shusuke Tomoshige1),

Hiroyuki Nakamura3), Minoru Ishikawa1), Shinichi Sato1,4)

1)Graduate School of Life Sciences, Tohoku University, 2)Cell Biology Center, Institute of Innovative

Research, Tokyo Institute of Technology, 3)Laboratory for Chemistry and Life Science, Institute of

Innovative Research, Tokyo Institute of Technology, 4)Frontier Research Institute for Interdisciplinary

Sciences, Tohoku University.

Email: nakane.keita.s1@dc.tohoku.ac.jp

Specific labeling of natural proteins with biorthogonal reactions is a powerful approach for the analysis of

living systems and the creation of novel protein-based biomaterials such as antibody-drug conjugates. However,

there are few variations in the amino acid residue-specific labeling reactions used for protein functionalization

and most of the labeling reactions target nucleophilic amino acid residues such as lysine and cysteine residues

using electrophilic reagents.

Histidine is an amino acid involved in biological phenomena such as catalysis of proton transfer in the active

center of enzymes and chelation to metal. However, there are only a few reports of histidine labeling reactions in

proteins, and in general, the electrophilic approach is a difficult challenge to develop selectivity in the presence

of other nucleophilic amino acid residues. Inspired by the cross-linking between oxidized histidine and

nucleophilic amino acid residues, we aimed to develop umpolung histidine labeling strategy, in which

electrophilically activated imidazole ring by oxidation is trapping by nucleophilic labeling reagent. In this

method, histidine residue was activated by Diels-Alder reaction with singlet oxygen (1O2) generated from a

photosensitizer, and the activated imidazole ring was attacked by 1-methyl-4-arylurazole (MAUra) as a

nucleophile. This histidine labeling proceeded selectively in close proximity to a photosensitizer by utilizing

microsecond-scale lifetime of 1O2 (Nakane, K.; Sato, S.; Niwa, T.; Tsushima, M.; Tomoshige, S.; Taguchi, H.; Ishikawa;

M.; Nakamura, H. J. Am. Chem. Soc. 2021, 143, 7726).

In recent years, techniques to label Fc region, in which the sequences are conserved among antibodies, has

attracted attention as the methods to functionalize antibodies. Here, we performed Fc-selective labeling of

antibody using photosensitizer-conjugated Fc binding peptide. As a result of analyzing the labeling site, the

histidine residues in Fc region of the antibody were identified.

THERAPEUTICALLY TARGETING CD64 IN ACUTE MYELOID LEUKEMIA VIA

SINGLE-CHAIN BASED ANTIBODY IMMUNOTOXIN

Ncembu S.1, Tai S.1, Barth S.2 & Harrison S.T.L1*

1Centre for Bioprocess Engineering Research (CeBER), Department of Chemical Engineering, Faculty of Engineering and the Built Environment, University of Cape Town, Private Bag X3, Rondebosch 7701, South Africa 2Institute of Infectious Disease and Molecular Medicine (IDM), Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Private Bag X3, Rondebosch 7701, South Africa

*sue.harrison@uct.ac.za

Cancer immunotherapy is a promising innovative and effective treatment for many forms of cancer. Among hematologic malignancies, acute myeloid leukemia (AML) remains an unmet medical need as it is primarily treated with chemotherapy, which is characterised by severe side effects. H22(scFv)- ETA' is an immunotoxin composed of a truncated toxin moiety; Pseudomonas exotoxin A (ETA’) that is linked to a humanised single-chain variable fragment (scFv) targeting CD4. H22(scFv)- ETA' has been shown in this study to be highly effective in selectively destroying CD64-positive dysfunctional myeloid tumour cells in AML. CD64 is highly expressed on monocytic blast cells in patients with AML and not in normal hematopoietic stem cells or non-hematopoietic tissues. The overexpression of CD64 and its rapid internalization make it a suitable target antigen for antibody-based targeted therapies. H22(scFv)- ETA' has only been produced in shake flasks, a scale that does not provide sufficient quantity to conduct preclinical and/or clinical studies. Therefore, the current phase of this study is focused on optimizing the productivity of H22(scFv)- ETA' and conducting a scale-up production from the shake flask to a 5 L stirred tank reactor (STR). This will enable us to conduct comprehensive preclinical studies to further evaluate the therapeutic efficacy of H22(scFv)- ETA' against AML. H22(scFv)- ETA' is recombinantly expressed in E. coli Bl21 (DE3) and purified by metal ion affinity chromatography and size exclusion chromatography. The volumetric mass transfer coefficient (kLa) is used as a scale-up criterion to achieve effective batch and fed-batch fermentation processes. The therapeutic efficacy of H22(scFv)- ETA' is evaluated by several biological assays, including binding assays using flow cytometry and cytotoxicity using Annexin-V bioassay. The development of successful scale-up production of H22(scFv)- ETA' is critical as it will provide insight into a process that can be established at pilot scale and eventually at commercial scale in the context of biopharmaceutical manufacturing.

1

Title: Next Generation Robotics for Chemogenomics Joseph Uche Ogbede1*, Marjan Barazandeh2, Guri Giaever2 and Corey Nislow1,2 1Genome Science & Technology Graduate Program, University of British Columbia, Canada 2Faculty of Pharmaceutical Science, University of British Columbia, Canada *presenting author; ucheogbede@gmail.com The discipline of Chemical Biology has been supported and accelerated by the introduction of new robotics that can support screens of increasing complexity. A key test bed for these devices has been the model organism Saccharomyces cerevisiae, aka bakers’ yeast. Our lab has focused on the use of yeast for drug assays and target discovery, including the HaploInsufficiency Profiling (HIP) and Homozygous Profiling (HOP) assays, which use the yeast Saccharomyces cerevisiae in investigating drug targets as well as the pathways involved in drug metabolism. Screening thousands of chemical compounds and collecting their growth parameters in a reproducible manner requires precision robotics. A major limitation of the conventional one-plate/one reader approach is its low throughput. Accordingly, we designed two high throughput robotic instruments, CG12 and GP1 for the whole organism, parallel chemical screens. The CG12 can read 12 microplates continually using a single reader- allowing for 1000+ growth assays/day. The GP1 is designed for pooled, genome-wide drug screens for continuous growth via robotic dilution, automatically transferring cells that have reached a certain optical density (OD) from one well to another in a 48-well or 96-well plate. We have applied the instruments to screen ~800 FDA approved drugs in multiple yeast strains and tested over 100 potential COVID19 therapeutics in a drug repurposing campaign. We also characterized genes and pathways that are involved in nitrosamines toxicity and uncovered the role of arginine metabolism. Furthermore, we built an easy-to-use software platform to collect and analyze these datasets. These new instruments are fast, reproducible and reliable and a powerful addition to the Chemical Biology toolkit.

Structural Basis of Toll-like Receptor 7 Antagonism with Small Molecule Modulators Sourav Pal1, Purbita Bandopadhyay2, Barnali Paul1, Dipika Sarkar1, Dipyaman Ganguly2, Dr. Arindam Talukdar1* 1. Department of Organic and Medicinal Chemistry, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C.

Mullick Road, Kolkata, 700032, WB, India 2. IICB-Translational Research Unit of Excellence, Department of Cancer Biology and Inflammatory Disorders,

CSIR-Indian Institute of Chemical Biology, CN6, Sector V, Salt Lake, Kolkata, 700091, WB, India E-mail: palsourav30@gmail.com Toll-like receptors (TLRs) are an essential component of our innate immunity. Aberrant

activation of the endosomal TLR7 has been implicated in numerous autoimmune disorders and is

an established therapeutic target in such scenario. We have implemented both structure-based

and ligand-based approaches in order to design novel TLR7 antagonists. We proposed a binding

hypothesis based on molecular docking analysis and chemical feature-based 3D-QSAR

pharmacophore model to correlate human TLR7 (hTLR7) antagonistic activity with structural

features in different chemotypes. The binding hypothesis of TLR7 antagonists having different

chemotypes revealed the significance of engaging different hydrophobic pockets, grooves and a

central cavity where ligand-receptor interactions with specific residues through hydrophobic and

hydrogen bond interactions which correlate with TLR7 antagonistic behaviour, paving the way

for the rational design using various chemotypes. Design and synthesis of quinazoline core-based

potent TLR7 antagonists validate the binding model that understands the effect of engagement of

these pockets as well as the boundaries of the chemical space associated with them, based on the

structural insight gained from the structural studies. By manipulating the substitution patterns at

C-2, C-4, and C-7 of the quinazoline ring, we were able to create a simple structure-activity

relationship (SAR) and discovered that these substitutions worked together to provide potent

human TLR7 inhibition. Besides the designing and synthesis of the molecular dataset, primary

human immune cell assays and cell-based reporter assays against human TLR7 were used to

determine the potency. The newly synthesized most potent hTLR7 antagonist has an IC50 value

of 1.03 ± 0.05 μM in TLR7-reporter HEK293 cells and was validated by a primary assay in

human plasmacytoid dendritic cells (pDC) (IC50pDC: 1.42 μM). The ligand-based drug designing

approach utilizes the 3D-QSAR model, which depicts the significant contribution of the

electrostatic potential and VdW interaction energy towards the TLR7 antagonism. Additionally,

the generated pharmacophore model shows the importance of hydrophobicity and hydrogen bond

acceptor/donors, ring aromaticity that is highly correlated between experimental and predicted

activity (Rtraining: 0.98 and Rtest: 0.90) and can be used to predict small-molecule hTLR7

antagonists. Both structure-based and ligand-based drug designing study provides a rational

design approach thus facilitating further development of novel small molecule hTLR7

antagonists based on different chemical scaffolds.

TLR7 Antagonist

IC50 = 1.03 µM

Gemcitabine Conjugated blockHPMA-based pH-sensitive Nanoconstruct for

Cancer Treatment Tarun Patel, Milan Paul, Kondapaneni Likhitha Purna, Swati Biswas, Balaram Ghosh*

Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad

Hyderabad, Telangana, India 500078.

Email ID: tkpatel51@gmail.com

Abstract

Polymeric nanocarriers have emerged as targeted drug delivery systems due to their better

physicochemical properties, biocompatibility, ease of preparation, scalability, and amenable

modification scopes. Nanoparticles are engineered with tumor-microenvironment sensitivity for

triggered on-demand release of drugs. Herein, we synthesized mPEG5k-b-N-(2-hydroxypropyl)

methacrylamide (HPMA)-gemcitabine (GEM) conjugate, where GEM is attached to the

bHPMA via pH-sensitive hydrazone linker. The conjugates were prepared by changing the chain

length of bHPMA to increase the nanoparticle's stability and GEM attachment. The conjugates

were characterized by UV, IR, NMR, and GPC analysis. The nanoparticles were characterized

for particles size, zeta potential, drug conjugation, stability by DSC, surface tension, and pH-

dependent drug release studies. The in vitro therapeutic efficacy of the NPs was tested in human

breast cancer cell lines, MCF-7 and MDA-MB-231. The results indicated that the pH-sensitive

optimized nanoparticles demonstrated increased cytotoxic response in both the cell lines (MDA-

MB-231(IC50-0.463 µg/ml) and MCF-7 (IC50-0.364 µg/ml) than the free GEM. The cellular

uptake study indicated that the conjugated micelles were internalized by MDA-MB-231 and

MCF-7 cell lines in a time-dependent manner. In summary,the pH-sensitive gemcitabine NPs

system could furtherbe explored as an improved treatment option than the conventional GEM-

chemotherapy in cancer.

Keywords: Cancer, gemcitabine, block-HPMA, pH, hydrazone.

Discovery of novel hydrazide chemotype-based HDAC3 selective inhibitors

with potent in-vivo anti-tumour activities.

Sravani Pulya, Tarun Patel, Milan Paul, Swati Biswas, Balaram Ghosh* Epigenetic Research Laboratory, Department of Pharmacy, Birla Institute of Technology & Science,

Pilani-Hyderabad, Hyderabad, Telangana, India 500078. Email ID: balaram@hyderabad.bits-pilani.ac.in

Keyword: Benzoyl-hydrazide, HDAC3 inhibitor, apoptosis, ROS, in-vivo antitumour activity.

Epigenetics has been attributed in the implications of several diseases in particular cancer. Post-translational modification (PTM) of histone proteins has been widely explored in cancer pathogenesis. Histone acetylation is one of the most studied histone PTMs that has been understood in different disease states including cancer. Hence, histone deacetylases (HDACs) have been reported to be a potential target for anticancer therapy. In specific, HDAC3, a well-validated isoform among the other isozymes is being targeted for its significant role in cancer progression. A number of HDAC inhibitors with different zinc binding groups (ZBG) have been developed pre-clinically and clinically as anticancer agents. Though several HDAC inhibitors (HDACis) have been identified, it is an unmet need to identify isoform-selective HDAC inhibitors (HDACis) in order to understand the mechanisms involved in HDAC inhibitor based cancer therapy where majority of clinically approved HDAC inhibitor drugs are nonselective. Here, we explored a novel ZBG with benzoyl hydrazide scaffold with various modifications on the cap region to develop selective HDAC3 inhibitors. We also evaluated in-vitro anti-proliferative activity against different cancer cell lines, pharmacokinetic profile and the in-vivo anti-tumour efficacy in 4T1-Luc tumour xenograft model with the lead compound 4e which was identified to be the most potent and selective HDAC3 inhibitor in isolated enzyme based biochemical assay with IC50 value of 15.41 nM and >10-fold selectivity over other class I HDAC isoforms and >3000-fold selectivity over class II HDACs tested. All the compounds in the series showed significant anti-proliferative activity against various cancer cell lines while 4e proved to be the most potent against 4T1 (IC50: 1.92 µM) and the least toxic (>80 fold selective towards 4T1 cancer cells over HEK-293 normal cells). Significant induction of acetylation on H3K9 and H4K12 was found both in-vitro and in-vivo when treated with 4e.

Cell cycle phase arrest at G2/M in B16F10 cells further confirmed the programmed cell death mechanism. The western blot analysis with isolated tumour tissue from 4e treated mice validated the reduced ‘cell proliferation and metastasis’ in-vivo. The excellent pharmacokinetic profile with t1/2 of 2.62 h at 25 mg/kg dose and significant reduction of tumour volume with no general toxicity in mice when treated with 4e in 4T1-Luc xenograft mouse model further suggested its significant anti-tumour activity and its potential to be translated as anticancer epigenetic drug.

Selective Covalent-Allosteric Inhibitors to Dissect Akt Isoforms Lena Quambusch‡1, Laura Depta‡1, Ina Landel1, Melissa Lubeck1, Tonia Kirschner1, Jonas Nabert1, Niklas Uhlenbrock1, Jörn Weisner1, Michael Kostka2, Laura M. Levy2, Carsten Schultz-Fademrecht3, Franziska Glanemann4,5, Kristina Althoff4,5, Matthias P. Müller1, Jens T. Siveke4,5 and Daniel Rauh1*. 1Faculty of Chemistry and Chemical Biology, TU Dortmund University and Drug Discovery Hub Dortmund (DDHD), Zentrum für Integrierte Wirkstoffforschung (ZIW), Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany 2Medicinal Chemistry, Taros Chemicals GmbH & Co. KG, Emil-Figge-Strasse 76a, 44227, Dortmund, Germany 3 Lead Discovery Center GmbH, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany 4 Bridge Institute of Experimental Tumor Therapy, West German Cancer Center, University Medicine Essen, Essen, Germany 5Division of Solid Tumor Translational Oncology, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), partner site Essen, Heidelberg, Germany lena.quambusch@tu-dortmund.de Signal transduction in cells is mainly proceeded by kinase-mediated downstream phosphorylation, thus the dysregulation of those enzymes has tremendous consequences and can cause severe diseases.[1] Isoforms of protein kinase Akt (Akt1/2/3) are involved in a myriad of essential processes including cell proliferation, survival, and metabolism.[2] However, their individual roles in health and disease have not been thoroughly evaluated. Thus, there is an urgent need for perturbation studies, preferably mediated by highly selective bioactive small molecules.[3] Here, we present a structure-guided approach for the design of structurally diverse and pharmacologically beneficial covalent-allosteric modifiers which enabled an investigation of the isoform-specific preferences and the important residues within the allosteric site of the different isoforms.[4],[5] The biochemical, cellular, and structural evaluations revealed interactions responsible for the selective binding profiles. The set of isoform-selective covalent-allosteric Akt inhibitors that emerged from this approach showed a conclusive SAR and broke ground for further development of selective probes to delineate the isoform-specific functions of Akt kinases.[6] [1] a) M. Rabiller, M. Getlik, S. Kluter, et al., Archiv der Pharmazie 2010, 343, 193-206; b) J. Lategahn,

M. Keul, D. Rauh, Angewandte Chemie 2018, 57, 2307-2313. [2] E. Gonzalez, T. E. McGraw, Cell cycle 2009, 8, 2502-2508. [3] P. Wu, M. H. Clausen, T. E. Nielsen, Pharmacology & therapeutics 2015, 156, 59-68. [4] J. Weisner, R. Gontla, L. van der Westhuizen, et al., Angewandte Chemie 2015, 54, 10313-10316. [5] L. Quambusch, I. Landel, L. Depta, et al., Angewandte Chemie 2019, 58, 18823. [6] L. Quambusch, L. Depta, et al., Nature Communications 2021, 12, 5297.

Development of novel o-hydroxy benzamide based HDAC3-selective inhibitors as

anticancer agents with promising in vivo potential.

Ganesh Routholla, Sravani Pulya, Tarun Patel, Milan Paul, Swati Biswas, Balaram Ghosh*

Department of Pharmacy, Birla Institute of Technology & Science, Pilani-Hyderabad

Hyderabad, Telangana, India 500078.

Email ID: ganeshncl7@gmail.com

Histone deacetylases (HDACs)are the enzymes that catalyse the removal of acetyl functional group from the lysine residues of both histone and non-histone proteins. There are 18 HDACs isoforms identified till date. These HDACs are classified into 4 types. Class I, Class II, Class III, Class IV. Class I HDACs contains HDAC1,2,3 & 8 whereas Class II HDACs contains HDAC4,5,7,9,6 & 10, Class IV contains a HDAC 11. The Class III belongs to sirtuin family (sirtuin 1-7). Generally, HDAC inhibitors contain a general pharmacophoric structure that includes CAP, LINKER and ZBG (zinc binding group). Based on their ZBG’s HDACis are chemically classified into 4 classes. 1) Hydroxamic acid, 2) Short chain fatty acid, 3) Benzamides, and 4) Cyclic Peptides. Here, we reported the design and synthesis of a small series of benzamide based HDAC inhibitors by incorporating the chemical features of few reported lead molecules those are at different phases of clinical or preclinical studies such as CI-994, BG-45 and RGFP966. All the newly synthesized molecules in the series were evaluated for their HDAC inhibitory potency against purified recombinant HDAC isoforms using fluorophore coupled biochemical assay. All the synthesized compounds were also screened against a set of different cancer cell lines. The lead compounds 11a and 12b showed good in-vitro anticancer activities with less cytotoxicity to normal HEK-293 cells and the compounds were also found to significantly inducing histone acetylation both in-vitro and in-vivo and also found to induce apoptosis with a G2/M phase arrest in B16F10 cells. Compound 11a exhibited potent anti-tumour efficacy in 4T1-Luc breast cancer xenograft mouse model in female Balb/c mice. It also showed significant tumour growth suppression with no general toxicity, and extended survival rates post-tumour resection. Compound 11a also significantly induced higher ROS generation leading to apoptosis. No considerable toxicity was noticed in major organs that were isolated from compound 11a-treated mice. Altogether, the findings suggest that the HDAC3 selective inhibitor 11a might be a potential lead for the clinical translation as an emerging drug candidate for the treatment of cancer.

Keywords: hydroxy benzamide, HDAC3 inhibitor, anti-cancer agent, apoptosis, in-vivo anti-tumour activity.

Inhibition of bacterial signal recognition particle system by antisense PNA:

A novel antibacterial strategy

Snehlata Saini[a], Sudipta Ghosh[b], and Ishu Saraogi*[a] [b] [a] Department of Biological Sciences, [b] Department of Chemistry,

Indian Institute of Science Education and Research Bhopal Bhauri, Bhopal Bypass Road, Bhopal-462066, Madhya Pradesh, India

(snehlatasaini@iiserb.ac.in)

Abstract: The upsurge of antibiotic resistance in pathogenic bacteria is one of the major global health concerns

[1]. To address this problem, new antibacterial strategies are required. In this direction, we have recently identified the bacterial signal recognition particle (SRP) pathway as a potential antibacterial target [2]. Bacterial SRP mediates the co-translational transport of nascent proteins to the plasma membrane, and is essential for bacterial cell survival [3-4]. Our approach aimed to inhibit an important RNA-protein interaction to disrupt the functional SRP cycle in bacteria. Antisense peptide nucleic acids (PNAs) were designed to target the bacterial 4.5S RNA-Ffh protein interaction (which makes a functional SRP complex). In vitro studies confirmed that the designed PNAs bound specifically to the target RNA, and inhibited the RNA-protein interaction in a dose-dependent manner, leading to the inhibition of SRP mediated GTP hydrolysis. In vivo studies showed that the cell permeable peptide conjugated PNA effectively inhibited E. coli AS19 bacterial cell growth in a dose-dependent manner. PNA mediated bacterial cell growth inhibition was rescued by over-expression of 4.5S RNA. This study confirms the cellular target of the designed PNA, and validates SRP as a potential system for antibacterial therapeutics.

Further, we explored the molecular details of the interaction between designed PNAs and RNA through various biophysical techniques [5]. One of the designed PNAs has a homo-pyrimidine sequence, capable of forming triplexes with homo-purine RNA. The other PNA having a mixed-base sequence can only form duplex structure. Biophysical assays at physiological pH revealed the formation of higher order structure in case of homo-pyrimidine PNA, whereas the mixed-base PNA was found to form a duplex structure with RNA. In vitro GTPase assay showed dose-dependent inhibitory effect of the homo-pyrimidine PNAs on SRP mediated GTP hydrolysis as compared to mixed-base PNA. Thus, our results show that the formation of higher order structures by homo-pyrimidine PNAs exhibit significant effect on RNA functionality than the mixed-base PNA.

In summary, we have proposed and validated SRP as a potential antibacterial target for the first time. Our results also suggest the importance of understanding the target site selection in order to develop better antisense molecules to target 4.5S RNA which will be a novel antibacterial target for multi-drug resistant bacteria. The approaches used here can also be generalized for discovery of other antibacterial targets for the development of broad-spectrum or even specific antibiotics.

References and Notes:

1. Chellat, M. F.; Raguz, L.; Riedl, R. Targeting Antibiotic Resistance. Angew. Chem. Int. Ed. Engl.2016, 55 (23), 6600–6626.

2. Ghosh, S.; Saini, S.; Saraogi, I. Peptide Nucleic Acid Mediated Inhibition of the Bacterial Signal Recognition Particle. Chem. Commun.2018, 54 (59), 8257–8260.

3. Keenan, R. J.; Freymann, D. M.; Stroud, R. M.; Walter, P. The Signal Recognition Particle. Annu. Rev. Biochem.2001, 70, 755–775.

4. Brown, S.; Fournier, M. J. The 4.5S RNA Gene of Escherichia Coli Is Essential for Cell Growth. J. Mol. Biol.1984, 178 (3), 533–550.

5. Saini, S.; Ghosh, S.; Saraogi, I. Deciphering the effect of designed homo-pyrimidine and and mixed-base PNAs on bacterial 4.5S RNA structure and functionality (Manuscript under preparation).

Discovery and Validation of Quinazolinone derivatives as COP1 modulator in

ameliorating Non-Alcoholic Fatty Liver Disease

Dipayan Sarkar,†# Saheli Chowdhuri,‡ Sunny Goon,† Partha Chakrabarti,‡ Arindam Talukdar†*#

†Department of Organic and Medicinal Chemistry, CSIR-IICB, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata - 700032, India

‡Cell Biology & Physiology Division, CSIR-IICB, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata - 700032, India Email id: atalukdar@iicb.res.in

#Academy of Scientific and Innovative Research, Ghaziabad-201002, India

Non-alcoholic fatty liver disease (NAFLD) is the most chronic liver disease in which >5 % steatosis has been occurred in absence of significant alcohol consumption, monogenic hereditary disorders, and steatogenic medications. NAFLD is subdivided into two classes based on histological outcome. As is evident, the most prominent feature of NAFLD is the deposition of excessive TAG (Triacylglycerol) in hepatocytes and therefore, deregulation of enzymes responsible for controlling intracellular lipid turnover and homeostasis may play an important role in NAFLD.1 A pivotal enzyme associated with the intracellular degradation of TAG is Adipose triglyceride lipase (ATGL) also known as patatin-like phospholipase domain-containing protein 2 (PNPLA2). Constitutive photomorphogenic 1 (COP1), also known as RFWD2, is a E3 ubiquitin ligase serves as a crucial regulator in mammalian embryonic development,cellular processes and the DNA damage response. In recent past, It is found that COP1 predominantly targets hepatic ATGL through ‘V-P’ motif recognition for degradation via Ubiquitin Proteasomal System and knock down of COP1 increases ATGL which in turn lowers the TAG content in liver.2 Here we report a new class of potent COP1 modulator for increasing ATGL activity in liver for NAFLD.3

Figure 1. Prototype of Design and Validation of new class of Quinazolinone scaffold The main objective of the study was to discover new chemical entity for COP1 modulator to improve ATGL activity. Our design is primarily based on quinazolinone core with urea linkage to mimic ‘V-P’ motif of ATGL. The SAR study revealed that specific and strategic substitutions on quinazolinone core at N-3, and aromatic ring containing urea linkage at C-6 position are important for interaction with COP1; stabilizing the enzyme and increase in ATGL activity. We found installation of ‘alkyether’ linker at N-3 position is important for activity. Thus our newly designed quinazolinone derivatives have been found to demonstrate improved activities of ATGL by modulating COP1 enzyme and could pave the way for the development of effective NAFLD medications.3 References

(1) J. C. Cohen, J. D. Horton, H. H. Hobbs, Science, 2011, 332(6037), 1519-23 (2) M. Ghosh, S. Niyogi, M. Bhattacharyya, M. Adak, D. K. Nayak, S. Chakrabarti , P. Chakrabarti, Diabetes,

2016, 65, 3561–3572 (3) Priority Application No.: 202011027502; International Application No.: PCT/IN2021/050621

Modulating Agonism and Antagonism in Toll-Like Receptor 7 and Structural Optimization Leading to Antagonists Relevant to Psoriasis

Dipika Sarkar, Deblina Raychaudhuri, Ayan Mukherjee, Biswajit Kundu, Bishnu Prasad Sinha, Dipyaman Ganguly, Arindam Talukdar*

Indian Institute of Chemical Biology, Jadavpur, Kolkata- 700032, India

*Corresponding e-mail: atalukdar@iicb.res.in Toll-Like Receptors (TLRs) are a class of germline-encoded pattern recognition proteins, which provide the first line of defense against pathogenic invasion by regulating innate immune system of the host. Among the 10 TLRs recognized in humans, endosomal TLRs play critical roles as "double edged sword" in identifying the pathogen derived nucleic acid as well as the extracellular nucleic acid of the host but the latter one trigger the systematic autoimmune and metabolic disorders by aberrantly activating endosomal TLRs. Among the endosomal TLRs, TLR7 and TLR9 are expressed in pDCs only and drive the production of type I interferon upon activation. When self-origin RNA & DNA molecules get access to endosomes, they abruptly activate TLR7/8/9 and initiate critical innate immune events which lead to the development of autoimmune disorders such as SLE, systemic sclerosis, psoriasis etc.1 Hence, inhibiting the activation of TLR7/9 with suitable antagonists could serve as novel pathogenic node in these disease context. To design a suitable TLR antagonist, we started our journey by developing a purine scaffold TLR7 antagonist which was rationally designed by dissecting the structural criterions for TLR7 activity. Computational studies exhibited that both TLR7 and TLR9 agonists and antagonists share overlapping binding region. Hence we selected a known purine TLR7 agonist as template and eventually discovered a single chemical switch with minimal structural modification at C2 position which can transform a purine agonist into a novel antagonist. The potency was further enhanced by incorporating a basic center at C6 position and we winded up with a clinically relevant TLR7 antagonist [IC50 (TLR7) = 4.7 µM] for having favourable pharmacokinetics with 70.8% oral bioavailability in mice and excellent selectivity over TLR8.2 The in vitro experiment was performed by using HEK293 cells that expressed exogenous human TLR7. Previously reported literatures suggested that due to the structural similarity of TLR7 and TLR9, the dual TLR7/9 antagonists could be more convenient clinically. Therefore, we further extended our strategy towards designing of dual and potent TLR7/9 antagonists through structure-activity relationship (SAR) development and illustrated that the modulation at C2, C6 and N9 position in the purine scaffold can lead to a robust dual TLR7/9 antagonists with promising in vivo pharmacokinetics with excellent ADME profile. The therapeutic window of the lead compound [IC50 (TLR9) = 0.08 M and IC50 (TLR7) = 2.16 M] in a preclinical murine model of psoriasis highlighted its potential as therapeutic successor in concerned autoimmune context.3

References:

1. D. Ganguly Trends Immnol. 2018, 39, 28-43. 2. A. Mukherjee et al. J. Med. Chem. 2020, 63, 4776-4789. 3. B. Kundu et al. J. Med. Chem. 2021, 64, 9279-9301.

Plex: A novel form of artificial intelligence (AI) for the identification of

compound targets and mechanism of action

Douglas W. Selinger & Timothy Wall

Plex Research, Inc., Cambridge, MA, USA

Web: www.plexresearch.com

Email: dselinger@plexresearch.com

Abstract

We have developed a novel form of AI, based primarily on search engine algorithms, which is useful for

the analysis of compound targets and mechanism of action (MoA). We have used this approach to

identify putative targets for compounds with unknown or unclear mechanisms, including phenotypic

screening hits, natural products, and marketed drugs.

A search engine AI approach has many advantages over traditional machine learning based approaches,

including:

Transparency: The data supporting any target inference can be easily identified, traced backed

to its original source, and evaluated. This transparency contextualizes all findings and allows

scientists to fully leverage their expertise when interpreting results. In chemical biology

applications, the structures of all analogs are shown, as well as their precise reported activity

against the putative target(s).

Concision: The most experimentally supported results are ranked first, regardless of how much

data is searched or how many additional results are returned.

Scale: Search engine algorithms can be tailored to work seamlessly and rapidly over multiple

large, heterogenous, and highly complex data sets, identifying instances where multiple data

sources converge on the same answer. For a given a chemical series of interest, for example,

Plex can identify if structurally related compounds converge on certain targets, mechanisms,

protein families, protein domains, pathways, patents, diseases, toxicities, etc.

Plex is powered by a massive knowledge graph of 700M nodes and edges covering all areas of

biomedical research, from pharmacology to omics data to clinical trial information. Analyses can be

initiated with chemical structures of interest, but also with gene lists from omics experiments, pathways,

biomarkers, or metabolites from metabolomics profiles. Multiple approaches can be used together, such

that a compound target can be pinpointed using a combination of chemical structure, transcriptional

perturbation data, and metabolomics profiling.

The flexibility of the approach lends itself to many applications, including compound target ID/MoA

analysis, safety assessment, biomarker discovery, disease indication discovery, precision medicine, and

many more.

A metal-free anion sensor to detect pyrophosphate and

its chemical relatives in water Chitra Shanbhag1 and Ishu Saraogi*1, 2

Department of Chemistry1 and Department of Biological Sciences2 Indian Institute of Science Education and Research, Bhopal, India

(Email: chitra16@iiserb.ac.in, ishu@iiserb.ac.in )

Abstract: Fluorescence-based chemosensors that recognize anions of physiological significance and biomedical relevance in water are of utmost importance. Among the several anions ubiquitous in biological system, pyrophosphate (PPi) anion has a pivotal role in regulating cellular metabolic processes. To selectively differentiate the PPi anion, the chemists have utilized a metal-ligand-based approach that enables the formation of a metal-chemosensor ensemble that strongly interacts with PPi to obtain a fluorescent response.1However, this method is an indirect approach to anion sensing as the sensor is metal-dependent, and the huge excess of metal used may be detrimental to enzyme-based application. In this regard, we report a serendipitously discovered water-soluble metal-free fluorescent PPi sensor that is composed of an amino acid (AA) coupled to fluorescent reporter 3-HNA (3-Hydroxy napthoic acid). The fluorescence emission intensity of the molecule is ~30-fold enhanced at 500 nm in the presence of PPi. The emission signal enhancement is also observed in the presence of phosphate and vanadate, which are chemically related to pyrophosphate anion in terms of geometry and basicity. Through proton NMR and fluorescence-based experiments, we have shown the sensing mechanism of the molecule is attributed to intermolecular proton transfer from the fluorescent chemosensor to the anion, depending on the basicity. Furthermore, we have shown the utility of the chemosensor to detect PPi in a routinely used bioanalytical application, polymerase chain reaction (PCR), to track DNA amplification. Overall, we have demonstrated a simple water-soluble metal-free chemosensor that detects biologically relevant chemically related anions.2

References:

1 S. Anbu, A. Paul, G. J. Stasiuk and A. J. L. Pombeiro, Coord. Chem. Rev., 2021, 431, 213744. 2 Shanbhag. C, and Saraogi.I (Manuscript under preparation)

Deciphering the role of a co-chaperone and a small molecule in

regulating E. coli DnaK function: An antibacterial approach

Tulsi Upadhyay1, Upasana S Potteth1, Vaibhav V Karekar2 , Yogesh M. Gangarde2 and Ishu Saraogi 1,2*

1Department of Biological Sciences, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal 462066, MP, India

2 Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal Bypass Road, Bhauri, Bhopal 462066, MP, India

E-mail: tulsi@iiserb.ac.in

The bacterial chaperone DnaK, a homologue of heat shock protein (Hsp70), actively holds or unfolds thermosensitive proteins and prevents their misfolding and aggregation [1a]. The DnaK ATPase cycle is stimulated synergistically by two essential co-chaperones, DnaJ and GrpE. The co-chaperone GrpE, a thermosensor, plays a central role as a nucleotide exchange factor and stimulates substrate release from DnaK. GrpE experiences significant structural changes during thermal stress that alter its interaction with DnaK and regulate the chaperone cycle [1b]. Interestingly, bacterial GrpE shows low evolutionary conservation among prokaryotes and eukaryotes, making GrpE an attractive antibacterial target. We have identified an essential structural feature of E. coli GrpE responsible for regulating its structural stability and influencing its interaction with DnaK. Biophysical and biochemical analyses suggest that idealizing the coiled-coil domain of GrpE homodimer by introducing hydrophobic residues enhances its interaction with DnaK. However, in vivo data suggests that the enhanced binding between GrpE and DnaK was detrimental to the protein folding cycle [2a]. We further applied the structural and functional understanding of the DnaK chaperone system to develop a structure-based inhibitor of DnaK function. Using an in-house small molecule library screening approach, we identified M7 as a potential inhibitor of DnaK [2b]. M7 likely binds at the DnaK substrate-binding domain and inhibits ATPase and luciferase refolding activity of DnaK. We find that M7 inhibits growth of several bacterial strains and biofilm formation of P. aeruginosa. SEM and confocal imaging suggest that M7 can permeate bacterial cells, and the observed filamentous growth phenotype is consistent with DnaK inhibition. In summary, our work demonstrates an important site in the co-chaperone GrpE, which could be a potential small molecule accessible site to be explored as a prospective antibacterial target. Additionally, our finding showcases M7 as a valuable lead for DnaK inhibition that can potentially be developed as an antibacterial molecule.

References:

1.(a) Sharma et al. Nat Chem Biol., 2010, 6(12), 914-20. (b) Siegenthaler et al. J Biol Chem. 2005, 280(15), 14395-401 2. (a) Upadhyay, T., Potteth, U. S., Karekar, V. V. & Saraogi, I. Biochemistry, 2021, 60, 1356–1367. (b) Upadhyay, T.; Gangarde Y. M.; Saraogi, I. “Identification of a small molecule inhibitor targeting the DnaK chaperone system” (manuscript in prep.)

Cloning and characterization of a new endolysin from P. acnes bacteriophage as a

potential enzybiotic candidate.

C. Varotsou1 and N.E. Labrou2

1,2Laboratory of Enzyme Technology, Department of Biotechnology, School of Applied

biology and Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855-

Athens, Greece

1christivar@hotmail.com, 2lambrou@aua.gr

The emergence of antimicrobial multidrug resistance (AMR) has risen worldwide and has led

to major concern for public health. Exploitation of lytic enzymes derived from

bacteriophages in fighting multidrug resistant (MDR) pathogenic bacteria is considered from

scientific community as a promising solution, since their bactericidal activity is immediate

and the development of antimicrobial resistance towards them is not feasible.

Propionibacterium acnes is a gram-positive human skin commensal that is associated with

acne and is considered as a MDR bacterial strain. In the present work, a putative endolysin

from a P. acnes phage (lysP) has been identified. The coding sequence of lysP has been

synthesized and cloned into a T7 expression vector providing a 6-His tag at C-terminal of the

protein. Expression of lysP has been studied in nine different E. coli strains under two

different expression conditions. LysP under optimal expression conditions was expressed as

insoluble protein in inclusion bodies (IBs). IBs were washed with non-ionic detergent and the

protein aggregates were solubilized and refolded using overnight dialysis. Protein analysis

and SDS-PAGE confirmed the purity of the refolded lysP. The optimal conditions for the lytic

activity of lysP were investigated with turbidity reduction assays. Enzyme activity was

measured and assessed with ten different microbial strains as substrates, under a wide pH

range and with the addition of different divalent metals. It was then determined the optimal

concentration of the metal that exhibited the best effect on the enzymatic activity. The lytic

activity of lysP was further confirmed with isolated peptidoglycan from P. acnes and

Remazol Brilliant Blue-labelled cells of P. acnes degradation assays. The enzymatic

degradation of the peptidoglycan of P.acnes cells was evaluated on agar plates and with

confocal microscopy. The results of the present study suggest that lysP could act as potential

antimicrobial factor.

Lipid peroxidation in the endoplasmic reticulum drives ferroptosis A. Nikolai von Krusenstiern1, Ryan N. Robson3, Fanghao Hu2, Fereshteh Zandkarimi2, Naixin Qian2, Verna M. Estes3, Marcel Dupont1, Mikhail S. Shchepinov4, Wei Min2, K. A. Woerpel3, Brent R. Stockwell1,2 1Department of Biological Sciences, Columbia University, New York, New York, 10027, USA 2Department of Chemistry, Columbia University, New York, New York, 10027, USA 3Department of Chemistry, New York University, New York, New York, 10003, USA 4Retrotope, Inc., Los Altos, California, 94022, USA Email: anv2117@cumc.columbia.edu Ferroptosis is a non-apoptotic iron-dependent cell death that has been implicated in several disease pathologies. Inducers of ferroptosis demonstrate promise as anti-cancer chemotherapeutics, while inhibitors could serve as drugs for neurodegenerative disorders and ischemia-reperfusion injury. Although it is established that peroxidation of unsaturated fatty acid chains in subcellular membranes drives ferroptosis, it is not known which membranes are required and how lipid peroxidation spreads. We explored the structure-distribution-activity relationship of ferroptosis-inhibiting and -inducing compounds, and identified the endoplasmic reticulum (ER) as an essential driving site of lipid peroxidation in ferroptosis. Stimulated Raman scanning (SRS) imaging was used to determine the subcellular distribution of various pro- and anti-ferroptotic fatty acids. Taking advantage of the Raman-active C-D vibration, we imaged the distribution of deuterated polyunsaturated and monounsaturated fatty acids, and determined that they accumulate in the ER, lipid droplets, and the plasma membrane. We further showed that the accumulation in lipid droplets does not play a role in their ability to modulate ferroptosis. To complement these studies, confocal fluorescence imaging was used to capture the subcellular distribution of FINO2, a ferroptosis inducer. FINO2 was found to accumulate in the ER and Golgi body. Interestingly, FINO2 analogs that redirected to the mitochondria or lysosomes were also able to potently induce ferroptosis, demonstrating that ferroptosis can be initiated in any organelle. However, death by any of these analogs was rescuable by compounds that protect the ER. Thus, blocking lipid peroxidation in the ER is sufficient to prevent ferroptosis, regardless of the initiating organelle. Finally, successive confocal fluorescence images were taken of cells treated with the four different classes of ferroptosis inducers, and stained to label the ER, plasma membrane, and lipid peroxidation. Across all inducers, morphological changes and peroxide formation were observed first in the ER before spreading later to the plasma membrane. Thus, we propose an ordered progression of lipid peroxidation in ferroptosis that accumulates first in the ER.