Chemical biology symposium 2023

Chemical biology symposium 2023 15 May 2023, London, United Kingdom

15 May 2023, London, United Kingdom Chemical biology symposium 2023 cbs2023

Book of abstracts

Registered charity number: 207890

Welcome to the 2023 Chemical biology symposium

Dear Colleagues, We are delighted that you have joined us for this hybrid edition of the Chemical biology symposium. Once again, we have set out to bring together the UK’s chemical biology community with leading scientists from around the world for this RSC Chemistry Biology Interface Community (CBIC) flagship event. Meeting in a hybrid space brings many benefits and opportunities, and we encourage you – whether you have joined in person or online – to participate fully in the sessions and network with each other throughout the programme. This past three years in particular has demonstrated the importance and impact of chemistry across the life sciences. Our outstanding programme of speakers, who are leaders in the field and known for pushing forward the boundaries of our discipline, exemplifies the breadth of chemical biology, the excitement, challenges and opportunities therein. We are also delighted to be showcasing the research from our community’s early career researchers and thank all of those who submitted poster abstracts. The high quality and innovative science presented in the posters underscores the strength and value of chemical biology research. We would like to thank you, the chemical biology community, for your enthusiastic support for this event. We are also grateful for the work of the 2023 Organising Committee from which this exciting programme of speakers was developed. On behalf of the Organising Committee and CBIC Council, we wish you a very enjoyable and engaging meeting. Rob Field Chair of the Organising Committee and Ali Tavassoli President of the RSC Chemistry Biology Interface Division

Organising committee

Rob Field (Chair) University of Manchester, UK

Federico Brucoli De Montfort University, UK

Helen Hailes University College London, UK

Jóhannes Reynisson Keele University, UK

Speaker biographies

Marius Clore NIDDK, National Institutes of Health, USA

Dr Clore’s research focuses on the development and application of nuclear magnetic resonance (NMR) to study the structure and dynamics of biological macromolecules and their complexes in solution. He is particularly interested in exploring fundamental questions associated with protein dynamics and macromolecular interactions. His research group is using NMR to detect and characterise short-lived, sparsely- populated states of macromolecules. Many important biological processes proceed through transient intermediate states that comprise only a fraction of the overall population of a molecular system. As a result, they are invisible (i.e. ‘dark’) to conventional biophysical techniques (including crystallography, cryo-electron microscopy and single molecule spectroscopies). The group’s research provides new insights into macromolecular recognition and assembly, and the effect of the invisible ‘dark’ state on some NMR observables so that its footprint is readily observed in measurements on the NMR visible species. His work on amyloid-β, huntingtin and Hsp40 has implications for the treatment of a range of neurodegenerative diseases associated with protein aggregation and amyloid formation. Marius Clore is a NIH Distinguished Investigator, a Member of the US National Academy of Sciences and a Fellow of the Royal Society. He has won numerous awards including the RSC Centenary and Khorana Prizes, and the Biochemical Society Centenary Award.

Claire Eyers University of Liverpool, UK

Professor Eyers’ team use analytical chemistry strategies, specifically based around a technique called mass spectrometry, to explore the protein components of cells and tissues. Mass spectrometry is a sophisticated strategy for determining mass, which the team use to work out the amount and sequence of proteins from biological, environmental or clinical samples. They develop different ways of using this technique to understand how biological systems respond to their environment or change as a result of disease. By defining changes in cellular protein profile under different conditions, the team can devise strategies to exploit these for therapeutic intervention, to develop hypotheses to understand biological drivers, or to use as markers of disease.

Speaker biographies

Emily Flashman University of Oxford, UK

Professor Flashman’s team look at the role of enzymes in plant and humans in response to reduced oxygen availability. The team explores how the structure and mechanism of these enzymes helps them control their rate of reaction with oxygen and therefore their ability to act as good oxygen sensors. In both humans and plants, these oxygen-sensing enzymes take oxygen from the atmosphere and transfer the oxygen atoms onto specific target proteins. This acts as a signal for the target proteins to be degraded by the cell. If oxygen levels reduce, the rate of enzyme activity decreases and the target proteins are stabilised. The consequence of this stabilisation is that cells adapt to the reduced oxygen availability, for example by switching to anaerobic metabolism. This system has been known for some time in humans, and inhibitors of the oxygen-sensing enzymes has led to treatments for anaemia. Excitingly, finding inhibitors for plant oxygen-sensing enzymes or engineering changes to their structure and mechanism could slow their activity and help plants survive flooded (low oxygen) conditions for longer. This will be important in generating crops that are more tolerant of stresses associated with climate change.

Kai Johnsson Max Planck Institute for Medical Research, Germany

Kai Johnsson is Director of the Department of Chemical Biology at the Max Planck Institute (MPI) for Medical Research, and Professor at the Institute of Chemical Sciences and Engineering of the École Polytechnique Fédérale de Lausanne (EPFL). His current research interests focus on the development of chemical approaches to visualize and manipulate biochemical activities in living cells. In the past he has introduced a number of widely used research tools and used these tools to make biological discoveries. He introduced methods to specifically label proteins in living cells (i.e. SNAP-tag and CLIP- tag), developed new fluorescent probes and sensors, and conducted studies on the mechanism of action of drugs and drug candidates. Kai Johnsson obtained his Diploma and PhD from the ETH Zürich in Switzerland. He joined the faculty of EPFL in 1999 and in 2017 became Director at the MPI for Medical Research. Kai Johnsson was Associate Editor of ACS Chemical Biology from 2005 to 2010 and since 2021 Executive Editor of the Journal of the American Chemical Society . He is member of the Editorial Advisory Board of Science and was member of the Research Council of the Swiss National Science Foundation from 2011-17. He received the Prix APLE for the invention of the year 2003 of EPFL, the Novartis Lectureship Award 2012/13, the Karl-Heinz Beckurts Prize 2016 and is elected member of EMBO.

Speaker biographies

Mauro Maccarrone University of L’Aquila, Italy

Dr. Enzymology and Bio-Organic Chemistry , is Professor and Chair of Biochemistry at the Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila (Italy). He is also Head of the Lipid Neurochemistry Unit at the European Center for Brain Research – IRCCS Santa Lucia Foundation, Rome. Published numerous highly cited full papers (citations = 22442, h-index = 79 according to Scopus). Invited speaker at more than 110 international congresses, Guest Editor of 16 theme-issues of scientific journals (including Drug Discovery Vol. 76 (2021) by RSC), holder of 9 granted patents. President of the International Cannabinoid Research Society (ICRS) in 2010-2011. Chair of the 2015 Gordon Research Conference on “Cannabinoid Function in the CNS”. Visiting Professor at University of Cambridge in 2017. Received various international awards, including the “2016 Mechoulam Award” for cannabinoid research and the “2020 Tu Youyou Award” for medicinal chemistry. Included by Stanford University in the “2022 World Top 2% Scientists’ List”, and listed among the “Top Italian Scientists”. Lydia Meyer-Turkson Former Sr Director Transactions J&J, USA Lydia Meyer-Turkson is the Global Early Innovation Partnering Leader for Oncology working out of the Boston Innovation Center. In her role, Lydia serves as the key point of contact for the oncology therapeutic area for interactions with the Janssen R&D (JRD) leadership to ensure external innovation opportunities are centrally coordinated and strategically aligned with business priorities. Lydia joined Johnson & Johnson Innovation in January 2022 from Inari Agriculture Inc, a Flagship Ventures company. In her prior role as Vice President of Business Development at Inari, she developed, led and executed the company’s partnering strategy for its gene editing and deep learning technologies in human therapeutics. She worked closely with the Leadership Team, External Innovation, Legal and Patent colleagues to define opportunities aligned to strategic goals and build the business case for partnering. Lydia brings 20 years of broad ranging business development experience working with R & D teams commercializing platform technologies across a range of in and outlicensing transactions, M&A and research collaborations in oncology and rare disease drug discovery, biomarkers and diagnostics. She has worked in the UK, Europe and US with CEO and Executive teams in roles at Horizon Discovery plc (aquired by Perkin Elmer), Evotec GmbH and Epistem plc helping lead business growth. Lydia earned her M.Phil (Masters) in Chemistry at Cardiff University and MBA at Manchester Business School, UK and is a Fellow of the Royal Society of Chemistry and member of the Integrated Chemistry- Biology Research Committee there. Lydia is passionate about socioeconomic diversity and inclusion and has undertaken volunteer roles as reviewer for the L’Oréal Women in Science Awards Committee for Sub-Saharan Africa and the Women in Cancer Research AACR Scholarship Awards Committee.

Speaker presentations

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Oxygen-sensing enzymes in plants and animals Emily Flashman University of Oxford, UK

Probing transient pre-nucleation oligomerization of huntingtin at atomic resolution by NMR Marius Clore NIDDK, National Institutes of Health, USA The impact of chemistry biology innovation on modern drug development past and present Lydia Meyer-Turkson Former Sr Director Transactions J&J , USA The challenge of developing selective drugs towards the complex endocannabinoid system Mauro Maccarrone University of L’Aquila, Italy

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Title TBC Claire Eyers University of Liverpool, UK

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Fluorescent and bioluminescent probes for imaging and diagnostics Kai Johnsson Max Planck Institute for Medical Research, Germany

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Poster presentations

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G-quadruplexes in microbial genomes: stabilization of LTR-III in HIV-1 as potential therapeutic targets Zainab Albader Imperial College London, UK

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Encapsulating cancer cells with peptide hydrogels Nouf Alzahrani University of Edinburgh, UK

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Controlled masking of redox cyclers: a novel β-Glucuronide-Triggered β-Lapachone prodrug for enhanced targeting Julie Becher University of Cambridge, UK Lighting up the bad bugs: fluorescent labelling of the bacterial resistance factor ArnT with commercial fluorophores Prachi Bendale Queen’s University Belfast, UK Discovery of specific inhibitors and activators of PADI4 to elucidate its biological role Teresa Bertran The Francis Crick Institute, UK

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Developing inhibitors of bacterial DNA-repair and SOS response pathways Jacob Bradbury University of Oxford, UK Novel fluorescent imaging and theranostic probes targeting mitochondria Federico Brucoli De Montfort University, UK

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Fragment-based drug discovery of SARS-CoV-2 methyltransferase nsp14 inhibitors Anna Lina Bula Latvian Institute of Organic Synthesis, Latvia

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Biocatalytic activity of cytochromes P450 for the valorization of terpenes Danilo Correddu University of Turin, Italy Structural characterization of Borrelia Burgdorferi protein BBA05 Laura Drunka Latvian Institute of Organic Synthesis, Latvia Biocatalytic reactions of Organosilyl Ethers using Silicatein-α Chisom Egedeuzu The University of Manchester, UK Good vibrations: small molecule Raman Optical Probes to Image metabolism in tissue microenvironments Ailsa Geddis University of Edinburgh, UK Investigation of the ligand topology for targeting multimeric G-quadruplex DNA Ariadna Gil Martínez Universidad de Valencia, Spain Double network hydrogel-based phantoms for single and multiphoton imaging Fizza Haseeb University of Edinburgh, UK Regioselective Oxidative carbon-oxygen bond cleavage by copper complexes Azza Hassoon University of Szeged, Hungary Understanding the foliar application of Amino Acids in Soybeans Bethany Henderson Durham University, UK RaPID development of macrocyclic peptides for challenging targets Catherine Hurd GSK/Francis Crick Institute, UK Investigating the interactions between Manganese Oxide Nanoparticles and key components of cellular membranes Travis Issler University of Calgary, Canada

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One trillion Photoswitchable Cyclic Peptides: shining light on questions in Chemical Biology Thomas Jackson Imperial College London/ The Francis Crick Institute, UK Novel bioorthogonal click-to-release systems for fluorogenic proximity labelling Dora Kern Research Centre for Natural Sciences, Hungary Two-photon fluorescent (nano) probes for a versatile intracellular detection and quantification of nitric oxide Maria J. Marin University of East Anglia, UK Orthogonal control of cell-free expression using photocaged nucleic acids Giacomo Mazzotti University of Oxford, UK De novo design and synthesis of EHE miniproteins that contain unnatural β-amino acid building blocks Natalia Miodowska Wrocław University of Science and Technology, Poland Unique ratiometric fluorescent probe, a derivative of IndiFluors for pH mapping during mitophagy Subrata Munan Shiv Nadar University, India Synthesis of Alkyne-tagged Ribitol-5-phosphate as metabolic labelling tools to study Muscular Dystrophy Lloyd Murphy University of York, UK Multimethod approach for a mechanistic understanding of bacterial adhesion on metal alloys Adam Mumford Swansea University, UK

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Discovery and characterisation of dual inhibitors of tryptophan 2,3-dioxygenase (TDO2) and indoleamine 2,3-dioxygenase 1 (IDO1) using virtual screening

Jóhannes Reynisson Keele University, UK

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Metal binding is not enough: characterisation of aminopeptidase PepA from Pseudomonas aeruginosa Martha Simpson The University of St Andrews, UK Stimulated raman scattering microscopy for evaluation of cancer drug biodistribution Craig Steven University of Edinburgh, UK Manganese negatively impacts the properties of biomimetic lipid membranes Kevin Sule University of Calgary, Canada Evaluation of small probes through in silico docking & screening against GDP-mannose dehydrogenase (GMD)c Alice Wahart Keele University, UK Using single-molecule spectroscopy to measure conformational changes in c-Met kinase with clinically arising resistance mutations Tamsin Wilcock University of Bath, UK Fluorescence-based strategies for monitoring/mapping biologically relevant species among bacteria: a new orientation Kai-Cheng Yan University of Bath, UK Rational design of potent Peptide Inhibitors of the PD-1:PD-L1 interaction for Cancer Immunotherapy Huawu Yin Imperial College London, UK

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Oxygen-sensing enzymes in plants and animals Emily Flashman

University of Oxford, United Kingdom emily.flashman@biology.ox.ac.uk.

All aerobic organisms need oxygen to survive and must have homeostatic mechanisms in place to adapt to a reduction in oxygen availability (hypoxia). In animals, these adaptation responses are driven by the Hypoxia- Inducible transcription Factors (HIF); HIF stability is controlled by oxygen-dependent HIF hydroxylase enyzmes whose kinetic properties control rates of reaction with oxygen, conferring a physiologically important sensitivity to the response to hypoxia. Plants are also susceptible to metabolic stress when oxygen availability is limiting. This can occur when plants are flooded or in certain developmental states. In a mechanism which is distinct yet parallel to the well-characterised HIF-mediated hypoxic response in animals, plants respond to hypoxia through the action of transcription factors that upregulate the expression of adaptive genes. The stability of these transcription factors is regulated by oxygen-sensing Plant Cysteine Oxidases (PCOs). I will describe our work defining how PCOs act on plant transcription factors to trigger their degradation in an oxygen-sensitive manner, as well as our efforts to engineer the activity of these enzymes to prolong the plant hypoxic response and improve flood tolerance. I’ll also describe how a homologous cysteine dioxygenase was revealed to act as a novel oxygen- sensing enzyme in humans, indicating that thiol dioxygenases may be an evolutionarily conserved mechanism of sensing and responding to reduced oxygen availability.

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Probing transient pre-nucleation oligomerization of huntingtin at atomic resolution by NMR G. Marius Clore NIDDK, National Institutes of Health, USA Huntington’s disease is a fatal, autosomal, neurodegenerative condition that arises from CAG expansion within exon-1 of the huntingtin gene that encodes a polyglutamine (polyQ) repeat. Although the huntingtin protein is very large (~350 kDa), proteolysis and/or incomplete mRNA splicing generates mutated N-terminal fragments encoded by exon-1 that aggregate to form neuronal inclusion bodies in pathological states. The N-terminal region of huntingtin encoded by exon 1, htt ex1 , comprises three distinct regions or domains: a 16-residue N-terminal amphiphilic sequence (htt NT ), a polyQ tract of variable length, and a proline rich domain (PRD) with two polyproline repeats of 11 (P 11 ) and 10 (P 10 ) residues. In this talk we will summarize out work on the earliest pre-nucleation transient oligomerization events involving htt ex1 using NMR experiments designed to probe rapidly exchanging systems (sub-millisecond time range) involving sparsely-populated excited states, and how reversible submillisecond tetramerization triggers much slower (minutes to hours time scale) nucleation and fibril formation. The importance of pre-nucleation tetramerization is evidenced by the fact that inhibition of tetramer formation blocks fibrillization. Tetramerization constitutes the molecular switch that increases the probability of occurrence of intermolecular polyQ contacts (by effectively increasing the local concentration of the polyQ tracts) and hence polyQ fibril formation. Blocking tetramer formation may provide a fruitful avenue for preventing or delaying the onset of Huntington’s disease. Inhibition of pre-nucleation oligomerization can be achieved in a number of ways: (a) perturbing the productive dimer and/or tetramer interface; (b) sequestration of htt ex1 through binding of the htt NT sequence to chaperones; and (c) allosteric, long-range inhibition by interaction of intracellular proline-binding proteins with the proline rich domain (PRD). References 1. Kotler, S.A., Tugarinov, V., Schmidt, T., Ceccon, A., Libich, D.S., Ghirlando, R., Schwieters, C.D. and Clore, G.M. (2019) Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR. Proc. Natl. Acad. Sci. U. S. A. 116 , 3562-3571. 2. Ceccon, A., Tugarinov, V., Ghirlando, R. & Clore, G.M. (2020) Abrogation of pre-nucleation, transient oligomerization of the Huntingtin exon-1 protein by human profilin-I. Proc. Natl. Acad. Sci. U.S.A. 117, 5844-5852. 3. Wälti, M.A., Kotler, S.A. & Clore, G.M. (2021) Probing the interaction of huntingtin exon-1 polypeptides with the chaperonin nanomachine GroEL. ChemBioChem 22, 1985-1991. 4. Ceccon, A., Tugarinov, V. & Clore, G.M. (2021) Quantitative exchange NMR-based analysis of huntingtin-SH3 interactions suggests an allosteric mechanism of inhibition of huntingtin aggregation . J. Am. Chem. Soc. 143, 9672-9681. 5. Ceccon, A., Tugarinov, V., Toricella, F. & Clore, G.M. (2022) Quantitative NMR analysis of the kinetics of pre-nucleation oligomerization and aggregation of pathogenic huntingtin exon-1 protein. Proc. Natl. Acad. Sci. U.S.A. 119 , e2207690119.

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The impact of chemistry biology innovation on modern drug development past and present Lydia Meyer-Turkson Former Sr Director Transactions J&J, USA Early human history of cancer treatment was limited to: surgery, radiation and chemotherapy. Then a little over 20 years ago, Targeted Therapies ushered in an era of precision oncology based on understanding unique molecular changes that drive the formation and spread of cancer cells. However, cancer is not a single disease rather it is many different diseases, each with diverse mechanisms and causes. This talk will give examples of how innovative chemistry/biology technologies like CRISPR gene editing, genomics and CAR-T cell therapies that harness the human immune system have impacted the work of drug hunters and will help to set future directions.

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The challenge of developing selective drugs towards the complex endocannabinoid system Mauro Maccarrone 1,2 1 University of L'Aquila, Italy, 2 European Center for Brain Research, IRCCS Santa Lucia, Foundation, Italy The cannabis (Cannabis sativa) derivative marijuana is the most widely used recreational drug in the Western world, and cannabis-derived Δ9-tetrahydrocannabinol (THC), cannabidiol and additional terpenophenolic substances – collectively termed phytocannabinoids – hold huge therapeutic potential to treat human diseases. Remarkably, our body has its own endocannabinoid system (ECS), made of a complex ensemble of bioactive lipids, their receptors, metabolic enzymes and transporters that can be hit by phytocannabinoids along with unexpected off-targets. Since the discovery of the first endocannabinoid anandamide (N-arachidonoylethanolamine) 30 years ago, distinct elements of the ECS have been the target of drug design programmes aimed at curing (or at least slowing down) a number of human diseases, both in the central nervous system and at the periphery. Here, the challenge of developing selective drugs towards the complex ECS will be discussed, in order to define whether ECS-active phytocannabinoids and ECS-oriented synthetic drugs should be considered the root of all evil, or a panacea for human health. References 1. Maccarrone M, Guzmán M, Mackie K, Doherty P, Harkany T. Programming of neural cells by (endo)cannabinoids: from physiological rules to emerging therapies. Nat Rev Neurosci. 2014 Dec;15(12):786-801. doi: 10.1038/nrn3846. 2. Maccarrone M, Bab I, Bíró T, Cabral GA, Dey SK, Di Marzo V, Konje JC, Kunos G, Mechoulam R, Pacher P, Sharkey KA, Zimmer A. Endocannabinoid signaling at the periphery: 50 years after THC. Trends Pharmacol Sci. 2015 May;36(5):277-96. doi: 10.1016/j.tips.2015.02.008. 3. Friedman D, French JA, Maccarrone M. Safety, efficacy, and mechanisms of action of cannabinoids in neurological disorders. Lancet Neurol. 2019 May;18(5):504-512. doi: 10.1016/S1474-4422(19)30032-8.

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Title TBC Claire Eyers University of Liverpool, UK

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Fluorescent and bioluminescent probes for imaging and diagnostics Kai Johnsson 1,2 1 Max Planck Institute for Medical Research, Germany 2 EPFL Lausanne, Institute of Chemical Sciences and Engineering, Switzerland; E-mail: johnsson@mr.mpg.de The topic of my presentation will be how a combination of protein engineering and synthetic chemistry can be exploited to generate fluorescent and bioluminescent probes for live-cell imaging. Specifically, I will review our attempts to introduce new fluorescent dyes and sensor proteins that permit to visualize biochemical activities in living cells with high spatial and temporal resolution. I will also discuss how these sensor proteins can be utilized for point-of-care therapeutic drug monitoring.

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G-quadruplexes in microbial genomes: stabilization of LTR-III in HIV-1 as potential therapeutic targets Zainab Albader 1 and Ramon Vilar 1 1 Imperial College London, UK b- CID: 01646940; Email: z.albader19@imperial.ac.uk. c-Supervisor; Email: r.vilar@imperial.ac.uk Guanine quadruplexes which is rich in guanine nitrogenous bases is a tetra-stranded structure that is fold due to the ability of guanines to display hydrogen bonds. These G-quadruplexes is further stabilized by electrostatic force between a mono cation (Na + or K + ) and the oxygen atoms of the guanine bases. Investigation of G-quadruplexes in microbial genomes are gaining increasing attention, especially with the challenges associated to antimicrobial resistance. Stabilization of G-quadruplexes present in gene promoter regions using small molecules can be a potential therapeutic target in microbial genomes. In this project, we aim to stabilize G-quadruplex structures in the gene promoter regions; thus, studying the effect of G-quadruplex formation in the unusual hybrid G-quadruplex structure LTR-III promoter in the HIV-1 virus. To stabilize LTR-III in HIV-1, we synthesized metal salphen complexes with different building blocks via click chemistry. Biophysical studies were conducted to evaluate the binding towards LTRIII and other G-quadruplexes. The metal salphen complexes are selectively bind to LTRIII with higher delta T values comparing to other G-quadruplexes and duplex DNA. CD spectra and FID assays were conducted for further understanding besides to docking to identify the bindings’ sites. References 1. Met. Ions Life Sci. 2018, 18, 325–349. 2. Comprehensive Supramolecular Chemistry II, 2017, 39–70.

3. Chem. Eur. J. 2019,25,417 –430. 4. Biol. Chem. 2001, 382, 621–628. 5. Nucleic Acids Res. 2006, 34, 5402–5415.

6. Drug Discovery. 2011,10, 261–275. 7. Trends Cell Biol. 2009, 19, 414–422. 8. Nucleic Acids Res. 2015, 43, 8627–8637. 9. Rev. Mol. Cell Bio. 2017, 18, 279–284.Nat. Rev. Chem. 2017, 1, 1–10. 10. Bioorg. Med. Chem. Lett. 2014, 24, 2602–2612. 11. Antimicrob. Chemother. 2014, 69, 3248–3258.Trends Microbiol. 2019, 27, 148–163. 12. Nucleic Acids Res. 2018; 7, 3270–3283. 13. Am. Chem. Soc. 2018, 140, 13654−13662. Med. Chem. 2013, 56, 6521−6530.

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Encapsulating cancer cells with peptide hydrogels Nouf Alzahrani, Annamaria Lilienkampf, and Mark Bradley University of Edinburgh, UK

Hydrogels are highly solvated, cross-linked, three-dimensional networks. They can be formed from natural biological molecules such as hyaluronic acid and collagen or synthetic molecules that include polyethylene glycol or polyacrylamides, while supramolecular hydrogelators include self-assembling peptides and monosaccharides. Peptide hydrogels can self-organize in water to form ordered nanofibers and generate a variety of well-ordered scaffolds. In biomedical research, peptide hydrogels are frequently employed as scaffolds for 3D cell culture, tissue engineering, and have also been used as an injectable drug/cell delivery tool. Hydrogel substrates are also being used in regenerative medicine as scaffolds to aid the healing of damaged or injured tissues. The aim of my project is to design and synthesise peptides that upon cleavage by proteases form hydrogels in situ , and lead to cellular encapsulation, leading to a reduction in cell proliferation and migration. In my poster I summarize the composition and structure of the peptide hydrogels, and the influencing factors for gelation.

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Controlled masking of redox cyclers: a novel β-Glucuronide- Triggered β-Lapachone prodrug for enhanced targeting Julie Becher, Gonçalo Bernardes, Lavinia Dunsmore, Nisita Dutta, Enrique Gil de Montes University of Cambridge, UK AIM: Ortho -quinone natural products have great therapeutic potential, but their clinical use has been limited due to systemic toxicity stemming from the redox activity of the ortho -quinone scaffold. This project discusses the development of a novel β-lapachone prodrug platform that detoxifies ortho -quinones while in circulation via C-alkylation, triggers site-specific release in the tumour microenvironment via β-glucuronidase activity, and controls drug release rate via para -hydroxybenzyl (PHB) self-immolative linker design. The optimized prodrugs will then be conjugated to a targeting carrier moiety for clinical use. MATERIALS AND METHOD: Four prodrug derivatives have been synthesized over 5 steps and characterized. Importantly, indium-mediated C-alkylation was utilized to attach the glucuronide linker to the ortho -quinone carbonyl under mild conditions. The pH-dependent pseudo-first-order drug release kinetics were then examined via HPLC assay, and preliminary cell viability assays were performed on a panel of cancer cell lines. RESULTS: Previously, our group published the discovery of para -aminobenzyl (PAB) C-alkylated ortho -quinone prodrugs that release via 1,6-elimination breaking a C-C bond in a pH-dependent manner. 1 Modelling shows the para -hydroxybenzyl (PHB) derivative should release 10,000X faster at physiological pH. PHB prodrug 11a was synthesized and released faster at physiological pH than the PAB derivative (t 1/2 =54±3hr vs. t 1/2 =13,000±10,000hr respectively) as expected. However, cell viability assays demonstrated incomplete restoration of toxicity due to slow drug release. To accelerate release, lowering the pKa of the PHB phenol was examined by making difluoro ( 11b ) and tetrafluoro ( 11c ) derivatives following a similar synthetic route. 11b (t 1/2 =25±2hr) released faster, while 11c (t 1/2 =1340±270hr) did not show improvement. Next, stabilizing the elimination transition state was explored via the methoxy derivative ( 11d ), which exhibited orders of magnitude faster release (t 1/2 =1.9±0.1hr). CONCLUSIONS: The release rate of a novel β-glucuronide-PHB-β-lapachone prodrug was greatly improved by modulating the pKa and transition state stabilization of the PHB linker. The first-order drug release half-life was improved 2X by lowering phenol pKa, while stabilizing the TS via addition of a methoxy group resulted in 28X faster ortho -quinone release. The synthesis of an optimized derivative combining the beneficial features of each linker and a click handle for carrier attachment is now underway. References 1. 1.Dunsmore, L., Navo, C.D., Becher, J.et al.Controlled masking and targeted release of redox-cyclingortho-quinones via a C–C bond-cleaving 1,6-elimination.Nat. Chem. 14 , 754–765 (2022). https://doi.org/10.1038/s41557-022-00964-7

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Lighting up the bad bugs: fluorescent labelling of the bacterial resistance factor ArnT with commercial fluorophores Prachi Bendale 1 and Abigail Hopkins 1 Sheiliza Carmali 1 , Mohammad Arefian 2 , Ben Collins 2 ,Mark Sutton 3 , Miguel A. Valvano 4 & Gerd K. Wagner 1 1 School of Pharmacy, Queen’s University Belfast, 2 School of Biological Sciences, Queen’s University Belfast, 3 UK Health Security Agency, 4 Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast Antimicrobial resistance (AMR) is a major threat to global health predicted to lead to 10 million deaths per year by 2050 if no action is taken [1]. Operationally simple technologies for the diagnostic labelling of bacterial resistance factors are therefore of great interest to study molecular, cellular, and environmental factors driving AMR development. One such resistance factor is the enzyme 4-amino-4-deoxy-l-arabinose transferase (ArnT). ArnT is involved in membrane lipopolysaccharide remodelling of Gram-negative pathogens such as Klebsiella pneumoniae , Pseudomonas aeruginosa , and Burkholderia cenocepacia , resulting in resistance to polymyxins, last-resort antibiotics employed to treat multidrug antimicrobial resistant infections [2]. In this project, we have investigated the use of commercially available fluorophores as chemical tools for the diagnostic labelling of ArnT. Analysis of sequence and structural data for ArnT from K. pneumoniae , P. aeruginosa , B. cenocepacia and Cupriavidus metallidurans identified several lysine residues as potential target sites for the covalent attachment of fluorescent probes. To exploit this opportunity, we assembled a small library of commercially available fluorophores with different lysine-reactive electrophiles and established a workflow for protein labelling. Relevant experimental parameters such as incubation time and fluorophore concentration were optimised using bovine serum albumin as a model protein. Application of our protocol to the B. cenocepacia ArnT [3] allowed the desired in-gel fluorescent detection of ArnT after electrophoretic separation. Protein mass spectrometry confirmed the covalent attachment of selected fluorophores and identified their attachment sites. Finally, we rationalised the observed reactivities using a recently developed algorithm for the prediction of site and sequence of protein modifications with lysine-reactive reagents [4]. Our results demonstrate that ArnT can be readily labelled with commercially available fluorophores, providing a basis for the rational development of bespoke labelling reagents for this enzyme as novel diagnostic tools in the fight against antimicrobial resistance. References 1. O’Neill (2016) Tackling Drug-Resistant Infections Globally: final report and recommendations 2. Petrou et al. (2018) Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation. Science , 351 (6273), 608-612. 3. Tavares-Carreon et al. (2015) Burkholderia cenocepacia and Salmonella enterica ArnT proteins that transfer 4-amino-4- deoxy-l-arabinose to lipopolysaccharide share membrane topology and functional amino acids. Sci. Rep. , 5, doi.org/10.1038/ srep10773 4. Patel, A. et al. (2022) Automated prediction of site and sequence of protein modification with ATRP initiators. PLoS One , doi. org/10.1371/journal.pone.0274606

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Discovery of specific inhibitors and activators of PADI4 to elucidate its biological role Teresa Bertran 1 , Bertran M.T. 1 , Walmsley R. 3 , Cummings T. 3 , Valle Aramburu I. 4 , Swanton T. 4 , Papayannopoulos V. 4 , Christophorou M. 3 , Walport L. J. 1, 1 Protein-Protein Interaction Laboratory, The Francis Crick Institute, London, UK, 2 Imperial College London, UK, 3 The Babraham Institute, Cambridge, UK , 4 Antimicrobial Defense Laboratory, The Francis Crick Institute, London, UK Peptidyl Arginine Deiminase 4 (PADI4) is a member of the peptidyl deiminase family of proteins that catalyse the post-translational modification of arginine to the non-canonical amino acid citrulline. PADI4 is involved in various biological processes and its dysregulation has been linked to various pathologies, including rheumatoid arthritis and a range of cancers; however, the molecular mechanisms that regulate PADI4 are poorly understood 1,2 . In consequence, the discovery of chemical tools that selectively modulate PADI4 activity is crucial to understand its biological function. The Random non-standard Peptide Integrated Discovery (RaPID) platform is a very powerful technology that enables us to screen > 1 trillion cyclic peptides against a target of interest 3,4 . We carried out three RaPID screens and identified macrocyclic peptides that bound to different conformations of PADI4 with nanomolar affinities. One of the peptides identified acts as a potent and selective PADI4 inhibitor both in vitro and in cells, while another one with no inhibitory effect was used as an affinity probe. In addition, we also identified a cyclic peptide that acts as a potent PADI4 activator at low calcium concentration. These peptides will be used as chemical tools to modulate PADI4 activity and hence elucidate its biological role. References 1. Witalison et al. Curr Drug Targets. 2015:16(7) 700-710.Christophorou M., R. Soc. Open. Sci. 2022, 9:220125

2. K. Bashiruddin et al., Curr. Opin. Chem. Biol. 2015 24, 131-138. 3. Passioura T and Suga H., Chem Commun, 2017, 53,1931.

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Developing inhibitors of bacterial DNA-repair and SOS response pathways Jacob Bradbury 1,2 , Timothy R. Walsh 2 , Thomas Lanyon-Hogg 1

1 Dept. of Pharmacology, University of Oxford, UK, 2 INEOS Oxford Institute, University of Oxford, UK

Antimicrobial resistance (AMR) has the potential to make many life-saving medical advances redundant, causing 10 million deaths per year by 2050. 1 New compounds with novel targets are required to address the challenge of AMR. 2 One such target is the DNA damage repair process which allows bacterial survival under stress from antibiotics or immune attack. Loading of the DNA repair enzyme AddAB onto damaged DNA facilitates double stranded break repair and upregulates the SOS response, which activates virulence, persistence, and resistance mechanisms. 4 IMP1700 inhibits AddAB, potentiating the DNA damaging antibiotic Ciprofloxacin (CFX) and inhibiting the SOS response in methicillin-resistant Staphylococcus aureus (MRSA). 3 IMP1700 contains a fluoroquinolone (FQ) motif associated with ‘black box’ toxicity and understanding of the structure-activity relationship (SAR) of this series is limited, both of which hinder future translation. To investigate the SAR of IMP1700 and develop improved lead compounds, over 90 analogues were synthesised (Fig. 1A), and two high-throughput assays were developed to assess compound activity. Firstly, compound growth inhibition was determined with and without half-minimum inhibitory concentration (MIC) of CFX to probe DNA- repair inhibition (Fig. 1B) and fold increase in potency with CFX (∆CFX) calculated. Secondly, SOS response inhibition after activation by CFX was measured using a reporter system expressing GFP under control of an SOS-responsive promoter (Fig. 1C). OXF077 showed greater synergy with CFX-induced DNA damage than IMP1700 (∆CFX = 260 and 106-fold, respectively) and stronger SOS inhibition (IC 50 = 75 and 132 nM, respectively). Interestingly, a selection of compounds, such as OXF030 and OXF031 showed only SOS response inhibition (IC 50 =455 and 607 nM, respectively) without strong synergy with CFX-induced DNA damage, suggesting the potential existence of a second target or mechanism of action (Fig. 1D). OXF030 also does not contain the CFX core associated with off-target ‘black-box’ toxicity. Overall, this work has uncovered a divergent SAR in DNA-repair and SOS response inhibitors, suggesting a new mechanism of action for this series which could provide a useful tool to combat the threat of AMR. Figure 1: A) DNA repair and SOS response inhibitors of interest. B) Growth inhibition with half MIC CFX (solid line) and without (dashed line). C) SOS response inhibition measured in GFP reporter assay. D) Comparison of ∆CFX and SOS response inhibition with 1.25 µM of compound.

References 1. O’Neill, Tackling drug-resistant infections globally: final report and recommendations , 2016. A. Cook and G. D. Wright, Sci Transl Med , 2022, DOI:10.1126/scitranslmed.abo7793. S. Q. Lim, K. P. Ha, R. S. Clarke, L. A. Gavin, D. T. Cook, J. A. Hutton, C. L. Sutherell, A. M. Edwards, L. E. Evans, E. W. Tate and T. Lanyon-Hogg, Bioorg Med Chem , 2019, DOI:10.1016/j.bmc.2019.06.025. Lanyon-Hogg, Future Med Chem , 2021, 13, 143–155. DOI:10.4155/fmc-2020-0310

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Novel fluorescent imaging and theranostic probes targeting mitochondria Marta Domínguez-Prieto, Nicoleta Moisoi and Federico Brucoli De Montfort University, UK

Delocalised lipophilic cations (DLCs) are small-molecules that can selectively accumulate in the mitochondria due to the difference in the negative mitochondrial transmembrane potential of cancer cells compared to normal cells. 1 Mitochondria play key roles in several biological processes, including cellular metabolism, generation of cellular chemical energy and overall cellular homeostasis. Dysfunction, or damage, occurring to these organelles is correlated with pathological conditions such as neurodegenerative disorders, cardiovascular diseases, cancer, obesity, and insulin resistance/type 2 diabetes. 2 There is currently much interest in the development of mitochondria-selective fluorescent probes 3 that can be used to monitor the activity of mitochondria within living cells in real-time and investigate mitochondria-associated dysfunctions in certain diseases. Moreover, one can capitalise on the avidity of cancer cells’ mitochondria for fluorescent DLCs and design fluorescent-dyes that function as mitochondria-targeting anti-cancer and diagnostic (“theranostic”) agents. In this work, we have prepared a focussed library of fluorescent probes, in which a series of aromatic and aliphatic heterocyclic rings were appended to the pyridinium moiety of ( E )-4-(1 H -indol-3-ylvinyl)- N -methylpyridinium iodide (F16), a known fluorescent DLC. 4 The resulting novel probes were characterised for optical properties ( i.e ., absorbance, fluorescence, quantum yield), and screened for anti-proliferative activity against cancer cells, i.e. , human bone osteosarcoma epithelial cells (U2OS), and normal cells, i.e. , human dermal fibroblasts (HDF 6C193). Staining of U2OS cells with the novel fluorescent probes at a concentration of 10 μM in water allowed for the visualisation of mitochondria through confocal microscopy. It was found that one of the probes effectively arrested the growth of the U2OS cells and showed low toxicity in normal fibroblast HDF up to a concentration of 50 µM. On the other hand, F16 was found to have very weak growth inhibition properties in the cancer cell lines used in this study even at the highest concentration, i.e., 100 µM. F16 selectively accumulates in the mitochondria of cancer cells dissipating proton gradient across mitochondrial membrane, but this dye has limited anti-proliferative activity and therapeutic efficacy and can be used as a cargo unit to deliver bioactive compounds to the mitochondria. Here, we have shown that connection of heterocyclic rings to the scaffold of F16 via short alkyl-chain spacers yield derivatives with improved anti-proliferative efficacy, albeit maintaining excellent imaging properties, compared to the parent compound. The probes are currently being evaluated for their ability to bind to predetermined structures within mitochondria with the aim of developing selective, fluorescent anti-cancer agents to track, detect and image these organelles. References 1. T Madak, J., Neamati, N., 2015. Membrane permeable lipophilic cations as mitochondrial directing groups. Current Topics in Medicinal Chemistry , 15 (8), pp.745-766. 2. Wang, H., et al. 2021. Recent advances in chemical biology of mitochondria targeting. Frontiers in Chemistry , 9 , p.683220. 3. Crawford H, et al. 2022. Mitochondrial Targeting and Imaging with Small Organic Conjugated Fluorophores: A Review. Chemistry – A European Journal. 28(72). doi:https://doi.org/10.1002/chem.202202366. 4. Fantin, V.R., et al. 2002. A novel mitochondriotoxic small molecule that selectively inhibits tumor cell growth. Cancer cell , 2 (1), pp.29-42.

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Fragment-based drug discovery of SARS-CoV-2 methyltransferase nsp14 inhibitors Anna Lina Bula, Iveta Kanepe, Diana Zelencova-Gopejenko, Laura Drunka, Kristaps Jaudzems Latvian Institute of Organic Synthesis, Latvia The Covid-19 pandemic, caused by the highly infectious SARS-CoV-2, has highlighted the urgent need to create effective antiviral therapies. The nonstructural protein 14 (nsp14) is a key component of the viral replication machinery. It is an S-adenosylmethionine-dependent guanine-N7 methyltransferase necessary for viral mRNA capping that ensures mRNA stability and translation, as well as host immune system evasion. It is therefore an appealing target for drug development. Using the DSI-poised library, we performed a ligand-observed NMR fragment screening of nsp14, specifically exploring the SAM binding pocket. The known SAM analog, sinefungin, was used in the competitive binding experiments. We identified 47 compounds that showed binding to nsp14, two of which were competitive binders. These fragments represent promising starting points for hit-to-lead optimization towards the development of potent and selective nsp14 inhibitors. This work was done as part of the Covid19-NMR consortium and was financially supported by the Latvian Council of Science, grant numbers:VPP-COVID-2020/1-0014 andVPP-EM-BIOMEDICĪNA-2022/1-0001. References 1. Y. Chen, et al. PNAS , 2009, vol. 106 (9 ), 3484-3489. 2. H. Berg, et al. Angew. Chem.Int.Ed. , 2022, vol. 134 (46) , 10.1002/ange.202205858.

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Biocatalytic activity of cytochromes P450 for the valorization of terpenes Danilo Correddu, Giovanna Di Nardo and Gianfranco Gilardi University of Turin, Italy

Cytochromes P450 are a group of heme-containing enzymes that play a significant role in the metabolism of xenobiotics, drugs, and endogenous compounds. Among them, self-sufficient P450s are enzymes that combine all their functional domains in a single polypeptide chain. They can perform regio- and stereo- selective oxidation of a wide range of organic compounds. 1 In recent years, their potential for biocatalysis in the valorization of terpenes has garnered significant attention. Terpenes are a class of natural compounds that are widely distributed in the plant kingdom and possess a variety of biological activities. They have also been identified as a valuable source of bio-based chemicals, including flavors, fragrances, and pharmaceuticals. In our works, we developed laboratory evolved P450 mutants with specific amino acid substitutions, leading to structural rearrangements of the enzyme, which gains flexibility and causes the rotation of a key residue in the active site. This results in enhanced and specific oxidation activity towards different substrates, including terpenes with the formation of valuable oxide derivatives. Their biocatalytic activity can be exploited in different systems including the use of whole cells or purified enzymes, highlighting the relevance of these enzymes for industrial applications. 2 References 1. Correddu, G. Di Nardo and G. Gilardi, Trends Biotechnol. , 2021, 39 , 1184–1207. 2. Correddu, S. H. Aly, G. D. Nardo, G. Catucci, C. Prandi, M. Blangetti, C. Bellomo, E. Bonometti, G. Viscardi and G. Gilardi, RSC Adv. , 2022, 12 , 33964–33969.

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Structural characterization of Borrelia Burgdorferi protein BBA05 Laura Drunka 1 and Kalvis Brangulis 2 1 Latvian Institute of Organic Synthesis, Latvia 2 Latvian Biomedical Research and Study Centre, Latvia Borrelia Burgdorferi is Lyme disease-causing agent in humans that is transferred to the host with the help of Ixodes ticks. About 10 % of its 1.5 mbp genome encodes lipoprotein genes, many of which are important either for survival of B. Burgdorferi or its ability to infect the host. Therefore lipoproteins such as BBA05 are important objectives in Lyme disease research. Previous research suggest that BBA05 might not be essential for survival of B. Burgdorferi but it is unclear if there are other proteins that are compensating the loss of BBA05 or its role in the life cycle is non-essential. This protein can not be found throughout full life cycle of B. Burgdorferi but is expressed when nymphs are fed with blood, suggesting that expression might be induced either by temperature or components of the blood. In our study, we produced recombinant BBA05 and solved the crystal structure to answer some questions about its function. BBA05 is made from two domains (amino acids 140-276 and 277-417) consisting of seven and eight α-spirals, respectively. Despite the relatively low sequence similarity, solved structure appears to be similar to some of other proteins encoded in lp54 plasmid (BBA64 for example), although it is not mentioned in the literature as a member of PFam54 paralogous gene family. This work was supported by the Latvian Council of Science projects VPP-EM-BIOMEDICĪNA-2022/1-0001 and lzp-2021/1-0068. References

1. Casjens, S.et. al. Molecular Microbiology, 2002, 35 (3) , 490-516 2. Xu, H.et. al. Infection and Immunity, 2010, 78 (1) , 100-107 3. Brangulis, K.et. al. Acta Crystallographica Section D, 2013, 69 (6) , 1099-1107

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Biocatalytic reactions of Organosilyl Ethers using Silicatein-α Chisom Egedeuzu 1,2 Matteo Trande 1,2 , Emily Sparkes 1,2 , Rachel Kettles 1,2 , Yuqing Lu 1,2 , Lu Shin Wong 1,2 1 Manchester Institute of Biotechnology, University of Manchester, UK, 2 Department of Chemistry, University of Manchester, UK email: chisom.egedeuzu@manchester.ac.uk Silicatein-α is an enzyme found in spicules of marine sponges with a catalytic triad similar to cathepsin L and it is able to catalyse the condensation of silicon-oxygen bonds, which these organisms use to polymerise silicic acid (dissolved silicates) into inorganic silica for incorporation into their skeletons [1]. Our laboratory has shown recently that silicatein-α from Suberites domuncula (the prototypical member of this family), is able to catalyse both bond hydrolysis and condensation for a range of model organosilyl ethers (i.e. compounds containing C– Si–O moieties), which alludes to the potential application of these enzymes in organosilicon chemistry [2,3,4]. Here, we report our latest results investigating the catalysis of silicatein-α in the context of synthetic chemistry. Specifically, we demonstrated the roles of the active site residues in catalysis (Si-O hydrolysis and silica formation); and a survey of silyl ether bond condensations from the corresponding alcohols and silanols. References 1. K. Shimizu and D. E. Morse, Methods Enzymol. , 2018, 605 , 429-455. 2. E. I. Sparkes, C. S. Egedeuzu, B. Lias, R. Sung, S. A. Caslin, S. Y. Tabatabaei Dakhili, P. G. Taylor, P. Quayle, L. S. Wong, Catalysts , 2021, 11 , 879. 3. S. Y. Tabatabaei Dakhili, S. A. Caslin, A. S. Faponle, P. Quayle, S. P. de Visser, L. S. Wong, Proc. Natl. Acad. Sci. , 2017, 114 , E5285-E5291. 4. E. I. Sparkes, R. A. Kettles, C. S. Egedeuzu, N. L. Stephenson, S. A. Caslin, S. Y. Tabatabaei Dakhili, L. S. Wong, Biomolecules , 2020, 10 , 1209.

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