WORKSHOP ON

DRUG DISCOVERY AND NANOMEDICINE

Porto Alegre

March 29 to 30, 2016

Federal University of Rio Grande do Sul

Institute of Chemistry

Av. Bento Gonçalves, 9500

Amphitheatre (43111, A212)

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Federal University of Rio Grande do Sul is pleased to held the Workshop on Drug

Discovery and Nanomedicine

President: Prof. Carlos Alexandre Netto

Vice President: Prof. Rui Vicente Oppermann

Center of Nanoscience and Nanotechnology

Director: Prof. Sílvia S. Guterres

Vice Director: Dr. Naira M. Balzaretti

Institute of Chemistry

Director: Prof. Nadya Pesce da Silveira

Vice Director: Dr. Henri Stephan Schrekker

Newton Fund/UK Deputy: Ms. Fernanda Silva

Workshop on Drug Discovery and Nanomedicine

Coordinators:

Prof. Adriana R. Pohlmann (UFRGS)

Dr. Nicholas Holliday (Univ. Nottingham)

Mentors:

Dr. Rafael Roesler (UFRGS)

Prof. Steven Charlton (Univ. Nottingham)

Our goal is to bring together a UK and Brazilian cohort of early career researchers to

take part in a workshop focusing on building links for future collaboration and enhancing

the researchers’ career opportunities. The workshop content is centred on drug

discovery and nanomedicine.

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PROGRAM

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Tuesday, March 29th

8:30 Opening Ceremony

9:00 Conference 1 Prof. Steven Charlton (University of Nottingham, UK)

The importance of target binding kinetics in drug discovery

9:45 Lecture 1 Dr. Brian Hudson (University of Glasgow, UK)

Unlocking the Therapeutic Potential of the FFA4 Long Chain Fatty Acid Receptor

10:15 Lecture 2 Dr. Dyeison Antonow (PUC-RS, Brazil)

Drug Delivery Systems: Targeted Therapies based on Antibody-Drug Conjugates (ADCs)

10:45 Pause

11h:10 Pharmacology 101 – session 1

11h:50 Lecture 3 Dr. Sophie Bradley (Medical Research Council Toxicology Unit, UK)

Targeting the M1 muscarinic acetylcholine receptor in neurodegeneration

12h:20 Discussion

1:00 Lunch

2:00 Conference 2 Dr. Rafael Roesler (UFRGS, Brazil)

Gastrin-releasing peptide receptors in the central nervous system: role in brain function and as a drug target

2:45 Lecture 4 Dr. Margaret Cunningham (University of Strathclyde, UK)

A multidisciplinary approach to thrombin receptor research: Targeting proteinase-activated receptors as an antiplatelet strategy

3:15 Lecture 5 Dr. Marcelo Bispo de Jesus (UNICAMP, Brazil)

Cellular Mechanisms in Nanomaterial Internalization, Intracellular Trafficking, and Toxicity

3:45 Pause

4:10 Pharmacology 101 – session 2

6:00 Return to the Hotel

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Wednesday, March 30th

9:00 Conference 3 Prof. Adriana R. Pohlmann (UFRGS, Brazil)

Polymeric nanocapsules as promising nanocarriers in therapeutics

9:45 Lecture 6 Dr. André Luís Branco de Barros (UFMG, Brazil)

Radiolabeled nanoparticles as alternative tool for cancer diagnosis and therapy

10:15 Lecture 7 Dr. Irene Clemes Kulkamp Guerreiro (UFRGS, Brazil)

Antifungal and antiretroviral drugs nanoencapsulation: Could it be an efficient strategy to improve actual therapy?

10:45 Pause

11:10 Pharmacology 101 – session 3

11:50 Lecture 8 Dr. Javier Hernández Gil (Imperial College London, UK)

Tumour-Targeted and Matrix Metalloproteinase-Responsive Iron Oxide Nanoparticles for Theranostic Applications

12:20 Discussion

1:00 Lunch

2:00 Conference 4 Dr. Nicholas Holliday (University of Nottingham, UK)

Shedding light on G protein coupled receptor pharmacology using fluorescence complementation and imaging approaches

2:45 Lecture 9 Dr. Elizabeth Rosethorne (University of Nottingham, UK)

Using Phenotypic Assays to Explore Ep4 Agonism in Airway Remodelling

3:15 Lecture 10 Dr. Ana Rosa Lopes Pereira Ribeiro (INMETRO, Brazil)

Biomineralization of Anatase Nanoparticles and its Implications in Bone cells Survival

3:45 Pause

4:00 Group competition – due diligence exercise and Final remarks

6:30 Return to the Hotel

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COORDINATORS AND MENTORS

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Coordinators

Associate Prof Nicholas D Holliday

Dr Nick Holliday was appointed Associate Professor of Molecular Pharmacology in 2013, after previously

holding a Lectureship (since 2011) and a five year research fellowship in the School of Life Sciences at

Nottingham. Following first class honours from the University of Cambridge (1994), Nick carried out his

PhD studies at King’s College London (1998), supported by a prize AJ Clark PhD studentship from the

British Pharmacological Society. It was during subsequent postdoctoral work in London that his interests in

peptide messengers regulating appetite and metabolism became focused on molecular mechanisms

underlying the signalling and regulation of their receptors, which led to the Nottingham appointment.

Since then, Dr. Holliday has built a research group investigating these and other G protein coupled

receptors (GPCRs), the largest class of drug targets in man. He has specialised in quantitative

pharmacological and signalling assays based on novel fluorescence techniques and automated imaging for

use in drug discovery, with 14 primary papers since 2010. His particular expertise in bimolecular

fluorescence complementation methods to study signalling partner interactions arose initially through

Medical Research Council funding establishing assays for GPCR association with arrestin proteins, and to

quantify pharmacology of defined receptor oligomers (Kilpatrick et al. (2010) Br J Pharmacol; Watson et al

(2012) Mol Pharmacol; Kilpatrick et al. (2015) Mol Pharmacol). Dr Holliday also pioneered developments of

this technology necessary to study signalling complexes using advanced imaging methods such as

fluorescence correlation spectroscopy (Kilpatrick et al (2012) Biochimica Biophysica Acta Mol Cell Res), and

adaptation of these techniques to membrane transporters and transcription factors (Evans et al (2011) Nuc

Acids Res; Haider et al (2011) PloS ONE; Alqahtani et al (2014) Open Biol; Wong et al (2015) Biochimica

Biophysic Acta, in press). This work was recognised by the prize award of the 2011 Bill Bowman lectureship

from the British Pharmacological Society, together with several invited reviews (e.g. Kilpatrick & Holliday

(2012) Methods Mol Biol; Sivertsen et al (2013) Br J Pharmacol; Stott et al (2015) Biochem Pharmacol in

press). It continues to support high calibre international academic collaborations (e.g. Mountford et al

(2014) Org Biomol Chem; Valentin-Hansen et al (2015) J Biol Chem), including as UK lead for CAPES Drug

Discovery Award held by Prof Roesler, together industrial funding (e.g. GSK, AstraZeneca, NovoNordisk).

Dr Holliday leads undergraduate pharmacology modules in pharmacy and neuroscience, and has trained 9

PhD students since 2009, three of which have won prize studentships or presentation prizes at

international conferences. He is on the editorial board of Pharmacology Research and Perspectives, and

also has been actively involved in public engagement, including an arts crossover project to explain the use

of imaging in pharmacology (“Hijacking Natural Systems”), funded by the Wellcome Trust.

Prof. Adriana R. Pohlmann

Adriana R. Pohlmann, Full Professor of Organic Chemistry at the Chemistry Institute of the Universidade

Federal do Rio Grande do Sul in Porto Alegre, Brazil, received her Graduation in Pharmacy (1985) and

Master in Chemistry (1991) at UFRGS, and Doctorate Degree in Therapeutic Chemistry at the University of

Paris V, France (1997). In 1998, she received the prestigious Roussel-Uclaf award for her Dissertation and a

Laureate Diploma from the College of Pharmaceutical and Biological Sciences, University of Paris V, France.

She served as Head of the Department of Organic Chemistry (1999-2001), as First Director of the Center of

Nanoscience and Nanotechnology at UFRGS (2006), as member of the Committee of the Postgraduate

Program in Chemistry (2001-2003; 2009-2011), Coordinator of the Post-Graduate Program on

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Pharmaceutical Nanotechnolgy (2013-2015) and as Vice-Director of the Institute of Chemistry (2003-2007).

She also served as the Vice-Director of the Brazilian National Nanotechnology Network, and Coordinator

of an international collaborative IBSA project between India, Brazil and South Africa both supported by the

Brazilian Ministry of Science and Technology. She is a recognized researcher at the National Council for

Scientific and Technological Development (CNPq/Brazil) leading the group: Micro- and nanoparticles for

therapeutics. She currently advises Graduate and Post-Graduate students in Chemistry, Pharmaceutical

Sciences and Pharmaceutical Nanotechnology. Her main research is focused on the organic chemistry

applied to drug nanocarriers, including polymeric nanocapsules and nanospheres, with the view of

understanding and controlling their sizes, shape, surface and physico-chemical properties. She has

published more than 200 peer-reviewed articles, 3 books and 19 book chapters. Her research group holds

50 patents and transferred 7 products to a Brazilian Company, reaching the market since 2009. She

currently serves as Ad-hoc reviewer for more than 25 International Scientific Journals, for Brazilian

Agencies to support scientific research, and as Associate Editor of the Journal of Nanoscience and

Nanotechnology and the Journal of Colloid Science and Biotechnology, Editorial Board member of the

Journal of Nanopharmaceutics and Drug Delivery and the Journal of Biomedical Nanotechnology

(American Scientific Publishers). She is also a member of the Brazilian Association of Pharmaceutical

Scientists and of the Brazilian Chemical Society.

Mentors

Prof Steven Charlton

Prof Charlton has recently joined the University of Nottingham where he is Professor of Molecular

Pharmacology and Drug Discovery. Prior to that he spent 16 years in the pharmaceutical industry, both at

SmithKline Beecham and Novartis. At Novartis he was Director of Molecular Pharmacology in Respiratory

Diseases, leading an assay development and compound profiling team of 30 scientists providing expert

opinion and support for GPCR, ion channel and enzyme projects. He has broad drug discovery experience,

ranging from target validation through to leading full lead optimisation programmes to successful clinical

proof of concept. He is interested in all aspects of the quantitative assessment of ligand-receptor

interactions, with a particular interest in the kinetics of ligand binding and signalling. Prof Charlton serves

as an editor of the British Journal of Pharmacology and is actively engaged in training new

pharmacologists, working closely with the British Pharmacological Society to organise scientific symposia

and teaching workshops. Prof Charlton was awarded Novartis Leading Scientist in 2007.

Associate Prof Rafael Roesler

Dr. Rafael Roesler, Associate Professor in the Department of Pharmacology, Institute for Basic Health

Sciences, Federal University of Rio Grande do Sul has published more than 190 articles, 11 book chapters

and holds 6 patents. Head, Cancer and Neurobiology Laboratory, University Hospital Experimental

Research Centre (CPE-HCPA) focuses his research interest on biology and pharmacology of

neurotransmitter and neuropeptide receptors; neurobiology and neuropharmacology of synaptic plasticity

and memory; brain tumour biology and pharmacology; cell signalling in brain function, brain disorders,

and cancer.

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ABSTRACTS

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The importance of target binding kinetics in drug discovery

Prof Steven Charlton, Molecular Pharmacology and Drug Discovery, University of Nottingham

Optimizing the receptor binding kinetics of new drugs can have significant benefits, ranging from

improved duration of action to enhanced efficacy through the insurmountable antagonism of dynamic

physiological systems. It is also becoming apparent that binding kinetics plays a role in the phenomenon of

biased agonism. Despite this, the kinetics of new receptor ligands are rarely measured early in the drug

discovery process, largely because current assays are technically difficult and relatively low-throughput.

This talk will introduce the potential clinical benefits of optimising binding kinetics and review the current

methods for measuring kinetic parameters. It will then describe the development of a novel approach

using time-resolved FRET in continuous read mode that is capable of simultaneously measuring the

kinetics of hundreds of compounds. This offers the potential for placing a kinetics assay at the top of a

screening cascade, negating the need to first run “IC50 curves” to assess affinity at the receptor. It also

presents the opportunity to screen fragment libraries at receptors in a kinetic mode. Finally, the talk will

end with a discussion on the concept of “micro pharmacokinetics” and how the local drug concentrations

around the receptor must be taken into consideration when interpreting the kinetics of new receptor

ligands.

Selected publications

Sykes DA, Bradley ME, Riddy DM, Willard E, Reilly J, Miah A, Bauer C, Watson SJ, Sandham DA, Dubois G,

Charlton SJ (2016) Fevipiprant (QAW039) a slowly dissociating CRTh2 antagonist with the potential for

improved clinical efficacy. Mol Pharmacol. mol.115.101832. [Epub ahead of print]

Klein Herenbrink C, Sykes DA, Donthamsetti P, Canals M, Coudrat T, Shonberg J, Scammells PJ, Capuano B,

Sexton PM, Charlton SJ, Javitch JA, Christopoulos A, Lane JR (2016) The role of kinetic context in apparent

biased agonism at GPCRs. Nat Commun. 24;7:10842

Rosethorne EM, Bradley ME, Gherbi K, Sykes DA, Sattikar A, Wright JD, Renard E, Trifilieff A, Fairhurst RA,

Charlton SJ (2016) Long Receptor Residence Time of C26 Contributes to Super Agonist Activity at the

Human β2 Adrenoceptor. Mol Pharmacol. mol.115.101253. [Epub ahead of print]

Sykes DA, Parry C, Reilly J, Wright P, Fairhurst RA, Charlton SJ (2014) Observed drug-receptor association

rates are governed by membrane affinity: The importance of establishing "micro PK/PD relationships" at

the β2-adrenoceptor. Mol Pharmacol 85(4):608-17

Sykes DA, Dowling MR, Leighton-Davies J, Kent TC, Renard E, Trifilieff A, Charlton SJ. (2012) The Influence

of Receptor Kinetics on the Onset and Duration of Action, and the Therapeutic Index of NVA237 and

Tiotropium. J Pharmacol Exp Ther. 343(2):520-8

Sykes DA, Dowling MR, Charlton SJ (2010) Measuring receptor target coverage: A radioligand competition

binding protocol for assessing the association and dissociation rates of unlabeled compounds. Curr.

Protoc. Pharmacol. 50:9.14.1-9.14.30

Vauquelin G, Charlton SJ (2010) Long-lasting target binding and rebinding as mechanisms to prolong in

vivo drug action. Br J Pharmacol 161(3):488-508

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Unlocking the Therapeutic Potential of the FFA4 Long Chain Fatty Acid Receptor

Dr Brian Hudson, Leadership Fellow, Institute of Molecular Cell and Systems Biology, University of Glasgow

http://www.gla.ac.uk/researchinstitutes/biology/staff/brianhudson/

Abstract: I am interested in the link between the fats obtained through diet and our health. In particular,

long chain fatty acids derived from dietary fats regulate two G protein-coupled receptors: FFA1 and FFA4.

My research focuses on building our understanding of these receptors, in particular FFA4, in order to

better understand how to target them for metabolic disease. This has led to the identification of the first

potent and selective FFA4 agonist, detailed characterization of its pharmacology, and defining the

molecular basis for its interaction with FFA4. To further refine our understanding of the therapeutic

potential of FFA4, my current focus is on developing biosensor technologies, based primarily on

bioluminescence resonance energy transfer, to assess complex aspects of FFA4 pharmacology; including:

ligand binding kinetics, receptor activation kinetics, active receptor conformations, and signaling bias. My

long-term goal is to use these biosensors to identify the optimal properties for FFA4 targeting

therapeutics.

Selected publications (FFA4 related marked with *):

*Prihandoko R., Alvarez-Curto, E., Hudson, B.D., Butcher, A.J., Ulven, T., Miller, A.M., Tobin, A.B., Milligan, G. Distinct

phosphorylation clusters determines the signalling outcome of the free fatty acid receptor FFA4/GPR120. (2016) Mol.

Pharmacol. Doi: mol.115.101949.

*Christiansen, E., Watterson, K.R., Stocker, C.J., Sokol, E., Jenkins, L., Simon, K., Grundmann, M., Petersen, R.K., Wargent,

E.T., Hudson, B.D., Kostenis, E., Ejsing, C.S., Cawthorne, M.A., Milligan, G., and Ulven, T. Activity of dietary fatty acids on

FFA1 and FFA4 and characterisation of pinolenic acid as a dual FFA1/FFA4 agonist with potential effect against

metabolic diseases. (2015) Br. J. Nutr. 113, 1677-1688.

*Hudson, B.D., Shimpukade, B., Ulven, T., and Milligan G. Defining the orthosteric binding pocket of free fatty acid

receptor 4 (GPR120). (2014) J. Biol. Chem. 289, 20345-20358.

*Butcher, A.J., Hudson, B.D.+, Shimpukade, B., Alvarez-Curto, E., Prihandoko, R., Ulven, T., Milligan, G., and Tobin, A.B.

Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the

Free Fatty Acid Receptor FFA4. (2014) J. Biol. Chem. 289, 18451-18465.

*Hudson, B.D., Shimpukade, B., Mackenzie, A.E., Butcher, A.J., Pediani, J.D., Christiansen, E., Heathcote, H., Tobin, A.B.,

Ulven, T., and Milligan, G. The pharmacology of a potent and selective agonist, TUG-891, demonstrates both potential

opportunity and possible challenges to therapeutic agonism of FFA4 (GPR120). (2013) Mol. Pharmacol. 84, 710-725.

Hudson, B.D., Due-Hansen, M.E., Christiansen, E., Hansen, A.M., Mackenzie, A.E., Murdoch, H., Pandey, S.K., Ward, R.J.,

Marquez, R., Tikhonova, I.G., Ulven, T., and Milligan, G. Defining the molecular basis for the first potent and selective

orthosteric agonists of the FFA2 free fatty acid receptor. (2013) J. Biol. Chem. 288, 17296-17312.

Hudson, B.D., Tikhonova, I.G., Pandey, S.K., Ulven, T., and Milligan, G. Extracellular ionic locks determine variation in

constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3.

(2012) J. Biol. Chem. 287, 41195-41209.

Hudson, B.D., Christiansen, E., Tikhonova, I.G., Grundmann, M., Kostenis, E., Adams, D.R., Ulven, T., and Milligan, G.

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between

species orthologs. (2012) FASEB J. 26, 4951-4965.

*Shimpukade, B., Hudson, B.D., Hovgaard, C.K., Milligan, G., and Ulven, T. Discovery of a potent and selective GPR120

agonist. (2012) J. Med. Chem. 55, 4511-4515.

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Drug Delivery Systems: Targeted Therapies based on Antibody-Drug Conjugates (ADCs).

Dr. Dyeison Antonow: Associate Professor at the Faculty of Medicine – PUCRS.

http://lattes.cnpq.br/2698938968819675

Abstract: Biological agents such as therapeutic technologies based on monoclonal antibodies (mAbs) are

established in the pharmaceutical market, especially in oncology. Trastuzumab (Herceptin®) and

cetuximab (Erbitux®) are good examples of this class of targeted therapies against cancer. Therapeutic

mAbs are selectively active against a variety of tumour types and considerably less toxic when compared

with cytotoxic agents from standard chemotherapy. However, these biological agents may have problems

related to resistance and are associated with a number of other challenges in the clinical setting. An

exciting development in the targeted therapy is the move towards antibody-drug conjugates (ADCs). In

general, ADCs are complex drug-delivery systems made of three main components: mAb, linker (which

can be self-immolative), and the drug itself, often called the “payload”. While biologically-inactive

themselves, these “warheads” or “payloads” can be released from the therapeutic antibody to form potent

cytotoxic compounds at the antibody-targeted tumour site. The first ADCs used molecules such as the

enediynes or maytansinoids that suffer from significant drawbacks including high molecular weight and

limited availability from natural sources. Even so, a number of ADCs have made to the clinic and they are

advancing rapidly through pharmaceutical industries pipelines. For example, in 2011 the FDA granted

accelerated approval to brentuximab vedotin (Adcetris®, Seattle Genetics) for two types of lymphoma.

More recently the FDA has approved ado-trastuzumab emtansine (Kadcyla®, Genentech/Roche), for

patients with HER2-positive metastatic breast cancer who had previously received treatment with

trastuzumab. Importantly, ADCs still have a number of regulatory challenges and is a very active area of

research. Highly innovative aspects of ADC-based therapies make them particularly attractive for

entrepreneurial initiatives and a number of companies have been exploring the ADC technology towards

clinical benefits. In this session, Dr. Antonow (PUCRS, Brazil) will discuss the use of natural products as

payloads, linker strategies, and basic aspects for the generation of ADCs as candidates for clinical

development.

Recommended reading:

Flygare, J. A.; Pillow, T. H.; Aristoff, P. Antibody-Drug Conjugates for the Treatment of Cancer. Chemical Biology &

Drug Design, 81, p. 113-121, 2013.

http://onlinelibrary.wiley.com/doi/10.1111/cbdd.12085/abstract;jsessionid=858C80080E0CD46D279B35BB52A266A2.f01t

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Bouchard, H.; Viskov, C.; Garcia-Echeverria, C. Antibody–drug conjugates—A new wave of cancer drugs, Bioorganic &

Medicinal Chemistry Letters, 24 (23), p. 5357-5363, 2014.

http://www.sciencedirect.com/science/article/pii/S0960894X1401066X

Antonow, D & Thurston, D. E. Synthesis of DNA-Interactive Pyrrolo[2,1-c][1,4]benzodiazepines (PBDs). Chemical

Reviews, 111 (4), p. 2815-2864, 2011.

http://pubs.acs.org/doi/abs/10.1021/cr100120f

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Targeting the M1 muscarinic acetylcholine receptor in neurodegeneration

Dr. Sophie Bradley, Career Development Fellow, MRC Toxicology Unit, Leicester

Abstract: Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s, and the less-common

prion diseases, are characterized by progressive neuronal atrophy and cognitive dysfunction. Muscarinic

acetylcholine receptors (mAChRs) regulate an array of CNS processes, including cognitive, behavioural and

motor functions. The M1 mAChR subtype is widely expressed post-synaptically in the cortex and the

hippocampus (areas known to be important in learning and memory) and here we show that M1 deficient

mice have a deficit in hippocampal-dependent learning and memory. In this study, we have used a well-

established mouse model of neurodegeneration to further explore the therapeutic potential of M1 mAChR

ligands in restoring cholinergic function and cognition in AD.

Prion-infected tg37 mice develop early pathological changes at 7 weeks post infection (wpi) with Rocky

Mountain Laboratory (RML) scrapie prion. At 9 wpi, mice display a decline in burrowing behaviour and an

abrupt reduction in synaptic proteins, rapidly followed by neurodegeneration, with 50% loss of

hippocampal pyramidal neurons by 10 wpi (Moreno et al., 2012). Choline acetyltransferase levels in the

hippocampus of prion-infected mice are reduced from 9 wpi, indicating degeneration of cholinergic

neurons. However, M1 mAChR expression and G-protein coupling at 9- and 10 wpi is maintained. Prion-

diseased mice display reduced fear conditioning responses at 9 wpi. This impairment in learning and

memory is rescued by xanomeline, an M1 and M4 orthosteric agonist, and also by BQCA and BQZ12, M1-

specific allosteric agonists. Furthermore, we show that xanomeline modulates postsynaptic activity and

AMPA receptor phosphorylation in the hippocampus of prion-diseased mice.

In conclusion, prion-infected mice undergo cholinergic degeneration in the hippocampus, which is

accompanied by a significant reduction in fear learning and memory. We show that targeting muscarinic

receptor activity, with both orthosteric and allosteric ligands, can rescue the cognitive deficit in prion-

diseased mice.

Selected publications:

Bradley, S.J., Bourgognon, J.M., Butcher, A.J., Sanger, H.E., Verity, N.C., Moreno, J.A., Molloy, C., Macedo-Hatch, T.,

Edwards, J.M., Wess, J., Pawlak, R., Read, D.J., Sexton, P.M., Broad, L.M., Mallucci, G.R., Christopoulos, A., Felder, C.C.,

Steinert, J.R. and Tobin, A.B. M1 muscarinic acetylcholine receptor allosteric modulators rescue memory loss in prion

neurodegeneration. J. Clin Invest., submitted.

Bradley, S.J., Wiegman C.H.C., Iglesias, M.M., Choi Kong, K.C., Butcher, A.J., Goupil, E., Bourgognon, J.M., Macedo-

Hatch, T., Russell, K., Laporte, S.A., Kostenis, E., Bouvier, M., Chung, K.F., Amrani, Y. and Tobin, A.B. (2016) Mapping

Physiological G protein-Coupled Receptor Signaling Pathways Reveals Role For Receptor Phosphorylation In Airway

Contraction. Proc. Natl. Acad. Sci., in press.

Butcher, A.J., Bradley, S.J., Prihandoko, R., Mogg, A., Bourgognon, J.M., Macedo-Hatch, T., Edwards, J.M., Bottrill, A.,

Challiss, R.A.J., Broad, L.M., Felder, C.C. and Tobin, A.B. (2016) Antibody biosensor detects G-protein coupled receptor

activation in vivo. J. Biol. Chem., in press.

Bradley, S.J. and Tobin, A.B. (2016) Design of next generation G protein coupled receptor-drugs - Linking novel

pharmacology and in vivo animal models. Annu. Rev. Pharmacol. Toxicol. 56: 535–559.

Bradley, S.J., Riaz, S.A. and Tobin, A.B. (2014) Employing novel animal models in the design of clinically efficacious

GPCR ligands. Curr. Opin. Cell Biol. 27 (100): 117-125.

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Gastrin-releasing peptide receptors in the central nervous system: role in brain function and as a

drug target

Prof. Rafael Roesler, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of

Rio Grande do Sul, Porto Alegre, RS, Brazil; Cancer and Neurobiology Laboratory, Experimental Research

Center, Clinical Hospital (CPE-HCPA), Porto Alegre, RS, Brazil

Abstract: Neuropeptides act as signaling molecules that regulate a range of aspects of brain function.

Gastrin-releasing peptide (GRP), a mammalian neuropeptide homolog of the amphibian peptide

bombesin, acts by binding to the GRP receptor (GRPR, also called BB2), a member of the G-protein

coupled receptor (GPCR) superfamily. GRPR signaling modulates memory formation, serving as a

component of the set of neurobiological systems underlying the enhancement of memory storage by

emotionally arousing information. In addition, some alterations in the GRP/GRPR system have been

described in patients with neurological disorders or brain tumors. Findings from preclinical models are

consistent with the view that drugs acting on the GRPR might ameliorate cognitive and social deficits

associated with neurological diseases and reduce the growth of brain cancer.

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A multidisciplinary approach to thrombin receptor research: Targeting proteinase-activated

receptors as an antiplatelet strategy.

Dr Margaret Cunningham, Chancellor Research Fellow, University of Strathclyde

https://pure.strath.ac.uk/portal/en/persons/margaret-rose-cunningham(a549beb6-badd-4784-a5b6-

e5986d394fcc).html

Abstract: The focus of our research is identifying targets for the development new or improved antiplatelet

therapies. One target of interest is the GPCR family called proteinase-activated receptors (PARs), which are

known to regulate thrombin activity in hemostasis and thrombosis. We have employed a variety of

approaches to identify regulatory pathways that could be exploited to target members of the PAR family

and limit the pro-thrombotic activity of thrombin. These approaches include SILAC mass spec-based

quantitative proteomics to identify PAR-protein and variant PAR-protein interacting complexes. Recently

we have identified patients harboring a novel PAR4 variant with defective receptor trafficking that result in

inhibition of platelet aggregation, activation and a mild bleeding phenotype. Follow-up proteomic analysis

has revealed critical changes in the variant PAR4 interactome and highlighted potentially new paradigms

for GPCR regulation which we are currently investigating. In addition to this, we are currently screening

new (and existing) synthetic modulators of PARs to assess their pharmacology and determine their use as

inhibitors of platelet activation and aggregation. The plan is to test these further on a recently customized

multi-panel biomimetic microfluidic thrombosis device (IC50-on-a-chip).

Selected publications (PAR starred):

*Cunningham, M.R., McIntosh, K.A., Bushell, T.J., Sloan, G., and Plevin, R. (2016) Proteinase-activated receptors

(PARs) as targets for antiplatelet therapy. Biochemical Society Transactions. DOI: 10.1042/BST20150282 Accepted

05/02/2016 (Review)

*Norman, J.E., Cunningham M.R., Jones M.J., Westbury, S.K., Walker, M, Mundell S.J. and Mumford A.D., (2016) The

p.Tyr157Cys variant in Protease Activated Receptor 4 reduces platelet functional responses and alters receptor

trafficking. ATVB (ref: ATVB/2015/207102R1). Accepted 22/02/2016.

*Stockley, J., Nisar, S.P., Leo, V.C., Sabi, E., Cunningham, M.R., Eikenboom, J.C., Lethagen, S., Schneppenheim, R.,

Goodeve, A.C., Watson, S.P., Mundell, S.J., Daly, M.E., and group, G.s.i.c.w.t.M.-V.s. (2015) Identification and

Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically

Diagnosed with Type 1 von Willebrand Disease. PLoS One. 10, e0143913. PMID: 26630678

*Nisar, S.P., Jones, M.L., Cunningham, M.R., Mumford, A.D., Mundell, S.J. and Group, U.G.S. (2014) Rare platelet G

protein-coupled receptor variants: What Can We Learn? Br J Pharmacol. PMID: 25231155

Nisar, S.P., Lordkipanidze, M., Jones, M.L., Dawood, B., Murden, S., Cunningham, M.R., Mumford, A.D., Wilde, J.T.,

Watson, S.P., Mundell, S.J., Lowe, G.C., and group, U.G.s. (2014) A novel thromboxane A2 receptor N42S variant

results in reduced surface expression and platelet dysfunction. Thromb Haemost. 111, 923-32. PMID: 24452735

*Cunningham, M.R., McIntosh, K.A., Pediani, J.D., Robben, J., Cooke, A.E., Nilsson, M., Gould, G.W., Mundell, S.,

Milligan, G. and Plevin, R. (2012) Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of

proteinase-activated receptor 4 (PAR4). J Biol Chem. 287, 16656-69. PMID: 22411985

19

Cellular Mechanisms in Nanomaterial Internalization,

Intracellular Trafficking, and Toxicity

Dr Marcelo Bispo de Jesus, Associate Professor of Biochemistry, University of Campinas

https://scholar.google.com.br/citations?user=PGvSnuQAAAAJ&hl=en

Abstract: Nanomaterials are expected to have a significant impact on medicine, although they still need to

overcome several challenges before they are widely used. Understanding the molecular interaction of

nanomaterials in the context of the cellular environment is crucial for the successful applications of

nanomaterials. Therefore, the groups’ primary research goals are directed towards understanding the

molecular basis of interactions between nanomaterials and eukaryotic cells (animal and plants). More

specifically we are interested in intracellular delivery of contents (e.g., gene delivey) and toxic effects of

nanomaterials to cells (nanotoxicity). In the past years we were acting on the following topics: endocytosis

and intracellular trafficking of nanoparticles, nanotoxicology (in vitro and in vivo), solid lipid nanoparticles

and non-viral transfection systems (gene delivery).

Selected publications:

Research articles:

Microfluidic Assembly of pDNA/Cationic Liposome Lipoplexes with High pDNA Loading for Gene Delivery. Balbino TA,

Serafin JM, Malfatti-Gasperini AA, de Oliveira CL, Cavalcanti LP, de Jesus MB, de La Torre LG. Langmuir, 2016.

PMID: 26814663

Silver Nanoparticles: A New View on Mechanistic Aspects on Antimicrobial Activity. Durán N, Durán M, de Jesus MB,

Seabra AB, Favaro WJ, Nakazato G. Nanomedicine, 2015. PMID: 26724539

Solid lipid nanoparticles as nucleic acid delivery system: Properties and molecular mechanisms. de Jesus MB, Zuhorn IS.

Journal of Controlled Release, 2015. PMID: 25578828

Reduced graphene oxide induces transient blood-brain barrier opening: an in vivo study. Mendonca M, Soares E, de

Jesus MB, Ceragioli H, Ferreira M, Catharino R, da Cruz-Hofling M. Journal of Nanobiotechnology, 2015. PMID:

26518450

Evaluation of the side effects of poly(epsilon-caprolactone) nanocapsules containing atrazine toward maize plants.

Oliveira HC, Stolf-Moreira R, Martinez CBR, Sousa GFM, Grillo R, de Jesus MB, Fraceto LF. Frontiers in Chemistry,

2015. PMID: 26539429

Nanoencapsulation Enhances the Post-Emergence Herbicidal Activity of Atrazine against Mustard Plants. Oliveira HC,

Stolf-Moreira R, Martinez CBR, Grillo R, de Jesus MB, Fraceto LF. PLoS ONE, 2015. PMID: 26186597

Book chapter:

"Cellular Mechanisms in Nanomaterial Internalization, Intracellular Trafficking, and Toxicity". de Jesus, M. B., Kapila, Y. L.

Nanomedicine and Nanotoxicology. 版, New York, NY, Springer New York, 2014, no. 9, pp. 201–227,

(Nanomedicine and Nanotoxicology, ISBN978-1-4614-8993-1.

20

Polymeric nanocapsules as promising nanocarriers in therapeutics

Prof. Adriana Raffin Pohlmann, Institute of Chemistry, Federal University of Rio Grande do Sul

http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4799738U0&idiomaExibicao=2

Abstract: Biodegradable nanocarriers have been studied as a promising alternative to

therapeutics. The control of size distribution, by using self-assembly methods of preparation,

affects the drug biodistribution and release. Some advantages of the nanoparticulate systems are

related to the drug targeting reducing side effects and increasing therapeutic index. The

presentation addresses the aspects of the synthesis of lipid-core nanocapsules, an original type

of carrier useful to encapsulate poorly water-soluble drugs, as well as their surface

functionalization by organometallic complex formation at the surface of multi-wall nanocapsules.

Examples of physico-chemical characterization and biological applications of surface-

functionalized lipid-core nanocapsules are discussed: i) LDL(-) molecular recognition aiming

atherosclerosis prevention, and ii) enzyme replacement therapy in pre-clinical model of

Mucopolysaccharidosis type I. (CNPq, CAPES, FAPERGS)

Selected publications:

FRACASSO, R et al. Evaluation of potential acute cardiotoxicity of biodegradable nanocapsules in rats by intravenous

administration. Toxicology Research, v. 5, p. 168-179, 2016.

MATHES, C. et al. Nanocarriers for optimizing the balance between interfollicular permeation and follicular uptake of

topically applied clobetasol to minimize adverse effects. Journal of Controlled Release, v. 223, p. 207-214, 2016.

DE BRUM, T. et al. Polymeric Nanocapsules and Lipid-Core Nanocapsules Have Diverse Skin Penetration. Journal of

Nanoscience and Nanotechnology (Print), v. 15, p. 773-780, 2015.

FONSECA, F. N. et al. Mucoadhesive Amphiphilic Methacrylic Copolymer-Functionalized Poly(-caprolactone)

Nanocapsules for Nose-to-Brain Delivery of Olanzapine. J Biomed Nanotechnol, v. 11, p. 1472-1481, 2015.

DIMER, F. A. et al . Nanoencapsulation Improves Relative Bioavailability and Antipsychotic Effect of Olanzapine in Rats.

J Biomed Nanotechnol, v. 11, p. 1482-1493, 2015.

FIGUEIRO, F. et al. Pharmacological Improvement and Preclinical Evaluation of Methotrexate-Loaded Lipid-Core

Nanocapsules in a Glioblastoma Model. J Biomed Nanotechnol, v. 11, p. 1808-1818, 2015.

MAYER, F.Q. et al. Laronidase-Functionalized Multiple-Wall Lipid-Core Nanocapsules: Promising Formulation for a

More Effective Treatment of Mucopolysaccharidosis Type I. Pharmaceutical Research, v. 32, p. 941-954, 2015.

BULCÃO, R P. et al. Do poly(epsilon-caprolactone) lipid-core nanocapsules induce oxidative or inflammatory damage

after in vivo subchronic treatment?. Toxicology Research, v. 4, p. 994-1005, 2015.

GARCIA, S et al. Caenorhabditis elegans as an alternative in vivo model to determine oral uptake, nanotoxicity, and

efficacy of melatonin-loaded lipid-core nanocapsules on paraquat damage. Int. J Nanomed, v. 10, p. 5093-5106, 2015.

BENDER, E. A. et al. New strategy to surface functionalization of polymeric nanoparticles: one-pot synthesis of scFv

anti-LDL(-)-functionalized nanocapsules. Pharmaceutical Research, v. 31, p. 2975-2987, 2014.

21

Radiolabeled nanoparticles as alternative tool for cancer diagnosis and therapy

Dr Andre Luis Branco de Barros, Assistant Professor of Radiopharmacy, Universidade Federal de Minas

Gerais

Abstract: Our group focuses on the development new nanoplatforms for detection and treatment of

cancer. In this specific field, we have been working with radiolabeled nanoparticles (liposomes, micelles,

solid lipid nanoparticles) as theranostic probes. In these sense, we have developed new formulations to

reduce side effects and improve antitumoral activity of drugs, moreover these formulations are able to

monitor tumor extent by scintigraphic images. For example, we have developed pH-sensitive liposomes

for specifically delivery the radiopharmaceutical (99mTc-HYNIC- -bombesin(7-14)) to tumor sites. This

approach have improved tumor uptake leading to high quality images, which may help in the diagnosis of

this malignancy. In another study, we develop radiolabeled Solid Lipid Nanoparticles (SLN) loading

doxorubicin as a theranostic probe. Tumor could be visualized by scintigraphic images and SLN improve

the efficacy of the treatment. Other nanoparticles have been studied in our group with promising results in

diagnosis and/or treatment of cancer.

Selected publications (NPY starred):

*de Barros ALB, Mota LG, Coelho MMA, Correa NCR, Goes AM, Oliveira MC, Cardoso VN (2015) Bombesin

Encapsulated in Long-Circulating pH-Sensitive Liposomes as a Radiotracer for Breast Tumor Identification. J Biomed

Nanotechnol 11:342-50.

*de Barros ALB, Ferraz KSO, Dantas TCS, Andrade GF, Cardoso VN, de Sousa BEM (2015) Synthesis, characterization,

and biodistribution studies of 99mTc-labeled SBA-16 mesoporous silica nanoparticles. Mat Sci Eng C 56:181-88.

*Fernandes RS, Mota LG, Kalbasi A, Werner TJ, Alavi A, Rubello D, Cardoso VN, de Barros ALB (2015) 99mTc-phytate as

a diagnostic probe for assessing inflammatory reaction in malignant tumors. Nucl Med Comm 36: 1042-48.

*Soares DCF; Sousa GF, de Barros ALB, Cardoso VN, Oliveira MC, Ramaldes GA (2015) Scintigraphic imaging and

increment in mice survival using theranostic liposomes based on Gadolinium-159. J Drug Del Sci Technol 30: 7-14.

* Al-Zaki A, Joh D, Cheng Z, de Barros ALB, Kao G, Dorsey J, Tsourkas A (2014) Gold-Loaded Polymeric Micelles for

Computed Tomography-Guided Radiation Therapy Treatment and Radiosensitization. ACS Nano 8: 104-12.

*de Barros ALB, Mota LG, Soares DCF, Souza CM, Cassali GD, Oliveira MC, Cardoso VN (2013) Long-Circulating, pH-

Sensitive Liposomes versus Long-Circulating, Non-pH-Sensitive Liposomes as a Delivery System for Tumor

Identification. J Biomed Nanotechnol 9: 1632-43.

*Soares DCF, Oliveira MC, de Barros ALB, Cardoso VN, Ramaldes GA (2011) Liposomes radiolabeled with 159Gd: In

vitro antitumoral activity, biodistribution study and scintigraphic image in Ehrlich tumor bearing mice. Eur J Pharm Sci

290-296.

22

Antifungal and antiretroviral drugs nanoencapsulation: Could it be an efficient strategy to

improve actual therapy?

Dr Irene Clemes Külkamp Guerreiro, Federal University do Rio Grande do Sul

http://lattes.cnpq.br/9385103078887175

Abstract: The focus of our research group is to understand how the nanoencapsulation could improve in

fact the therapy of fungal and HIV infections. Besides the promises related to the applications of

nanotechnology to drugs, we need proofs of the better action of the nanoencapsulated drugs. We aimed

to find the enhancement of the therapy of fungal infections, mainly related to drug action and

microorganisms resistance. We have also been working on the development of pharmaceutical dosage

forms suitable for children use. We have been using different approaches to prepare and characterize

nanotechnological formulations, and to prove the therapy enhancement reached by the

nanoencapsulation. As examples, we can highlight the improvement of ketoconazole and fluconazole drug

action, overcoming fluconazole resistance, and the nanoencapsulation of saquinavir reaching a liquid

formulation with taste masking.

Selected publications:

Svetlichny G, Kulkamp-Guerreiro IC, Cunha SL, Silva FEK, Pohlmann AR, Fuentefria AM, Guterres SS (2015). Solid lipid

nanoparticles containing copaiba oil and allantoin: development and role of nanoencapsulation on the antifungal

activity. Die Pharmazie 70:155-164.

Fuentefria AM, Jesus RS, Back PI, Oliveira TV, Knecht H, Kulkamp-Guerreiro IC (2014). Ketoconazole-Loaded Poly(ε-

Caprolactone) Nanocapsules for Improved Antifungal Activity Against Candida spp. Latin American Journal of

Pharmacy 33:1589-1595.

23

Tumour-Targeted and Matrix Metalloproteinase-Responsive Iron Oxide Nanoparticles for

Theranostic Applications

Dr Javier Hernández-Gil, Research Associate, Department of Chemistry, Imperial College London

http://www.imperial.ac.uk/people/j.hernandez-gil

Abstract: The application of nanotechnology in medicine offers extraordinary opportunities to yield

advances in the diagnosis and treatment of cancer. To achieve these goals, inorganic nanoparticle (iNPs)

have attracted considerable interest due to their unique and versatile electronic, optical, plasmonic and

magnetic properties. Nowadays, it seems clear that modern iNPs can be more than just “imaging” or

“therapeutic” probes and, combine both features to enable detection and treatment of disease in a single

procedure (“theranostic” agents). However, these iNP-based systems still need to tackle issues such as

insufficient sensitivity, limited spatial and temporal resolution, efficient tumour targeting, undesirable off-

target activities and, simple preparation/purification before being real candidates for clinical translation.

Through a “multimodal imaging” approach, nanoparticles can help to overcome the intrinsic limitations of

each imaging modality towards diagnosing/staging the disease. On the other hand, through a “multimodal

therapy” design, nanoparticles can exhibit a synergistic cancer cell killing effect by exploiting different

complementary drug payloads, cellular targets and “off-target” effects. At the workshop, I will present our

last efforts to develop a “theranostic” agent that upon an enzyme-controlled stimulus offers the possibility

of dual-modality imaging and drug release in cancerous tissue.

The oral presentation will start introducing our initial proof-of-concept in this regard: two sets of

superparamagnetic iron oxide NPs that selectively aggregate in the presence of cancer-specific matrix

metalloproteinases (MMP). The self-assembled nanoclusters induce a magnetic resonance (MR) signal

amplification and therefore in vivo cancer detection. Then, I will discuss ongoing results on developing the

second-generation system that aims to overcome detected limitations from the original design such as

large hydrodynamic size, sub-optimal coupling rates and, insufficient PEG length. The new nanoconstructs

can be also easily radiolabelled with 68Ga for positron emission tomography (PET). The dual-mode MR-PET

probe can enable high spatial resolution (MR) and extreme sensitivity (PET) in a single unit, improving

tumour detection and in vivo tracking. Finally, I will show our strategy to provide the constructs with

therapeutic properties. Through surface functionalization, one set of iNPs will contain gatekeepers that

specifically respond to the other set of iNPs upon the MMP-triggered self-assembly process and therefore,

leading to on-demand release of therapeutic molecules. I believe these nanoconstructs hold considerable

promise as new delivery vehicles for developing more selective and/or efficient materials for drug delivery.

Selected publications (initial proof-of-concept starred):

*Gallo J, Kamaly N, Lavdas J, Stevens E, Nguyen, Q-D, Wylezinska-Arridge, M, Aboagye, EO, Long, NJ (2014) CXCR4-

Targeted and MMP-Responsive Iron Oxide Nanoparticles for Enhanced Magnetic Resonance Imaging. Angew Chem

Int Ed 53: 9550-9554. DOI: 10.1002/anie.201405442

Hernández-Gil J, Cobaleda-Siles M, Zabaleta Azpiroz A, Salassa L, Calvo J, Mareque-Rivas JC (2015) An Iron Oxide

Nanocarrier Loaded with a Pt(IV) Prodrug and Immunostimulatory dsRNA for Combining Complementary Cancer

Killing Effects. Ad Health Mat 4:1034-1042. DOI: 10.1002/adhm.201500080

Ruggiero E, Hernández-Gil J, Mareque-Rivas JC, Salassa L (2015) Near infrared activation of an anticancer PtIV complex

by Tm-doped upconverting nanoparticles. Chem. Commun 51: 2091-2094. DOI: 10.1039/c4cc07960d

24

Shedding light on G protein coupled receptor pharmacology using fluorescence

complementation and imaging approaches

Dr Nick Holliday, Associate Professor of Pharmacology, University of Nottingham

http://www.nottingham.ac.uk/life-sciences/people/nicholas.holliday

Abstract: Our group focuses on the mechanisms driving the signalling, intracellular trafficking and ligand

pharmacology of GPCRs for peptides such as neuropeptide Y and ghrelin, and nutrients such as free fatty

acids – all of which are implicated in the control of appetite, body weight and insulin secretion. We use a

variety of approaches, especially high content fluorescent imaging and quantitative analysis of fluorescent

ligand binding, receptor endocytosis and receptor interaction with associated signalling proteins. Recently

for example, we have applied bimolecular fluorescence complementation (BiFC) to study interactions

between GPCRs and signalling partners known as arrestins, and also to examine the function of particular

receptor homo- and heterodimers. This is a route to explore the pharmacology of particular receptor

heterodimers selectively and quantitatively (for example, the NPY Y1/Y5 dimer proposed to regulate

appetite), and it has also allowed investigation of key signalling complexes using advanced imaging

methods such as fluorescence correlation spectroscopy.

Selected publications:

Wong KW, Briddon SJ, Holliday ND, Kerr ID (2016) Plasma membrane dynamics and tetrameric organisation of ABCG2

transporters in mammalian cells revealed by single particle imaging techniques. Biochim Biophys Acta 1863: 19-29.

PMID: 26453803

Stott LA, Hall DA, Holliday ND (2016) Unravelling intrinsic efficacy and ligand bias at G protein coupled receptors: a

practical guide to assessing functional data. Biochem Pharmacol 101,1-12.PMID: 26478533)

Kilpatrick LE, Humphrys LJ, Holliday ND (2015) A G protein-coupled receptor dimer imaging assay reveals selectively

modified pharmacology of neuropeptide Y Y1/Y5 receptor heterodimers. Mol Pharmacol 87: 718-32. PMID: 25637604

Mountford SJ, Liu M, Zhang L, Herzog H, Groenen M, Holliday ND, Thompson PE (2014) Synthetic routes to the

Neuropeptide Y Y1 receptor antagonist 1229U91 and related analogues for SAR studies and cell-based imaging. Org

Biomol Chem 28:3271-81. PMID: 24733083

Kilpatrick LE, Briddon SJ, Holliday ND (2012) Fluorescence correlation spectroscopy, combined with bimolecular

fluorescence complementation, reveals the effects of β-arrestin complexes and endocytic targeting on the membrane

mobility of neuropeptide Y receptors. Biochim Biophys Acta 1823:1068-81. PMID: 22487268

Watson SJ, Brown AJ, Holliday ND (2012) Differential signaling by splice variants of the human free fatty acid receptor

GPR120. Mol Pharmacol 81:631-42. PMCID: PMC3336805

Sivertsen B, Lang M, Frimurer TM, Holliday ND, Bach A, Els S, Engelstoft MS, Petersen PS, Madsen AN, Schwartz TW,

Beck-Sickinger AG, Holst B (2011) Unique interaction pattern for a functionally biased ghrelin receptor agonist. J Biol

Chem 286:20845-60. PMCID: PMC3121491

Kilpatrick LE, Briddon SJ, Hill SJ, Holliday ND (2010). Quantitative analysis of neuropeptide Y receptor association with

beta-arrestin2 measured by bimolecular fluorescence complementation. Br J Pharmacol 160:892-906. PMCID:

PMC2901518

25

Use of phenotypic assays to explore Gs-coupled receptor agonism in airway remodelling.

Dr Elizabeth M Rosethorne, Snr Research Fellow, University of Nottingham

Abstract: One of the major research themes in our group is the use of human primary cells to explore

GPCR pharmacology. My specific interests include the use of phenotypic assays to identify novel targets for

disease, the exploration of biased signalling and the role of signalling kinetics in determining agonist

efficacy. My current research is focussed on the role of the second messenger cAMP in the inhibition of the

airway remodelling observed in respiratory diseases such as pulmonary fibrosis and severe asthma. The

reason for this is that in addition to the acute bronchoconstriction observed in patients with asthma, there

are a number of studies demonstrating that severe, persistent asthma is selectively associated with

increased airway smooth muscle and fibroblast accumulation resulting in pathological airway remodelling.

Here we have used isolated primary human cells to investigate the potential for Gs-coupled receptor

agonists to inhibit the proliferation and differentiation of airway smooth muscle cells and fibroblasts. We

have also demonstrated that the anti-proliferative effects of Gs-coupled receptors may not be solely

dependent on global cAMP accumulation. This highlights the importance of monitoring the kinetics and

localisation of intracellular signals, as well as multiple intracellular signalling pathways when profiling novel

compounds, as endpoint population second messenger assays may not always predict phenotypic

outcomes.

Selected peer reviewed publications:

Rosethorne EM, Bradley ME, Gherbi K, Sykes DA, Sattikar A, Wright JD, Renard E, Trifilieff A, Fairhurst RA, Charlton SJ

(2016) Long Receptor Residence Time of C26 Contributes to Super Agonist Activity at the Human β2 Adrenoceptor.

Mol Pharmacol. pii: mol.115.101253. [Epub ahead of print]

Rosethorne EM, Bradley ME, Kent TC, Charlton SJ (2014) Functional desensitization of the β2 adrenoceptor is not

dependent on agonist efficacy. Pharm Res & Perspectives 3(1): e00101 doi: 10.1002/prp2.101

Nijmeijer S, Vischer HF, Sirci F, Schultes S, Engelhardt H, de Graaf C, Rosethorne EM, Charlton SJ, Leurs R (2013)

Detailed analysis of biased histamine H₄ receptor signalling by JNJ 7777120 analogues. Br J Pharmacol. 170:78-88.

Nijmeijer S, Vischer HF, Rosethorne EM, Charlton SJ, Leurs R (2012) Analysis of multiple histamine H₄ receptor

compound classes uncovers Gαi protein- and β-arrestin2-biased ligands. Mol Pharmacol. 82:1174-82.

Rivero G, Llorente J, McPherson J, Cooke A, Mundell SJ, McArdle CA, Rosethorne EM, Charlton SJ, Krasel C, Bailey CP,

Henderson G, Kelly E (2012). Endomorphin-2: a biased agonist at the μ-opioid receptor. Mol Pharmacol. 82:178-88.

Rosethorne EM and Charlton SJ (2011) Agonist-Biased Signalling at the Histamine H4 Receptor: JNJ7777120 Recruits

Beta-Arrestin Without Activating G Proteins. Mol Pharmacol. 79:749.

Kong KC, Butcher AJ, McWilliams P, Jones D, Wess J, Hamdan FF, Werry T, Rosethorne EM, Charlton SJ, Munson SE,

Cragg HA, Smart AD, Tobin AB (2010). M3-muscarinic receptor promotes insulin release via receptor

phosphorylation/arrestin-dependent activation of protein kinase D1. Proc Natl Acad Sci USA 107(49):21181.

McPherson J, Rivero G, Baptist M, Llorente J, Al-Sabah S, Krasel C, Dewey WL, Bailey CP, Rosethorne EM, Charlton SJ,

Henderson G, Kelly E (2010) μ-opioid receptors: correlation of agonist efficacy for signalling with ability to activate

internalization. Mol Pharmacol. 78(4):756.

Rosethorne E, Turner R, Fairhurst R and Charlton S (2010) Efficacy Is a Contributing Factor to the Clinical Onset of

Bronchodilation of Inhaled β2 Adrenoceptor Agonists. Naunyn-Schmiedeberg's Archives of Pharmacology 382:255-

263.

Poulin B, Butcher A, McWilliams P, Bourgognon JM, Pawlak R, Kong KC, Bottrill A, Mistry S, Wess J, Rosethorne EM,

Charlton SJ, Tobin AB (2010) The M3-muscarinic receptor regulates learning and memory in a receptor

phosphorylation/arrestin-dependent manner. Proc Natl Acad Sci USA 107(20):9440.

26

Biomineralization of Anatase Nanoparticles and its Implications in Bone Cells Survival

Dr Ana Ribeiro, Researcher Scientist, Metrology Division Applied to Health Sciences, National Institute of

Metrology Quality and Technology

Abstract: Dentistry and orthopedics are undergoing a revolution in order to provide more reliable,

comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in

dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based

implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic

ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions.

Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles

is still very limited. Taking this into consideration, this study focuses on the internalization of titanium

dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. In

the complex biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and

ions) resulting in a kind of Trojan horse that facilitates their internalization by cells.

Selected publications:

Ana Rosa Ribeiro, Mirian Noemi Pinto Vidal, Fernando Oliveira, Sara Gemini-Piperni Luis Augusto Rocha, José Mauro

Granjeiro, Nanotechnology approaches to enhance dental implants: human health implications, Implant News

2015;12(5):643-55.

A.R. Ribeiro, F. Oliveira, L.C. Boldrini, P.E. Leite, P. Falagan-Lotsch, W.F. Zambuzzi, B. Fragneaud, A.P.C. Campos, C.P.

Gouvêa, B.S. Archanjo, C.A. Achete, E. Marcantonio Jr., L.A. Rocha, J.M. Granjeiro, Micro-arc oxidation as a tool to

develop multifunctional calcium rich surfaces for dental implants applications, Journal Materials Science and

Engineering 54 (2015)196-206.

Oliveira, Fernando, Ribeiro, Ana R., Perez, Geronimo, Archanjo, Bráulio S., Gouvea, Cristol P., Araújo, Joyce R., Campos,

Andrea P.C, Kuznetsov, Alexei, Almeida, Clara M., Maru, Márcia M., Achete, Carlos A., Ponthiaux, Pierre, Celis, Jean-

pierre, Rocha, Luis A, Understanding Growth Mechanisms and Tribocorrosion Behaviour of Porous TiO2 Anodic Films

Containing Calcium, Phosphorous and Magnesium. Applied Surface Science. 341 (2015) 1 – 12.

Damasceno, J. C., Balottin, L. B., Ribeiro, A. R, Granjeiro, J. M. Nanometrologia – desafios para a regulação sanitária.

Vigilância Sanitária em Debate: Sociedade, Ciência & Tecnologia, 1 (2013) 104 - 114.

A. R. Ribeiro, S. Gemini, L. Teixeira, H. Cruz, F. Oliveira, A. L. Rossi, J. Werckmann, K. Anselme, T. Shokhufar, L. A.

Rocha, J.M. Granjeiro, Trojan-Like Internalization of Anatase Nanoparticles by Osteoblast Cells. Submitted to Nature

Scientific Reports, accepted with revisions.

A. R Ribeiro, P. E. Correa, P. Falagan Lotsch, F.Benetti, H. C. Budtz, N. R. Jacobsen, Dana Kühnel, Maureen R Gwinn,

J.M. Granjeiro, Engineered Nanomaterials risk assessment and hazard identification to human health: current

knowledge and future challenges. Submitted to Nanotoxicology Journal, under revision.

L. Laquieze, A. R. Ribeiro, J.M. Granjeiro, Alternative methods and cosmetics safety: Update on the regulatory and

technological aspects. Submitted to Altex

27

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