Biological Chemistry Group

Biological Chemistry Group
Prevention, Detection and Treatment of Amyloid Diseases Biological Chemistry Group Coimbra Chemistry Center, Chemistry Department, FCT Coimbra University . U C . UNIVERSIDADE DE COIMBRA Ciência 2017 Lisbon, July

Amyloid Diseases Insoluble protein aggregates in cellular and extracellular fluids images/Image/Eva/Beta_amyloid.jpg Different proteins (and different locations) lead to distinct Diseases transthyretin – senile systemic amyloidosis familial amyloid polyneuropathy familial amyloid cardiomyopathy b-amyloid – Alzheimer disease amylin – type 2 diabetes prion – bovine spongiform encephalopathy (BSE) “mad cow disease”, scrapie and Creutzfeldt-Jakob disease a-synuclein – Parkinson disease Amyloid diseases are due to the presence of protein aggregates in celular and extracellular fluids. When formed in the brain they compromisse neuronal function and lead to a decline in cognition. Different proteins are known to form amyloid aggregates, and they do so in distinct locations in the body, thus leading to distinct diseases. Transthyretin is involved in several diseases, depending on the mutation occurring in the protein which affects the place where the aggregates are accumulated. Alzheimer’s disease is due to the formation of b-amyloid aggregates in the brain, and the aggregation of a-synuclein leads to Parkinson disease. Some types of diabetes are also due to the formation of amyloid aggregates, in this case amylin, and prions are proteins that were very popular some decades ago when it was found that they could induce the aggregation of other proteins and the disease could therefore be contagious. known popularly as "mad cow disease“, scrapie in sheep and Creutzfeldt-Jakob disease (CJD) in humans.

Why are they formed? Globular proteins have irregular surfaces and are flexible Globular proteins are formed by several elements of secondary structure, namely a-helix and b-sheets, they are dynamic and their surface is irregular. They interact favorably with the solvent, with several small molecules and with other proteins. The dynamic nature of the structure of globular proteins means that occasionally they unfold partially, in a reversible way. Several conditions may favor this process, such as oxidative stress, interaction with cell membranes or interaction with some metals. Partially unfolded states have hydrophobic regions exposed to the aqueous media and this may lead to aggregation. If the structure of the intermediates is rich in beta-sheet, they tend to form very stable and rigid aggregates, the amyloid fibrils, fibers or plaques. Partial unfolding Intermediates in degradation Oxidative stress Interaction with membranes Interaction with metals Hydrophobic and rigid structures Fibrils, fibers and plaques

[18F]-Florbetapir ([18F]-AV-45)
Current Diagnosis Advanced stages - Based on symptoms Definitive diagnosis - only post-mortem Non-invasive diagnosis from imaging: PET/SPECT, MRI commonly used to image cancer metastasis (PET/SPECT) associate with b-amyloid fibers [18F]-Florbetapir ([18F]-AV-45) Current diagnosis is based on symptoms and therefore can only be done at advanced stages of the diseases. In fact, because the symptoms are similar for several amyloid diseases, the definitive diagnosis is only obtain post-morten. Non-invasive diagnosis tools would allow the identification of the disease at earlier stages. This has potentially a strong impact on the life quality of the patients as it allows guided intervention preventing the advancement of the disease. PET (positron emission tomography) and SPECT (single-photon emission computed tomography) are imaging techniques based on the detection of gamma rays, either emitted directly by a radioactive tracer (SPECT) or generated through the annihilation of the positron emitted by the radioactive tracer (PET). A common application of PET/SPECT is based on the measurement of glucose accumulation in tissues with high metabolic activity, for example to detect tumors or damaged brain regions. Glucose labelled with 18F is then the tracer. If the radionuclide is incorporated in molecules which associate specifically with amyloid fibers, this technique may be used in the eary diagnosis of amyloid diseases. The lifetime of radionuclides is relatively small (20 min for 11C and 110 min for 18F), which imposes severe difficulties in the synthesis and storage/manipulation of the tracer molecules.

 ionizing radiation  spatial resolution
Current Diagnosis PET/SPECT already in clinical use in the case of AD (b-amyloid) and PD (a-synuclein) [18F]-Florbetapir [99mTc]-BAT-Bp2 (SPECT) [18F] DOPA (PET) (PET) Nevertheless, PET is already being used in clinical essays to detect amyloid fibers of b-amyloid (Alzeimer’s disease), 11C-PiB ou Florbetapir, and to detect a-synuclein (Parkinson disease) using 18F-DOPA. Pre-Clinical essays have also been performed with 99mTC (a metastable nuclear isomer of technetium-99 used in SPECT, with a half life for gamma emission of 6 h), which in conjunction with thioflavin derivatives associates with amyloid aggregates. The major advantage of PET is its high sensitivity. However, PET (and SPECT) has several disadvantages. The use of ionizing radiation is a major concern, and there a small but significant cases of cancer that could be attributed to the repeated use of this method of diagnosis. Another disadvantage is the low spatial resolution. MRI is a widely used technique to obtain images of tissues, organs or even whole bodies. It is cheaper than PET or SPECT and much safer as it does not require the use of ionizing radiation. The signal in MRI is originated from the nuclei of hydrogen atoms of water and organic matter, which behave differently under a magnetic field depending on their environment. Autoradiography of mice + [99mTc]BAT-Bp2 and thioflavin-S staining PET  sensitivity  ionizing radiation  spatial resolution MRI  cost  no ionizing radiation  spatial resolution

T1w MRI of transgenic mouse
Current Diagnosis MRI  not enough contrast to detect amyloid aggregates (contrast agents needed) Peptide Iron Oxide T2w MRI of transgenic mouse pre-contrast of brain section USPIO-PHO Perl’s iron staining Larbanoix et al. Neurobiology of Aging 31 (2010) 1679–1689 USPIO-PHO Pre-clinical tests (BBB disrupted by osmotic stress ) Gd-DTPA - putrescine Ab1-42 immunostaining 6-month-old control mouse APP/PS1 Transgenic mouse J. Poduslo et al., Neurobiol. Dis., 2002, T1w MRI of transgenic mouse However, the contrast generated by the hydrogen nuclei from water is not sufficient to detect amyloid aggregates (except when they are naturally enriched in paramagnetic ions). There is therefore the need to administer contrast agents to enhance the distinct behavior of hydrogen nuclei in the different environments. The most common contrast agents are lanthanides (most commonly Gadolinium, Gd3+), or iron nanoparticles. However, the bioavailability of those contrast agents is poor and they cannot permeate the BBB. The use of MRI to detect amyloid aggregates is therefore only in the pre-clinical stage, because it requires disruption of the BBB (for example by osmotic stress using mannitol). In vivo studies using transgenic mouse have shown that Gd(III) complexes and iron oxide nanoparticles conjugated with peptides with high affinity for amyloid aggregates, allow the use of MRI to detect amyloid aggregates.

Treatment Genetic factors – Organ Transplant
Not an option if the organ is the brain… – Other forms of Gene Therapy Low efficiency of delivery and incorporation in the genome In General - Stabilization of the native structure Case of Tafamidis… - Destabilization of the aggregates Small intermediates may be even more toxic… The strategy followed to treat amyloid diseases depends on the factor that have originated the disease. If the factors are of genetic origin – a mutation in the protein that reduces its stability leading to its aggregation – one effective strategy is to replace the organ where the protein is synthesized. This has been the strategy followed in “doença dos pezinhos” with liver transplant. Certainly this is not an option if the protein is being synthesized in the brain… Other less invasive forms of gene therapy include the delivery of the specific gene to the relevant cells. This approach is very promising but currently limited by the low efficiency of delivery and incorporation of the therapeutic genes into the genome. Gene therapy is useless when the disease is not due to a defective gene, but rather to the “normal” aging of the best genes available. In this case, the stabilization of the native tertiary structure of the relevant protein is a general strategy that may prevent (or at least slow down) further progression of the disease. Tafamidis is a drug currently used in the treatment of TTR dependent amyloid diseases. Another strategy may be the destabilization of the amyloid aggregates. This strategy may be required in later stages of the disease, but it is a high risk approach as smaller aggregates may be even more toxic than the larger ones.

Areas of Intervention New Drugs to prevent amyloid formation
New Probes to detect amyloid aggregates at early stages Open areas of intervention that are priority for the next 3 years include: The development of new drugs that interact specifically and stabilize the native structure of the distinct proteins involved in the amyloid diseases. Development of more sensitive and specific probes to detect amyloid aggregates earlier and allow a more effective treatment. A challenge common to the two above areas of intervention is the availability of the drug/probe at the target site. This is particularly relevant for the case of brain diseases such as Alzheimer's or Parkinson. Strategies to improve drug/Probe bioavailability

The Biological Chemistry Group
Contribution from The Biological Chemistry Group @ CQC-UC Biomedical NMR and Imaging Biomembrane Structure and Function Bioinorganic Chemistry Computational & Systems Biology Protein Biophysical Chemistry Open areas of intervention that are priority for the next 3 years include: The development of new drugs that interact specifically and stabilize the native structure of the distinct proteins involved in the amyloid diseases. Development of more sensitive and specific probes to detect amyloid aggregates earlier and allow a more effective treatment. A challenge common to the two above areas of intervention is the availability of the drug/probe at the target site. This is particularly relevant for the case of brain diseases such as Alzheimer's or Parkinson.

Protein Structure and Ligand Interactions
Daniela Vaz Zaida Almeida Rui Brito Protein Structure and Ligand Interactions Pedro Cruz - Molecular mechanisms of amyloid formation - Protein folding and stability - Protein-ligand interactions - Protein-protein interactions NMR, CD, Computation - Citizen science The biological chemistry group includes several independent researchers that have their own laboratories and scientific objectives. As a team, the members of the biological chemistry group have identified some common objectives for the next years, to gain synergy from the know-how of the distinct members and laboratories. The overall common objective is to prevent, detect and treat amyloid diseases. This will be achieved through some specific objectives, one of which is to understand the factors that control protein structure. This has been the major area of research of Professor Rui Brito and his team, and NMR is one of the major techniques used. Professor Carlos Geraldes is an expert in NMR being an active member in the establishment and development of the NMR facilities in Coimbra. The team from Professor Rui Brito laboratory involved in this research area includes the PhD Daniela Vaz, and the fellows Zaida Almeida and Pedro Cruz. Specific objectives include the elucidation of the molecular mechanisms of amyloid formation, which depend on protein folding and stability of the native structure. The stability of the native structure depends on the interactions established with small molecules (ligands) as well as with other proteins. The methodologies used include experimental tools, such as NMR and CD, and also computational tools, and the involvement of the society is strongly encouraged through programs that use computing power from society volunteers or were the volunteers may even be directly involved in data analysis. Collaborations - Javier Sancho, Univ Zaragoza, Spain - Christina Redfield, Univ Oxford, UK - Helena Rebelo Andrade, FF-UL - Pedro Castanheira, UC-Biotech - Carlos Serpa, CQC, UC 10

Drug Discovery and Development
Rui Brito Carlos Simões Andreia Cunha - Discovery and rational design of: - inhibitors of amyloid formation - amyloid binders (imaging/diagnostic) Cândida Silva - Machine learning models in drug discovery Another common objective is the discovery and development of new drugs effective in the prevention and treatment of amyloid diseases. This area of research is also headed by Professor Rui Brito. Together with his team members, PhDs Carlos Simões and Cândida Silva, and the Master student Andreia Cunha, their objective is to design and develop new inhibitors of amyloid formation and molecules with a high affinity for the amyloids to be used in imaging and diagnosis of amyloid diseases. To achieve this goal it is necessary to use computational tools, from which machine learning tools are or great utility when considering large amounts of data. The team not only uses those approaches but is also involved in the development of faster and more reliable tools to be used in drug discovery. In addition to the computational tools, the discovery and development of new drugs requires the synthesis of the most promising drug candidates and the in vitro and in vivo characterization of their efficacy. The tools and know-how available at the group is complemented with the expertize from collaborators, namely Teresa Pinho e Melo from the CQC for the synthesis of the compounds. Professor Rui Brito and his team have been very active in this area of research during the last years, and founded the spin-off BSIM2 in 2011, which is dedicated to drug discovery and finding therapeutic solutions for TTR amyloid diseases. Collaborations - Maria João Saraiva, I3S, UP - Teresa Pinho e Melo, CQC, UC Drug discovery spin-off: therapeutic solutions for ATTR 11

MRI (Gd), PET (68Ga), SPECT (111In), luminescence (Eu, Tb, Yb)
Margarida Castro Carlos Geraldes Detection of amyloid fibers Development of more efficient contrast agents for MRI and PET – PIB conjugates MRI (Gd), PET (68Ga), SPECT (111In), luminescence (Eu, Tb, Yb) M3+ Ab targeting function Imaging reporter Ana Marguerita Metelo Mariana Laranjo Alexandre Oliveira Collaborations Mariette Pereira, Mário Calvete and Sara Pinto, Catalysis & Fine Chemistry Laboratory Hugh Burrows and Licinia Justino, Photochemistry Laboratory Coimbra Chemistry Center: Centre de Biopysique Moléculaire- CNRS Orléans, France - Éva Tóth The development of contrast agents for MRI and PET has been one of the research areas of the laboratories of “Bioinorganic Chemistry” and “Biomedical NMR Imaging”, headed by Professors Margarida Castro and Carlos Geraldes. The contrast agents have been mostly based on lanthanides and iron nanoparticles, the specificity to amyloids being given by conjugation with PiB. The contrast agents developed include the imaging reporter, in this case the trivalent cation stabilized by the chelator (DO3A), which is linked to the targeting function. The tri-valent cation may be selected to give contrast in MRI (Gd3+), PET (68Ga3+), SPECT (111In) or luminescence microscopy (such as Europium or Terbium). This work involves several methodologies and expertize, being pursuit in collaboration with several members of the Coimbra Chemistry Center (namely the Catalysis & Fine Chemistry Laboratory for the synthesis, and the Photochemistry Laboratory for characterization of the contrast agents), and with several groups abroad from which the collaboration with the laboratory of Eva Toth is the most significant and involves the distinct aspects of the development of the contrast agents. During the last years the group has developed a family of compounds varying in the structure of the chelating group, linker and targeting function, with changes in the overall charge, hydrophobicity and distance between the reporter and the targeting group.

Interaction with isolated and aggregated Ab1-40
SPR STD NMR 1H-15N HSQC NMR Ex-vivo co-localization with amyloid deposits in AD human brain slices mPET studies on mice with 68Ga-L1-3 control transgenic Ex-vivo auto-radiography [18F]-AV-45 [68Ga]-L3 mouse Control transgenic Human AD brain Thioflavin-S [68Ga]-L1 The properties of the probes as contrast agents have been characterized and the Gadolinium chelates show high relaxivity. Their interaction with b-amyloid aggregates was characterized by SPR and STD NMR and they showed to have moderate affinity. Europium conjugates were found to bind specifically to amyloid places in ex-vivo essays with brain slices of Alzeimer’s patients post-morten. Gallium 68 conjugates have also been studied as contrast agents in PET. In vivo studies using mice showed that on both control and transgenic mice showing the phenotype of Alzeimer disease, the majority of the contrast agent administered accumulated in the liver. A small fraction reached the brain and the signal in the most relevant regions was somewhat higher for the case of the transgenic mice. On the other hand, the interaction with the isolated peptides was characterized by 1H-15N HSQC NMR. The interaction of the Gd(III) chelates with the peptide lead to an increase in the width of the signals originated from some of the peptide groups, but the association constant was very small, thus indicating that those contrast agents selectively associate with the amyloid aggregates. The localization of the imaging probes was further characterized by auto-radiography where the increased spatial resolution may elucidate on whether the accumulation regions are in fact the amyloid aggregates. The imaging probes used were thioflavin and 18F-AV-45. The ligands from the family of compounds developed by this laboratory with 68Ga has the contrast agent have also been characterized and the signal was increased in the transgenic mouse and in the brain with Alzeimer’s disease. However, the signal was not from amyloid-rich regions for the case of 68Ga probes, in contrast with the location of thioflavin and 18F-AV-45, which are known to be able to cross the BBB and selectively associate with the amyloid aggregates. The conclusion was that the Ga ligands could not permeate efficiently the BBB and were accumulated only in the brain capillaries. Further changes in the structure of the contrast agents are needed to optimize their permeation through the BBB. GPOL 2010

Understanding Membrane Permeation
Mª João Moreno Luís Loura Understanding Membrane Permeation Molecular Dynamics simulations Equilibrium & Biased Membrane Model Systems Hugo Filipe Mª Cristiana Neves All atom & Coarse grained Jaime Samelo Filipe Gomes Joana Cunha Partition/Permeation k+ k- kf Fluorescence & Calorimetry To increase the permeability through the BBB without its disruption it is necessary to understand the factors that control and limit permeation through biological membranes. This is one of the main research objectives of the Biomembranes laboratory at CQC. This goal is being approached using several methodologies, namely Molecular Dynamics simulations. Professor Luís Loura leads this area of research, in collaboration with other members of the Biomembranes laboratory, namely myself, the PhD Hugo Filipe and more recently the graduation student Maria Cristiana Neves. The work is also done in collaboration with Ilpo Vattulainen from Finland and with João Paulo Ramalho from the University of Évora. MD simulations may be used to characterize the equilibrium position and orientation of the molecule of interest in the membrane, and to obtain information regarding perturbation of the membrane by the solute. Biased simulations are also being performed, where the solute is constrained at given position in the bilayer. Fluctuations around the defined position allow obtaining the Gibbs free energy as a function of the solute position in the bilayer, from which one may obtain the equilibrium association between the aqueous phase and the lipid bilayer, as well as the rate constants for crossing the energy barriers. In some cases, imposing long residence times for the solute at high energy positions lead to local perturbation of the membrane and may the affect the results obtained. A less intrusive strategy would be to allow for an unconstrained sampling of the whole system by the solute. This is not however possible with all atom simulations due to the excessive computing power required. One way to go around this limitation is using coarse grained simulations, where each molecule is represented by a small number of beads which correspond to several atoms. The simulations may now be easily extended to several microseconds allowing the characterization of the rate constants without imposing constraints to the system. In this example cholesterol remained in its equilibrium position for several hundreds on ns, and spontaneously translocated into the opposite leaflet in about 2 ns. Collaborations - Ilpo Vattulainen, Tampere, Finland - João Paulo Ramalho, UE - Luís Arnaut, CQC-UC - Winchil Vaz, CEDOC, UNL - Margarida Bastos, UP - Adrian Velazquez-Campoy,BiFi,Spain - Júlia Mora, Univ Cordoda,Argentina

Understanding Membrane Permeation
Mª João Moreno Luís Loura Cells & Cell Membranes P-glycoprotein Specificity Experimental studies & MD simulations Hugo Filipe Cristiana Pires Permeation through Cell monolayers Caco-2 & BBB endothelial cells ZO Phaloidin DAPI To increase the permeability through the BBB without its disruption it is necessary to understand the factors that control and limit permeation through biological membranes. This is one of the main research objectives of the Biomembranes laboratory at CQC. This goal is being approached using several methodologies, namely Molecular Dynamics simulations. Professor Luís Loura leads this area of research, in collaboration with other members of the Biomembranes laboratory, namely myself, the PhD Hugo Filipe and more recently the graduation student Maria Cristiana Neves. The work is also done in collaboration with Ilpo Vattulainen from Finland and with João Paulo Ramalho from the University of Évora. MD simulations may be used to characterize the equilibrium position and orientation of the molecule of interest in the membrane, and to obtain information regarding perturbation of the membrane by the solute. Biased simulations are also being performed, where the solute is constrained at given position in the bilayer. Fluctuations around the defined position allow obtaining the Gibbs free energy as a function of the solute position in the bilayer, from which one may obtain the equilibrium association between the aqueous phase and the lipid bilayer, as well as the rate constants for crossing the energy barriers. In some cases, imposing long residence times for the solute at high energy positions lead to local perturbation of the membrane and may the affect the results obtained. A less intrusive strategy would be to allow for an unconstrained sampling of the whole system by the solute. This is not however possible with all atom simulations due to the excessive computing power required. One way to go around this limitation is using coarse grained simulations, where each molecule is represented by a small number of beads which correspond to several atoms. The simulations may now be easily extended to several microseconds allowing the characterization of the rate constants without imposing constraints to the system. In this example cholesterol remained in its equilibrium position for several hundreds on ns, and spontaneously translocated into the opposite leaflet in about 2 ns. Collaborations - Lino Ferreira, UCBiotech - Luís Arnaut, CQC-UC - Suresh Ambudkar, NIH, USA

Integrative Approach to Improve Permeation
Armindo Salvador Integrative Approach to Improve Permeation Mª João Moreno Kinetic model including distribution in the blood and permeation through a cell monolayer Hugo Filipe Future inclusion of P-glycoprotein Intracellular organelles Metabolism For that we count with the precious help from Armindo Salvador, and his expertize in kinetic modeling. A simple model including distribution of the solute among binding agents in the blood and interaction of the free form with the membrane of a cell monolayer has already been developed. During the next triennium our objective is to increase the complexity of the model to include the efflux by P-gp, sequestration by intracellular organelles, and eventually metabolism. The analysis of the results for a large set of solutes will lead to the establishment of QSPR for the kinetic parameters, which inserted in the kinetic model provide predictions for their permeability through cell monolayers such as the BBB. Structure QSPR Kinetic parameters Kinetic model Permeability predictions

Joining the expertize from all team members
Armindo Salvador Carlos Geraldes Margarida Castro Mª João Moreno Luís Loura Rui Brito Daniela Vaz Design and develop drugs and contrast agents with improved bioavailability To prevent, detect and threat Amyloid diseases

Thank you for your attention
Acknowledgements Thank you for your attention . U C . UNIVERSIDADE DE COIMBRA

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