Expertise and Resources
For this project we have brought together the experience of medical chemists, pharmacologists, and molecular & cellular biologists, which allow us to cover all key phases in the development of new compounds with potential therapeutic activity for the treatment of Alzheimer’s disease. The consortium has broad experience in molecular modelling, design and synthesis of multitarget compounds that induce NRF2, neuroprotective drug development, and the study of the molecular basis underpinning neurodegenerative diseases.
Computational Microscopy Unit (COMICU)
The Computational Microscopy Unit (COMICU) employs computational molecular simulation models at different resolution scales to tackle a wide range of biological questions. The main research focus is the development of novel therapeutics for neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis and ictus). Applications range from the prediction of computer-aided molecular design to biophysical properties.
Our in silico techniques include:
– Virtual Screening
- Structure based in silico screening
- Ligand based in silico screening
– Drug design
- De novo drug design
- Drug re-design based on the nature of the active zone
– Drug Repositioning
- Repositioning and finding other potential targets for a given compound.
– Molecular docking
– Homology Modelling
– Atomistic and Coarse-Graining Molecular Dynamics
– 3D DNA/RNA Structure Prediction
We have implemented a computerized sample storage system for pharmacological screening (“chemistry library”) in which all the studies carried out for each of the compounds are recorded.
Chemical Synthesis
We have the experience and resources for the synthesis and purification of high quality organic compounds. Our equipment includes:
- Microwave Synthesizer for chemical reactions that saves time and energy-costs (eg., Estrada Valencia et al. Eur. J. Med. Chem. 2018, 156, 534-553).
- Equipment for solid phase synthesis for obtaining compounds with speed, simplicity, high efficiency and throughput (eg., Monjas et al. Eur. J. Med. Chem. 2017, 130, 60-72).
- Equipment for parallel synthesis (carrousel) that increases the speed in the search of the most suitable experimental conditions for a given reaction.
- Automated preparative chromatography equipment (IsoleraBiotage) for the fast separation and purification of new compounds. In only 10-15 minutes and with a reduced volume of solvents, this equip allows obtaining final compounds with the required purity (> 95%) for biological evaluation.
- Analytical HPLC equip for determining purity of compounds.
- Semi-preparative HPLC equip for separation of complex mixtures in aqueous media.
- HPLC-MS that allows the rapid determination of both purity and identification of compounds by combining a HPLC equip and a mass spectrometer.
- Five NMR equips (from 300 to 600 MHz) for the structural characterization of new compounds
- High Resolution Mass Spectrometer (HRMS) for determining the exact molecular mass of a given compound.
We have also created a chemical library of nearly 1000 products with privileged structures to act in the nervous system.
In vitro Pharmacological Evaluation
For this project the consortium has developed and tuned up several in-house protocols for in vitro assessments, in addition to some other wide used molecular techniques:
- Surface plasmon resonance (SPR) equipment for studying protein-protein and protein- small molecule interactions
- Experimental methods for the evaluation of biological (LOX5, QR2, ORAC, BACE1, GSK3beta, MAOA/B) and drug-like properties (pKa, logP, solubility in physiological-like media).
- In vitro evaluation of the CNS permeation of new compounds by PAMPA-BBB methodology (Rodríguez-Franco et al. J. Med. Chem. 2006, 49,459-462). In vitro screening of inducing compounds based on a chemical genetic approach with fluorescent proteins (patent pending).
- Evaluation in AREc32 cells that express a reporter luciferase NRF2dependent (Sci Rep 2017, 7: 45701).
- Evaluation of neuronal and hepatic toxicity in human cell lines SHSY5Y and HepG2.
- Neuroprotective and antineuroinflammatory effect assessment in SHSY5Y cells and two toxicity models: (i) Okadaic acid and (ii) combination of rotenone and oligomycin A. To measure the antineuroinflammatory component, we use primary cultures of rat glia exposed to proinflammatory stimulus LPS, using the Griess technique to measure the generation of nitrites (In-house procedure, Sci Rep 2017, 7: 45701).
- Therapeutic potential evaluation in organotypic hippocampal cultures. This model allows reducing the number of animals needed, the economic costs and human efforts. (In-house procedure, Buendia et al., Mol Neurobiol, 2016 Jul; n53 (5): 33383348).
In vivo models.
- AAVTAU, based on the adeno-associated neuronal expression of TAU protein (P301L) under the control of synapsin1 promoter (AAV6TAU (P301L)). This model has three main advantages: a) it allows studying the neurodegenerative process in response to a high expression of TAU, b) they can be used in diverse genetic backgrounds, and c) they enable shorter pharmacological studies (about 4 weeks) compared to transgenic mice where the drug has to be applied for several months. We have two variants within this approach:
–AAVTAUIH: Stereotaxic delivery in the right hippocampus (LastresBecker et al., Brain 137 (Pt 1): 7891.2014).
–AAVTAUICV: Intracerebroventricular Stereotaxic delivery (on-going publication). - APPTAU+ NRF2: C57 / bl / 6 mouse expressing APP (V717I) and TAU (P301L) proteins under the control of the Thy1 neuronal promoter. These mice express both transgenes in the presence (ATNrf2 + / +) or absence (ATNrf2 -/-) of NRF2 (In-house developed, Autophagy. 2016, 2, :1902; Redox Biol. 2017, 13, 444).
Other analysis.
- Evaluation of the transcriptional signature of NRF2 by analyzing the levels of mRNA and proteins of the most representative NRF2 target genes.
- Electrophysiological analysis of the perforating hippocampal pathway (Redox Biol. 13:444451. 2017)
- Cognitive tests: objects recognition, Morris water maze.
- Inmmunohistochemical and inmmunofluorescence tests from brain samples.
