HUMAN VDR BINDING ASSAY. This assay will be carried out in the Centro de Investigación en Medicina Molecular (CIMUS) in collaboration with Prof. Román Pérez-Fernández (Department of Physiology, School of Medicine, University of Santiago). Binding affinity to VDR is evaluated using a 1,25D assay kit under manufacturer conditions (Polarscreen Vitamin D receptor competitor assay, Red, catalogue no. PV4569, Invitrogen). This kit is a fluorescence polarization (FP)-based competition assay that provides a sensitive and robust method for high-throughput screening of potential vitamin D receptor ligands. VDR is added to a fluorescent VDR ligand to form a receptor/tracer complex resulting in a high polarization value. This complex is then added to individual test compounds in microwell plates. Competitors displace the tracer from the complex, causing the fluorescent ligand to tumble more rapidly during its fluorescence lifetime and resulting in a low polarization value. The polarized fluorescence is measured in a 384-well black plate during 200 ms/well using a Mithras LB 940 (Berthold Technologies). All compounds are evaluated within the range from 10−11 M to 10−5 M. IC50 values are calculated using average of measured values. The activity of each compound is also shown as percentage, in which the activity of the natural hormone 1,25D is normalized to 100%. For an example, see: Carballa DM, Seoane S, Zacconi F, Pérez X, Rumbo A, Alvarez-Díaz S, Larriba MJ, Pérez-Fernández R, Muñoz A, Maestro M, Mouriño A, Torneiro M. Synthesis and Biological Evaluation of 1α,25-Dihydroxyvitamin D3 Analogues with a Long Side Chain at C12 and Short C17 Side Chains. J. Med. Chem. 2012, 55, 8642–8656.
HUMAN DBP BINDING ASSAY. Will be performed at the laboratories of Prof. Mieke Verstuyf (Department of Endocrinology -LEGENDO-Leuven Un). [3H]1,25(OH)2D3 and 1,25(OH)2D3 or its analogues are added in 5 íL of ethanol into glass tubes and incubated with h-DBP (0.18 íM) in a final volume of 1 mL (0.01 M Tris-HCl buffer and 0.154 M NaCl, pH 7.4) for 3 h at 4 °C. Phase separation is then obtained by the addition of 0.5 mL of cold dextran-coated charcoal.
Biological assays: Prof. Román Pérez-Fernández (Departament of Physiology, Faculty of Medicine, CIMUS-Un Santiago). Prof. Mieke Verstuyf (Departament Endocrinology-Leuven Un), Prof. Alberto Muñoz (Institute of Biomedical Investigations-CSIC-Madrid). Prof. José Castillo (Clinical Neurosciences Research Laboratory of the Hospital Clínico Universitario).
TRANSACTIVATION ASSAYS (LUCIFERASE REPORTER ASSAYS). CIMUS: Transactivation assays will be accomplished to evaluate in vivo the effect of 1,25D analogues. MCF-7 human adenocarcinoma cells, cultured as previously described (J. Med. Chem. 2012, 55, 8642), are transfected using JetPEI transfection reagent (PolyPlus Transfection, Illrich, France). Transfections are performed in triplicate using 1 μg of pCYP24A1-Luc plasmid (kindly provided by Dr. Aranda, Instituto de Investigaciones Biomédicas Alberto Sols, Madrid). This vector encoding the luciferase gene under control of a consensus vitamin D response element (24-hydroxylase promoter, CYP24A1), and it is very responsive to the 1,25D treatment. After incubation for 24 h in DMEM supplemented with 10% of charcoal-stripped FCS, culture medium are replaced to phenol red free DMEM containing 10% FCS for 24 h with each analogue at several concentrations (1 × 10−11 to 1 ×10−6 M). Cells are then treated during 10 min with luciferin potassium salt (100 mg/L) (Regis Technologies, Morton Grove, IL), and bioluminescence images acquired with the In Vivo Imaging System (IVIS, Caliper Life Sciences, Alameda, CA, USA), quantified as total photon counts, and processed by Living Image software (Caliper Life Sciences). The EC50 values derived from dose−response curves and represent the analogue concentration capable of increasing the luciferase activity by 50%. The luciferase activity ratio is the average ratio of the EC50 for the analogue to the EC50 for 1,25D. ENSAYOS Occasionally the transactivation assays and related assays will also be carried out by Dr Alberto Muñoz’s research group (Instituto de Investigaciones Biométicas-Alberto Sols-Madrid). These assays include: (a) Induction of the transcriptional activity of VDR. (b) Inhibition of cell proliferation and phenotype changes. (c) Study of cellular levels of VDR. (d) Induction target genes of vitamin D.
CELL DIFFERENTIATION, INHIBITION OF CELL PROLIFERATION. CIMUS: Differentiation assay are performed in MCF-7 cells after treatment with 1,25D (as control) and the different analogues at several doses (10-11-10-6 M). MCF-7 cell morphology are evaluated by microscopy, and proteins involved in celular diferentiation (e.g. E-cadherin) are evaluated by Western blot. Cell proliferation experiments are carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, where MTT is reduced to purple formazan by the mitochondria of living cells. Increase in cell number are detected by augmented MTT metabolization. Calcium determination are carried out in female CD-1 mice (age matched, between 6 and 8 weeks) obtained from Charles River Laboratories (L’Arbresle, France). The analogues are dissolve in sesame oil and administer intraperitoneally (0.3 μg/kg) every other day for three weeks. Calcium measurement are determined a day after the last dose using the QuantiChom Calcium Assay Kit (BioAssay Systems, Hayward, CA, USA) following the manufacturer’s guidelines. The analogues described in objetive A will be tested by the group of Prof. Mieke Verstuyf (Department of Endocrinology-LEGENDO, Un Lovaina) with whom we have established a conjoint reserach program.
ALZHEIMER. The project will be developed at the laboratory of Prof. José Castillo (Clinical Neurosciences Research Laboratory of the Hospital Clínico Universitario) in collaboration with the group of Prof. Antonio Mouriño (Department of Organic Chemistry of the University of Santiago de Compostela).
The present project will be developed in 2 different work packages (WP). In this section of the project we will first describe the defined WP, and at the end we will describe all the experimental procedures that are necessary to carry out the project.
This project is organized in 2 WP:
- WP1 (Target Validation - screening): To test that 1,25D analogues induce protective effects in an in vitro model of AD.
The objective of this WP1 is to identify 1,25D analogues that induce protective effects in an in vitro model of AD. To achieve this objective we have used 2 in vitro models of AD: Abeta1-42 exposure in cortical/hippocampal culture and human neuroblastoma SH-SY5Y cells stably transfected with human amyloid precursor protein (APP) bearing the Swedish mutation APPsw.
The following milestones are expected for WP 1:
Milestone 1: Identification of 1,25D analogues that induce protective effects in an in vitro model of AD.
- WP2 (Target Validation – preclinical study): To test that those 1,25D analogues, selected from the in vitro study, modify the underlying pathogenic mechanisms associated to AD in an in vivo model of AD.
The objective of this WP2 is to identify which of the 1,25D analogues, selected from the in vitro study, modify the underlying pathogenic mechanisms associated to AD in an in vivo model of AD without side effects. To achieve this objective our in vivo experimental design will consist of APP/PS1 transgenic mice subjected to a 90-day treatment period (control, selected 1,25D analogues from in vitro study, n = 10, each group) followed by memory and learning tests. The follow-up period will be 6 months. The hippocampi and cerebral cortices of the left brain hemispheres will be examined for protein expression of APP695 and APP threonine (Thr)668, the key enzymes, β-secretase 1 (BACE1), and presenilin 1 (PS1). The right hippocampi and cerebral cortices were examined via immunohistochemistry (IHC) for Aβ plaque distribution. Furthermore, we will perform a noninvasive in vivo MRI study for detection of amyloid plaques.
The following milestones are expected for WP2:
Milestone 1: Selection of the best 1,25D analogue that modify the underlying pathogenic mechanisms associated to AD in an in vivo model of AD without side effects.
Milestone 2: To obtain a preclinical candidate compound for AD.
CALCEMIC EFFETCS. These assays will be carried out in the Centro de Investigación en Medicina Molecular (CIMUS) in collaboration with Prof. Román Pérez-Fernández (Department of Physiology, School of Medicine, University of Santiago). Calcium determinations are carried out in female CD-1 mice (age matched, between 6 and 8 weeks) obtained from Charles River Laboratories (L’Arbresle, France). The analogues are dissolved in sesame oil and administer intraperitoneally (0.3 μg/kg) every other day for three weeks. Calcium measurement are determined a day after the last dose using the QuantiChom Calcium Assay Kit (BioAssay Systems, Hayward, CA, USA) following the manufacturer’s guidelines.
STRUCTURAL ELUCIDATION. Crystallizations of the complexes ligand-VDR(Moras) and structural determinations will be carried out by Prof. Dino Moras (IGBMC-Illkirch, France). The data will be collected at Grenoble (ESRF). Personnel from the USC will occasionally move to the IGBMC.
MOLECULAR MODELLING. The conformers of each analogue will be generated by molecular dinamics employing the Discover module of Insight II. The generated conformations will be coupled to the binding domain of the VDR crystallographic structure using as anchoring points the hydroxyl groups. Occasionalyy no anchoring points will be used. The structural conformations will be optimized by energy minimization of the complex employing the CHARMM program. The affinity of the ligand for the receptor will be calculated on the basis of interaction energies of the analogue with the receptor. The Gold program will be used for a quick view of the most stable conformations of the target 1,25D analogues into the LBD(VDR).