Overview of Research Capabilities of the Radioisotope and Functional Analysis Laboratory (RFAL) at CDT-CARD

 

In the field of drug candidate selection in preclinical in vitro research methodology:

affinity studies of new compounds (structures) for specific biological targets

(e.g., G-protein-coupled receptors, ion channels, nuclear receptors, membrane transporters) are conducted using radioreceptor methods or fluorescent ligand techniques. This includes both screening studies and detailed characterization, with determination of K_i and IC₅₀ values

 

Examples of Research Activities:

Study of Specific Receptors:

Serotonin receptors: 5-HT1A, 5-HT2A, 5-HT2C, 5-HT6, 5-HT7

Dopamine receptors: D1, D2, D3

Histamine receptors: H1, H3, H4

Cannabinoid receptors: CB1, CB2

Muscarinic receptor: M1

Opioid receptors: mu, delta, kappa

Monoamine transporters: SERT (serotonin), DAT (dopamine), NET (norepinephrine)

Adrenergic receptors: α1, α2, β

GABAergic receptors: GABA-A, GABA-B, BZD

Glutamatergic receptors: AMPA, NMDA (MK-801 and glycine sites), mGluR

Voltage-gated sodium channels (VGSC)

Binding Kinetics Studies:

Determination of binding kinetics parameters (k_on, K_off, and K_D) for compounds targeting selected receptors using radioreceptor methods.

Membrane Receptor Density Analysis:

Assessment of changes in membrane receptor density induced by tested compounds through in vitro and ex vivo studies, with determination of B_max and K_d values.

Allosteric Modulation Studies:

Identification and characterization of allosteric modulation of compounds using radioreceptor methods and in vitro electrophysiological studies, including screening and detailed analysis with EC₅₀/IC₅₀ determination.

Functional Assessment of Transporters:

Evaluation of compound effects on the activity of cellular transporters (e.g., SERT, DAT, NET, B⁰AT2) using isotopic methods and solid-phase scintillation techniques.

Ion Channel Studies:

Assessment of ion channel activation or inhibition using functional electrophysiological methods and alternative fluorescence-based techniques.

Examples:

Specific Channels and Receptors:

hERG (Kv11.1)

GABAA (α1β2γ2)

NMDA-2A

 

Enzyme Inhibition Studies:

Measurement of enzymatic inhibitor activity (e.g., phosphatases, phosphodiesterases, monoamine oxidases, kinases such as Cdk5) using recombinant enzyme preparations.

 

Signal Transduction Studies:

Investigation of signal transduction processes to evaluate the functional activity of receptor ligands, including:

Determination of intrinsic activity of GPCR ligands.

Assessment of functional selectivity based on activation of specific intracellular signaling pathways.

 

Advanced Detection Technologies in Signal Pathway Analysis:

Utilization of homogeneous, ultra-sensitive detection methods such as AlphaScreen, AlphaLISA, FRET, TR-FRET, fluorescence detection, luminescence, or cell imaging techniques (e.g., HCS, flow cytometry) to study various signaling pathways, including:

 

• PI3K/Akt/mTOR pathway: 4E-BP1, Akt, BAD, c-Myc, GSK-3ß, mTOR, p70S6K, PDGF, S6RP, SHP-1, SHP-2
• JAK/JAK2/STAT pathway: c-Myc, STAT1-STAT6
• NFκB/TBK1 pathway: IKKα, NFκB p65, PKC μ, TBK1
• MAPK pathway: ATF-1, ATF-2, c-Myc, ERK1/2, JNK1/2/3, MEK1, p38 MAPK, Raf1
• TGFß/BMP/SMAD pathway: c-Myc, SMAD1-SMAD3
• Adenylyl cyclase (AC) pathway: cAMP
• β-Arrestin signaling

 

Additional Examples of Research Focus and Capabilities:

Serotonin Receptors (5-HT):

5-HT1A: cAMP, Ca²⁺, ERK1/2, β-arrestin, GTPγ[S³⁵]

5-HT2A: Ca²⁺, cAMP

5-HT6: Ca²⁺, cAMP, ERK1/2

5-HT7: cAMP

Dopamine Receptors (D):

D1: cAMP

D2: Ca²⁺, cAMP, ERK1/2, β-arrestin

Histamine Receptors (H):

H3: cAMP, ERK1/2, β-arrestin

H4: cAMP, ERK1/2, β-arrestin

Opioid Receptor (μ):

β-arrestin, ERK1/2, cAMP, Ca²⁺

Adrenergic Receptors:

cAMP, Ca²⁺

Adenosine Receptors:

A1: cAMP

A2A: cAMP

A2B: cAMP

Muscarinic Receptor:

Ca²⁺

Glucagon-like peptide-1 receptor: β-arrestin

 

Extended Pharmacological Characterization:

In vitro and ex vivo studies of new compounds for:

Neuroprotective properties

Antioxidant activity

Anti-inflammatory effects

Anticancer potential

 

Cell Phenotype Interaction Studies:

 

Investigation of new compounds and materials’ effects on cell phenotypes in primary cell culture models and immortalized cell lines.

Primary cell models: Differentiated pluripotent stem cells (neuronal or cardiomyocyte-like), murine and human hepatocytes, human fibroblasts, and murine embryonic fibroblasts.

Immortalized cell lines (collection of several continuous lines):

Neuronal models: PC-12, SH-SY5Y, IMR-32, Neuro-2a

Cancer models: HepG2, PC3, MCF-7, Caco-2

Immune and inflammatory models: Jurkat, RAW 264.7, HL-60

Cardiac models: HL-1

General cellular models: HeLa, Caco-2

Research on the Impact of Compounds and Materials on Cellular and Molecular Processes:

Neuronal Growth and Development:

Analysis of neuron outgrowth using advanced cell imaging techniques.

Gene Expression Analysis:

Measurement of gene expression levels using real-time PCR (qPCR).

Apoptosis Studies:

Detection of apoptosis processes through: Caspase 3/7 and 8/9 assays, Annexin V staining, TUNEL assay

 

Antioxidant Markers and Mitochondrial Function:

Measurement of reactive oxygen species (ROS) and mitochondrial potential to evaluate oxidative stress.

Protein Marker Expression:

Analysis of intracellular and extracellular protein markers, including inflammatory markers and cytokine production.

Cellular Processes: cell migration assays, assessment of phagocytic activity, studies of cell differentiation.

Safety Profile Evaluation of New Compounds:

Mutagenicity testing (Ames test)

Multiparametric cytotoxicity assays on panels of cell lines and primary cells to evaluate: neurotoxicity, cardiotoxicity, hepatotoxicity.

Specific tests include:

Cell proliferation and morphology (MTS assay, PrestoBlue).

Intracellular accumulation of phospholipids and lipids (phospholipidosis markers; LipidTOX stains).

Mitochondrial function and toxicity: Depolarization of the inner mitochondrial membrane (MitoTracker).

Cell membrane damage (LDH assay, calcein-AM staining).

Organotypic Cultures:

Use of organotypic cultures to study: neurodegeneration processes, neurotoxicity, anticancer potential.

Protein Overexpression Studies:

Production of recombinant proteins in eukaryotic and prokaryotic systems.

 

 

Should you be interested in research on other biological targets, we offer the possibility of tailoring our services on demand to meet your specific needs.