Drug/Target Validation BMS 409 (Lecture 2) Biological Target validation September/29/ 2016

How to validate the identified Drug/Target Target validation is the process by which the predicted molecular target (e.g.: protein or nucleic acid )of a small molecule is verified. It may include: 1- Determining the SAR of analogs of the drug candidate 2- Generating a drug-resistant mutant of the assumed target 3- knockdown or overexpression of the target 4- Monitoring the known signalling systems downstream of the presumed target.

Drug/Target Validation It is also the confirmation of biologically activity of the target. It employs in vitro and in vivo bio-assays. Verify a link between target and disease ??? Have evidence that modulating the target produces the desire effect Wrong Hypothesis Failure of the drug (high cost) Fully validation occurs only when the desired effect seen in patients

Types of different assays Biochemical Assays Functional Assays (Bioassays) Biophysical Assays Analytical in vitro procedures used to detect, quantify, study the binding or activity of a biological molecule and/or the affinity of the test compound (ligand) to specific target as receptors and enzymes e.g.: Radio-active ligand binding assay, Fluorescence techniques They are techniques/methods having HTS capabilities. They need sophisticated instruments e.g.: X-ray Crystallography, NMR, Surface Plasmon Resonance , chromatography Imaging techniques Cell –Based assays that measure the function of the selected target with or without the drug???. [Within the cell ] It measures the biological activity of the target in cells and tissues and how they respond to the tested compound.

Target Identification Test the compound to see its modulation on the target (effect on target biological activity) Target Identification Develop an assay to measure its biological activity Measure chemical characteristics of the hit compound [ex-vivo(body part, organ, organ part), Pharmacological evaluation in vivo HTS(sensitive, stable, reproducible, robust, suitable for screening large number of samples) Hit compound is re-screened to exclude false positives Generally, validation starts with 1- Biochemical assays 2-cell based assay/ ex-vivo (functional assays) 3- in vivo assessment (animal models)

Assay Development 1- Selection or developing an assay? 2- Technique/Method/Assay evaluation? Selection or developing an assay? 1- Nature of the target: protein, DNA, RNA, glycans 2- Lab infrastructure 3-Experience of the lab scientists 4- Nature of the test compound (Radio-labeled, Fluorescent, colorimetric)

Technique/Method/Assay evaluation? Proper Method selection and its evaluation are extremely essential steps in providing highly accurate results, as the results will affect all the downstream applications. So, proper selection and evaluation of any bio-analytical method/assay should fulfill specific characteristics to be approved for using in the lab.

Characteristics of Analytical Methods/or factors affecting the assay Non-analytical Factors or characteristics Reliability Factors or characteristics Relate to the performance of the method used Equipments Workload Sample handling Personal skills Cost per test Standardization methods Reagents used (fresh or not) Procedures required for each method - Pre, post & analytical variables - Documentation - Data Interpretation Accuracy Precision Specificity Sensitivity

Precision (Repeatability) Accuracy (Trueness) It defines how close the results approaches the absolute or standard substance. Results from every test performed are compared to Standard that have undergone multiple evaluation and compared to the best testing standards available that have been already established before (The Gold Standard) Precision (Repeatability) It is the reproducibility of an analytical method If the test is repeated for the same target, the same results are obtained

Specificity of an assay (test) Sensitivity of an assay (test) Relates to how good the assay is at discriminating between the requested or tested analyte and other interfering substances in the sample ( False positive) Sensitivity of an assay (test) It is a measure of how little of the target, the method can detect The determination of the detection limit of the assay (False negative)

Quality Control It represents the steps, techniques and procedures taken by the laboratory to monitor the performance of the laboratory, to ensure that the tests(assays) are performed correctly and reliable i.e. Set of rules that is used to verify the reliability of test results Also, it is the laboratory system that deals with the study of the source of results variation, recognizing them & minimizing the analytical errors Quality Control of the various methods are essential for evaluating and performance of the developed assays

Biological Fluorescence IC: Radiationless internal conversion to ground state

Förster Resonance Energy Transfer: FRET This mean the radiationless energy transfer between a donor-acceptor pair between two proteins or a protein and fluorophore, or two fluorophores.

FRET Ex Em R < 1.5 R0 R >> 1.5 R0 R0 = critical transfer distance, for CFP-YFP R0 = 5.0 nm 1.0 0.8 0.6 0.4 0.2 0.0 600 550 500 450 400 350 Absorbance, fluorescence Wavelength, nm Abs. Em. CFP YFP

FRET can be used in cells to study Protein-protein interactions Interaction CFP YFP FRET NO FRET Conformational CFP YFP Conformational changes Change FRET NO FRET CFP YFP Proteolysis Proteolytic processing FRET NO FRET

Fluorescence Quenching Quenching: refers to any process that lowers or fade the fluorescence intensity of a fluorophore or fluorescent protein. It occurs when the quencher absorption wavelength overlaps with the fluorophore emission wavelength.

Quenching can be either dynamic or static: Dynamic: occurs by colloidal of the quencher with the fluorophore during the lifetime of the excited state. Upon contact , the fluorophore returns to the ground state without emitting a photon. In general, dynamic quenching occurs without any parameter change in the fluorophore, which is without a photochemical reaction. Few example of dynamic quencher are Oxygen, Acryl amide, Nitrogen oxide, Iodide, Halogens. Static: a complex is formed between the fluorophore and the quencher. The complex is non-fluorescent and exists much longer than the complex between a fluorophore and a dynamic quencher. Often, static quenching is permanent. Static quenchers such as:Nitrogen bases, Nicotinamide, Heavy metals, Guanine.

Radiolabelled ligand Binding Assays It is used mainly to measure the binding of a ligand to a drug/target. Evaluate the binding of a ligand to its target using specific radioligand. Simple, easily automated for HTS (it can measure many compounds at the same time. The purpose of the assay is to assess the proportion of a ligand binding to a target at any given ligand concentration. Requirements for a radioligand binding assay 1- Sufficient amount of target protein to enable detection of binding Problem, why?? So, select a tissue or cells that overexpress the target protein. 2- Availiability of the radiolabelled ligands (available commercially) Problem, why?? Limits the types of targets under study. 3- Availiability of instruments to measure radioactivity. 4- Design a method for separation the bound ligand from the free ligand. The most common used radio ligands: Tritium 3H, iodinie-125 125I and sulphur-35 35S

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