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

2. 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.

3. 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

4. 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.

5. 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)

6. 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)

7. 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.

8. 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

9. 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

10. 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)

11. 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

12. Biological Fluorescence
IC: Radiationless internal conversion to ground state

13. 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.

14. 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

15. 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

16. 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.

17. 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.

18. 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 I and sulphur-35 35S

19. Thank You