Biological Screening of Drug Activities Lecture 3 PDF

Biological screening of Drug Activities Dr. Maha Abdollah Lecture 3 Anti-cancer Bioassays part I (in vitro)  IDENTIFY DRUG TARGET WHO PRIORITY MEDICINE Which pharmacological...

Biological screening of Drug Activities Dr. Maha Abdollah Lecture 3 Anti-cancer Bioassays part I (in vitro)  IDENTIFY DRUG TARGET WHO PRIORITY MEDICINE Which pharmacological classes are in desperate need for new drugs? Gap 1 Antimicrobials and Treatment(s) exist but will Influenza pandemic soon become ineffective Gap 2 WHO Priority Treatment(s) exist but the pharmaceutical delivery CVS, Cancer, HIV/AIDS, Medicine and mechanism or formulation is Depression Pharmaceutical gaps not appropriate for the target population Gap 3 Acute Stroke, Treatment does not exist OR Osteoarthritis, Alzheimer's is not sufficiently effective. and dementia 2  Hallmarks of Cancer Watch the video and write down the main properties of cancer cells Video link: http://www.youtube.com/watch?v=8LhQllh46yI 3  How Does Cancer Develop? cell Image from http://www.bbc.co.uk/schools/gcsebitesize/science/edexcel_pre_2011/genes/dnarev1.shtml Accelerator = oncogene Cancer arises from normal cells that start to divide uncontrollably (i.e. gone crazy!) Brakes = Tumour suppressor gene 4  Accelerator = oncogene Definition of Cancer Brakes = Tumour suppressor gene ✓ A term for diseases in which abnormal cells divide without control and can invade nearby tissues. ✓ If we imagine a normal cell is like a car, its movement (division) is governed by the gas (oncogenes) and the brakes (tumour suppressor genes) then a cancer cells is like a car which lost its brakes leading to a speeding car down a cliff (i.e. uncontrollable cell division)! 5  Cancer treatment Conven onal therapies Novel therapies Local treatments Systemic treatments Watch the video: http://www.youtube.com/watch?v=8LhQllh46yI Image from http://cancersystemsbiology.net/predicting.html 6  Goal of Cancer Therapy Cure: Cancer disappears and does not come back (uncommon) Control: shrink the tumour mass and stop cancer from growing and spreading. So it can be treated as a chronic disease Palliation: in advanced cancers; when the aim is to relieve symptoms to improve the quality of life Adjuvant: in combination with other types of treatments 7  What is the Main Therapeutic Effect of Anticancer Agents? Cancer arise form a single malignant cell, the therapeutic goal of cancer chemotherapy may require killing all actively growing tumour cells. Antineoplastics or Anticancer drugs are the drugs that prevent or inhibit the proliferation of neoplasms. Achievement of a therapeutic effect by combination therapy. 8  What parameter(s) will an in vitro bioassay assess to test the potential efficacy of a new anti-cancer drug? Choose all that apply Open Quizizz and let’s play… What parameter(s) will an in vitro bioassay assess to test the potential efficacy of a new anti-cancer drug? Choose all that apply 1. Cell viability 2. Cell death 3. Cytotoxicity 4. Inhibition in cell proliferation 5. Inhibition of cell death  Disease Models How to mimic a biological system? MOLECULAR LEVEL Molecular level Cellular level (in vitro) Organ/tissue level (ex-vivo) Organism level (in vivo) Why are these terms in italic? Because they are in Latin In vivo = in a living body Ex vivo = outside the living body In vitro = in glass 12 Types of cell death H2O H2O 13 (Cell Homicide) (Cell Suicide=programmed cell death) 14 It much more complicated…! For reference only…Don’t study! 15  VIDEOS OF CELLS UNDERGOING CELL DEATH See videos on e-learning Apoptosis 16 T-cytotoxic cells eating a cancer cells  How to know (measure) if a cell is dead or dying? Membrane integrity Cellular Functions (e.g. ATP production, enzyme activity..etc. ) DNA integrity Cell morphology 17  CYTOTOXICITY (VIABILITY) BIOASSAYS In vitro cytotoxicity assays are assays that measure whether a test compound is toxic to cells in culture, usually by determining the number of viable cells remaining after a defined incubation period. The desired approach is to use a convenient and cost-effective method that predicts in vivo toxicity by measuring a surrogate marker to indicate the viable cell number compared to untreated controls 18  Cytotoxicity (viability) Bioassays 1. Membrane integrity 2. Functional metabolic assays (Mitochondrial function) 3. DNA labelling 4. Morphological assays 19  The major criteria employed in viability assay Extra info Extra info Extra info 20  1- Morphological Assays Large-scale, morphological changes that occur at the cell surface, or in the cytoskeleton, can be followed and related to cell viability. Damage can be identified by large decreases in volume secondary to losses in protein and intracellular ions of due to altered permeability to sodium or potassium. Necrotic cells: nuclear swelling, chromatin flocculation, loss of nuclear basophilia Apoptotic cells: cell shrinkage, nuclear condansation, nuclear fragmentation 21  2- Functional Metabolic Assays Most famous examples: tetrazolium reduction, resazurin reduction, protease markers, and ATP detection assays. These assays measure some aspect of general metabolism or an enzymatic activity as a marker of viable cells. 22 2- Functional Metabolic assays (cont.) Under most standard culture conditions, incubation of the substrate with viable cells will result in generating a signal that is proportional to the number of viable cells present. Signal can be colorimetric, florescent or luminescent signal Cell When cells die, they rapidly lose the ability to convert the substrate viability reagent to product. Mitochondria 23  2- FUNCTIONAL METABOLIC ASSAYS (CONT.) TETRAZOLIUM & RESAZURIN REDUCTION ASSAYS A variety of tetrazolium compounds have been used to detect viable cells. e.g. MTT, MTS, XTT. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was the first homogeneous cell viability assay developed for a 96-well plates. Viable cells with active metabolism convert MTT into a purple colored formazan product with an absorbance 570 nm. When cells die, they lose the ability to convert MTT into formazan, thus color formation serves as a useful and convenient marker of only the viable cells. Colored 24 Procedure 25  Extra info-read NCI-60 Human Tumor Cell Lines Screen The screen was implemented in fully operational form in 1990 and utilizes 60 different human tumor cell lines to identify and characterize novel compounds with growth inhibition or killing of tumor cell lines. It is designed to screen up to 3,000 small molecules (synthetic or purified natural products) per year for potential anticancer activity. The operation of this screen utilizes 60 different human tumor cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney cancers. 26  3- MEMBRANE INTEGRITY ASSAYS DYE EXCLUSION ASSAYS This is generally accomplished by a dye exclusion stain, where cells with an intact membrane are able to exclude the dye while cells without an intact membrane take up the coloring agent. The trypan blue dye exclusion assay is the most commonly used and accepted method for the measurement of cell viability It relies on the alteration in membrane integrity as determined by the uptake of dye by dead cells, thereby giving a direct measure of cell viability Stain : Trypan Blue (stains only dead/non-viable cells as it can penetrate their cell membrane) Device used : Hemocytometer: Principledevice originally invented to count blood cells Live 27 Dead Trypan Blue dye Exclusion Methods  4- DNA Based Assays DNA-binding dye labelling Some fluorochromes (fluorescent dyes) have the ability to bind to the DNA of the cell Some dyes can penetrate the intact cell membrane and therefore can stain both live Extra info and dead cells (e.g. Hoechst, DAPI, Acridine orange). Depending on the morphology of the stained DNA; a cell is identified as dead or alive While other dyes can only penetrate the the membrane of dead cells (e.g. PI or EtBr) and therefore only stain dead cells 28 (similar to trypan blue) Nuclei of Cells Stained With Hoechst Square: intact nuclei with condensed chromatin Arrows: examples of fragmented DNA of dying or dead cells 29 Nuclei of cells stained with PI Rituximab is an antibody used to treat blood cancer Lymphoma cells were suspended in 4 μL of culture medium containing Alexa 488–labeled rituximab ( Alexa 488 is a dye which makes the antibody green) and propidium iodide (red). The alteration of cellular viability from immediately after the addition of the treatment was observed by the incorporation of PI (red stain) into the nuclei of dead cells. 30 http://clincancerres.aacrjournals.org/content/15/10/3624  The Power of Experimental Controls!! Something might seem simple at a first look but will take more time and effort to complete than expected.  Open Quizizz and let’s play again… A herbal extract with potential anti-cancer effect is being screened using the MTT assay. The extract is not water soluble so the researcher dissolved it in DMSO (organic solvent). Suggest a positive and a negative control for this study.  Choose all that apply A negative control could A positive control could be........ be....... 1. DMSO 1. Bleach (Clorox) 2. Saline 2. Ethanol 3. Water 3. Detergent (Fairy liquid Soap) 4. Olive oil 4. Saline 5. Ethanol 5. Water