Principles of Biological Assays and Lead Compound Identification Flashcards
biochemical assays
identify how compounds interact with an isolated target in an artifical environment
what kind of assay is a receptor binding assay?
biochemical assay
what kind of assay is an enzymatic activity assay?
biochemical assay
what kind of assay looks at protein-protein interactions?
biochemical assay
cell-based assays:
analyze a measurable effect of a given target within the cell, its natural environment
what kind of assay looks at signal transduction pathways?
cell-based assay
an analytical assay looks at
binding –> does your compound bind?
a functional assay looks at
enzyme activity –> does your compound have an effect on the system?
name a protein type that would be difficult to study with a biochemical assay
membrane protein
what quantity of protein is required to do a high throughput screen?
milligram quantities
what are two types of biochemical assays?
analytical and functional
cell-based screens are predominantly _____ assays
functional
to determine binding to a membrane bound receptor, you can
express the receptor of interest in an alternate cell line through DNA construct/ vector and measure binding/ displacement of fluorescent analogue
when would you use a cell-based analytical assay?
when determining binding to a membrane bound receptor
why might you clone a vector into a cell of interest?
to study mutated versions of protein of interest in cell culture, if there is not enough protein of interest in cell of interest, to study cell surface binding for membrane proteins, because reporter output makes for an easier experiment
in a vector, you want the ____ of your protein of interest to cause the transcription of ____
promoter/ reporter protein
for an anti-proliferative assay, you measure _______. If a cell is alive, it converts ____ to ___ via enzymatic reduction. If the cell is dead, no reduction takes place and ____ light is absorbed
the absorption of purple light/ Yellow MTS/ purple formazan/ yellow
Amplex red can be converted to resorufin by horseradish peroxidase in the presence of _______. This is only produced, through a series of reactions, if ____ is made available by the _______ of the protein of interest. If there is no red color, ______
H2O2/ phosphate/ ATPase activity/ the ATPase activity of the protein of interest was inhibited by the drug
chemiluminescence:
substrate absorbs energy from an exothermic chemical reaction. Relaxation to ground state produces quantifiable light emission
bioluminescence:
light is produced through enzymatic activity - luciferase - catalyzes chemical reaction that results in light production
the dual luciferase assay system is a ______ assay
cell-based
in the dual luciferase assay system, the constitutive _______ luciferase serves as a _____
Renilla/ positive control
________ luciferase is only present upon activation of the pathway of interest
firefly
a fluorophore is excited from its ground state by
absorbing a photon
___________ cause the excited molecules to lose energy and return to ground state
collisions
as molecules return to ground state they emit
a photon
each fluorophore has
characteristic exitation and emission wavelengths
caspases
proteases that are activated in cells and regulate apoptosis
caspases _____ to ________
cleave specific proteins/ initiate or continue “death cascade”
a ______ assay can check the activity of a capsase
fluorescence
fluorophore excited with polarized light emits _______ light
polorized
________ of the fluorophore results in depolarization
increased rotation
the degree of depolarization of the light from a fluorophore directly correlates to
the amount of binding of the fluorophore/ligand complex to a receptor molecule. If your ligand of interest is attached to the fluorophore, polarized output would mean that it had bound to a receptor. If your fluorophore was attached to a ligand known to bind the receptor and you introduced a new ligand to see if it could compete for binding sites, depolarization would mean that your new ligand had bound and some of the initial ligand was now free and undergoing rapid rotation.
if you were checking for activity of a kinase, you might use an antibody to _____ bound to a tracer. If your kinase was active, you would expect the tracer to give _____ light
ADP/ depolarized
morphine is a
natural product
synthetic compounds can be formed by using
combinatorial libraries, high-throughput screening, traditional chemical synthesis
herceptin is a
biological compount (antibody)
the most prevalent categories of FDA Approved Drugs 1981 - 2006 are
synthetic, natural product derived, biological
you can use _____ to do preliminary assessment of metabolism and toxicity of new drug candidates
high-throughput screening
you can use ____ for ______ - a search for compounds that modulate a new biochemical or cellular target
high-throughput screening/ de novo discovery
how many compounds are in a high-throughput screening compound library?
100,000 - 3 million
for high-throughput screening, it is important to use _____ structural scaffolds
diverse
“Drug-like” properties:
no more than 5 H bond donors (OH or NH), no more than 10 H bond acceptors (N or O), molecular weight less than 500 g/mol, log P less than 5
small molecules in a library for screening should have these properties:
MW less than 350 g/mol, log P of about 3
attrition rate = ______/______ = ____
of compounds synthesized and tested/ # of compounds approved by FDA = about 1000
approximately ___ percent of drug candidates are halted because of toxicity
30
high-content screening:
form of HTS that analyzes images of cells following compound treatment
standard output of high-content screening is
fluorescence (GFP)
you can view movement of proteins within the cell with
high-content screening
you can view morphology of cells with
high-content screening
you can view integrity of cytoskeleton with
high-content screening
you can view DNA content with
high-content screening
you can see the shape and localization of organelles with
high-content screening
In Vivo high-throughput screening uses __. Issues are that they don’t have __, ____ or ____, they have some differences in ____, and ___ is not characterized.
zebrafish/ lungs/ prostate/ breasts/ skin/ CYP450
the degree of depolarization of the light from a fluorophore directly correlates to
the amount of binding of the fluorophore/ligand complex to a receptor molecule.
the dual luciferase assay system is a ______ assay
cell-based
in the dual luciferase assay system, the constitutive _______ luciferase serves as a _____
Renilla/ positive control
________ luciferase is only present upon activation of the pathway of interest
firefly
a fluorophore is excited from its ground state by
absorbing a photon
___________ cause the excited molecules to lose energy and return to ground state
collisions
as molecules return to ground state they emit
a photon
each fluorophore has
characteristic exitation and emission wavelengths
caspases
proteases that are activated in cells and regulate apoptosis
caspases _____ to ________
cleave specific proteins/ initiate or continue “death cascade”
a ______ assay can check the activity of a capsase
fluorescence
fluorophore excited with polarized light emits _______ light
polorized
________ of the fluorophore results in depolarization
increased rotation
the degree of depolarization of the light from a fluorophore directly correlates to
the amount of binding of the fluorophore/ligand complex to a receptor molecule. If your ligand of interest is attached to the fluorophore, polarized output would mean that it had bound to a receptor. If your fluorophore was attached to a ligand known to bind the receptor and you introduced a new ligand to see if it could compete for binding sites, depolarization would mean that your new ligand had bound and some of the initial ligand was now free and undergoing rapid rotation.
if you were checking for activity of a kinase, you might use an antibody to _____ bound to a tracer. If your kinase was active, you would expect the tracer to give _____ light
ADP/ depolarized
morphine is a
natural product
synthetic compounds can be formed by using
combinatorial libraries, high-throughput screening, traditional chemical synthesis
herceptin is a
biological compount (antibody)
the most prevalent categories of FDA Approved Drugs 1981 - 2006 are
synthetic, natural product derived, biological
you can use _____ to do preliminary assessment of metabolism and toxicity of new drug candidates
high-throughput screening
you can use ____ for ______ - a search for compounds that modulate a new biochemical or cellular target
high-throughput screening/ de novo discovery
how many compounds are in a high-throughput screening compound library?
100,000 - 3 million
for high-throughput screening, it is important to use _____ structural scaffolds
diverse
“Drug-like” properties:
no more than 5 H bond donors (OH or NH), no more than 10 H bond acceptors (N or O), molecular weight less than 500 g/mol, log P less than 5
small molecules in a library for screening should have these properties:
MW less than 350 g/mol, log P of about 3
attrition rate = ______/______ = ____
of compounds synthesized and tested/ # of compounds approved by FDA = about 1000
approximately ___ percent of drug candidates are halted because of toxicity
30
high-content screening:
form of HTS that analyzes images of cells following compound treatment
standard output of high-content screening is
fluorescence (GFP)
you can view movement of proteins within the cell with
high-content screening
you can view morphology of cells with
high-content screening
you can view integrity of cytoskeleton with
high-content screening
you can view DNA content with
high-content screening
you can see the shape and localization of organelles with
high-content screening
In Vivo high-throughput screening uses __. Issues are that they don’t have __, ____ or ____, they have some differences in ____, and ___ is not characterized.
zebrafish/ lungs/ prostate/ breasts/ skin/ CYP450
antisense oligonucleotides:
single strand of DNA or RNA that is complementary to chosen sequence
mRNA must be ______ for translation into protein
single-stranded
a DNA/RNA hybrid will be ________
degraded by RNase H
RNase H is a
non-specific endonuclease
__________ are chemically modified antisense oligonucleotidses that are more stable than standard synthetic oligonucleotides
morpholino
_____ are catalytic RNA molecules. They have a _______flanked by _________. They ____
ribozymes/ catalytic core/ arms complementary to desired mRNA sequence/ cleave RNA messages
RNA Interference: ___________: short RNA sequences (18-26 nucleotides) - natural mechanism to silence gene transcription
miRNAs
pre-miRNAs are exported ______ and processed by ______ into miRNAs
to the cytoplasm/ Dicer
miRNAs work in concert with _______ to prevent protein expression
the RNA - silencing complex (RISC)
_____ are part of the natural cellular mechanism to regulate gene transcription
miRNAs
_____ are synthetic RNA sequences analogous to ____
siRNA/ miRNA
______ prevent activity of specific genes in vitro and/or in vivo
siRNAs
______ are not yet stable enough to be great drugs
siRNA
when creating knockout mice, you introduce __________ by _______ to _______. Cells carrying the target gene are isolated using ____.
a targeting vector for gene of interest/ electroporation/ mouse embryonic stem cells/ positive negative selection
what is a caveat to knockout mice?
most disease states are controlled by more than one gene/protein
