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Fundamentals of Bio Orgo > Principles of Biological Assays and Lead Compound Identification > Flashcards

Principles of Biological Assays and Lead Compound Identification Flashcards

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Biology 101 flashcards
1
Q

biochemical assays

A

identify how compounds interact with an isolated target in an artifical environment

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2
Q

what kind of assay is a receptor binding assay?

A

biochemical assay

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3
Q

what kind of assay is an enzymatic activity assay?

A

biochemical assay

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4
Q

what kind of assay looks at protein-protein interactions?

A

biochemical assay

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5
Q

cell-based assays:

A

analyze a measurable effect of a given target within the cell, its natural environment

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6
Q

what kind of assay looks at signal transduction pathways?

A

cell-based assay

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7
Q

an analytical assay looks at

A

binding –> does your compound bind?

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8
Q

a functional assay looks at

A

enzyme activity –> does your compound have an effect on the system?

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9
Q

name a protein type that would be difficult to study with a biochemical assay

A

membrane protein

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10
Q

what quantity of protein is required to do a high throughput screen?

A

milligram quantities

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11
Q

what are two types of biochemical assays?

A

analytical and functional

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12
Q

cell-based screens are predominantly _____ assays

A

functional

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13
Q

to determine binding to a membrane bound receptor, you can

A

express the receptor of interest in an alternate cell line through DNA construct/ vector and measure binding/ displacement of fluorescent analogue

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14
Q

when would you use a cell-based analytical assay?

A

when determining binding to a membrane bound receptor

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15
Q

why might you clone a vector into a cell of interest?

A

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

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16
Q

in a vector, you want the ____ of your protein of interest to cause the transcription of ____

A

promoter/ reporter protein

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17
Q

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

A

the absorption of purple light/ Yellow MTS/ purple formazan/ yellow

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18
Q

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, ______

A

H2O2/ phosphate/ ATPase activity/ the ATPase activity of the protein of interest was inhibited by the drug

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19
Q

chemiluminescence:

A

substrate absorbs energy from an exothermic chemical reaction. Relaxation to ground state produces quantifiable light emission

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20
Q

bioluminescence:

A

light is produced through enzymatic activity - luciferase - catalyzes chemical reaction that results in light production

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21
Q

the dual luciferase assay system is a ______ assay

A

cell-based

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22
Q

in the dual luciferase assay system, the constitutive _______ luciferase serves as a _____

A

Renilla/ positive control

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23
Q

________ luciferase is only present upon activation of the pathway of interest

A

firefly

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24
Q

a fluorophore is excited from its ground state by

A

absorbing a photon

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25
Q

___________ cause the excited molecules to lose energy and return to ground state

A

collisions

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26
Q

as molecules return to ground state they emit

A

a photon

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27
Q

each fluorophore has

A

characteristic exitation and emission wavelengths

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28
Q

caspases

A

proteases that are activated in cells and regulate apoptosis

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29
Q

caspases _____ to ________

A

cleave specific proteins/ initiate or continue “death cascade”

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30
Q

a ______ assay can check the activity of a capsase

A

fluorescence

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31
Q

fluorophore excited with polarized light emits _______ light

A

polorized

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32
Q

________ of the fluorophore results in depolarization

A

increased rotation

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33
Q

the degree of depolarization of the light from a fluorophore directly correlates to

A

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.

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34
Q

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

A

ADP/ depolarized

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35
Q

morphine is a

A

natural product

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36
Q

synthetic compounds can be formed by using

A

combinatorial libraries, high-throughput screening, traditional chemical synthesis

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37
Q

herceptin is a

A

biological compount (antibody)

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38
Q

the most prevalent categories of FDA Approved Drugs 1981 - 2006 are

A

synthetic, natural product derived, biological

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39
Q

you can use _____ to do preliminary assessment of metabolism and toxicity of new drug candidates

A

high-throughput screening

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40
Q

you can use ____ for ______ - a search for compounds that modulate a new biochemical or cellular target

A

high-throughput screening/ de novo discovery

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41
Q

how many compounds are in a high-throughput screening compound library?

A

100,000 - 3 million

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42
Q

for high-throughput screening, it is important to use _____ structural scaffolds

A

diverse

43
Q

“Drug-like” properties:

A

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

44
Q

small molecules in a library for screening should have these properties:

A

MW less than 350 g/mol, log P of about 3

45
Q

attrition rate = ______/______ = ____

A

of compounds synthesized and tested/ # of compounds approved by FDA = about 1000

46
Q

approximately ___ percent of drug candidates are halted because of toxicity

A

30

47
Q

high-content screening:

A

form of HTS that analyzes images of cells following compound treatment

48
Q

standard output of high-content screening is

A

fluorescence (GFP)

49
Q

you can view movement of proteins within the cell with

A

high-content screening

50
Q

you can view morphology of cells with

A

high-content screening

51
Q

you can view integrity of cytoskeleton with

A

high-content screening

52
Q

you can view DNA content with

A

high-content screening

53
Q

you can see the shape and localization of organelles with

A

high-content screening

54
Q

In Vivo high-throughput screening uses __. Issues are that they don’t have __, ____ or ____, they have some differences in ____, and ___ is not characterized.

A

zebrafish/ lungs/ prostate/ breasts/ skin/ CYP450

55
Q

the degree of depolarization of the light from a fluorophore directly correlates to

A

the amount of binding of the fluorophore/ligand complex to a receptor molecule.

56
Q

the dual luciferase assay system is a ______ assay

A

cell-based

57
Q

in the dual luciferase assay system, the constitutive _______ luciferase serves as a _____

A

Renilla/ positive control

58
Q

________ luciferase is only present upon activation of the pathway of interest

A

firefly

59
Q

a fluorophore is excited from its ground state by

A

absorbing a photon

60
Q

___________ cause the excited molecules to lose energy and return to ground state

A

collisions

61
Q

as molecules return to ground state they emit

A

a photon

62
Q

each fluorophore has

A

characteristic exitation and emission wavelengths

63
Q

caspases

A

proteases that are activated in cells and regulate apoptosis

64
Q

caspases _____ to ________

A

cleave specific proteins/ initiate or continue “death cascade”

65
Q

a ______ assay can check the activity of a capsase

A

fluorescence

66
Q

fluorophore excited with polarized light emits _______ light

A

polorized

67
Q

________ of the fluorophore results in depolarization

A

increased rotation

68
Q

the degree of depolarization of the light from a fluorophore directly correlates to

A

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.

69
Q

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

A

ADP/ depolarized

70
Q

morphine is a

A

natural product

71
Q

synthetic compounds can be formed by using

A

combinatorial libraries, high-throughput screening, traditional chemical synthesis

72
Q

herceptin is a

A

biological compount (antibody)

73
Q

the most prevalent categories of FDA Approved Drugs 1981 - 2006 are

A

synthetic, natural product derived, biological

74
Q

you can use _____ to do preliminary assessment of metabolism and toxicity of new drug candidates

A

high-throughput screening

75
Q

you can use ____ for ______ - a search for compounds that modulate a new biochemical or cellular target

A

high-throughput screening/ de novo discovery

76
Q

how many compounds are in a high-throughput screening compound library?

A

100,000 - 3 million

77
Q

for high-throughput screening, it is important to use _____ structural scaffolds

A

diverse

78
Q

“Drug-like” properties:

A

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

79
Q

small molecules in a library for screening should have these properties:

A

MW less than 350 g/mol, log P of about 3

80
Q

attrition rate = ______/______ = ____

A

of compounds synthesized and tested/ # of compounds approved by FDA = about 1000

81
Q

approximately ___ percent of drug candidates are halted because of toxicity

A

30

82
Q

high-content screening:

A

form of HTS that analyzes images of cells following compound treatment

83
Q

standard output of high-content screening is

A

fluorescence (GFP)

84
Q

you can view movement of proteins within the cell with

A

high-content screening

85
Q

you can view morphology of cells with

A

high-content screening

86
Q

you can view integrity of cytoskeleton with

A

high-content screening

87
Q

you can view DNA content with

A

high-content screening

88
Q

you can see the shape and localization of organelles with

A

high-content screening

89
Q

In Vivo high-throughput screening uses __. Issues are that they don’t have __, ____ or ____, they have some differences in ____, and ___ is not characterized.

A

zebrafish/ lungs/ prostate/ breasts/ skin/ CYP450

90
Q

antisense oligonucleotides:

A

single strand of DNA or RNA that is complementary to chosen sequence

91
Q

mRNA must be ______ for translation into protein

A

single-stranded

92
Q

a DNA/RNA hybrid will be ________

A

degraded by RNase H

93
Q

RNase H is a

A

non-specific endonuclease

94
Q

__________ are chemically modified antisense oligonucleotidses that are more stable than standard synthetic oligonucleotides

A

morpholino

95
Q

_____ are catalytic RNA molecules. They have a _______flanked by _________. They ____

A

ribozymes/ catalytic core/ arms complementary to desired mRNA sequence/ cleave RNA messages

96
Q

RNA Interference: ___________: short RNA sequences (18-26 nucleotides) - natural mechanism to silence gene transcription

A

miRNAs

97
Q

pre-miRNAs are exported ______ and processed by ______ into miRNAs

A

to the cytoplasm/ Dicer

98
Q

miRNAs work in concert with _______ to prevent protein expression

A

the RNA - silencing complex (RISC)

99
Q

_____ are part of the natural cellular mechanism to regulate gene transcription

A

miRNAs

100
Q

_____ are synthetic RNA sequences analogous to ____

A

siRNA/ miRNA

101
Q

______ prevent activity of specific genes in vitro and/or in vivo

A

siRNAs

102
Q

______ are not yet stable enough to be great drugs

A

siRNA

103
Q

when creating knockout mice, you introduce __________ by _______ to _______. Cells carrying the target gene are isolated using ____.

A

a targeting vector for gene of interest/ electroporation/ mouse embryonic stem cells/ positive negative selection

104
Q

what is a caveat to knockout mice?

A

most disease states are controlled by more than one gene/protein

Fundamentals of Bio Orgo (9 decks)

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