Principles of Biological Assays and Lead Compound Identification Flashcards Preview

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Flashcards in Principles of Biological Assays and Lead Compound Identification Deck (104):
1

biochemical assays

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

2

what kind of assay is a receptor binding assay?

biochemical assay

3

what kind of assay is an enzymatic activity assay?

biochemical assay

4

what kind of assay looks at protein-protein interactions?

biochemical assay

5

cell-based assays:

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

6

what kind of assay looks at signal transduction pathways?

cell-based assay

7

an analytical assay looks at

binding --> does your compound bind?

8

a functional assay looks at

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

9

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

membrane protein

10

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

milligram quantities

11

what are two types of biochemical assays?

analytical and functional

12

cell-based screens are predominantly _____ assays

functional

13

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

14

when would you use a cell-based analytical assay?

when determining binding to a membrane bound receptor

15

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

16

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

promoter/ reporter protein

17

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

18

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

19

chemiluminescence:

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

20

bioluminescence:

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

21

the dual luciferase assay system is a ______ assay

cell-based

22

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

Renilla/ positive control

23

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

firefly

24

a fluorophore is excited from its ground state by

absorbing a photon

25

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

collisions

26

as molecules return to ground state they emit

a photon

27

each fluorophore has

characteristic exitation and emission wavelengths

28

caspases

proteases that are activated in cells and regulate apoptosis

29

caspases _____ to ________

cleave specific proteins/ initiate or continue "death cascade"

30

a ______ assay can check the activity of a capsase

fluorescence

31

fluorophore excited with polarized light emits _______ light

polorized

32

________ of the fluorophore results in depolarization

increased rotation

33

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.

34

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

35

morphine is a

natural product

36

synthetic compounds can be formed by using

combinatorial libraries, high-throughput screening, traditional chemical synthesis

37

herceptin is a

biological compount (antibody)

38

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

synthetic, natural product derived, biological

39

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

high-throughput screening

40

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

high-throughput screening/ de novo discovery

41

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

100,000 - 3 million

42

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

diverse

43

"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

44

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

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

45

attrition rate = ______/______ = ____

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

46

approximately ___ percent of drug candidates are halted because of toxicity

30

47

high-content screening:

form of HTS that analyzes images of cells following compound treatment

48

standard output of high-content screening is

fluorescence (GFP)

49

you can view movement of proteins within the cell with

high-content screening

50

you can view morphology of cells with

high-content screening

51

you can view integrity of cytoskeleton with

high-content screening

52

you can view DNA content with

high-content screening

53

you can see the shape and localization of organelles with

high-content screening

54

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

55

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.

56

the dual luciferase assay system is a ______ assay

cell-based

57

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

Renilla/ positive control

58

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

firefly

59

a fluorophore is excited from its ground state by

absorbing a photon

60

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

collisions

61

as molecules return to ground state they emit

a photon

62

each fluorophore has

characteristic exitation and emission wavelengths

63

caspases

proteases that are activated in cells and regulate apoptosis

64

caspases _____ to ________

cleave specific proteins/ initiate or continue "death cascade"

65

a ______ assay can check the activity of a capsase

fluorescence

66

fluorophore excited with polarized light emits _______ light

polorized

67

________ of the fluorophore results in depolarization

increased rotation

68

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.

69

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

70

morphine is a

natural product

71

synthetic compounds can be formed by using

combinatorial libraries, high-throughput screening, traditional chemical synthesis

72

herceptin is a

biological compount (antibody)

73

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

synthetic, natural product derived, biological

74

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

high-throughput screening

75

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

high-throughput screening/ de novo discovery

76

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

100,000 - 3 million

77

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

diverse

78

"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

79

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

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

80

attrition rate = ______/______ = ____

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

81

approximately ___ percent of drug candidates are halted because of toxicity

30

82

high-content screening:

form of HTS that analyzes images of cells following compound treatment

83

standard output of high-content screening is

fluorescence (GFP)

84

you can view movement of proteins within the cell with

high-content screening

85

you can view morphology of cells with

high-content screening

86

you can view integrity of cytoskeleton with

high-content screening

87

you can view DNA content with

high-content screening

88

you can see the shape and localization of organelles with

high-content screening

89

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

90

antisense oligonucleotides:

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

91

mRNA must be ______ for translation into protein

single-stranded

92

a DNA/RNA hybrid will be ________

degraded by RNase H

93

RNase H is a

non-specific endonuclease

94

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

morpholino

95

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

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

96

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

miRNAs

97

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

to the cytoplasm/ Dicer

98

miRNAs work in concert with _______ to prevent protein expression

the RNA - silencing complex (RISC)

99

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

miRNAs

100

_____ are synthetic RNA sequences analogous to ____

siRNA/ miRNA

101

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

siRNAs

102

______ are not yet stable enough to be great drugs

siRNA

103

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

104

what is a caveat to knockout mice?

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