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endo y3s1 > IC7 ADME of macromolecules > Flashcards

IC7 ADME of macromolecules Flashcards

1
Q

timeline of structural proteins

A

long lifetime; do not require high turnover

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2
Q

timeline of regulatory proteins

A

short lifetime

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3
Q

reason for degradation of regulatory protein

A

once signal transmitted results in response to environmental change, regulatory protein no longer required

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4
Q

importance of protein degradation

A
  1. Ensures proper regulation of cell signalling pathways via normal protein turnovers
  2. Remove misfolded & damaged proteins that can lead to abnormal cellular activities
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5
Q

problem of accumulating misfolded & damaged proteins

A

deviation from normal activity
results in disease

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6
Q

methods of protein degradation

A
  1. Lysosomal degradation (10-20%)
  2. Proteasomal degradation (80-90%)
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7
Q

key step in protein degradation
(before degradation can occur)

A

Endocytosis

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8
Q

types of endocytosis

A

Phagocytosis
Pinocytosis
Receptor-mediated endocytosis

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9
Q

particles involved in phagocytosis

A

large solid particles

cell debris, dead cells, protein aggregates, pathogenic microorganisms , particulate non-living matter

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10
Q

how does phagocytosis work

A

large solid particles phagocytosed into cells as phagosomes

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11
Q

particles involved in pinocytosis

A

Fluids & solutes dissolved in fluids

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12
Q

how does pinocytosis work

A

Fluids & solutes dissolved in fluids ingested by budding of small vesicles from cell membranes

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13
Q

receptor-mediated endocytosis

specific molecules involved with specific receptors

A

hormones, metabolites, proteins & some viruses

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14
Q

how does receptor-mediated endocytosis work

A

Molecules to be taken up = usually ligands (in ECF) recognized by receptors expressed on the cell membrane of cells.

Binding of extracellular macromolecules with receptors → triggers activation & folding of plasma membrane → internalised into coated vesicles → fusion with endosomes

Contents in endosomes sent to lysosomes for degradation or recycled to plasma membrane

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15
Q

lysosomal degradation

process

A

Proteolysis (cleavage of peptide bonds) in lysosomes

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16
Q

lysosomal degradation

specificity

A

Non-specific → proteins degraded regardless of identity; as long as in lysosomes

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17
Q

lysosomal degradation

molecules involved

A

Higher eukaryotes: only membrane-associated proteins & alien proteins (non-intracellular proteins) internalised by endocytosis

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18
Q

proteasomal degradation

proteasome involved

A

26S proteasome

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19
Q

proteasomal degradation

specificity

A

Specific process → for most ubiquitinated & some non-ubiquitinated proteins

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20
Q

proteasomal degradation

molecules involved

A

recombinant proteins that can be recognised

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21
Q

proteasomal degradation

process

A

(a) Ubiquitin tagging → (b) delivery of substrate to proteasome → (c) proteasome degradation

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22
Q

proteasomal degradation

structure of 26S proteasome

A

composed of a 20S core (cylindrical) particle capped by 19S regulatory particles at one or both ends.

20S core particle made up of 4 heptameric rings assembled to form cylindrical structure
2 outer rings = 2 ⍺ subunits
2 inner rings = 2 β subunits

Inner rings house a central cavity (hollow) containing proteolytic active sites
Present on the walls of rings
Protease activity ⇒ cleaves peptide bonds

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23
Q

proteasomal degradation

26S proteasome purpose

A

specific degradation of regulatory protein & removal of damaged proteins

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24
Q

process of protein in proteasome

entry

A

Proteins enter via top 19S regulatory particle

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25
Q

process of protein in proteasome

unfolding of protein upon entry (purpose)

A

Unfolding of proteins important before translocation into 20S core to ensure ability to fit into narrow entrance of channel (13 Å)

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26
Q

process of protein in proteasome

unfolding of protein upon entry (method)

A

Ubiquitin molecules removed by deubiquitinating enzymes (DUBs) into monomers

Removes ubiquitin tagged on protein 1 by 1;
monomers escape from proteasome & recycled to label other protein substrates (ubiquitinate other proteins)

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27
Q

process of protein in proteasome

role of proteasome

A

engages protein substrate → polypeptide unfolds, translocate into degradation channel ⇒ hydrolysation of protein into short peptides of 3-25 amino acids

Complete removal of ubiquitin tag = entrance to 20S core opens

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28
Q

process of protein in proteasome

exit of protein

A

Hydrolysed proteins exit via bottom 19S regulatory particle

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29
Q

proteasomal degradation

19S regulatory particle (structure)

A

Contains ATPase subunits, gates entrance to degradation channel

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30
Q

proteasomal degradation

19S regulatory particle (purpose)

A

Hydrolyses ATP to provide energy for removal of Ub, protein unfolding & transfer of unfolded protein into 20S core particle

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31
Q

proteasomal degradation

(a) ubiquitin tag purpose

A

proteolysis by 26S proteasome only selective towards protein marked by ubiquitin

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32
Q

proteasomal degradation

(a) ubiquitin tag
polyubiquitin chain (definition)

A

Multiple ubiquitin tagged onto protein; protein now recognised by proteasome

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33
Q

proteasomal degradation

(a) ubiquitin tag
monoubiquitination (definition)

A

attachment of one Ub to protein

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34
Q

proteasomal degradation

(a) ubiquitin tag
monoubiquitination (purpose)

A

predominant regulatory modification → post-translational; hence protein cannot be targeted for proteasomal degradation; can be activated/ inactivated to carry out cellular function

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35
Q

proteasomal degradation

(a) ubiquitin tag
minimal signal required for proteosome targetting

A

chain of 4 Ub monomers linked through Lys48

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36
Q

proteasomal degradation

(a) delivery of substrates to proteasome
routes

A
  1. Substrates bind directly to proteasomes by interacting with 19S regulatory particle subunits
  2. Substrates brought to proteasome by adaptor proteins that bind both proteasome & polyubiquitin chains on the substrate to deliver it for degradation.
  3. Some protein substrates are degraded by proteasome without being ubiquitinated ⇒ rare
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37
Q

proteasomal degradation

(a) delivery of substrates to proteasome
routes
1. interactions with 19S regulatory particle subunits: how it occurs

A

19S subunits recognises polyubiquitin tag (PT)

Proteins to be degraded must be in close proximity to 26S proteasome

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38
Q

proteasomal degradation

(a) delivery of substrates to proteasome
routes
2. purpose of adaptor proteins

A

Helps to bring PT & proteasomes close to each other

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39
Q

Chemical based drugs

MW

A

<1000Da

40
Q

Chemical based drugs

synthesis & purity

A

Chemically synthesised & purified to homogeneity

41
Q

Chemical based drugs

modifications

A

drastic changes in activity & new drugs for new uses (improvement in use)

42
Q

Chemical based drugs

side effects

A

May have off-target effects [SE]; due to small size of chemicals

43
Q

biologics

MW

A

larger, typically in kDa

44
Q

biologics

synthesis & purity

A

Derived from living sources: human & animal tissues, cells & microorganisms

Difficult to sequence & fold properly

Not easily characterised & refined to high degree of purity

45
Q

biologics

modifications

A

same name even with modifications

Amino acid in recombinant protein can be substituted with another amino acid/ chemically modified

46
Q

biologics

SE

A

Behave more predictability with lesser SE

provides basis for targeted therapy

47
Q

types of biopharmaceutical products

A

recombinant proteins, monoclonal Ab, nucleic acid-based products

48
Q

routes of administration of biologics

A

Common: IV, IM, SC
Others: oral, nasal, transmucosal, transdermal

49
Q

Role of biologics in clinical devices & diagnostics

A

Enzymes → glucose test strips, urine test strips
Ab → immunoassay

50
Q

Challenges of biopharmaceutical products

I, D, D, P&V

A
  1. Immunogenicity
  2. Proteins susceptible to denaturation & protease degradation in biological fluids (in extracellular fluids) upon administration
  3. Proteins susceptible to degradation by intracellular degradation systems
  4. Distribution of proteins to tissues limited by permeability (porosity) of vasculatures
51
Q

Challenges of biopharmaceutical products

(1) Immunogenicity
how impurities derived

A

Recombinant proteins require use of CHO cells as host to make proteins
CHO host cells may make own endogenous proteins → considered impurity (not human proteins)

Important to remove endogenous proteins; but in reality might retain in formulation

52
Q

Challenges of biopharmaceutical products

(2) Denaturation & protein degradation
Proteins involved

A

MW of proteins > 200 kDaltons

53
Q

Challenges of biopharmaceutical products

(2) Denaturation & protein degradation
how proteins will be eliminated

A

phagocytosis (by neutrophils & monocytes)

54
Q

Challenges of biopharmaceutical products

(3) intracellular degradation systems

A

lysosomal degradation → fusion of proteins with lysosomes

intracellular proteases → non-specific processes

ubiquitin-proteasomal degradation → specific processes

55
Q

ADME of biopharmaceutical products: absorption

reasons for poor systemic absorption of proteins

A

poor protein stability & permeability

56
Q

ADME of biopharmaceutical products: absorption

factors causing poor protein stability

A

(1) acidity of gastric fluids [pH 1~2; can denature proteins]

(2) digestive enzymes

57
Q

ADME of biopharmaceutical products: absorption

factors causing poor permeability

A

(3) mucus layer lining entire GIT

(4) intestinal epithelium overall carry negative charges & tight junctions exist between epithelial cells to restrict absorption of hydrophilic peptides/proteins

58
Q

ADME of biopharmaceutical products: absorption

problem of innate immunity

A

Mucosal epithelia consists of immune cells of first line of defence: monocytes, neutrophils, mast cells, dendritic cells

Administered peptides/ proteins can be recognised as foreign particles ⇒ degraded
(Larger MW/ size = larger chance of recognition by immune cells)

59
Q

ADME of biopharmaceutical products: absorption

transport mechanism for proteins

A

diffusion and convection

60
Q

ADME of biopharmaceutical products: absorption

transport mechanism for proteins: diffusion - movement

A

Movement of single particles from high (site of injection) to low concentrations

61
Q

ADME of biopharmaceutical products: absorption

transport mechanism for proteins: convection - movement

A

Collective bulk movement of large mass of particles in a fluid

Flux is fluid-driven by motion of bulk fluid

62
Q

ADME of biopharmaceutical products: absorption

transport mechanism for proteins: diffusion - factors affecting rate

A

Inversely related to MW/ size of proteins
1. Larger MW/ size = slower rate of diffusion ⇒ not for large proteins
2. Smaller proteins diffuses more effectively from site of injection → blood capillary

63
Q

ADME of biopharmaceutical products: absorption

transport mechanism for proteins: convection - factors affecting rate

A

not limited by MW; unless protein molecules extremely large & gets entrapped in ECM

Influenced by steric hindrance & charge interactions

64
Q

ADME of biopharmaceutical products: absorption

transport of large proteins - movement

A

by convection

Difficult to pass through tight endothelial cells to enter blood stream

65
Q

ADME of biopharmaceutical products: absorption

transport of large proteins - absorption

A

mostly via lymphatic system → drain into lymph nodes & larger lymphatic vessels ⇒ lymphatic vessels merge with BV for proteins to enter circulatory system

66
Q

ADME of biopharmaceutical products: absorption

transport of large proteins - characteristics of lympathic capillaries

A

Lymphatic capillaries lack well-defined basement membrane
Clefts exists between endothelial cells ⇒ more permeable to proteins

67
Q

ADME of biopharmaceutical products: absorption

transport of large proteins - purpose of lymph nodes

A

consist of T cells → can recognise large proteins as foreign & cause degradation of proteins via innate immunity

68
Q

ADME of biopharmaceutical products: absorption

transport of small proteins - movement

A

by diffusion

69
Q

ADME of biopharmaceutical products: absorption

transport of small proteins - absorption

A

via both circulatory & lymphatic systems

70
Q

ADME of biopharmaceutical products: absorption

transport of small proteins - factors influencing absorption

A

Perfusion (blood flow throughout tissue)

71
Q

ADME of biopharmaceutical products: absorption

rate limiting factors of absorption

A
  1. Interstitial fluid transport rate affected by disease/ physiologic differences
  2. Lymphatic transport rate affected by disease
72
Q

ADME of biopharmaceutical products: distribution
purpose of protein binding

A
  1. improves circulation t1/2 of protein drugs
  2. allows more efficient delivery of protein drugs to target tissues
73
Q

ADME of biopharmaceutical products: distribution

tissue distribution of protein drugs

A

from the circulation → interstitial fluid of tissues → into tissues

74
Q

ADME of biopharmaceutical products: distribution

(1) how it improves t1/2 of proteins

A

Protein bound (usually albumin) protects proteins from recognition by circulating immune cells & hence being attacked

75
Q

ADME of biopharmaceutical products: distribution

movement of proteins across vascular barrier

A

movement across endothelial cells or between endothelial cells.

76
Q

ADME of biopharmaceutical products: distribution

2 pore model; types of pores & movement

A

Small pores → via diffusion (PS)

Large pores → via phase convection (J)

77
Q

ADME of biopharmaceutical products: metabolism

method

A

Via proteolysis by proteolytic enzymes (activated proteases)

78
Q

ADME of biopharmaceutical products: metabolism

location

A
  1. Interstitial fluid (ECF) in tissues/ organs
  2. On cell surfaces
  3. Intracellularly once protein drugs are taken up into cells
79
Q

ADME of biopharmaceutical products: metabolism

how it works in interstitial fluids

A

Proteases released by activated immune cells & other cell types → involved in proteolysis

Immune cells present in ECF → involved in phagocytosis & proteolysis

80
Q

ADME of biopharmaceutical products: elimination

methods

A

Proteolytic degradation → intracellular/ extracellular; via proteases

Renal (glomerular) filtration → dominates renal excretion of protein

81
Q

ADME of biopharmaceutical products: elimination

factors affecting renal excretion

M, C, SR, T

A

Cut-off molecular weight of protein

Charge of protein

Shape and rigidity of protein

Tubular reabsorption

82
Q

ADME of biopharmaceutical products: elimination

factors affecting renal excretion: MW of proteins

A

proteins > ~50 kDa cannot get filtered & hence renal eliminated → too big to pass through renal glomerular barrier

83
Q

ADME of biopharmaceutical products: elimination

factors affecting renal excretion: charge of protein

A

positively charged proteins have higher renal filtration than negatively charged proteins of same size due to negative charges on glomerular basement membrane

84
Q

ADME of biopharmaceutical products: elimination

factors affecting renal excretion: shape & rigidity of protein

A

affect how well proteins undergo glomerular filtration

Lesser filtration = longer t1/2 of drug (retains in body)

85
Q

ADME of biopharmaceutical products: elimination

factors affecting renal excretion: tubular reabsorption

A

tubular epithelium have net negative charge → positively charged proteins get more reabsorbed

(in relation to higher renal filtration) Need to consider whether filtration/ reabsorption more significant; higher filtration = more net loss of protein

86
Q

Improving PK profiles of proteins

G, P, S

A
  1. Glycosylation of proteins
  2. PEGylation of proteins
  3. increase size (MW) of proteins
87
Q

Improving PK profiles of proteins

  1. Glycosylation of proteins: method
A

Addition of glycans (carbohydrates) to specific amino acids in a protein

Glycosylation pattern (different types of glycans attached to different amino acids, straight vs branched chain) can vary
Formation of larger protein chain

88
Q

Improving PK profiles of proteins

  1. Glycosylation of proteins: outcomes
A

affect activity: Glycans may be required for enhanced receptor binding

increase t1/2 of proteins: likely due to large size limiting glomerular filtration & poorer substrates to proteolysis
Allows for increased circulation half-time by increasing size of protein/ modifying binding to glycoprotein receptors

89
Q

Improving PK profiles of proteins

  1. Glycosylation of proteins: pros & cons
A

+: Human IgGs contain N-linked glycans at Asn297

+/-: Engineering antibodies containing high mannose glycans are rapidly eliminated compared to other glycosylated antibodies.

90
Q

Improving PK profiles of proteins

  1. Glycosylation of proteins: N-linked glycans
A

removal of fucose improves affinity of binding of Fc domain in IgG to Fc receptor ⇒ increased effectiveness of binding in defucosylated Ab
Greeter clinical efficacy

91
Q

Improving PK profiles of proteins

  1. Glycosylation of proteins: rapid elimination of engineered Ab with high mannose glycans
A

(+) Improves recognition of Ab by mannose receptors ⇒ immune cells will phagocytose & remove Ab
Helps to (priming immune system) trigger immune cells of innate immunity better, increasing the activity

(-) Might cause the t1/2 of the mABs injected to be lower

92
Q

Improving PK profiles of proteins

  1. PEGylation of proteins: types
A

PEG with free hydroxyl at both ends

methoxylated PEG
(mPEG) with hydroxyl at one or both ends methoxylated

93
Q

Improving PK profiles of proteins

  1. PEGylation of proteins: PEG conjugation
A

Reactive functional groups of activated PEG/mPEG attached to sites

94
Q

Improving PK profiles of proteins

  1. PEGylation of proteins: how it increases t1/2
A

Increase in size of conjugated protein

Decrease elimination by proteolysis

Decrease elimination by action of Ab & activated immune cells

95
Q

Improving PK profiles of proteins

  1. PEGylation of proteins: types of configurations
A

Linear or branched PEG/mPEG polymers conjugated to protein drugs → give rise to PEGylated proteins of different extended half-lives

96
Q

Improving PK profiles of proteins

  1. Increase in size (MW) via fusion proteins: purpose
A

Larger protein = slower clearance; longer t1/2

97
Q

Improving PK profiles of proteins

  1. Increase in size (MW) via fusion proteins: how it works
A

Fusion proteins with Fc domain of antibody or albumin fused to a therapeutic protein to utilise FcRn-mediated recycling → increase t1/2 of therapeutic protein → enhance circulation half-lives

endo y3s1 (16 decks)

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