IC7 ADME of macromolecules Flashcards
(97 cards)
1
Q
timeline of structural proteins
A
long lifetime; do not require high turnover
2
Q
timeline of regulatory proteins
A
short lifetime
3
Q
reason for degradation of regulatory protein
A
once signal transmitted results in response to environmental change, regulatory protein no longer required
4
Q
importance of protein degradation
A
- Ensures proper regulation of cell signalling pathways via normal protein turnovers
- Remove misfolded & damaged proteins that can lead to abnormal cellular activities
5
Q
problem of accumulating misfolded & damaged proteins
A
deviation from normal activity
results in disease
6
Q
methods of protein degradation
A
- Lysosomal degradation (10-20%)
- Proteasomal degradation (80-90%)
7
Q
key step in protein degradation
(before degradation can occur)
A
Endocytosis
8
Q
types of endocytosis
A
Phagocytosis
Pinocytosis
Receptor-mediated endocytosis
9
Q
particles involved in phagocytosis
A
large solid particles
cell debris, dead cells, protein aggregates, pathogenic microorganisms , particulate non-living matter
10
Q
how does phagocytosis work
A
large solid particles phagocytosed into cells as phagosomes
11
Q
particles involved in pinocytosis
A
Fluids & solutes dissolved in fluids
12
Q
how does pinocytosis work
A
Fluids & solutes dissolved in fluids ingested by budding of small vesicles from cell membranes
13
Q
receptor-mediated endocytosis
specific molecules involved with specific receptors
A
hormones, metabolites, proteins & some viruses
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
15
Q
lysosomal degradation
process
A
Proteolysis (cleavage of peptide bonds) in lysosomes
16
Q
lysosomal degradation
specificity
A
Non-specific → proteins degraded regardless of identity; as long as in lysosomes
17
Q
lysosomal degradation
molecules involved
A
Higher eukaryotes: only membrane-associated proteins & alien proteins (non-intracellular proteins) internalised by endocytosis
18
Q
proteasomal degradation
proteasome involved
A
26S proteasome
19
Q
proteasomal degradation
specificity
A
Specific process → for most ubiquitinated & some non-ubiquitinated proteins
20
Q
proteasomal degradation
molecules involved
A
recombinant proteins that can be recognised
21
Q
proteasomal degradation
process
A
(a) Ubiquitin tagging → (b) delivery of substrate to proteasome → (c) proteasome degradation
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
23
Q
proteasomal degradation
26S proteasome purpose
A
specific degradation of regulatory protein & removal of damaged proteins
24
Q
process of protein in proteasome
entry
A
Proteins enter via top 19S regulatory particle
25
process of protein in proteasome
unfolding of protein upon entry (purpose)
Unfolding of proteins important before translocation into 20S core to ensure ability to fit into narrow entrance of channel (13 Å)
26
process of protein in proteasome
unfolding of protein upon entry (method)
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)
27
process of protein in proteasome
role of proteasome
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**
28
process of protein in proteasome
exit of protein
Hydrolysed proteins exit via bottom 19S regulatory particle
29
proteasomal degradation
19S regulatory particle (structure)
Contains ATPase subunits, gates entrance to degradation channel
30
proteasomal degradation
19S regulatory particle (purpose)
Hydrolyses ATP to provide energy for removal of Ub, protein unfolding & transfer of unfolded protein into 20S core particle
31
proteasomal degradation
(a) ubiquitin tag purpose
proteolysis by 26S proteasome only selective towards protein marked by ubiquitin
32
proteasomal degradation
(a) ubiquitin tag
polyubiquitin chain (definition)
Multiple ubiquitin tagged onto protein; protein now recognised by proteasome
33
proteasomal degradation
(a) ubiquitin tag
monoubiquitination (definition)
attachment of one Ub to protein
34
proteasomal degradation
(a) ubiquitin tag
monoubiquitination (purpose)
predominant regulatory modification → post-translational; hence protein cannot be targeted for proteasomal degradation; can be activated/ inactivated to carry out cellular function
35
proteasomal degradation
(a) ubiquitin tag
minimal signal required for proteosome targetting
chain of 4 Ub monomers linked through Lys48
36
proteasomal degradation
(a) delivery of substrates to proteasome
routes
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
37
proteasomal degradation
(a) delivery of substrates to proteasome
routes
1. interactions with 19S regulatory particle subunits: how it occurs
19S subunits recognises polyubiquitin tag (PT)
Proteins to be degraded must be in close proximity to 26S proteasome
38
proteasomal degradation
(a) delivery of substrates to proteasome
routes
2. purpose of adaptor proteins
Helps to bring PT & proteasomes close to each other
39
Chemical based drugs
MW
<1000Da
40
Chemical based drugs
synthesis & purity
Chemically synthesised & purified to homogeneity
41
Chemical based drugs
modifications
drastic changes in activity & new drugs for new uses (improvement in use)
42
Chemical based drugs
side effects
May have off-target effects [SE]; due to small size of chemicals
43
biologics
MW
larger, typically in kDa
44
biologics
synthesis & purity
Derived from living sources: human & animal tissues, cells & microorganisms
Difficult to sequence & fold properly
Not easily characterised & refined to high degree of purity
45
biologics
modifications
same name even with modifications
Amino acid in recombinant protein can be substituted with another amino acid/ chemically modified
46
biologics
SE
Behave more predictability with lesser SE
provides basis for targeted therapy
47
types of biopharmaceutical products
recombinant proteins, monoclonal Ab, nucleic acid-based products
48
routes of administration of biologics
Common: IV, IM, SC
Others: oral, nasal, transmucosal, transdermal
49
Role of biologics in clinical devices & diagnostics
Enzymes → glucose test strips, urine test strips
Ab → immunoassay
50
Challenges of biopharmaceutical products
| I, D, D, P&V
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
Challenges of biopharmaceutical products
(1) Immunogenicity
how impurities derived
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
Challenges of biopharmaceutical products
(2) Denaturation & protein degradation
Proteins involved
MW of proteins > 200 kDaltons
53
Challenges of biopharmaceutical products
(2) Denaturation & protein degradation
how proteins will be eliminated
phagocytosis (by neutrophils & monocytes)
54
Challenges of biopharmaceutical products
(3) intracellular degradation systems
lysosomal degradation → fusion of proteins with lysosomes
intracellular proteases → non-specific processes
ubiquitin-proteasomal degradation → specific processes
55
ADME of biopharmaceutical products: absorption
reasons for poor systemic absorption of proteins
poor protein stability & permeability
56
ADME of biopharmaceutical products: absorption
factors causing poor protein stability
(1) acidity of gastric fluids [pH 1~2; can denature proteins]
(2) digestive enzymes
57
ADME of biopharmaceutical products: absorption
factors causing poor permeability
(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
ADME of biopharmaceutical products: absorption
problem of innate immunity
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
ADME of biopharmaceutical products: absorption
transport mechanism for proteins
diffusion and convection
60
ADME of biopharmaceutical products: absorption
transport mechanism for proteins: diffusion - movement
Movement of single particles from high (site of injection) to low concentrations
61
ADME of biopharmaceutical products: absorption
transport mechanism for proteins: convection - movement
Collective bulk movement of large mass of particles in a fluid
Flux is fluid-driven by motion of bulk fluid
62
ADME of biopharmaceutical products: absorption
transport mechanism for proteins: diffusion - factors affecting rate
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
ADME of biopharmaceutical products: absorption
transport mechanism for proteins: convection - factors affecting rate
not limited by MW; unless protein molecules extremely large & gets entrapped in ECM
Influenced by steric hindrance & charge interactions
64
ADME of biopharmaceutical products: absorption
transport of large proteins - movement
by convection
Difficult to pass through tight endothelial cells to enter blood stream
65
ADME of biopharmaceutical products: absorption
transport of large proteins - absorption
mostly via lymphatic system → drain into lymph nodes & larger lymphatic vessels ⇒ lymphatic vessels merge with BV for proteins to enter circulatory system
66
ADME of biopharmaceutical products: absorption
transport of large proteins - characteristics of lympathic capillaries
Lymphatic capillaries lack well-defined basement membrane
Clefts exists between endothelial cells ⇒ more permeable to proteins
67
ADME of biopharmaceutical products: absorption
transport of large proteins - purpose of lymph nodes
consist of T cells → can recognise large proteins as foreign & cause degradation of proteins via innate immunity
68
ADME of biopharmaceutical products: absorption
transport of small proteins - movement
by diffusion
69
ADME of biopharmaceutical products: absorption
transport of small proteins - absorption
via both circulatory & lymphatic systems
70
ADME of biopharmaceutical products: absorption
transport of small proteins - factors influencing absorption
Perfusion (blood flow throughout tissue)
71
ADME of biopharmaceutical products: absorption
rate limiting factors of absorption
1. Interstitial fluid transport rate affected by disease/ physiologic differences
2. Lymphatic transport rate affected by disease
72
ADME of biopharmaceutical products: distribution
purpose of protein binding
1. improves circulation t1/2 of protein drugs
2. allows more efficient delivery of protein drugs to target tissues
73
ADME of biopharmaceutical products: distribution
tissue distribution of protein drugs
from the circulation → interstitial fluid of tissues → into tissues
74
ADME of biopharmaceutical products: distribution
(1) how it improves t1/2 of proteins
Protein bound (usually albumin) protects proteins from recognition by circulating immune cells & hence being attacked
75
ADME of biopharmaceutical products: distribution
movement of proteins across vascular barrier
movement across endothelial cells or between endothelial cells.
76
ADME of biopharmaceutical products: distribution
2 pore model; types of pores & movement
Small pores → via diffusion (PS)
Large pores → via phase convection (J)
77
ADME of biopharmaceutical products: metabolism
method
Via proteolysis by proteolytic enzymes (activated proteases)
78
ADME of biopharmaceutical products: metabolism
location
1. Interstitial fluid (ECF) in tissues/ organs
2. On cell surfaces
3. Intracellularly once protein drugs are taken up into cells
79
ADME of biopharmaceutical products: metabolism
how it works in interstitial fluids
Proteases released by activated immune cells & other cell types → involved in proteolysis
Immune cells present in ECF → involved in phagocytosis & proteolysis
80
ADME of biopharmaceutical products: elimination
methods
Proteolytic degradation → intracellular/ extracellular; via proteases
Renal (glomerular) filtration → dominates renal excretion of protein
81
ADME of biopharmaceutical products: elimination
factors affecting renal excretion
| M, C, SR, T
Cut-off molecular weight of protein
Charge of protein
Shape and rigidity of protein
Tubular reabsorption
82
ADME of biopharmaceutical products: elimination
factors affecting renal excretion: MW of proteins
proteins > ~50 kDa cannot get filtered & hence renal eliminated → too big to pass through renal glomerular barrier
83
ADME of biopharmaceutical products: elimination
factors affecting renal excretion: charge of protein
positively charged proteins have higher renal filtration than negatively charged proteins of same size due to negative charges on glomerular basement membrane
84
ADME of biopharmaceutical products: elimination
factors affecting renal excretion: shape & rigidity of protein
affect how well proteins undergo glomerular filtration
Lesser filtration = longer t1/2 of drug (retains in body)
85
ADME of biopharmaceutical products: elimination
factors affecting renal excretion: tubular reabsorption
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
Improving PK profiles of proteins
| G, P, S
1. Glycosylation of proteins
2. PEGylation of proteins
3. increase size (MW) of proteins
87
Improving PK profiles of proteins
1. Glycosylation of proteins: method
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
Improving PK profiles of proteins
1. Glycosylation of proteins: outcomes
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
Improving PK profiles of proteins
1. Glycosylation of proteins: pros & cons
+: Human IgGs contain N-linked glycans at Asn297
+/-: Engineering antibodies containing high mannose glycans are rapidly eliminated compared to other glycosylated antibodies.
90
Improving PK profiles of proteins
1. Glycosylation of proteins: N-linked glycans
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
Improving PK profiles of proteins
1. Glycosylation of proteins: rapid elimination of engineered Ab with high mannose glycans
(+) 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
Improving PK profiles of proteins
2. PEGylation of proteins: types
PEG with free hydroxyl at both ends
methoxylated PEG
(mPEG) with hydroxyl at one or both ends methoxylated
93
Improving PK profiles of proteins
2. PEGylation of proteins: PEG conjugation
Reactive functional groups of activated PEG/mPEG attached to sites
94
Improving PK profiles of proteins
2. PEGylation of proteins: how it increases t1/2
Increase in size of conjugated protein
Decrease elimination by proteolysis
Decrease elimination by action of Ab & activated immune cells
95
Improving PK profiles of proteins
2. PEGylation of proteins: types of configurations
Linear or branched PEG/mPEG polymers conjugated to protein drugs → give rise to PEGylated proteins of different extended half-lives
96
Improving PK profiles of proteins
3. Increase in size (MW) via fusion proteins: purpose
Larger protein = slower clearance; longer t1/2
97
Improving PK profiles of proteins
3. Increase in size (MW) via fusion proteins: how it works
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
