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Pharmacology > Molecular Aspects > Flashcards

Molecular Aspects Flashcards

1
Q

What are the characteristics of small molecules?

A

4 characteristics:
-High affinity and selectivity for the target.
-Few adverse effects.
-Low production costs.
-Administered in a single daily oral dose.

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2
Q

Small molecules vs biologics?

A

Small molecules are ideal drugs, and are the most abundant and important drug class.

Biologics include monoclonal Ab and vaccines.

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3
Q

Types of binding, in order of weakest -> strongest

A

VDW/hydrophobic -> weakest
H-bonds
Ionic bonds
Disulfide bonds/covalent bonds -> strongest

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4
Q

Compare receptor core and polar residues for drugs

A
  • As drug receptors are proteins, they have a complex tertiary structure which defines its function and its sensitivity to drugs.
  • The side chains of amino acids define their interactions.
  • The receptor core is mostly hydrophobic, and specific polar residues dictate drug binding.
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5
Q

What 2 factors can drug-receptor interactions be defined by and describe them?

A

Affinity and efficacy.

Affinity = strength of drug interaction with receptor (fitting the lock)
Efficacy = strength of drug-bound receptor to produce a response (turning the key)

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6
Q

Which has affinity and affinity/efficacy? Agonist or Antagonist

A

agonist: has affinity and efficacy as a response is produced.
antagonist: has affinity but no/zero efficacy as there is no response.

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7
Q

Hyperbolic curve vs sigmoidal

A

Sigmoidal = S shaped
Hyperbola = Steep rise which flattens

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8
Q

What is the occupation theory?

A

-drug effect is proportional to the number of receptors occupied by drug.
-Maximal effect occurs when all receptors are occupied.

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9
Q

What are the assumptions of the occupation theory?

A

3 assumptions for drug-receptor interaction: reversible, bimolecular (1 agonist molecule binds 1 receptor), and at equilibrium.

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10
Q

What is the law of mass action?

A

Rate of a chemical reaction is directly proportional to the product of reactant conc.

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11
Q

At equilibrium, forwards rate

A

= backwards rate

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12
Q

What is Kd?

A
  • Dissociation constant.
  • This measures the affinity a drug has for a receptor.
  • The higher the Kd value, the weaker the binding and the lower the affinity.
    -Half receptor occupancy.
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13
Q

Bmax=

A

total no of drug binding sites

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14
Q

Kd in relation to Bmax

A

Kd = Bmax/2

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15
Q

How can drug binding experiments be measured experimentally?

A

Using labelled ligands which are either fluorescent or radioactive.

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16
Q

Saturation binding experiments

A
  • First, the source of receptor is incubated with radioligands, and allowed to bind.
  • The reactions are then separated. Pores are large enough for the free unbound ligand to pass through, but not large enough for the receptor-bound ligand to pass through (since this is a large complex).
  • The radioactivity in the filters is then quantified.
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17
Q

Scatchard plot =

A

The most common linear transformation of binding data

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18
Q

IC50

A

-measurement of displacement potency -conc. of drug required to reduce effect by 50%.

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19
Q

Why does IC50 not equal Ki

A

IC50 also takes into account a few other parameters:

  • [A*] – determines extent of receptor occupancy
  • Kd – affinity of drug A* for the same site
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20
Q

How to change IC50 into Ki?

A

use Cheng-Prusoff equation.

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21
Q

EC50

A

the concentration of agonist required to produce a half-maximal response.

22
Q

What are the exceptions to the occupation theory?

A

Number 1: some agonists acting on the same receptor elicit different maximal responses even at maximal receptor occupancy. This discrepancy between receptor occupancy and maximal effects of different agonists required the introduction of the intrinsic activity term (a).
Number 2: some agonists produce a maximal response while only occupying a small fraction of the receptor population.

23
Q

Based on the occupation theory, EC50 =

A

Kd

24
Q

Spare receptors

A

= when receptors are free due to maximal occupancy already.
-detectable when EC50 (agonist concentration required to produce half maximal effect) is less than the KD (concentration of drug required to occupy half the receptor population).

25
Q

What are the types of antagonism?

A
  • Competitive
  • Non-competitive
  • Mixture
  • Physiological

Competitive:
- Antagonist binds to the same site on the receptor as agonist
- Reversible
- Can be overcome by high [agonist]
- Straightforward to analyse quantitatively using a Schild Plot
- Curve shifts to the right
- EC50 increases

Non-competitive:
- Occurs when an antagonist binds irreversibly or from binding of antagonist to a site distinct from agonist binding site (allosteric effect).
- Irreversible/low dissociation rates
- Cannot be overcome by high [agonist]
- Curve drops down
- Minimal effect on EC50

Mixture:
- Typically indicates receptor reserve/spare receptors

Physiological:
- Describes interaction of two drugs exerting opposing effects in vivo e.g., in cardiac muscle, Epinephrine (acting on b-adrenoceptors) – INCREASES contraction, whereas Acetylcholine (muscarinic acetylcholine receptors) - DECREASES contraction.
- Each drug binds a different receptor.

26
Q

What is inverse agonism?

A
  • Inverse agonists reduce the basal activity of receptors.
  • The existence of inverse agonists suggests that receptors have basal activity even in the absence of agonist
27
Q

What type of drug target are GPCRs?

A

Small molecules. 33% of small molecule drug targets

28
Q

GPCR structure?

A
  • Common core domain of 7 membrane-spanning alpha helices, hence called 7TMRs.
  • 3 intracellular loops (used for the initiation and regulation of downstream signalling) & 3 extracellular loops connect the helices.
29
Q

How many families are there for GPCR?

A

7TMRs divided into 5 families. Family 1 is the largest group. 1a – ligands bind in cavity within common core. Examples: catecholamines (epinephrine, dopamine). 1b – short peptide ligands bind to N-terminal domain and EC loops, C-terminus of peptides also bind cavity in common core. Examples: chemokines, thrombin. 1c – glycoprotein hormones bind to large N-terminal domain which contacts EC loops to activate receptors. These are large agonists. Examples: thyroid stimulating hormone.

30
Q

General GPCR interaction

A

1) Extracellular agonist binds to receptor.

2) The agonist-bound receptor interacts with G protein which is on the inner leaflet of the plasma membrane.

3) G protein then interacts with effector enzyme.

31
Q

G protein structure

A
  • Heterotrimeric (3 subunits): alpha, beta, gamma.
  • Attached to membrane by lipid modifications on alpha and gamma.
  • The alpha subunit has intrinsic GTPase activity. Alpha subunit binds GDP or GTP and displays GTPase activity critical for function.
32
Q

detailed G-protein activation

A

An agonist binding to a receptor attracts the GDP-bound G protein. This catalyses the release of GDP from the G protein alpha-subunit. As GTP is present in higher concentrations in the cell, it is able to bind to the G protein and activate it. The G protein dissociates into a GTP-a and BY complex. (The agonist then dissociates from the receptor as it is no longer needed). GTP-a and BY complexes bind and activate effectors. After a certain length of time, the GTP dissociates into GDP due to intrinsic GTPase activity of alpha subunit. The GDP-a and BY dissociates from the effector, and G protein trimer reforms. Basal state re-achieved.

33
Q

G protein classification

A

Gs: increases adenylyl cyclase and Ca
Gi: decreases adenylyl cyclase and increases K
Gq, G11: increases phospholipase C

34
Q

What type of molecule is PKA

A

serine/threonine directed kinase

35
Q

G protein independent signalling

A
  • Agonist bound receptors are substrates for GRK (G-protein-coupled receptor kinase) which phosphorylate GPCRs at the serine and threonine residues.
  • This phosphorylation triggers the binding of the arrestin protein.
  • 2 consequences: 1) reduction in cAMP = blocks Gs/adenylyl cyclase; 2) increase in MAPK pathway

The implications this has on drug action include the inflammation of airways in the case of B2 agonists.

36
Q

Smooth muscle contraction is directed by

A

by the phosphorylation of myosin light chain.

37
Q

Inhibition of smooth muscle contraction: SABA

A
  • Inhibition of smooth muscle contraction is done by 3 activities of PKA:

1) PKA phosphorylates MLCK (which is responsible for phosphorylating and activating MLC) —> deactivation.

2) PKA phosphorylates MYPT (which is responsible for dephosphorylating MLC and therefore reducing its ability to interfere with the actin cytoskeleton) —> activation.

3) PKA phosphorylates hsp20 which lowers the ability of phosphorylated MLC to induce contraction.

38
Q

General GPCR pathway

A

Hormone binds to 7TMR
This recruits the G protein in its heterotrimeric form
When it binds, GDP is released.
GTP binds to the alpha subunit as it is present in higher conc in the cell
When GTP binds the G protein is activated
Then, the alpha subunit dissociates from the BY complex
GTP bound alpha subunit binds to effector enzymes which form secondary messengers
The second messengers bind to and activate/inactivate enzymes leading to phosphorylation eg.

39
Q

GCPR pathway for cyclic AMP

A

adenylyl cyclase converts ATP -> cAMP.

cAMP binds to PKA and activates it

PKA phosphorylates substrates leading to their activation/inactivation

40
Q

What type of drug targets are RTK?

A

Small molecules. 3%

41
Q

RTK structure?

A
  • Comprises 3 components:

1) Ligand binding domain

2) Single transmembrane helix

3) Cytoplasmic domain - which contains protein tyrosine kinase activity.

42
Q

Which RTK domain contains protein tyrosine kinase activity?

A

Cytoplasmic domain

43
Q

RTK activation

A

1) Inactive/basal state – no ligand, no tyrosine kinase activity.

2) Ligand binding promotes receptor dimerisation. Dimerisation has TWO effects:

-Allows ATP to bind to each tyrosine kinase domain
-Allows one of the ATP-bound tyrosine kinases to phosphorylate tyrosines on the other partner in the complex (transphosphorylation) —> RTK ACTIVE

3) Phosphorylated tyrosines function as docking sites for intracellular signalling proteins (i.e., SH2 domains) which relay and amplify downstream signal.

44
Q

How many downstream signalling molecules bind simultaneously to active RTK dimer?

A

Approx. 10-20 downstream signalling molecules bind simultaneously to an active dimer, components include PI3K and GEFs for small G-proteins such as Ras.

45
Q

What is SH2 domain and how many binding sites does it have?

A

Src homology 2 (SH2) domains are protein modules (ofapproximately 100 amino acids) found in many proteins involved in tyrosine kinase signalling cascades. Their function is to bind tyrosine-phosphorylated sequences in specific protein targets.

2 binding sites in SH2 domain:
P-Tyr binding site = common to all SH2 domains & binding site for side chain of AA 3 positions down from P-Tyr = variable between different SH2 domains and confers specificity.

46
Q

What is Ras?

A
  • Part of GTPase superfamily that includes heterotrimeric G-proteins
  • Cycles between GTP-bound/GDP-bound states
  • Intrinsic GTPase activity
  • Permanently activating Ras mutations found in many forms of cancer as Ras controls the MAP kinase pathway.
47
Q

Ras activation

A

1) Inactive Ras is in GDP-bound OFF state at plasma membrane

2) Phosphorylated Tyr’s act as docking sites for SH2 domain on an adaptor protein Grb2

3) Grb2 interacts with a Ras guanine nucleotide exchange factor SOS

4) Membrane-localised SOS promotes release of GDP, allowing GTP to bind. GTP-bound Ras = ACTIVE

5) GTP-bound Ras recruits a MAP-kinase-kinase-kinase called Raf (Ser/Thr kinase) to the plasma membrane.

6) Raf phosphorylates/activates a MAP-kinase-kinase called MEK. MEK phosphorylates/activates MAP kinase/ERK

7) MAP kinase/ERK phosphorylates and alters the activity of multiple enzymes and transcription factors to stimulate cell proliferation

48
Q

Targeting kinases in cancer:

3 drug types?

A

1) Therapeutic Ab preventing receptor activation

2) Tyrosine kinase inhibitors preventing enzyme activity

3) Raf inhibitors which inhibit the downstream signalling pathways.

49
Q

drugs for breast cancer

A

Herceptin/Trastuzumab = Monoclonal Ab. Binds and inactivates the extracellular domain of HER2 to block autophosphorylation.

Lapatinib = Small molecule. Binds to the ATP-binding site of the HER2-selective Tyrosine Kinase Inhibitor (TKI) to block autophosphorylation.

50
Q

drug for malignant melanoma

A

Vemurafenib: Small molecule. Orally available selective inhibitor of Val600Glu B-Raf that binds to the ATP-binding site. Approved for treatment of metastatic melanoma.

51
Q

Sildenafil MOA

A

GTP converts into cGMP by guanylyl cyclase.
PDE5 (phosphodiesterase type 5) inhibitor which prevents the breakdown of cGMP to GMP. This means cGMP levels stay high and can cause activation of the cyclic-GMP-dependent kinase. This phosphorylates/activates MLC by MYPT and inactivates MLCK -> smooth muscle relaxation -> erection

Pharmacology (10 decks)

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