LECTURE 4: ENZYME KINETICS & INHIBITION

Question 1

SPR:
1) what is a surface plasmon

Answer 1

1) Generates when a laser hits a gold surface

Question 1

SPR:
1) what happens to the laser that hits the gold surface

2) what is it used to measure

3) what is the principle behind using SPR for measuring x

4) what is the change in angle proportional to

Answer 1

1) The energy put in with the laser on the gold surface will dissipate because:
- At the correct specific angle, there will be energy absorbed

2) Used to measure drug protein interactions - the interaction comes from the interaction between an immobilized ligand and a flowing analyte

3) Binding of a molecule to the surface will change the refractive index
♣ Changes the angle of the light (refractive index) where there is a dip in energy can be detected
♣ Determines the change in angle

4) This change of angle is proportional to:
o The change in angle of the signal
o Bound mass: how many copies of the molecule is bound and size of molecule

Question 2

SPR Sensorgram:
1) what is a sensogram

2) What are the 3 main phases when using SPR to measure interactions

3) what happens in each of the phases and the RU of the different phases

Answer 2

1) - A sensogram is the resonance unit (RU) measured as a function of time

(RU is how response is measured)

2) Injection of analyte (association phase), steady state, (dissociation phase)

3)

ASSOCIATION PHASE: Analyte starts associating with the immobilized ligand
o increase of signal that is RU
- Function of both kon and koff

STEADY STATE:
o Association rate Equals dissociation constant
♣ Still injecting analyte
♣ How much is bound to immobilized ligand = how much dissociates

DISSOCIATION PHASE:
o Injection if buffer
o (decrease of signal RU)
♣ Analyte dissociates
♣ Function of koff only

Question 3

SPR Kinetic Analysis:
1) what can an SPR sensogram be fitted to and what does this mean

2) what kinetic parameters

3) how are these parameters determined

4) what does a sensogram with injections of different concentrations of analyte overlaid and fitted together show the same of

Answer 3

1) exact kinetic parameter, we can extract certain kinetic parameters from sensogram

2) kon and koff

3) koff:
♣ determined first from dissociation constant

kon:
♣ back-calculated from the association phase using:
* previously determined koff
* known [analyte]
o Range of concentration should be used to obtain more accurate parameters

4) o All decays fit the same single kinetic dissociation rate constant (koff)
♣ Therefore, kon should be the same too

Question 4

SPR Equilibrium Analysis:
1) sometimes dissociations are quick, when is this

2) how can we use SPR data to determine Kd independelty of kinetic parameters + equation

Answer 4

1) when dissociation rates are faster than the SPR sampling rate

2) Only look at equilibrium part (Req)
♣ Reach a steady state
* DISCCOATION = ASSOCIATION
* Independent of kinetic rate constant

Question 5

SPR Equilibrium Analysis:
1) response at equilibrium is a function of what (the equation)

2) what does Rmax depend on

3) how can you make the Req curve

Answer 5

1) the analyte concentration and Kd and Rmax

2) Rmax is dependent on the # of immobilized ligands on the sensor chip

3) Plot intensity on y-axis of the plateau, it will create a curve of the Req equation

Question 6

SPR: Stoichiometry
1) what is Rmax

2) what is SPR response proportional to

3) how can you calculate Rmax

Answer 6

1) maximum ligand response you can get by injecting the analyte

2) The mass of molecules bound to surface

3)

Question 7

What are 3 ways to use SPR data:

Answer 7

1) SPR sensogram –> RU

2) Kinetic analysus

3) equilibrium analysis (Req)

4) Rmax + stoichiometry

Question 8

Enzyme Kinetics:
1) what are enzymes
2) whats theur advantage
3) what is the # of conversion of ligands/second

Answer 8

1) Enzymes are catalysts enhancing reaction rates

2) Reactions that take hours/years take a few seconds with enzymes

3) kcat = number of conversions of ligands per second

Question 9

Michaelis-Menton Kinetics:

1) what can be described by the Michaelis-Menton equation

2) what are the assumptions of the model (3)

3) what is the equation

Answer 9

1) Enzyme kinetics

2)
o Free diffusion

o Enzyme concentration much smaller than substrate concentration OR KM

o Irreversibility: [S]&raquo_space; [P] or ∆G &laquo_space;0

3)

Question 10

Michaelis-Menton Kinetics:

1) what is the equation for
a) V0
b) Vmax
c) KM
d) Kd

2) what happens if koff&raquo_space; kcat

Answer 10

1)

2) if koff&raquo_space; kcat then Km ~ Kd
o kcat would be negligible

Question 11

Michaelis-Menton Kinetics:

1) what is the Vmax and what is it proportional to

2) what is the Michaelis constant

3) what is the Michaelis constant in terms of numbers

Answer 11

1) Vmax is the max reaction rate at saturating substrate concentration
o Proportional to the [enzyme] and enzyme turnover rate kcat

2) KM = michaelis constant

3) It is ½ Vmax

Question 12

Competitive Enzyme Inhibition:

1) what does this inhibition involve and what are the possible complexes formed

2) why is it easier to produce /design this type if inhibitor

3) what changes in kinetics when this type of inhibitor is added to an assay and what is the change proportional to

4) what doesnt change in kinetics

Answer 12

1) Competitive inhibitor excludes binding of the natural substrate
o Only 2 possible complexes can form
♣ E: S or E: I

2) - Easier to make this type of inhibitor cause we already know what a natural ligand that fits looks like

3) Addition of competitive inhibitor to an enzymatic rate determination assay –> increase in KMapp (shifts to the right when increasing competitive inhibitor)
o The change is proportional to the ratio of [I] and the Ki (inhibition constant)

4) No change to Vmax

Question 13

Competitive Enzyme Inhibition:
1) what is the Vknot equation for this type of inhibition

2) what is the KMapp for this inhibition

3) what is the Ki for this type if inhibition

Answer 13

Question 14

Non-Competitive Inhibition:

1) what does this type of inhibitor bind

2) what happens when it binds to enzyme:substrate complex

3) what changes in kinetics when this type of inhibitor is added to an assay and what is the change proportional to

4) what doesnt change in kinetics

Answer 14

1) A non-competitive inhibitor binds both free enzyme and the enzyme: substrate complex (E: S)

2) The ternary complex (E: S: I) cannot transform the product substrate (non-productive)

3) Addition of a non-competitive inhibitor to enzymatic rate determination assay decrease in apparent Vmax
o The change is proportional to the ratio of [I] and inhibition constant Ki

4) no change to Km

Question 15

Non-Competitive Enzyme Inhibition:
1) what is the Vknot equation for this type of inhibition

2) what is the VMaxapp for this inhibition

3) what is the Ki for this type if inhibition

Answer 15

Question 16

Uncompetitive Inhibition:

1) what does this type of inhibitor bind

2) what happens when it binds to enzyme:substrate complex

3) what changes in kinetics when this type of inhibitor is added to an assay and what is the change proportional to

4) what happens @ high [substrate]

Answer 16

1) Uncompetitive inhibitor binds only to the E: S complex

2) The ternary complex (E: S: I) cannot transform the substrate (non-productive)

3) Addition of uncompetitive inhibitor to enzyme rate determination assay –> change shape of reaction curve
o Because inhibitory effect is proportional to the [substrate]
o However hard to see difference in graph compared to non-competitive

4) @ high [substrate]
o the net effect is a reduction in the apparent Vmax
♣ This change is proportional to the ratio of [I] and the inhibition constant KI

Question 17

NON/UNCOMPETITIVE Mechanisms

1) what is unique about these mechanisms

2) explain under conditions

3) how do these conditions compare to endogenous concentration of the substrate

4) what is the result of these conditions on inhibition for non/un competitive vs competitive

Answer 17

1) NON/UNCOMPETITIVE Mechanisms can be more Potent than Competitive Mechanisms i.e. - Uncompetitive inhibitors are more potent than competitive inhibitors
o A lot more inhibition for same concentration of inhibitor

2) @ high [S] ([S] > Km), the non/uncompetitive mechanisms will achieve greater inhibition than the competitive mechanisms for the same [I] and Ki ratio

3) Endogenous substrate:
o Typically: [S] ≥ Km

4) Under this condition, 10x more competitive inhibitors are required to achieved levels of inhibition that match non-uncompetitive mechanisms
♣ Assuming equal Ki values

Question 18

EXAMPLE:
1) explain an example of synergy (drugs)

2) explain what each does

3) what does synergy mean in this scenario

Answer 18

1) AZT + Nevirapine

2) AZT: competes with nucleotide binding

Nevirapine: Prevents the domain movements required for the RT activity of HIV RT (allosteric inhibitor)

3) These 2 drugs synergize and can bind at the same time
o Therefore, even more potent inhibition

Question 19

Quantifying Synergy:

1) how do you Quantify Synergy and what is its definition

2) what does synergy mean

3) why is it important to have synergy

Answer 19

1) We can graphically represent + quantify synergy using an isobologram
o Correlation plot between doses of 2 drugs required to achieved a pre-determined effect (ex: EC50)

2) ≥2 drugs can synergize
i.e. display a combined effect greater than the sum of their individual effects

3) synergy allows a decreased dose to be given per druge so Decreases dose .˙. decreases side effect and decreases resistance to drug

Question 20

Mechanisms of Synergy:
1) Whats an example of a synergistic mechanism

2) whats a more common mechanism

Answer 20

1) o2 inhibitors for the same enzyme can work through different mechanisms (comp vs noncomp)
♣ Combined leads to an enhanced action of the other drug

2) target 2 enzymes that converge on same pathway

Question 21

EXAMPLE of target 2 enzymes that converge on same pathway (MEK kinase)

Answer 21

• MEK kinase inhibitor (trametinib) shows to inhibit KRAS-mutant cancer cell lines by preventing MAPK activation
  • BUT exhibits strong toxicity + resistance
• Through siRNA screening, it was found that FGFR1 mediates adaptive resistance to trametinib
  • .˙. treatment with ponatinib (FGFR1 inhibitor) dramatically potentiates effect of trametinib

Question 22

Occupancy + Biological Effect:

1) what is EC50

2) what does a change in efficacy + potency show on a dose responds curve

3) is the Relationship between receptor or enzyme occupancy AND biological response linear?

Answer 22

1) EC50: [drug] where 50% of therapeutic effect is obtained

2)
o Max effect = efficacy
o Shift = potency

3) may not be linear

Question 23

EXAMPLE OF Occupancy + Biological Effect being NON-LINEAR

Answer 23

EX: effector may be activated downstream of a receptor through signaling cascade
♣ Amplification through 2nd messenger (cAMP) will lead to a non-linear relationship
♣ Not a direct impact of occupancy and the biological response

Question 24

SPARE RECEPTORS:
1) what is a spare receptor

2) what is its relevance

3) whats do spare receptors change in invitro vs in vivo effects

4) what is Kd (graphically)

5) in a system with spare receptors, how does the EC50 and Kd compare

Answer 24

1) receptors that remain unbound when an agonist is producing its maximal biologic response.

2) Spare receptors likely play a role in amplifying signal duration and intensity.

3) Lead to greater activity in vivo than expected from in vitro effect (EC50 < Ki)

4) Kd = concentration at which 50% of receptors are bound

5) the EC50 is lower than the Kd
o indicating that to achieve 50% of maximal effect, less than 50% of the receptors must be activated (because there are many available)

Question 25

SPARE RECEPTOR EXAMPLE:
1) calcitonin receptor inducing histamine release
a) what is the difference between the H+ release graph vs the receptor occupancy curve

b) how does this show spare receptors

Answer 25

a) Curve for H+ (histamine) release is shifted to the left compared to receptor occupancy curve

b) Where achieving 50% response (H+ release) requires less than 50% of the receptors to be active (receptor occupancy)

Question 26

SPARE RECEPTOR EXAMPLE:
2) H+ stimulates contraction of guinea pig ileal smooth muscle

a) what is gd-121

b) what does the increase of GD-121 (in moderate concentrations) result in and how does this show graphically

c) what happens at high [GD-121]

Answer 26

1) GD-121 is an irreversible inhibitor of the H+ receptor

2) EC50 (of % H+ response) drops as GD-121 increases
* At some [I], max response was still achieved but potency declined
* EC50 shifts to right and max response still achieved (therefore spare receptors available)

c) @ high concentration, decreases efficacy and max response

Question 27

Therapeutic Index:
1) what is TD50

2) what is the TI and its equation

3) what number indicates a safe TI and what does this mean between the 2 factors

Answer 27

1) TD50: dose where 50% of a toxic effect is obtained
2)
TI = Ratio between highest dose where no toxicity and effective therapeutic dose (depending on endpoint)

TI = TD50/ED50

3) Bigger TI is safer
♣ Therefore bigger distance between TD50 and ED50

Question 28

EXAMPLE OF TI: hERG K+ channel
1) what does inhibition of this channel result in

2) how does this impact the testing of other drugs

3) what does the ratio of free plsama concentration (Cmax) and hERG IC50 need to be to provide a safety margin

Answer 28

1) inhibition of cardiac hERG K+ channel –> fatal arrhythmias

2) All drugs must be tested for hERG off-target inhibition before clinical trial

3)
o The ratio between free plasma concentration (Cmax) and hERG IC50 should be ≥30 to provide a safety margin and minimize arrhythmias