Quantification of drug-target interactions Flashcards
Define the Molecular mechanisms of action
The interactions between a drug and a target that leads to a specific outcome
What parameters must be considered When measureing MMOA?
The kinetics (Reversible versus irreversible binding) The Binding Site (Orthosteric vs allosteric) The functional effect (activation vs inhibition)
During reversible equilibrium binding of a drug to its target ligand or receptor, Kd and Ki are ____, Inhibition can be ___ or ____, and potential competition can come from ____, ____, or ____
equal
orthosteric
allosteric
Enzyme
Protein:protein interaction inhibitor
Receptor antagonist
Define competitive inhibition
Neither the ligand nor the drug can bind at the same time
Define uncompetitive inhibition
The inhibitor binds the enzyme/receptor only when the natural ligand is bound (potency is proportional to the physiological concentration of the ligand)
Define noncompetitive inhibition
The inhibitor reduces the activity of the enzyme/receptor and binds to an allosteric site, whether it has the ligand bound or not
Define agonism
Binding of compound activates the receptor or enzyme (can be full or partial)
Define allosteric modulation
Binding of compound will modify the activity of a receptor or enzyme, thereby shifting its substrate selectivity profile
How do you calculate the Kd for receptor:binding at equilibrium? What is the unit?
k_off/k_on
Molar
The lower the number, the stronger the interaction
What is receptor occupancy and how is it calculated during Receptor:ligand binding at equilibrium?
The fraction of receptors bound over all receptors
Y = [R:L]/[Rtotal]
Where [Rtotal] = [Rfree] + [R:L]
or
If the receptor concentration is much lower than the ligand concentration of Kd (often the case):
Y = [Ltotal]/([Ltotal] + Kd)
What property is seen when plotting Receptor occupancy over ligand concentration semilogarithmically?
All concentrations show the same curve, just shifted left/right
Define cooperativity
When the binding of a ligand influences binding at another site (oligomeric receptors display this behaviour often)
Can be positive or negative cooperativity
How is cooperativity modelled mathematically?
Y = [L]/([L] +Kd)
Raise the two [L]s and Kd by the Hill coefficient n
A Hill coefficient above 1 shows positive cooperativity
”” below 1 shows negative cooperativity (rare)
How do you calculate Receptor occupancy during reversible competitive inhibition?
Y = [RL]/[Rtotal] or Y = [L]/([L]+K_L(1 + [I]/K-I)) or [L]/([L] + K_app)
How do you calcualte receptor occupancy during irreversible competitive inhibition?
Y = [RL]/[Rtotal] or Y = [L]/([L] + K_L) or [R_unmodified]/[R_total]
When is K_app = [L]?
When Y = 0.5
What is the K_internal of a two-state model of receptor activation? what is the equation?
The rate at which a receptor switches from an inactive form to its active form
K_int = [R^A]/[R^I]
How d you calculate apparent K_d in a two-state model for an agonist?
K_appD = Kd(1+ 1/Kint)
How d you calculate apparent K_d in a two-state model for an inverse agonist?
K_appD = Kd(1+K_int)
What is a partial agonist and how do you calculate its apparent K_d?
An agonist that binds to both active an inactive forms of a two-state receptor (can be positive or negative depending of affinity)
K_appD = (Kint +1)/((K_int/K_A) + (1/K_I))
Where
K_A = [R^A][A]/[R^A:A]
K_I = [R^A][A]/[R^I:A]
At saturating concentrations, a full agonist will lead to _____ whereas an inverse agonist will lead to _____
A partial agonist will lead to ____, and a neutral antagonist leads to ______
Full receptor activation
Full inactivation
Partial activation or inactivation
Competitive inhibition (antagonizes the action of another agonist)
Why does Y not start at 0 in dose response curves?
Even without ligand there’s always an equilibrium between active/inactive forms
Where is K_app on a dose-response graph?
Halfway between minimal response and maximal response (the inflection point of the curve)
What is the enzymatic k_cat
The rate at which an enzyme catalyzes a substrate into its product
How do you use the Michaelis-Menten equation and what is it?
It relates the initial rate of product formation (v0) to the substrate concentration
v0 = k_cat[E:S] = d[P]/dt (slope of velocity/conc)
When do you use the Briggs-Haldane approximation?
When the enzyme concentration is much smaller than substrate concentration
What is K_M?
The substrate concentration at which the rate is equal to V_max/2 (most efficient)
How do competitive, noncompetitive and uncompetitive inhibition affect K_M, if at all?
Competitive: shifts rightwards as [I] increase
Noncompetitive: K_M stays the same but V_max lowers as [I] rises
Uncompetitive: same as noncompetitive
Why don’t humans develop noncompetitive inhibitors instead of competitive inhibitors (noncompetitive works better)?
Because we don’t always know the allosteric binding site, often discovered by chance
What is residence time?
The time the ligand spends in complex with the receptor
1/k_off
What is residence time proportional to?
Half life
Why shouldn’t you use K_I to infer potency?
K implies equilibrium, which is hardly the case in the human body
What is one way to increase residence time?
“locking” the receptor:drug complex in another state
eg E + I <=> EI <=> EI*
What is the ED50, the TD50, and what is their relation to the therapeutic index?
ED is efective dose for 50% of population
TD is toxic dose for 50% of population
TI = TD/ED
Why wouldn’t the relationship between receptor occupancy and effect be linear?
Eg an amplifying cascade
How do you calculate an isobologram?
Measure the eg ED50 for two separate drugs
draw a dashed line from each ED50 on a graph of dose of B over dose of A (dashed line represents nointeraction, additive effect
Fix one dose and vary dose of other until same level of effect is reached (typically below dashed line)
What is the combination index and how is it calculated?
How much of a synergy is present between two drugs
Conc of drug A in combination with drug B required for effect normally seen at conc X / conc X + “” drug B “” drug A
How can Ponatinib, a drug that typically has no effect on cancer cells, be used to treat cancer cells?
In combination with trametinib (effective at high/toxic doses), lowers effective dose 5-fold
What are the two mechanisms to measure interactions between molecules learned in class?
Surface Plasmon Resonance
Fluorescence polarization
How does SRP work?
Polarized light is shone through a prism onto a gold film with surface coated by receptors (“ligands”) and a flow of liquid above it with ligands (“analytes”)
At a certain angle, light will be absorbed by the molecule
This shows as a decrease in intensity of the reflected light
You can measure this light and find out exactly at which angle the light was absorbed
The change in angle is exactly proportional to the bound mass
How can you determine k_on from SPR?
At the dissociation phase you can measure k_off because there can only be dissociation
During association phase, function of both k_on and k_off (can use known k_off to find k_on)
Why do SRP at varying concentration of “ligand”?
To find the (constant) K_D
What is R_Max?
The highest response you can get at the highest analyte concentration
When is the binding isotherm equation useful in SRP?
When the dissociation rates are faster than the SPR sampling rate
How can you calculate the stoichiometry of a drug:receptor interaction using SRP?
Rmax
Ri
Stoichiometric ratio
You’ll always have two and need to find the other
Rmax is the ratio of MWs of the analyte over the ligand multiplied by the initial rate and stoichiometric ratio
How does fluorescence anisotropy work?
Polarized light is shone on a molecule, and depending on the size it will move and depolarize the light of not move (if it’s bigger) and keep polarization
What property of a fluorescent label needs to be considered and why?
Lifetime
Lifetime determines the shift in polarization based on change in molecular weight (smaller lifetimes for smaller changes in MW)
