BCH3052 Lecture 19: Protein Protein Interactions

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25 May 2018

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Lecture 19 – Protein-Protein Interactions

Why we need to Measure Binding Interactions?

• In order to have a complete understanding of the system

• Discover drug targeting a receptor in body

o Measure interaction in vitro

o Need to occupy receptor in order to inhibit its activity – need to

determine how much drug is needed to bind to receptor

Relationship between Strength of Binding (affinity) and Amount Bound

• Don’t memorise equations

• Single site binding: P + L  PL (1:1)

• System is at equilibrium

o Not in body but controllable in test tube

o If you know how much protein is in there – amount of free: total

amount – amount in complex

• Get Kd – if know how much protein and ligand is in there → can determine

amount of protein ligand complex

o Measure of strength of binding

o Concentration of free and bound protein/ligand

o Units of Kd same as concentration

• Concentration of PL complex is related to the total amount of ligand put in

o Pt = [P] + [PL]

o If Pt >> Kd (case 1) ** EXAM (shapes of curves)

▪ Protein binds all the available ligand until it is saturated

▪ Protein concentration is higher than binding affinity

• Lower Kd = higher affinity (strong binding)

• Tight binding

▪ As you add ligand – binding is so tight: every molecule (ligand)

added binds to protein until you run out

of protein

▪ Binding curve:

• X axis: concentration of Ligand

added

• Y axis: concentration of PL

complex

• Flat line

o If Pt << Kd (case 2)

▪ Protein binding is incomplete, even at high

ligand concentration

▪ Binding is weak – not all ligand binds to

protein

▪ Binding curve

▪ Hyperbolic curve

▪ Logarithmic scale: tighter binding shifted

to left, weaker binding shifted to right

Logarithmic

Scale

Linear Scale

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Document Summary

Relationship between strength of binding and amount bound. If given a ligand concentration and know kd for interaction and know it is case. 2: look at protein concentration to know it is true, from ligand concentration predict ratio of bound protein to free protein ([pl]/[p], estimate what proportion of protein is bound vs free state, 10x higher in ligand conc. Methods used to measure binding: enzyme assays (1) know: pt,lt (2) measure pl (3) determine kd, take known concentrations of protein and ligand measure what is bound determine kd concentration but use variable ligand concentrations. Repeat experiment: keep constant pt: read off from graph (pl vs lt): when 50% bound gives estimate of kd, fit a line of best fit from data gives kd value, experimental methods. Chemical methods: free and bound species are first, binding causes some separative then quantitated independently, radioligand binding, gel shift, elisa, e. g.

Related Questions

Dr Vlad is testing the effects of two drugs named D1 and D2 on a protein P found in the blood. He does several tests to characterize how the drugs interact with protein P. He tests them separately and determined that one protein P molecule has only one binding site for D1. Similarly, one protein P interacts only with one molecule of D2.

a) (2pts) First, Dr Vlad, tests the interaction of D1 with P in his lab. At equilibrium: the concentration of free D1 is 10 mM, and the concentration of free P is 50 mM

Given that the affinity constant is for this interaction is Kd=40 mM under these experimental conditions (pH salt, temperature, etc), what is the concentration of complex PD1 at equilibrium?

b (2pts) Then Dr Vlad adds more D1 drug is added to the mixture. When the equilibrium is established again, the concentration of free D1 is now 40 mM. What percentage of P remains free at this new equilibrium? Explain. Note: you need to know that the experimental conditions (pH, salt, temperature, etc) in part b are the same as in part a

c (2pts) The experimental conditions used by Dr Vlad in the lab reflect very closely the conditions in the blood, he can therefore use his data to calculate what happens in the blood. Using his available data, what is the concentration of free drug D1 in the blood when 50% of the protein P in the blood is bound to the drug? Explain your reasoning.

d (2pts) Now Dr Vlad considers the same protein P binding to drug D2. The experimental conditions are the same as those used for D1 above (points a and b).

P and D2 are mixed together: at equilibrium, the concentration of free P is 100 mM and the concentration of free D2 is 100 nM, the concentration of PD2 complex is 10 mM.

Which drug D1 or D2 has the highest affinity for protein P? ________

Explain how you get to that answer

e) (2pts) Dr Vlad accumulated lots of data on how D1 and D2 bind to P. In addition to Kd values (defined or determined above), he also found that the ka value for D1 binding to P is 10^-6 M^-1s^-1 and the ka value for D2 binding to P is 10^-5 M^-1s^-1. Which drug has the fastest association rate for P? And how much faster is that rate compared to the other drug? Explain briefly without calculations.

Multiple Choice questions 2 points each

1. Creatinine clearance is a commonly used estimate of:

A)Glomerular filtration rate (mL/min)

B)Tubular secretion (mL/min)

C)Total renal blood flow (mL/min)

D)Renal reabsorption (cc/min)

E)Steady state muscle breakdown rate (mcg/min)

2. Which of the following is not a measure of exposure?

a) Peak concentration

b) Time of maximum concentration

c) Area under the concentration time curve

d) Concentration measured 2 hours after dose administration

3. A clinically impactful drug interaction based upon inhibition of cytochrome P450 is most likely when:

a)The drug is metabolized by multiple enzymes

b)The drug has a narrow therapeutic index

c)There is limited variation in interindividual enzyme activity in the involved enzyme

d)The drug is a substrate of P-glycoprotein

e)All of the above

2. Why are dose-response (or concentration-response) analyses recommended for drug approval by a regulatory agency?

a) To increase the potential for adverse reactions in clinical trials in order to fully characterize the safety profile

b) To prolong the length of time it takes for drug approval

c) To maximize the likelihood that optimal drug dose will be prescribed after approval

d) To reduce the costs of clinical trials

e) To explore all possible disease state indications at time of initial approval

5. Which of the following is true of inhaled delivery of an aerosolized drug suspension?

a)An ideal drug would have low first pass hepatic metabolism to maximize systemic absorption

b)In contrast to milled powders, drugs in suspension are dosed in partial pressures

c)Efficiency of drug delivery by multi-dose inhalers is highly dependent upon patient technique

d)Typical multi-dose inhalers produce a very homogenous particle size of drug

e)Nebulized delivery of drug by face mask is not appropriate for children, demented or obtunded patients

6. The apparent volume of distribution (Vd):

a)Does not represent an actual physiologic volume

b)Can never be higher than total body water volume

c)Can be determined using only the half life of a drug

d)Will be large in a drug that is highly bound to serum proteins

e)Is usually small in drugs that require a loading dose

7. Which of the following characteristics of a drug make it most amenable to removal by hemodialysis?

a)Large volume of distribution

b)Large molecular weight

c)Strong protein binding

d)Good water solubility

8. Which is the best predictor of need for dosage adjustment in hepatic disease?

Child Pugh score

Portal splanchnic pressure (as measured by peripheral heart rate)

Degree of fibrosis and shunt on ultrasound

Levels of liver function tests

There is no single marker of hepatic dysfunction

9. Fexofenadine is not metabolized but is a P-glycoprotein (Pgp) substrate. How does St. Johnâs Wort cause decreased serum fexofenadine levels?

Inhibition of gut Pgp

Induction of gut PgP

Induction of CNS Pgp

Inhibition of biliary Pgp

None of the above

10. Which of the following is the best measure of renal function?

a)age

b)random urine creatinine concentration

c)spot plasma creatinine concentration

d)estimated creatinine clearance (Cockroft Gault)

e)Child-Pugh

11. Which pharmacokinetic parameter is the best measure of total systematic drug exposure after an oral dose?

a)Cmax

b)Elimination half life

c)Absorption rate constant

d)Area under the curve

e)Clearance

12. Which of the following is a mechanism that would cause a drug to have zero order elimination in humans?

a)Ultra-metabolizer genotype

b)Enterohepatic recirculation

c)High serum protein binding

d)Saturation of the prime metabolic enzyme

e)Sustained release version of a drug

13. Phase II metabolic metabolism:

a)Involves the covalent binding of a polar side chain

B)Always follows phase 1 metabolism

c)Employs breaking of aromatic ring structures

d)Is not subject to pharmacogenetic variation

e)Occurs exclusively pre-systemically, in the gut lumen

14. Which is not a high profile FDA Initiative?

a)Orphan Disease Act

b)Childhood Vaccine Act

c)Rare Disease Act

d)Breakthrough Drug Designation

15 In explaining risk posed by pharmaceutical R&D, which is not a development risk:

a)Patient population

b)Clinical endpoint

c)Predictability of trials

d)Competitive advantage

16. The key pivotal trials undertaken as part of the IND are part of which phase of the clinical drug development process?

a)Phase I

b)Phase II

c)Phase III

d)Phase IV

17. Which is not an EU Regulatory Process

a)Centralized Procedure

b)Certificate of Pharmaceutical Product Procedure

c)De-Centralized Procedure

d)Mutual Recognition

18. The role of experimental medicine strategies is to

a)Explore disease and drug biology to support go/no go decisions

b)Generate and fully validate biomarkers for FDA approval

c)Primarily to co-develop diagnostic tests to be filed with the NDA (new drug application)

d)Harmonize FDA and EMA phase 1 requirements

e)Define the pharmacokinetics of exploratory drugs in healthy volunteers or selected patient populations

BCH3052 Lecture 19: Protein Protein Interactions

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