1

Life Sciences 1aLecture Slides Set 10Fall 2006-2007Prof. David R. Liu

N N

HO OH

O

O

NH

O

HN

N

S S

N

Lectures 17-18: The molecular basis of drug-protein binding:

HIV protease inhibitors

1. Drug development and its impact on HIV-infected patients

2. Energetic dissection of a small molecule binding to a protein

a. Enthalpy changes upon binding

b. Entropy changes upon binding

3. Case studies of saquinavir and ritonavir, two small-molecule HIV

protease inhibitors

a. Fill hydrophobic pockets with hydrophobic groups

b. Provide complementary hydrogen bond donors and acceptors

c. Mimic the transition state of a reaction

d. Maximize the rigidity of the drug

e. Displace bound water molecules Required: Lecture NotesMcMurray p. 808-810, 640-642

Lecture Readings

2

Impact of Anti-HIV Drugs• 1990s: anti-HIV drugs transform HIV infection from a short

death sentence to a chronic (but very serious) illness• 13 FDA-approved drugs inhibit HIV reverse transcriptase; 9

drugs inhibit HIV protease (first approved December, 1995)• Mortality rate of U.S. patients with advanced AIDS:

• 29% per year in 1995• 9% per year in mid-1997

• 1997-2003: Death rate from AIDS in Europe falls 80%• Gains primarily attributed to combination therapy involving

HIV protease inhibitors + other antiretroviral agents

Drug Development is Very Difficult

• Total cost to develop a drug = ~$1 billion + ~10-15 years

3

1) Potency (affinity)

Successful Drugs Must Satisfy ManyChemical and Biological Requirements

+Keq = Ka = 1÷Kd

drug-protein complexdrug protein target

targetnon-target non-target non-target

2) Specificity (toxicity, immunogenicity)

oralcellular

3) Bioavailability

k inactive or toxic4) Biostability     5)   Economics

Lectures 17-18a: The molecular basis of drug-protein binding:

HIV protease inhibitors

1. Drug development and its impact on HIV-infected patients

2. Energetic dissection of a small molecule binding to a protein

a. Enthalpy changes upon binding

b. Entropy changes upon binding

3. Case studies of saquinavir and ritonavir, two small-molecule HIV

protease inhibitors

a. Fill hydrophobic pockets with hydrophobic groups

b. Provide complementary hydrogen bond donors and acceptors

c. Mimic the transition state of a reaction

d. Maximize the rigidity of the drug

e. Displace bound water molecules

4

H

O H

HO

H

H

O

H

solvatedsubstrate

solvatedproteinbindingpocket

protein-substratecomplex

Enthalpy Changes (ΔH) Involving WaterUpon Drug-Protein Binding

H

O H

water moleculesreleased into bulk

solvent

• Interactions with water can play crucial roles in binding!

H O

H

H

O

H

H

O H

HOH

Drug-Protein BindingEnthalpy (H) Balance Sheet

• Loss of some protein-water interactions: ΔHP-W > 0*• Loss of some drug-water interactions: ΔHD-W > 0** These losses are minimized when the drug and protein

binding pocket are more hydrophobic• Gain of some drug-protein interactions: ΔHD-P < 0**

• Van der Waals   •   Hydrogen bonding • Ionic bonding** These gains are maximized when D & P are complementary• Gain of water-water interactions: ΔHW-W < 0

Keq = Ka = 1÷Kd

drug-protein complexdrug (D) protein (P)water (W)

H2O H2O

water (W)

H2O

H2O

5

Loss of Entropy Upon Binding

+

• Two freely rotating and translating molecules uponbinding form one complex

• Both the protein and drug often become more rigid uponbinding, leading to additional entropy loss

Releasing Water Molecules into “BulkSolvent” is Entropically Favorable

H

O H

HO

H

HO

H

HO

H

H

OH

HO

H

H

OH

H

OH

H

OH

H

O H HO

H

HO

H

H

OH

H

OH

+

H

O H

HO

H

HO

H

HO

H

HO

H

HO

H

H

OH

H

OH

H

OH

+

H O

H

H O

H

H O

H

HOH

HO

H

• Recall: the increase in entropy as water molecules are releasedinto “bulk solvent” is the basis of the hydrophobic effect

bulk water

6

Drug-Protein BindingEntropy (S) Balance Sheet

• Protein loses translational and rotational entropy: ΔSP < 0• Drug loses translational and rotational entropy: ΔSD < 0• Protein and drug rigidity increases: ΔSD < 0*, ΔSP < 0*

* To minimize this loss, pre-rigidify the drug• Bound water gains entropy when released: ΔSW > 0**

** To maximize this gain, design the drug to displace boundwater molecules wherever possible

Keq = Ka = 1÷Kd

drug-protein complexdrug (D) protein (P)water (W)

H2O H2O H2O

water (W)

H2O

Changes in Free Energy and EntropyUpon Drug-Protein Binding

ΔG = ΔH – TΔSG = Free energyH = Enthalpy (heat)T = Temperature in KelvinS = Entropy (disorder)

• In general, for ΔG of binding to be negative (favoring binding):Favorable enthalpic interactions (ΔHP-D < 0) between theprotein and drug and favorable changes in the entropy ofwater (ΔSwater > 0) must overcome…Unfavorable entropy loss in the protein and drug (ΔSP andΔSD < 0), as well as the loss of enthalpic interactionsbetween water and the protein or small molecule (ΔHP-Wand ΔHD-W> 0)

7

Lectures 17-18a: The molecular basis of drug-protein binding:

HIV protease inhibitors

1. Drug development and its impact on HIV-infected patients

2. Energetic dissection of a small molecule binding to a protein

a. Enthalpy changes upon binding

b. Entropy changes upon binding

3. Case studies of saquinavir and ritonavir, two small-molecule HIV

protease inhibitors

a. Fill hydrophobic pockets with hydrophobic groups

b. Provide complementary hydrogen bond donors and acceptors

c. Mimic the transition state of a reaction

d. Maximize the rigidity of the drug

e. Displace bound water molecules

Two HIV Protease Inhibitors

ritonavir(Abbot)

saquinavir(Hoffmann-La Roche)

OH

N

H

H

O NH

CH3CH3CH3

HN

O

NH

O

N

O

NH2

O

HN

OH

NH

O

O

N

SNH

O

N

CH3

N

S

H3C

H3C

8

Hydrophobic Surface Complementarity:Saquinavir and HIV Protease

OH

N

H

H

O NH

CH3CH3CH3

HN

O

NH

O

N

O

NH2

Pro 81

Val 82Ile 84

Leu 23 • The hydrophobic groupsof saquinavir fit preciselyinto hydrophobic bindingpockets in HIV protease

Hydrophobic Surface Complementarity:Saquinavir and HIV Protease

OH

N

H

H

O NH

CH3CH3CH3

HN

O

NH

O

N

O

NH2

Pro 81

Val 82Ile 84Leu 23

• Filling hydrophobicpockets increases Vander Waals interactions(ΔHD-P < 0) andincreases thedisplacement of water(ΔSW > 0)

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Pro 81

Val 82Ile 84

Leu 23

Hydrophobic Surface Complementarity:Saquinavir vs. the “Runner-Up” Candidate

Pro 81

Val 82Ile 84

Leu 23

OH

N

H

H

O NH

CH3CH3CH3

HN

O

NH

O

N

O

NH2

No hydrophobic group tocomplement binding pocket

saquinavir

OH

N

O NH

CH3CH3CH3

HN

O

NH

O

N

O

NH2

runner-up

~10-fold worsebinding than

saquinavir

• Removing one of the hydrophobic pocket-filling groups ofsaquinavir (only 4 carbons!) greatly reduces binding potency

Hydrophobic Surface Complementarity:Ritonavir and HIV Protease

Animation rendered by Brian Tse

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Leu 23

Ile 84

Val 82

Pro 81

Hydrophobic Surface Complementarity:Ritonavir and HIV Protease

O

HN

OH

NH

O

O

N

SNH

O

N

CH3

N

S

H3C

H3C

• The hydrophobicgroups of ritonaviralso complementthe hydrophobicbinding pockets inHIV protease

Leu 23Ile 84

Val 82 Pro 81

Hydrophobic Surface Complementarity:Ritonavir and HIV Protease

• Multiple structurescan fill the samepocket, especiallywith someenzyme flexibility

O

HN

OH

NH

O

O

N

SNH

O

N

CH3

N

S

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HIV Protease Changes Shape SlightlyWhen Binding Saquinavir vs. Ritonavir

Leu 23Ile 84

Val 82 Pro 81

HIV Protease + Ritonavir

Pro 81

Val82

Ile 84Leu 23

HIV Protease Changes Shape SlightlyWhen Binding Saquinavir vs. Ritonavir

HIV Protease + Saquinavir

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Hydrogen Bonding: Saquinavir andHIV Protease

N

H

H

OHN

OH

O

NHO

N

CH3

CH3CH3

NH

H H

O

N

O

H

O NH2

N N

H H

Ile50 Ile50'

• Saquinavircomplementshydrogen bonddonors providedby the enzyme,enhancingfavorable(negative) ΔHD-P

HN

O

O

N

S

NH

O

N

CH3N

S

H3C

H3C

HO

O

H H

O

N

O

H

N N

H H

Ile50 Ile50'

Hydrogen Bonding: Ritonavir andHIV Protease

• H-bonds between small molecules and proteins help tooffset the penalty of giving up H-bonds to water upon binding

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O

HN

O

O

H

O

OH

O H

‡

Substrate, Transition State, andIntermediate of HIV Protease

O

HN

O

O

O

OH

O HH

transition state tetrahedralintermediate

O

HN

O

O

H

O

O

substrate

enzyme

δ–

δ–

O

HN

OH

NH

O

O

N

SNH

O

N

CH3

N

S

H3C

H3C

Transition State Analogs

tetrahedral transitionstate mimic

O

HN

HH

better mimic, butchemically unstable

O

C

HH

H H

OH

N

H

H

O NH

CH3CH3CH3

HN

O

NH

O

N

O

NH2

saquinavirritonavir

O

HN

O H

transition state

• Enzymes can bind transition states mimics very potently

14

Rigid Versus Flexible Inhibitors

Dupont-Merck inhibitor

cyclic, rigidcore

N N

HO OH

O

O

NH

O

HN

N

S S

N

more potentbinding

• Rigidity reduces entropy lost upon binding (less negative ΔSD)

More flexible variant

N N

HO

OHO

ONH

O

HN

N

S

S

N

N N

HO

OHO

ONH

O

HN

N

S

S

N

N N

HO OH

O

O

NH

O

HN

N

S S

N

weakerbinding

N N

H H

Ile50 Ile50'

N N

HO OH

O

O

NH

O

HN

N

S S

N

N N

HO OH

O

O

NH

O

HN

N

S S

N

Water-Liberating Inhibitors

H H

O

N N

H H

Ile50 Ile50'

+

+H H

O

Dupont-Merck inhibitor

liberated water

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Replacing a Bound Water MoleculeWith a Small-Molecule Group

Ile 50

water

HIV protease + substrate

Ile 50carbonyl oxygenreplaces water

HIV protease + Dupont-Merck inhibitor

• The Dupont-Merck inhibitor replaces the bound water in theHIV protease active site with a carbonyl oxygen

• Releasing bound water is entropically favorable (ΔSw > 0)

Key Points: Molecular Basis of Drug Binding• Drug development has had a major impact on society and

on the lives of patients infected with HIV

• Effective drugs must meet several chemical and biologicalrequirements, including potent binding to a target

• The combination of enthalpic and entropic changes thatoccur upon small molecule-protein binding ultimatelydetermines the binding potency (Kd) of a drug

• HIV protease inhibitors bind favorably by (i) fillinghydrophobic pockets with complementary hydrophobicgroups, (ii) providing hydrogen bonding partners,(iii) mimicking the amide hydrolysis transition state, (iv)being rigid, and (v) releasing bound water molecules