Life Sciences 1a Lecture Slides Set 10 Fall Prof. David R. Liu. Lecture Readings. Required: Lecture Notes McMurray p , O NH

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1 Life ciences 1a Lecture lides et 10 Fall Prof. David R. Liu Lectures 17-18: The molecular basis of drug-protein binding: IV protease inhibitors 1. Drug development and its impact on IV-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 IV 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 Lecture Readings Required: Lecture otes McMurray p ,

2 Impact of Anti-IV Drugs 1990s: anti-iv drugs transform IV infection from a short death sentence to a chronic (but very serious) illness 13 FDA-approved drugs inhibit IV reverse transcriptase; 9 drugs inhibit IV protease (first approved December, 1995) Mortality rate of U.. patients with advanced AID: 29% per year in % per year in mid : Death rate from AID in Europe falls 80% Gains primarily attributed to combination therapy involving IV protease inhibitors + other antiretroviral agents Drug Development is Very Difficult Total cost to develop a drug = ~$1 billion + ~10-15 years 2

3 uccessful Drugs Must atisfy Many Chemical and Biological Requirements 1) Potency (affinity) + K eq = K a = 1 K d drug protein target 2) pecificity (toxicity, immunogenicity) drug-protein complex non-target target non-target non-target 3) Bioavailability cellular oral 4) Biostability k 5) Economics inactive or toxic Lectures 17-18a: The molecular basis of drug-protein binding: IV protease inhibitors 1. Drug development and its impact on IV-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 IV 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 3

4 Enthalpy Changes (Δ) Involving Water Upon Drug-Protein Binding solvated protein binding pocket proteinsubstrate complex solvated substrate water molecules released into bulk solvent Interactions with water can play crucial roles in binding! Drug-Protein Binding Enthalpy () Balance heet 2 2 K eq = K a = 1 K d 2 2 water (W) drug (D) protein (P) drug-protein complex water (W) Loss of some protein-water interactions: Δ P-W > 0* Loss of some drug-water interactions: Δ D-W > 0* * These losses are minimized when the drug and protein binding pocket are more hydrophobic Gain of some drug-protein interactions: Δ D-P < 0** Van der Waals ydrogen bonding Ionic bonding ** These gains are maximized when D & P are complementary Gain of water-water interactions: Δ W-W < 0 4

5 Loss of Entropy Upon Binding + Two freely rotating and translating molecules upon binding form one complex Both the protein and drug often become more rigid upon binding, leading to additional entropy loss Releasing Water Molecules into Bulk olvent is Entropically Favorable + + bulk water Recall: the increase in entropy as water molecules are released into bulk solvent is the basis of the hydrophobic effect 5

6 Drug-Protein Binding Entropy () Balance heet 2 2 K eq = K a = 1 K d 2 2 water (W) drug (D) protein (P) drug-protein complex water (W) Protein loses translational and rotational entropy: Δ P < 0 Drug loses translational and rotational entropy: Δ D < 0 Protein and drug rigidity increases: Δ D < 0*, Δ P < 0* * To minimize this loss, pre-rigidify the drug Bound water gains entropy when released: Δ W > 0** ** To maximize this gain, design the drug to displace bound water molecules wherever possible Changes in Free Energy and Entropy Upon Drug-Protein Binding ΔG = Δ TΔ G = Free energy = Enthalpy (heat) T = Temperature in Kelvin = Entropy (disorder) In general, for ΔG of binding to be negative (favoring binding): Favorable enthalpic interactions (Δ P-D < 0) between the protein and drug and favorable changes in the entropy of water (Δ water > 0) must overcome Unfavorable entropy loss in the protein and drug (Δ P and Δ D < 0), as well as the loss of enthalpic interactions between water and the protein or small molecule (Δ P-W and Δ D-W > 0) 6

7 Lectures 17-18a: The molecular basis of drug-protein binding: IV protease inhibitors 1. Drug development and its impact on IV-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 IV 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 IV Protease Inhibitors saquinavir (offmann-la Roche) 2 C 3 C3 C 3 3 C 3 C C 3 ritonavir (Abbot) 7

8 ydrophobic urface Complementarity: aquinavir and IV Protease 2 C 3 C3 C 3 Leu 23 Ile 84 Pro 81 Val 82 The hydrophobic groups of saquinavir fit precisely into hydrophobic binding pockets in IV protease ydrophobic urface Complementarity: aquinavir and IV Protease 2 C 3 C3 C 3 Leu 23 Ile 84 Val 82 Pro 81 Filling hydrophobic pockets increases Van der Waals interactions (Δ D-P < 0) and increases the displacement of water (Δ W > 0) 8

9 ydrophobic urface Complementarity: aquinavir vs. the Runner-Up Candidate o hydrophobic group to complement binding pocket 2 C 3 C3 C 3 2 C 3 C3 C 3 saquinavir runner-up Leu 23 Pro 81 Leu 23 ~10-fold worse binding than saquinavir Pro 81 Ile 84 Val 82 Ile 84 Val 82 Removing one of the hydrophobic pocket-filling groups of saquinavir (only 4 carbons!) greatly reduces binding potency ydrophobic urface Complementarity: Ritonavir and IV Protease Animation rendered by Brian Tse 9

10 ydrophobic urface Complementarity: Ritonavir and IV Protease 3 C 3 C C 3 The hydrophobic groups of ritonavir also complement the hydrophobic binding pockets in IV protease Leu 23 Val 82 Ile 84 Pro 81 ydrophobic urface Complementarity: Ritonavir and IV Protease C 3 Multiple structures can fill the same pocket, especially with some enzyme flexibility Leu 23 Ile 84 Val 82 Pro 81 10

11 IV Protease Changes hape lightly When Binding aquinavir vs. Ritonavir Leu 23 Ile 84 Val 82 Pro 81 IV Protease + Ritonavir IV Protease Changes hape lightly When Binding aquinavir vs. Ritonavir Leu 23 Ile 84 Val 82 Pro 81 IV Protease + aquinavir 11

12 ydrogen Bonding: aquinavir and IV Protease Ile50 Ile50' 2 C 3 C 3 C 3 aquinavir complements hydrogen bond donors provided by the enzyme, enhancing favorable (negative) Δ D-P ydrogen Bonding: Ritonavir and IV Protease Ile50 Ile50' 3 C 3 C C 3 -bonds between small molecules and proteins help to offset the penalty of giving up -bonds to water upon binding 12

13 ubstrate, Transition tate, and Intermediate of IV Protease substrate δ δ enzyme transition state tetrahedral intermediate Transition tate Analogs C transition state tetrahedral transition state mimic better mimic, but chemically unstable 3 C 3 C C 3 2 C 3 C3 C 3 ritonavir saquinavir Enzymes can bind transition states mimics very potently 13

14 Rigid Versus Flexible Inhibitors Dupont-Merck inhibitor More flexible variant cyclic, rigid core more potent binding weaker binding Rigidity reduces entropy lost upon binding (less negative Δ D ) Water-Liberating Inhibitors Ile50 Ile50' + Dupont-Merck inhibitor Ile50 Ile50' + liberated water 14

15 Replacing a Bound Water Molecule With a mall-molecule Group Ile 50 water Ile 50 carbonyl oxygen replaces water IV protease + substrate IV protease + Dupont-Merck inhibitor The Dupont-Merck inhibitor replaces the bound water in the IV protease active site with a carbonyl oxygen Releasing bound water is entropically favorable (Δ w > 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 IV Effective drugs must meet several chemical and biological requirements, including potent binding to a target The combination of enthalpic and entropic changes that occur upon small molecule-protein binding ultimately determines the binding potency (K d ) of a drug IV protease inhibitors bind favorably by (i) filling hydrophobic pockets with complementary hydrophobic groups, (ii) providing hydrogen bonding partners, (iii) mimicking the amide hydrolysis transition state, (iv) being rigid, and (v) releasing bound water molecules 15