Lecture 3: Targeting of enzymes

Question 1

Potency

Answer 1

-inhibition of specific function in vitro
-often used as antagonist of cell receptor
-measures inhibition

Question 2

Dose-response curve

Answer 2

-measures potency
-IC50 = concentration at which 1/2 the max biological response is inhibited
-backwards S curve

Question 3

Cheng-Prusoff Equation

Answer 3

KI = IC50 / (1+ ([S]/KM))

Question 4

Enzymes

Answer 4

-stabilize transition state of catalyzed reaction
-lower free energy of transition state

Question 5

Transition state analogue

Answer 5

-compound closely resembling transition state
-should bind tightly to enzyme active-site to lead to an effective therapeutic

Question 6

Binding curves

Answer 6

-fraction of protein bound vs [ligand]
-ex. hemoglobin

Question 7

Heliobacter pylori

Answer 7

-gram neg bacteria
-cause >85% ulcers

Question 8

MTAN (5’-methylthioadenosine nucleosidase)

Answer 8

-antibiotic target
-part of biosynthetic pathway for menaquinone
-reaction has a dissociative transition state

Question 9

TS analogue

Answer 9

-an antibiotic design

Question 10

Gaining high affinity of TS analogue

Answer 10

-vary R group and test activity

Question 11

TS analogue affinity

Answer 11

-high
-KI < 200pM

Question 12

Irreversible Inhibitors

Answer 12

-compounds that mod and inactivate enzyme
-covalent bonds

Question 13

irreversible inhibitor strategy

Answer 13

-mimic natural substrate and bind to active site –> utilize binding specificity of the target enzyme for selectivity
-react with surface residues not necessarily catalytic residues

-mimic, bind, react

Question 14

Dihydrofolate reductase

Answer 14

-irreversible inhibitor
-mimics the biological substrate dihydrofolate with the addition of reactive group

Question 15

Aspirin

Answer 15

-irreversible inhibitor of COX
-acetylates a SERINE residue near the active site where the products block substrate
-acyl enzyme + salicyclic acid

Question 16

cyclo-oxygenases

Answer 16

COX
-homodimer
-heme active site
-aspirin blocks access to active site

Question 17

Mechanism-based irreversible inhibitors

Answer 17

-utilize enzyme properties to generate active species
-avoids side effects of highly reactive compounds
-aka: suicide, trojan horse, enzyme-activated substrate inhbitors (EASI)

Question 18

Mech based irreversible inhibitors mechanims

Answer 18

E+I <-> EI —> EI* -> E-I*
KI kcat Kinact
E+I* (Kdis)

Question 19

Kdis

Answer 19

slower than Kinact

Question 20

Myeloperoxidase (MPO)

Answer 20

-heme-containing enzyme from neutrophils
-kill microorganisms
-use H2O2 to form reactive species = oxidative damage

Question 21

Myeloperoxidase (MPO) as a therapeutic target

Answer 21

-contributes harmfully to protein damage (cystic fibrosis, atrial fib, sepsis)
-implicated in oxidative stress (arthritis, parkinsons, alzheimers)

Question 22

2-thioxanthines (TX2)

Answer 22

-proposed mech based inhibitor of MPO

Question 23

MPO inactivation by TX2

Answer 23

-covalently attached to heme via thioether bond between exocyclic sulfur of TX2 ring and one of the heme methyl groups
-crystal structure
-redox reactions in heme that leads to covalent link of inhibitor to heme = kill enzyme

Question 24

HIV protease

Answer 24

-cleaves Gag into 3 functional proteins from HIV
-homodimer and aspartic protease
-cleave peptide bond

Question 25

HIV protease goal against resistance

Answer 25

-design compounds that bind HIV protease with high affinity that DOES NOT lead to mutations of protease

Question 26

Darunavir (DRV)

Answer 26

-backbone and catalytic asp binding
-purdue prof
-potential therapeutic for patients with HIV variant resistant to other antiviral agents

Question 27

HIV protease-DRV complex

Answer 27

-targeting essential to avoid resistance
-backbone H-bonds interactions with Catalytic Asp residues

Question 28

HMG-CoA reductase

Answer 28

HMG-CoA –> mevalonate + CoA
deacylation

-cholesterol syntheesis

Question 29

mevalonate

Answer 29

-precursor to cholesterol
-commited step in cholesterol biosynthesis

Question 30

Statins as HMG-CoA reductase inhibitor (properties)

Answer 30

-competitive (note structure)
-lower cholesterol
-Merck Research 1978

Question 31

atorvastatin (lipitor)

Answer 31

statin
-best selling drug in world 2009
-pfizer

Question 32

Mevalonate moiety of HMG-CoA

Answer 32

-interacts with loop of HMG-CoA reductase, helix La 10 and 11 fold over substrate
-ternary strucure

Question 33

Statin recognition by HMG-CoA reductase (induced fit)

Answer 33

-substrate induces fit of helix L11 NOT drug binding
-statin-bound structure accomodates bulky aromatic groups of statin because residues near C-terminus of L10 and 11 are disordered and form a shallow groove

Question 34

Statins: substrate-analogue inhibitors

Answer 34

KI values less than 100nm

Question 35

A transition-state inhibitor has high affinity for the targeted enzyme because

Answer 35

it mimics the putative transition state of the enzyme reaction.

Question 36

Darunavir is a potent drug against HIV infection in drug-experienced patients infected with resistant HIV strains
because

Answer 36

Darunavir interacts with the polypeptide backbone and catalytic residues from each subunit

Question 37

Atorvastatin (see figure) is a highly successful drug prescribed to lower cholesterol. Statins bind the active site of
HMG-CoA reductase and block its enzyme activity because

Answer 37

part of the statin molecule resembles the mevalonate moiety of HMG-CoA.