Lecture 3: Targeting of enzymes Flashcards
Potency
-inhibition of specific function in vitro
-often used as antagonist of cell receptor
-measures inhibition
Dose-response curve
-measures potency
-IC50 = concentration at which 1/2 the max biological response is inhibited
-backwards S curve
Cheng-Prusoff Equation
KI = IC50 / (1+ ([S]/KM))
Enzymes
-stabilize transition state of catalyzed reaction
-lower free energy of transition state
Transition state analogue
-compound closely resembling transition state
-should bind tightly to enzyme active-site to lead to an effective therapeutic
Binding curves
-fraction of protein bound vs [ligand]
-ex. hemoglobin
Heliobacter pylori
-gram neg bacteria
-cause >85% ulcers
MTAN (5’-methylthioadenosine nucleosidase)
-antibiotic target
-part of biosynthetic pathway for menaquinone
-reaction has a dissociative transition state
TS analogue
-an antibiotic design
Gaining high affinity of TS analogue
-vary R group and test activity
TS analogue affinity
-high
-KI < 200pM
Irreversible Inhibitors
-compounds that mod and inactivate enzyme
-covalent bonds
irreversible inhibitor strategy
-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
Dihydrofolate reductase
-irreversible inhibitor
-mimics the biological substrate dihydrofolate with the addition of reactive group
Aspirin
-irreversible inhibitor of COX
-acetylates a SERINE residue near the active site where the products block substrate
-acyl enzyme + salicyclic acid
cyclo-oxygenases
COX
-homodimer
-heme active site
-aspirin blocks access to active site
Mechanism-based irreversible inhibitors
-utilize enzyme properties to generate active species
-avoids side effects of highly reactive compounds
-aka: suicide, trojan horse, enzyme-activated substrate inhbitors (EASI)
Mech based irreversible inhibitors mechanims
E+I <-> EI —> EI* -> E-I*
KI kcat Kinact
E+I* (Kdis)
Kdis
slower than Kinact
Myeloperoxidase (MPO)
-heme-containing enzyme from neutrophils
-kill microorganisms
-use H2O2 to form reactive species = oxidative damage
Myeloperoxidase (MPO) as a therapeutic target
-contributes harmfully to protein damage (cystic fibrosis, atrial fib, sepsis)
-implicated in oxidative stress (arthritis, parkinsons, alzheimers)
2-thioxanthines (TX2)
-proposed mech based inhibitor of MPO
MPO inactivation by TX2
-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
HIV protease
-cleaves Gag into 3 functional proteins from HIV
-homodimer and aspartic protease
-cleave peptide bond
HIV protease goal against resistance
-design compounds that bind HIV protease with high affinity that DOES NOT lead to mutations of protease
Darunavir (DRV)
-backbone and catalytic asp binding
-purdue prof
-potential therapeutic for patients with HIV variant resistant to other antiviral agents
HIV protease-DRV complex
-targeting essential to avoid resistance
-backbone H-bonds interactions with Catalytic Asp residues
HMG-CoA reductase
HMG-CoA –> mevalonate + CoA
deacylation
-cholesterol syntheesis
mevalonate
-precursor to cholesterol
-commited step in cholesterol biosynthesis
Statins as HMG-CoA reductase inhibitor (properties)
-competitive (note structure)
-lower cholesterol
-Merck Research 1978
atorvastatin (lipitor)
statin
-best selling drug in world 2009
-pfizer
Mevalonate moiety of HMG-CoA
-interacts with loop of HMG-CoA reductase, helix La 10 and 11 fold over substrate
-ternary strucure
Statin recognition by HMG-CoA reductase (induced fit)
-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
Statins: substrate-analogue inhibitors
KI values less than 100nm
A transition-state inhibitor has high affinity for the targeted enzyme because
it mimics the putative transition state of the enzyme reaction.
Darunavir is a potent drug against HIV infection in drug-experienced patients infected with resistant HIV strains
because
Darunavir interacts with the polypeptide backbone and catalytic residues from each subunit
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
part of the statin molecule resembles the mevalonate moiety of HMG-CoA.
