Proteins as Drug Targets Flashcards
Amino Acids & Proteins
Only L-Amino Acids are Present in proteins
- 1* Structure
- sequence of AA’s joined by peptide bond
- 2* Structure
- folding into alpha helices / beta sheets
- Functions:
- Structural / Mechanical (keratin/collagen)
- Coordinated motion
- ability to contract (actin)
- Catalysis of chemical rxns
- Transport / Storage (hemoglobin)
- Immune protection (AB’s)
- Signals (hormones/peptides/insulin)
- transmembrane receptors
- Control of gene expression
NonPolar Aliphatic R-Groups
plus Cysteine (-SH) for some ppl
Polar, Uncharged Groups
Aromatic R Groups
POSITIVEly charged R Groups
NEGATIVELY charged R groups
Flexibility of Peptide Chain
Only rotations about the N-Ca (phi) and Ca-C1 (psi) bonds are allowed.
shown by Ramachandran Plot
Alpha Helix Structure
- Integrity maintained by the H-Bonds between n & n-4 residues
- More stable in hydroPHOBIC environments
- but in general LESS stable than beta sheets
- RIGHT HANDED
Beta Sheet Structure
Can be Antiparallel or Parallel
-
ANTIparallel beta sheets are more preferred (more HB’s)
-
connected by B-turns
- = frequent motifs recognized by other proteins
- Drugs try to MIMIC beta turns
- IkBa transcription regulator
- GLY / PRO residues found on beta turns
-
connected by B-turns
Why proteins w/ a large # of CYS are more rigid?
- Cysteines -SH can crosslink to form DISULFIDE BONDS
- –> more crosslinking –> more rigid
- –> Covalent bond that are stable at high temps
- Can still be destablized
*
Which Bond in Peptides is Shorter?
Ca-N or C1-NH
C1-NH bond (peptide)
has partial double bond character
overlap of 2p nonbonding orbital with nitrogen group
Ramachandran Plot
-
Contour Plot showing potential enrgies of peptide fragments
- as a fxn of the angle of rotation about 2 flexible bonds
- Ca-N & Ca-C
- Shows that combinations of these angles are more FAVORABLE than others
- Ex. Antiparallel B-sheets > parallel beta sheets
- RIGHT handed alpha helix > left handed
- Ex. Antiparallel B-sheets > parallel beta sheets
Reversible Enzyme Inhibitor
Inibition of enzyme activity that is REVERSIBLE
typically NON-covalent
Irreversible Enzyme Inhibitor
Inhibits enzyme for an EXTENDED period of time
typically, but NOT always, covalent
Enzyme INACTIVATOR
IRREVERSIBLE inhibitor
- Typically associated w/ Covalent / irreversible modification of an enzyme active site
- Acetylation of Ser530 of COX-2 by ASPIRIN
Enzyme INHIBITOR
Slows or Blocks enzyme catalysis
- Associated with reversible alteration of enzyme catalysis
- Ex. Blockade of the active site by SALICYLIC ACID residue in COX-2
- from Aspirin
- aspirin does BOTH, inactivates and inhibits
Why are Enzymes the most promising Protein Targets
for drug design?
- Enzymes are PURIFIED more easily than receptor proteins
- CRYSTALLIZED 3d structures are easier to establish
- Enzyme inhibitors may look like substrates
- Only can use enzyme mechanism for inhibitor design
Ideal Enzyme target
-
for foreign organism or abberant cell:
-
–> enzyme that is ESSENTIAL FOR GROWTH
- but not needed in humans
-
–> enzyme that is ESSENTIAL FOR GROWTH
-
Ideally want a target that only has 1 ligand
- But this is very rare
- most drugs are promiscuous
Catalytic Efficiency of enzymes result from…
Very tight binding of the TRANSITION STATE
Orthosteric
Non-Allosteric Interactions
- Enzyme may have multiple binding sites
- There can be more than one substrate
Allosteric Interaction
- Enzyme NEEDS the allosteric ligand to be bound @ the allo site
- –> so that the actual substrate can bind
- Binding modulates the structure of the substrate binding site
Binding to Catalytic Site
- Free energy of the complex (bound enzyme) should be LOWER than the SUM of each component
- Low Complex Energy = Tighter Binding***
- ***Needs only to be low enough to provide significant enzyme occupupency at phys. ligand conc.
Enzymes are NOT optimized for the tightest binding of substrates….
INHIBITORS ARE
Why does it not make sense for an enzyme to bind the ligand much more tightly than [L]?
When L = 0.1 –> 11% of the protein is bound
When L = 10Kd –> protein is already 91%
There is already complete binding at 10kd, not much different from going to 100kd or 1000kd
KD
SMALLER the KD = Greater Binding Affinity
equilibrium dissociation constant, measures binding affinity
Activation energy barrier for catalyzed reaction is
much SMALLER than the non-catalyzed
- This diffrence is from the very TIGHT binding of the transition state
-
NO POTENTIAL ENERGY MINIMUM
- –> no finite life time
-
NO POTENTIAL ENERGY MINIMUM
- For drug design:
- We simply try to MIMIC transition states
Transition State Mimics
can reproduce TS’s…
-
Geometry
- sp3/sp2
-
Electronic state
- presence of -/+ charges resulting from partial bond breaking
-
Longer Seperation
- between interactions
-
Specific Conformation
- substrate adopts the reaction trajectory
- TSAI’s require knowledge of enzyme mechanism
TSAI’s as drugs:
Biochemical Mech. of HMG CoA Reductase
- HMG CoAR = NADH dependent reductase
- Converts HMG-CoA –> Mevalonate
- Step 1/1:
- Reduction of HMG w/ NADPH via TETRAHEDRAL INTERMEDIATE (TI)
- first aldehyde then alcohol
- Transition state of 1st rxn is “late”
- reproduces ost of the feature of the TI
- Reduction of HMG w/ NADPH via TETRAHEDRAL INTERMEDIATE (TI)
- Stereoselective
- Interaction of -OH with the LYS side chain is KEY
HMG-CoA Reductase Inhibitors
- Statins - discovered as a TSAI of HMG-CoA
- Typically, (Enzyme-substrate-cofactor complex)
- LYS make HB bond to C=O group of HMG
- With statin (enzyme-inhibitor complex)
-
-OH of inhibitor makes a STRONGER HB to NH3 of lysine
- –> 104 INCREASE in POTENCY
-
-OH of inhibitor makes a STRONGER HB to NH3 of lysine
Induced Fit
mechanism that Ensures substrate specificity
- Complex ligand binding process that involves:
- Changes in the structure of the Protein and/or Ligand
-
Ligand –> Protein conformation change
- Provide best alignment with interacting residues
- Ligand –> Binds to a form of the protein that @ conformational EQ
- Ex. in hexokinase rxn:
-
Enzyme is NOT active until it’s bound with both ATP & Glucose
- If it were HK would xfer P–>H2O INSTEAD of glucose
-
Enzyme is NOT active until it’s bound with both ATP & Glucose
Themotoga Maritima
Maltotriose
- Maltotriose –> (Gram negative bacteria ) enzyme
- –> Inhibits the bacteria of a necesary nutrient
- = strategy of development of Antibacterias
- Conformational change occurs
Conformational Change in
Tyrosine Kinases
-
TK’s become enzymatically ACTIVE
- after ligand binding
- –> Autophosphoralation on TYROSINE residue
- –> Conformational change of FGFR1 Kinase Domain
- –> ACTIVE conformation
- –> Conformational change of FGFR1 Kinase Domain
- Both active / inactive conformations are targets of drug design
- 20 TK inhibitors are in clinical use
Both Enzyme & Substrate conformations
are altered upon complex formation
Both conformations are altered
-
Ligand
- –> adopts conformation that is FURTHER along the rxn coordinate
- better positioned to enter the TRANSITION State
- –> MINIMIZE the activation energy barrier
- Ex. bacterial enzyme’s biosyntheisis of histadine
IC50
inhibition constant
- Concentration of INHIBITOR that reduces activity of a protein to
- HALF MAXIMAL VALUE
- depends on the measurement conditions
- For Kinase Inhibition
- @ the IC50 = rate of kinase is 50%
- NOT A PHYSICAL CONSTANT
Ki
inhibition constant
Dissociation constant of Kinase Inhibitor Complex
characterizes the AFFINITY of ATP along w/ KD
- Measuured by monitoring rates of kinase rxn in the presence of a present concentration of INHIBITOR
- varying concentrations of ATP
- PHYSICAL CONSTANT
Competitive Inhibitor
- Drug that displaces ATP from its binding site
- synonomous to ANTAGONIST for RECEPTORS
Cheng-Prusoff Equation
Shows the relationship between IC50 & Ki
- IC50 = Ki when [L] = 0
- does not occur experimentally
- b/c rate of enzymatic rxn can not be ZERO
- does not occur experimentally
-
IC50 > Ki
- when ligand concentrations [L] are > 0
-
you can not use IC50 for DIFF drugs as a measure of their potency
- UNLESS measurements were carried out w/ the same [L]
MDR P1
Multiple Drug Resistance Protein 1
-
AN EFFLUX PUMP / USES ATP
- PUMP DRUG OUT OF THE CELL
- Realtively structure non-selective
- AS LONG AS MOLECULE IS HYDROPHOBIC
- Removes both drugs and non-therapeutic hydrophobic molecules
