Proteins as Drug Targets

Question 1

Amino Acids & Proteins

Answer 1

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

Question 2

NonPolar Aliphatic R-Groups

Answer 2

plus Cysteine (-SH) for some ppl

Question 3

Polar, Uncharged Groups

Answer 3

Question 4

Aromatic R Groups

Answer 4

Question 5

POSITIVEly charged R Groups

Answer 5

Question 6

NEGATIVELY charged R groups

Answer 6

Question 7

Flexibility of Peptide Chain

Answer 7

Only rotations about the N-Ca (phi) and Ca-C1 (psi) bonds are allowed.​

shown by Ramachandran Plot

Question 8

Alpha Helix Structure

Answer 8

• 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

Question 9

Beta Sheet Structure

Answer 9

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

Question 10

Why proteins w/ a large # of CYS are more rigid?

Answer 10

• Cysteines -SH can crosslink to form DISULFIDE BONDS
  
  • ​–> more crosslinking –> more rigid
  • –> Covalent bond that are stable at high temps
  • Can still be destablized
    *

Question 11

Which Bond in Peptides is Shorter?

Ca-N or C1-NH

Answer 11

C1-NH bond (peptide)

has partial double bond character

overlap of 2p nonbonding orbital with nitrogen group

Question 12

Ramachandran Plot

Answer 12

• 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

Question 13

Reversible Enzyme Inhibitor

Answer 13

Inibition of enzyme activity that is REVERSIBLE

typically NON-covalent

Question 14

Irreversible Enzyme Inhibitor

Answer 14

Inhibits enzyme for an EXTENDED period of time

typically, but NOT always, covalent

Question 15

Enzyme INACTIVATOR

Answer 15

IRREVERSIBLE inhibitor

• Typically associated w/ Covalent / irreversible modification of an enzyme active site
• Acetylation of Ser530 of COX-2 by ASPIRIN

Question 16

Enzyme INHIBITOR

Answer 16

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

Question 17

Why are Enzymes the most promising Protein Targets

for drug design?

Answer 17

• 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

Question 18

Ideal Enzyme target

Answer 18

• for foreign organism or abberant cell:
  
  • ​–> enzyme that is ESSENTIAL FOR GROWTH
    
    • ​but not needed in humans
• ​​Ideally want a target that only has 1 ligand
  
  • But this is very rare
  • most drugs are promiscuous

Question 19

Catalytic Efficiency of enzymes result from…

Answer 19

Very tight binding of the TRANSITION STATE

Question 20

Orthosteric

Answer 20

Non-Allosteric Interactions

• Enzyme may have multiple binding sites
  
  • There can be more than one substrate

Question 21

Allosteric Interaction

Answer 21

• 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

Question 22

Binding to Catalytic Site

Answer 22

• 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.

Question 23

Enzymes are NOT optimized for the tightest binding of substrates….

Answer 23

INHIBITORS ARE

Question 24

Why does it not make sense for an enzyme to bind the ligand much more tightly than [L]?

Answer 24

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

Question 25

**K_D**

Answer 25

*SMALLER* the K_D = **Greater Binding Affinity** equilibrium dissociation constant, measures binding affinity

Question 26

**Activation energy barrier** for catalyzed reaction is much SMALLER than the non-catalyzed

Answer 26

* This diffrence is from the very **TIGHT binding of the transition state** * **NO POTENTIAL ENERGY MINIMUM** * --\> no finite life time * For drug design: * We simply try to **MIMIC** transition states

Question 27

**Transition State Mimics** can reproduce TS's...

Answer 27

* **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***

Question 28

**TSAI's as drugs:** **Biochemical Mech. of HMG CoA Reductase**

Answer 28

* 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** * **Stereoselective** * Interaction of -OH with the **LYS** side chain is **KEY**

Question 29

**HMG-CoA Reductase Inhibitors**

Answer 29

* **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

Question 30

**Induced Fit** mechanism that Ensures substrate specificity

Answer 30

* 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

Question 31

**Themotoga Maritima** **Maltotriose**

Answer 31

* Maltotriose --\> (Gram negative bacteria ) enzyme * --\> Inhibits the bacteria of a necesary nutrient * = strategy of development of **Antibacterias** * **​Conformational change occurs**

Question 32

Conformational Change in ## Footnote **Tyrosine Kinases**

Answer 32

* **TK's** become enzymatically ACTIVE * after ligand binding * --\> Autophosphoralation on **TYROSINE** residue * --\> **Conformational change of FGFR1 Kinase Domain** * **--\> ACTIVE conformation** * Both active / inactive conformations are targets of drug design * 20 TK inhibitors are in clinical use

Question 33

Both Enzyme & Substrate conformations are altered upon complex formation

Answer 33

**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

Question 34

**IC₅₀** inhibition constant

Answer 34

* 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**

Question 35

**K_i** inhibition constant

Answer 35

Dissociation constant of **Kinase Inhibitor Complex** characterizes the AFFINITY of ATP along w/ K_D * Measuured by monitoring rates of kinase rxn in the presence of a present concentration of INHIBITOR * + varying concentrations of ATP * **PHYSICAL CONSTANT**

Question 36

**Competitive Inhibitor**

Answer 36

* Drug that **displaces ATP** from its **binding site** * * _synonomous to **ANTAGONIST** for RECEPTORS_

Question 37

**Cheng-Prusoff Equation**

Answer 37

Shows the relationship between **IC₅₀ & K_i** * IC50 = Ki when [L] = 0 * does not occur experimentally * b/c rate of enzymatic rxn can not be ZERO * **IC₅₀ \> K_i** * **_​**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]*

Question 38

**MDR P1** Multiple Drug Resistance Protein 1

Answer 38

* **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