Enzymes as Therapeutic Targets

Question 1

General principles of drug design

Answer 1

1. use knowledge of protein structure and enzyme mechanism to design enzyme inhibitors for use as drugs
2. best inhibitors often mimic the TS conformation-competitive inhibitors
3. overall strategy-model active site and active site conformation at TS
   - computer model molecules predicted to fit enzyme conformation
   - synthesize these molecules and a large number of derivatives with slight structural differences
   - test designed for inhibition of:
   - purified enzyme
   - enzyme in cells
   - function of target enzyme in animal models
   - function of target enzyme in humans

Question 2

serine proteases

Answer 2

• 3 H bonded amino acids at active site-asp, his, ser
• serine in active site forms a covalent acyl enzyme intermediate
• catalytic strategies:
• preferential binding of TS
• covalent catalysis
• acid/base catalysis
• electrostatic catalysis

Question 3

aspartyl proteases

Answer 3

-2 H bonded asp at active site
-active site is formed from two homologous domains of protein, each of which provides one asp
-catalytic strategies-acid base catalysis
-some preferential binding of TS
HIV protease is one of these

Question 4

mechanism of aspartyl protease

Answer 4

• protein folds and makes deep cleft
• HIV protease is a homo dimer that folds
• active site has 2 asp, one carboxyl is protonated and the other isn’t when the substrate binds
• the deprotonated Asp acts as a base and accepts H from water so water can attack the substrate
• water attacks peptide bond and forms tetrahedral TS
• the protonated Asp (from beginning) acts as acid and donates H to breakdown TS and release of split products
• proton is shuttled to initial spot

Question 5

overall strategy for designing HIV proteases

Answer 5

• HIV protease is an aspartyl protease that is essential for maturation
• design inhibitors that bind the active site of the HIV protease

Question 6

problems in inhibitor design

Answer 6

can’t inhibit other aspartyl proteases in the body

-active site is hydrophobic, drugs must be hydrophilic enough to allow delivery in the body

Question 7

successes in HIV protease inhibitors

Answer 7

• at least 7 different inhibitors now on market
• HAART-highly active anti retroviral therapy-combination of HIV protease inhibitors and other anti HIV drugs
• extremely effective at reducing viral RNA levels and increasing CD4 levels

Question 8

major HIV inhibitors

Answer 8

• reverse transcriptase
• integrase
• protease

Question 9

function of HIV protease

Answer 9

• cleaves the polyprotein that is the translation product of the integrated viral DNA to release individual viral proteins essential for maturation and infectivity of the virus
• loss-no mature/infectious virus
• enzymes are inactive if not cleaved from polyprotein

Question 10

structure of HIV protease

Answer 10

• aspartyl protease
• homodimer-two subunits each 1/2 size of most proteases
• each subunit contributes 1 asp to the active site
• 22kDA per dimer
• limited homology except near active site

Question 11

specificity of HIV protease

Answer 11

• does not have absolute substrate specificity-must cleave at several different sequences in polyprotein
• large, hydrophobic, active site crevice
• formation of multiple hydrophobic contacts help dictate specificity
• asp not involved in specificity
• flaps allow entry of substrate that fold down to sequester it from the aq environment

Question 12

natural cleavage sites

Answer 12

• between Phe and pro, or Phe and tyr

- these have been incorporated into inhibitors

Question 13

design TS analogs

Answer 13

• design inhibitors that look enough like substrates to allow recognition by the enzyme
• introduce a non-hydrolyzable bond where peptide bond would be
• peptides cleaved by the aspartyl proteases go through testrahedryl transition state-incorporate that geometry into inhibitors

Question 14

synthesize inhibitors and modified forms and test

Answer 14

• inhibitory properties (Ki) for purified HIV protease
• inhibition of virus production by infected cell culture
• pharmacological properties
• water solubility
• stability
• inhibition of other human proteases
• effectiveness and toxicity in animal and human models

Question 15

when all inhibitors look the same

Answer 15

1. all inhibitors will bind at the active site
2. all inhibitors will have some structural similarity
   - can lead to resistance

Question 16

enzyme based inhibitor design

Answer 16

• starts from enzyme structure and designs molecules that might “fit” based on computer modeling
• not as successful as initial strategy for HIV protease inhibitors but has been very useful for a number of other targets
• refinement of inhibitor structures has used information about enzyme structure

Question 17

clinical problems with HIV protease inhibitors

Answer 17

1. resistance
2. pharmacokinetics
3. accessing reservoirs of virus
4. cost and availability
5. side effects/long term toxicity
6. patient compliance
7. when to initiate treatement

Question 18

resistance

Answer 18

• high error rate by RT and large number of virus particles synthesized daily imply that virtually every possible viral sequence will have been synthesized in a patient within a very short time
• some of these sequences will encode viral proteins that can perform their normal function in viral propagation, but not longer bind the inhibitor tightly
• these viruses can multiply in presence of anti HIV drug

Question 19

acquisition of resistance

Answer 19

• A and B can’t grow
• C and D are resistant
• D can not die and multiply (4-7 mutations)
• D will predominate population
• C can’t replicate-mutation that allows to to avoid inhibitor also harms normal function
• still dangerous because replicates at low levels
• can make new variants-that is drug resistant and better at dividing
• can arise in patient after treatment
• need two treatments

Question 20

pharmacokinetics

Answer 20

-problem with getting the drug to the virus

Question 21

Hep C

Answer 21

• infects 2% of the general US population and 90% of long term injected drug users
• transmitted through blood, new infections now decreasing in developed, but not developing nations
• often asymptomatic for many years
• causes long term liver damage in up to 20% of infected people-cirrhosis, can be followed by liver cancer or liver failure
• current therapy is pegylated interferon and ribavirin have serious side effects and aren’t very effective
• latest therapy is HCV protease inhibitor in combination therapy
• life cycle resembles HIV

Question 22

regulation of enzyme activity

Answer 22

1. allosteric regulation
2. regulation by reversible covalent modification
3. regulation by irreversible covalent modification
4. regulation by protein-protein interactions

Question 23

regulation of enzyme availability

Answer 23

1. regulation of enzyme synthesis
2. regulation of enzyme degradation
3. compartmentalization of enzyme activity
4. differential activities of isozymes

Question 24

allosteric enzymes

Answer 24

• operate at control points in metabolic pathways
• usually the enzyme at the rate limiting step-regulated and for feedback
• don’t follow michaelis menten
• have pos modulators-inc affinity (dec K0.5)
• and neg modulators-dec affinity (inc K0.5)
• K0.5 is concentration of substrate giving half maximal activity

Question 25

aspartate transcarbamoylase (ATCase)

Answer 25

- catalyzes first step in synthesis of CTP - CTP is allosteric inhibitor-preferentially binds and stabilizes T state - ATP is allosteric activator-binds and stabilizes R state