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Enzymes > Enzymes as therapeutic Targets for drug design > Flashcards

Enzymes as therapeutic Targets for drug design Flashcards

1
Q

Increased knowledge of protein structure and enzyme mechanism has allowed for what design?

A
  • inhibitor design - earlier approaches relied on trial and error
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2
Q

What are the best inhibitors?

A

ones that mimic the transition state of the substrate

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3
Q

What does the catalytic triad of a serine protease consist of?

A

His, Aspartate, serine

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4
Q

What is the basic mechanism of a serine protease?

A

It forms a covalent acyl enzyme intermediate - breaks down a complicated reaction into two easier steps

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5
Q

What are the catalytic strategies employed by a serine protease?

A
  • preferential binding of transition state
  • covalent catalysis
  • acid-base catalysis
  • electrostatic catalysis
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6
Q

What forms the active site of a typical aspartyl protease?

A

-two homologous domains of protein - each of which provides one aspartate (H)

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7
Q

What are the catalytic strategies of aspartyl proteases?

A
  • acid-base catalysis

- preferential binding of the transition state

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8
Q

Why is HIV protease an exception to aspartyl proteases?

A

It acts as a homodimer with each subunit contributing an aspartyl (rather than two domains in a single polypeptide)

It is half the size of typical eukaryotic proteases

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9
Q

Are the aspartates close together in primary structure, in an aspartyl protease?

A

no! They are brought together through protein folding

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10
Q

What is the mechanism of aspartyl proteases, including HIV protease?

A
  1. two aspartates - one protonated, one deprotonated
  2. one aspartate is a base - abstracts a proton from water
  3. activated water forms a tetrahedral transition state
  4. the other aspartate acts as an acid and donates a H+ to breakdown the intermediate
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11
Q

Is the HIV protease essential to the HIV virus?

A

Yes - for viral maturation

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12
Q

What is the biggest problem in designing HIV protease inhibitors?

A

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

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13
Q

What treatment is highly successful in treating AIDS?

A

HAART - highly active anti-retroviral therapy -combo of inhibitors of:

  • HIV protease
  • reverse transcriptase
  • integrase
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14
Q

What is HAART successful in doing?

A
  • reducing viral RNA levels

- inc CD4 cell levels

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15
Q

What is the function of a reverse transcriptase?

A

It makes the first DNA strand from a ssRNA as a template in order to integrate the viral genome into the host genome

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16
Q

What is the function of integrase?

A

catalyzes the integration of the dsDNA

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17
Q

What is the function of the HIV-1 Protease

A

cleaves the polyprotein (translation product) - release of viral proteins essential for maturation and infectitvity (such as RT and integrase)

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18
Q

What is the specificity of the HIV protease?

A
  • NOT absolute sequence
  • large active site crevice - highly hydrophobic
  • formation of multiple tight hydrophobic contacts with amino acids around the active site
19
Q

Which are the active site aspartates in HIV protease?

A

Asp25 and Asp25’

20
Q

What is a function of the conformational change when substrate binds to HIV protease?

A

It has flaps that sequester the substrate from water (except the oriented H20 involved in the rxn)

21
Q

What is the shape of the transition state in all aspartyl protease reactions?

A

tetrahedryl

22
Q

How should you design transition state analogs? (3 steps)

A
  1. inhibitors which look like substrates - recognition
  2. introduce a non-hydrolyzable bond where peptide bond would be
  3. incorporate tetrahedryl geometry into these inhibitors
23
Q

What are some ways that you can test the effectiveness of an inhibitor?

A
  • Ki
  • virus production by an infected cell culture
  • pharmacological properties
  • water solubility
  • stability
  • inhibition of other similar enzymes (other aspartyl proteases in this case)
  • effectiveness and toxicity in animals and humans
24
Q

What are two results of using substrate-based design for HIV protease inhibitors?

A
  1. all inhibitors bind at enzyme’s active site

2. all inhibitors have some structural similarity

25
Q

Is substrate-based design or enzyme-based design more prominent?

A

substrate-based

26
Q

What is the concept of enzyme-based design?

A

computer programs pick known molecules that are predicted to “fit” into the active site

27
Q

Was enzyme-based design useful in designing HIV protease inhibitors?

A

No - but useful for other targets

28
Q

What are some clinical problems with HAART and HIV protease inhibitors?

A
  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 treatment
29
Q

Why do patients become resistant to HIV-1 drugs?

A
  • High error rate of RT
  • large number of virus particles made

-some of these sequences will encode viral proteins that can perform normal function and not bind inhibitor

30
Q

The HIV-1 protease in resistant virus must be able to: (2)

A
  1. insensitive to drug - high Ki

2. able to carry out normal catalytic activity efficiently - kcat/Km= WT

31
Q

Is a virus variant that is not able to replicate rapidly, but still able to replicate, in the presence of a protease inhibitor dangerous?

A

Yes - the production of new mutations in the viral genome can still occur

32
Q

What is a solution to resistance to HIV-protease inhibitors?

A

Combinations of anti-HIV drugs against different targets - HAART

33
Q

What is the first direct antiviral against HCV?

A

HCV protease inhibitors

34
Q

What are two broad categories of enzyme regulation?

A
  1. regulation of enzyme activity

2. regulation of enzyme availability

35
Q

How is the activity of an allosteric enzyme regulated?

A
  1. by levels of its own substrate
    or
  2. levels of other activating or inhibitory modulators
36
Q

What is K0.5?

A

concentration of substrate giving half -maximal activity - aprox equivalent to km

37
Q

What is the effect of an allosteric activator on k0.5?

A

activators decrease K0.5

38
Q

What is the effect of an allosteric inhibitor on K0.5?

A

inhibitors increase k0.5

39
Q

Do allosteric enzymes follow typical Michaelis-Menten equations?

A

No0o0o

40
Q

Even though there is a strong resemblance between hemoglobin and allosteric enzymes, and myoglobin and michaelis-menten enzymes, what are you actually looking at?

A

Hemoglobin and Myoglobin: substrate binding

Allosteric and Michaelismenten: reaction rates

41
Q

What is the general model for the regulation of allosteric enzymes?

A

-Binding at an allosteric site changes conformation of the enzyme so that other sites are affected

42
Q

CTP is an allosteric _____ of ATCase

A

Inhibitor

  • stabilizes low affinity conformation -Tstate
    • active sites less available
    • shifts RIGHT
43
Q

ATP is an an allosteric _____ of ATCase

A

activator

  • stabilizes the high affinity conformation - R state
    • active sites more accessible
    • Shifts LEFT
44
Q

Are CTP or ATP substrates of ATCase?

A

NOO - must bind at other locations other than the active site (allosteric enzyme DUuUuH)

Enzymes (4 decks)

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