Enzymes as therapeutic Targets for drug design Flashcards
Increased knowledge of protein structure and enzyme mechanism has allowed for what design?
- inhibitor design - earlier approaches relied on trial and error
What are the best inhibitors?
ones that mimic the transition state of the substrate
What does the catalytic triad of a serine protease consist of?
His, Aspartate, serine
What is the basic mechanism of a serine protease?
It forms a covalent acyl enzyme intermediate - breaks down a complicated reaction into two easier steps
What are the catalytic strategies employed by a serine protease?
- preferential binding of transition state
- covalent catalysis
- acid-base catalysis
- electrostatic catalysis
What forms the active site of a typical aspartyl protease?
-two homologous domains of protein - each of which provides one aspartate (H)
What are the catalytic strategies of aspartyl proteases?
- acid-base catalysis
- preferential binding of the transition state
Why is HIV protease an exception to aspartyl proteases?
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
Are the aspartates close together in primary structure, in an aspartyl protease?
no! They are brought together through protein folding
What is the mechanism of aspartyl proteases, including HIV protease?
- two aspartates - one protonated, one deprotonated
- one aspartate is a base - abstracts a proton from water
- activated water forms a tetrahedral transition state
- the other aspartate acts as an acid and donates a H+ to breakdown the intermediate
Is the HIV protease essential to the HIV virus?
Yes - for viral maturation
What is the biggest problem in designing HIV protease inhibitors?
The active site is hydrophobic, but drugs must be hydrophilic enough to allow delivery in the body
What treatment is highly successful in treating AIDS?
HAART - highly active anti-retroviral therapy -combo of inhibitors of:
- HIV protease
- reverse transcriptase
- integrase
What is HAART successful in doing?
- reducing viral RNA levels
- inc CD4 cell levels
What is the function of a reverse transcriptase?
It makes the first DNA strand from a ssRNA as a template in order to integrate the viral genome into the host genome
What is the function of integrase?
catalyzes the integration of the dsDNA
What is the function of the HIV-1 Protease
cleaves the polyprotein (translation product) - release of viral proteins essential for maturation and infectitvity (such as RT and integrase)
What is the specificity of the HIV protease?
- NOT absolute sequence
- large active site crevice - highly hydrophobic
- formation of multiple tight hydrophobic contacts with amino acids around the active site
Which are the active site aspartates in HIV protease?
Asp25 and Asp25’
What is a function of the conformational change when substrate binds to HIV protease?
It has flaps that sequester the substrate from water (except the oriented H20 involved in the rxn)
What is the shape of the transition state in all aspartyl protease reactions?
tetrahedryl
How should you design transition state analogs? (3 steps)
- inhibitors which look like substrates - recognition
- introduce a non-hydrolyzable bond where peptide bond would be
- incorporate tetrahedryl geometry into these inhibitors
What are some ways that you can test the effectiveness of an inhibitor?
- 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
What are two results of using substrate-based design for HIV protease inhibitors?
- all inhibitors bind at enzyme’s active site
2. all inhibitors have some structural similarity
Is substrate-based design or enzyme-based design more prominent?
substrate-based
What is the concept of enzyme-based design?
computer programs pick known molecules that are predicted to “fit” into the active site
Was enzyme-based design useful in designing HIV protease inhibitors?
No - but useful for other targets
What are some clinical problems with HAART and HIV protease inhibitors?
- resistance
- pharmacokinetics
- accessing reservoirs of virus
- cost and availability
- side-effects/ long-term toxicity
- patient compliance
- When to initiate treatment
Why do patients become resistant to HIV-1 drugs?
- 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
The HIV-1 protease in resistant virus must be able to: (2)
- insensitive to drug - high Ki
2. able to carry out normal catalytic activity efficiently - kcat/Km= WT
Is a virus variant that is not able to replicate rapidly, but still able to replicate, in the presence of a protease inhibitor dangerous?
Yes - the production of new mutations in the viral genome can still occur
What is a solution to resistance to HIV-protease inhibitors?
Combinations of anti-HIV drugs against different targets - HAART
What is the first direct antiviral against HCV?
HCV protease inhibitors
What are two broad categories of enzyme regulation?
- regulation of enzyme activity
2. regulation of enzyme availability
How is the activity of an allosteric enzyme regulated?
- by levels of its own substrate
or - levels of other activating or inhibitory modulators
What is K0.5?
concentration of substrate giving half -maximal activity - aprox equivalent to km
What is the effect of an allosteric activator on k0.5?
activators decrease K0.5
What is the effect of an allosteric inhibitor on K0.5?
inhibitors increase k0.5
Do allosteric enzymes follow typical Michaelis-Menten equations?
No0o0o
Even though there is a strong resemblance between hemoglobin and allosteric enzymes, and myoglobin and michaelis-menten enzymes, what are you actually looking at?
Hemoglobin and Myoglobin: substrate binding
Allosteric and Michaelismenten: reaction rates
What is the general model for the regulation of allosteric enzymes?
-Binding at an allosteric site changes conformation of the enzyme so that other sites are affected
CTP is an allosteric _____ of ATCase
Inhibitor
- stabilizes low affinity conformation -Tstate
- active sites less available
- shifts RIGHT
ATP is an an allosteric _____ of ATCase
activator
- stabilizes the high affinity conformation - R state
- active sites more accessible
- Shifts LEFT
Are CTP or ATP substrates of ATCase?
NOO - must bind at other locations other than the active site (allosteric enzyme DUuUuH)
