Enzymes as therapeutic Targets for drug design Flashcards
(44 cards)
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
