Enzymes as Therapeutic Targets Flashcards
General principles of drug design
- use knowledge of protein structure and enzyme mechanism to design enzyme inhibitors for use as drugs
- best inhibitors often mimic the TS conformation-competitive inhibitors
- 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
serine proteases
- 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
aspartyl proteases
-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
mechanism of aspartyl protease
- 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
overall strategy for designing HIV proteases
- HIV protease is an aspartyl protease that is essential for maturation
- design inhibitors that bind the active site of the HIV protease
problems in inhibitor design
can’t inhibit other aspartyl proteases in the body
-active site is hydrophobic, drugs must be hydrophilic enough to allow delivery in the body
successes in HIV protease inhibitors
- 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
major HIV inhibitors
- reverse transcriptase
- integrase
- protease
function of HIV protease
- 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
structure of HIV protease
- 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
specificity of HIV protease
- 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
natural cleavage sites
- between Phe and pro, or Phe and tyr
- these have been incorporated into inhibitors
design TS analogs
- 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
synthesize inhibitors and modified forms and test
- 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
when all inhibitors look the same
- all inhibitors will bind at the active site
- all inhibitors will have some structural similarity
- can lead to resistance
