Unit 3 - Week 2 Flashcards
what do the best drug inhibitors look like?
mimic the transition state conformation (since preferential binding to transition state)
- such inhibitors are competitive inhibitors
- create large number of derivatives with slight structural differences
what designed inhibitors should be tested for (4 things)
- purified enzyme
- enzyme in cells
- function of target enzyme in animal models
- function of target enzyme in humans
serine protease VS aspartyl proteases
- active site H-bonds and functions
- catalytic strategies
3 H-bonded AA (catalytic triad of asp, his, ser) VS 2 H-bonded asp
ser in active site forms covalent acyl enzyme intermediate VS 2 homologous domains of PRO
both have preferential binding of transition state and acid-base catalysis, but only SP has covalent and electrostatic catalysis
HIV protease mechanism
aspartyl protease (exception b/c homodimer) essential for viral maturation
- asp X carboxyl is protonated (activates H2O to attack peptide bond), Y is deprotonated when substrate binds (base catalysis)
- creates tetrahedral transition state (highest peak of diagram) when H2O attacks
- asp Y acts as acid to breakdown intermediate (acid catalysis), donating H+ to newly formed amino group
- shuffling of H+ from X to Y restores protease to original state
problems in inhibitor design of HIV protease inhibitors
- can’t inhibit other aspartyl proteases in body
- active site is hydrophobic, but drugs must be hydrophilic enough to be delivered throughout body
successes in HIV protease inhibitors
7-10 different HIVPIs on market
-combined HAART or ART has been responsible for transforming death sentence to manageable disease
HAART/ART
(highly active) antiretroviral therapy
- combination of HIV protease inhibitors and other anti-HIV drugs
- extremely effective in reducing viral RNA levels and increasing CD4 cell levels
3 enzymes targeted for HIV therapy
- reverse transcriptase - makes DNA strand using ssRNA as template
- integrase - catalyzes integration of dsDNA into host DNA
- HIV-1 protease - processing of viral polyPRO crutial for maturation/infectivity of virus
HIV-1 protease function
cleaves polyPRO that is translation product of integrated viral DNA to release individual viral PRO essential for maturation/infectivity of virus
- must cleave several different sequences to process polyPRO
- inhibition results in formation of immature virions were not competent for further infection (since both integrase and reverse transcriptase were not released)
HIV-1 protease structure
approximately half the size of typical aspartyl proteases, and symmetrical homodimer
- limited sequence homology except for sequences at/near active site
- 3D structure similar to other aspartyl proteases
HIV-1 protease specificity
does not have absolute sequence specificity, although all proteases have some degree
- large active site crevice that is highly hydrophobic
- multiple tight hydrophobic contacts
- asp 25 and asp 25’ in active site give specificity due to multiple interactions with AA around them
- flaps allow entry of substrate, then fold down on substrate to sequester it from aqueous environment
HIV-1 protease inhibitor target sites
in vivo: natural cleavage sites between phe and pro, or phe and tyr
in vitro: use peptide that can be cleaved efficiently between beta-naphthylalanine (similar to phe) and pro
-cleavage product formation is detected by chromatography
substrate-based inhibitor design
- starts from sequences of known substances (must have specificity for enzyme)
- insert non-hydrolyzable bond where peptide bond would be (resemble transition state)
- peptides cleaved by aspartyl proteases go thru tetrahedryl transition state to incorporate tetrahedryl geometry into inhibitors
what to test inhibitors for
- Ki for purified HIV-1 protease
- inhibition of virus production by infected cell culture
- pharmacological properties
- water solubility
- stability
- inhibition of other human aspartyl proteases
- effectiveness and toxicity in animal/human models
what does predominant use of substrate-based design for virtually all HIV protease inhibitors mean?
- all inhibitors bind at enzyme active site
2. all inhibitors have some structural similarity
structure-based inhibitor design VS enzyme-based inhibitor design
SBID: starts from substrate structures, and is predominant strategy used
EBID: start s from enzyme structure and designs molecules that might “fit” based on computer modeling (may have no obvious resemblance, but can conform to active site)
-not as effective as initial strategy for HIV protease inhibitors, but useful for toher things
-refinement of inhibitor structures has used information about enzyme structure
clinical problems with HIV-protease inhibitors and HAART (7 problems)
- resistance
- pharmacokinetics - getting drug to virus
- accessing reservoirs of virus
- cost/availability
- side-effects/long-term toxicity - liver damage
- patient compliance
- when to initiate treatment (used to wait until CD4 levels <500 cells/mm3)
why do patients become resistant to HIV-1 drugs?
high error rates of RT and large number of virus particles made daily
-some sequences encode viral PRO that can perform normal function in viral propagation, but no longer bind inhibitor tightly, thus multiply even if drug
requirements for an HIV-1 protease-resistant virus
- replicate at high levels
- insensitive to drug (high Ki)
- able to carry out normal catalytic activity with reasonable efficiency
solution to HIV-1 protease inhibitor resistance
shut down viral replication as completely as possible via combos of anti-HIV drugs against different targets (HAART/ART)
hepatitis C latest therapy
HCV (serine) protease inhibitor (teleprevir, boceprevir) in combination therapy
-viral life-cycle resembles HIV-1
regulation of enzyme activity (4) and regulation of enzyme availability (4)
- allosteric regulation
2/3. regulation by reversible AND irreversible covalent modification - regulation by PRO-PRO interactions
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5/6. regulation of enzyme synthesis AND degredation - compartmentalization of enzyme activity
- differential activities of isozymes
allosteric enzymes
frequently operate at control points in metabolic pathways (rate-limiting steps; feedback inhibition)
- modulated by levels of own substrate, or other activating/inhibitory molecules
- DON’T follow Michaelis-Menten, but have multiple active sites and subunits
- -either activate or inhibit (binding changes conformation of enzyme so binding to other sites is affected)
K0.5 and relationship to allosteric modulators
concentration of substrate giving half-maximal activity (similar to Km, but related equations don’t count b/c not related to Michaelis-Menten)
- allosteric activators decrease K0.5
- allosteric inhibitors increase K0.5
