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
apsartate transcarbamoylase (ATCase)
catalyzes first step in synthesis of CTP for RNA synthesis
- classic allosteric enzyme (feedback inhibition: high CTP slows down ATCase, high ATP vice versa)
- due to 6 regulatory and 6 catalytic subunits arranged in rings
ATCase allosteric properties
CTP - allosteric inhibitor (preferentially binds and setabilizes a low-affinity conformation of ATCase - T state)
ATP - allosteric activator (preferentially binds and setabilizes high affinity conformation of ATCase - R state)
NEITHER CTP NOR ATP ARE SUBSTRATES FOR ATCASE, so must bind at locations other than active site
ways for regulation by reversible covalent modification
- causing conformational change that affects catalysis
- altering cellular localization of the enzyme
- altering interactions with other PRO
- commonly phosphorylation, methylation, acetylation, glutathionylation, ubiquitination
glycogen phosphorylase
reversible covalent modification
- catalyzes degradation of glycogen into glucose-1-phosphate
- needs to be phosphorylated to become active, via glycogen phosphorylase kinase
- -reversed by phosporylase phosphatase
regulation by irreversible covalent modification
enzymes are made in inactive form (zymogen) and activated irreversibly in time/place needed
-kept in zymogen granules
enteropeptidase and subsequent cascade
cleaves trypsinogen –> trypsin
-trypsin then cleaves chymotrypsinogen et al
rapid activation of all; irreversible
maintenence of blood volume requires what 3 rapid responses to blood vessel injury? and are they reversible or irreversible?
- rapid activation of blood coagulation - irreversible covalent modifications
- localization of clot to site of injury - reversible covalent modifications)
- rapid termination after clot formation to prevent thrombosis - irreversible covalent modifications
factor VIIIa
modulator PRO (not an enzyme) that is needed for the clotting cascade -missing in hemophiliacs
localizations of blood clots mechanism
clotting PRO are converted to gamma-carboxyglutamates via vit K-dependent enzyme
-the subsequent GLA modification allows interaction with Ca and binding to phospholipid membranes to localize
inhibition of coagulation
dicoumarol (coumadin; vit K analog), warfarin
-competitive inhibitors of vit K-dependent enzymes for GLA, to prevent the modification and subsequent binding to Ca and phospholipid membranes
how is clotting terminated?
opposing cascade with other serine protease inhibitors
-TPA converts plasminogen to plasmin, which causes fibrins to hydrolyze clot
tissue plasminogen activator (TPA)
serine protease that activates plasminogen –> plasmin, which allows fibrins to hydrolyze clots
-administered therapeutically for heart attack/stroke within hours
regulation by PRO-PRO interactions
while zymogen activation is irreversible, activated proteases can be turned off by interaction with inhibitor proteins
pancreatic trypsin inhibitor
inhibits inappropriately activated digestive proteases in pancreas
anti-thrombin (AT III)
serpin that inactivates thrombin and other proteases (by binding active sites) of clotting cascade to arrest inappropriate clotting, as long as heparin is present to stabilize protease-inhibitor complex
alpha-antitrypsin (or alpha-antiproteinase)
elastase inhibitor that protects tissues from neutrophilic elastase
anti-thrombin deficiency
AD genetic deficiency
- excessive, inappropriate clotting (thrombosis) in legs/lungs
- observed after serious injury or oral contraceptives
- may be fatal, so treat with long-term anticoagulants
a1-antitrypsin defiency
very common due to too much elastase in lung (from MPs recruited to lungs) –> COPD/emphysema (smoking, infection)
- can’t inhibit elastase, causes SOB
- severe deficiency also causes liver disease (misfolding in ER)
PRO kinase A
regulated by interaction of cAMP with regulatory subunits
-cAMP binding releases inhibition by regulatory subunits and activates catalysis
calmodulin
shows Ca2+ dependent interactions with multiple enzymes
how can enzymes be used as diagnostic tools?
- diagnostic measurement of enzyme levels
- measurement of substrate or metabolite levels
- diagnosis of tissue damage or tumors by isozyme distribution
all are measured over time
usage of lactate dehydrogenase for enzymatic assay
measure product (NADH) formation directly via absorbance at 340 nM (rate of appearance = enzyme activity)
- has greater rate of absorbance than NAD precursor
- requires excess concentration of lactate added to serum
why do we start enzyme assays at saturating [substrate]?
because both [S] and [P] are linear with time under these conditions, and Vmax is directly proportional to total [E]
SGPT (ALT; ala aminotransferase) usage in enzymatic assays
liver enzyme that converts glutamate and pyruvate to alpha-ketoglutarate and alanine
- products not measured easily, so use coupled enzyme assay with alpha-detoglutarate dehydrogenase to make succinate and NADH
- requires excess of secondary materials
use of enzymes to measure metabolite or drug levels
fast, accurate, cheap quantification of small molecule in complex mixture (serum, urine)
- use a large excess of enzyme specific for the substance measured to convert substrate completely to product in a short time
- measure amount of product formed, directly or indirectly
- cons: other factors in sample can interfere with enzyme activity
measurement of blood glucose levels
coupled enzyme assay detecting colored NADPH
- G6P, ATP, NADP, hexokinase are added in excess
- easy dipstick measurement b/c [NADPH] proportional to [glucose]
non-plasma-specific enzymes appearing in plasma and how to detect
damage to tissue of origin or because of “spillover” due to overproduction in tissue of origin or tumor in tissue
- level of enzyme activity
- timing of appearance of activity
- presence of tissue-specific isozymes
diagnostic use of alanine aminotransferase (ALT, SGPT)
viral hepatitis
diagnostic use of amylase
acute pancreatitis
diagnostic use of lipase
acute pancreatitis
diagnostic use of creatine kinase
muscle disorders and myocardial infarction
-is more common in skeletal
diagnostic use of lactate dehydrogenase isozyme 5
liver diseases
diagnostic use of phosphatase acid VS phosphatase alkaline (isozymes)
acid: metastatic carcinoma of prostate
base: various bone disorders, obstructive liver disease
enzyme levels in plasma following myocardial infarction
peaks of creatine kinase, aspartate aminotransferase, and lactate dehydrogenase
-timing of rise/fall are characterstic of MI, so must measure levels at multiple times
isozymes
different forms of enzyme that carry out same RXN
- have different AA sequences, diff chemical properties, and diff enzymatic characteristics
- may have specific expression in different tissues, or specific pattern of expression during development
how can different isozymes be distinguished? (4 ways)
- charge differences (electrophoresis)
- specific monoclonal Abs
- differences in enzymatic properties
- inhibitor sensitivities
lactate dehydrogenase isozymes (the ones in heart and muscle/liver)
heart: LDH-1 - alpha tetramer
lung: LDH-5 - beta tetramer
(other 3 are different subunits, elsewherei n body)
creatine kinase isozymes (in heart and muscle)
heart only: CK-2 - beta-alpha subunits
skeletal/cardiac muscle: CK-3 - alpha dimer
separation of LDH isozymes by electrophoresis
they will migrate to the negative end
use of LDH and CK in MI diagnosis
CK-2 and LDH-1 (heart) in plasma
-timing of appearance: both enzyme activities appear transiently after attack and fade away
use of LDH and CK in muscular dystrophy diagnosis
CK-3 (from muscle) is present in plasma, with a long-term rise over weeks
