Transcript Slide 1

Enzyme Inhibition
There are several motivations to study enzyme inhibition:
• Distinguish among the different potential mechanisms in
multisubstrate reactions
• Relative binding affinity of competitive inhibitors  active site
structure research in the absence of 3-D structure info
• Control mechanisms in biology: balance of protease NZs and their
inhibitors in a tissue  help to achieve homeostasis
• Commercial applications:
– Insecticides, weed killers
– Drugs
Enzyme Inhibition
• Since most clinical drug therapy is based on inhibiting the activity of
enzymes, analysis of enzyme inhibition kinetics is fundamental to
the modern design of pharmaceuticals
– Well-known examples of such therapy include the use of methotrexate
in cancer chemotherapy to semi-selectively inhibit DNA synthesis of
malignant cells
– the use of aspirin to inhibit the synthesis of prostaglandins which are at
least partly responsible for the aches and pains of arthritis
– the use of sulfa drugs to inhibit the folic acid synthesis that is essential
for the metabolism and growth of disease-causing bacteria
• In addition, many poisons (such as cyanide, carbon monoxide and
polychlorinated biphenols (PCBs)) produce their life- threatening
effects by means of enzyme inhibition
Enzyme Inhibition
• Reversible inhibition: the effect of an inhibitor can be reversed by
decreasing the concentration of inhibitor
• Irreversible inhibition: there is no reversal of inhibition on
decreasing the inhibitor concentration: an example of enzyme
inactivation
– e.g. Cyanide: by covalently binding mitochondrial cytochrome oxidase, it
inhibits all the reactions associated with electron transport
– Penicillin for bacterial peptidase
• The distinction between reversible and irreversible inhibition is not
absolute and may be difficult to make if the inhibitor binds very
tightly to the enzyme and is released very slowly (tight-binding
inhibitors)
Mixed Inhibition
1. Always possible
2. Not possible with
3.
4.
uncompetitive inhibitors
Not possible with competitive
inhibitors
Not possible with competitive
or uncompetitive inhibitors
A noncompetitive inhibitor is capable of all four reactions, but the
classical noncompetitive inhibitor, as opposed to a mixed one, is a
special case. With these inhibitors Ks and Ks' are equal to each
other, as are Ki and Ki'
Mixed Inhibition
When [E]0<< [I]0:
Simplified cases: Competitive inhibition
•
Both the substrate and inhibitor
compete for binding to the same
form of the enzyme: free form
 ESI complex is not formed
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•
The inhibition is most noticeable at
low [S] but can be overcome at
sufficiently high [S]
Vmax remains unaffected
Attaining Vmax requires higher [S] in the presence of competitive
inhibitor
 Apparent Km is increased
Simplified cases: Competitive inhibition
Kapp
To obtain accurate data:
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•
•
At [S] = Km  check broad range of [I]
Choose [I] that yield between 30 % to
75 % inhibition
Measure vo values as a function of [S]
at several fixed [I]
Simplified cases: Competitive inhibition
• Competitive inhibitors are especially attractive as clinical modulators
of enzyme activity because they offer two routes for the reversal of
enzyme inhibition
1. like all kinds of reversible inhibitors, a decreasing concentration of the
inhibitor reverses the equilibrium
2. since substrate and competitive inhibitors both bind at the same site,
raising [S], while holding [I] constant, provides the second route for
reversal of competitive inhibition
Examples of competitive inhibitors
2,3-biphosphoglycerate
–
Inhibits its own formation by inhibiting biphosphoglycerate
mutase
 Metabolic regulation by product inhibition
Examples of competitive
inhibitors
• Malonate vs succinate
Enzyme: succinate
dehydrogenase
• Krebs and his
colleagues used
malonate to
investigate the TCA
cycle
Examples of competitive
inhibitors
Sulphonamides: widely used in medicine to limit bacterial
growth
Antifreeze: ethylene glycol competes for active site of
alcohol dehydrogenase
– Ethylene glycol is metabolized to oxalic acid, which crystallizes in
kidneys and causes renal failure
– Can be treated by alcohol infusion
– A new treatment, approved by the FDA in December 1997, uses
the alcohol dehydrogenase inhibitor 4-methylpyrazole (trade name
= Antizol). Unlike ethanol, 4-methylpyrazole is not a substrate for
the enzyme, is therefore not metabolized
Simplified cases: Noncompetitive inhibition
• Since noncompetitive inhibitors do
not interfere in the binding of the
substrate (the dissociation
constant of ES and ESI have the
same value Ks)
 Km is not affected
• However, increasing [S] can not abolish the inhibition  (ESI)
complex are formed and these are incapable of progressing to
reaction products
• The effect of a noncompetitive inhibitor is to reduce [ES] that can
advance to product
• Since Vmax = k2[Et], and the concentration of competent Et is
diminished by the amount of ESI formed
 Vmax is decreased
Simplified cases: Noncompetitive inhibition
When Ki ≈ Ki’
• Reciprocal plot
Examples of noncompetitive inhibitors
• Heavy metals like lead, mercury (breaks disulfide bonds), chromium
will act as non-competitive inhibitors
• Mono-amine oxidase (MAO) inhibitors that are used as antidepressants: They covalently react with the enzyme in the liver and
effectively remove it.
– There are many potent drug interactions with MAO inhibitors. One of
these is tyramine, a compound that is present in red wine and aged
cheeses
– MAO inhibitors and tyramine (blocks neurotransmitter reuptake in the
brain)  hypertensive crisis
– Patients are still subject to hypertension for as long as two weeks after
discontinuing the drug
JNK-ATP Noncompetitive Inhibitors
JNK-ATP Noncompetitive Inhibitors
Simplified cases: Uncompetitive inhibition
• The ES complex dissociates the
substrate with a dissociation
constant equal to Ks, whereas
the ESI complex does not
dissociate it (i.e has a Ks value
equal to zero)
 Km is decreased
• Increasing [S] leads to increasing [ESI] (a complex incapable of
progressing to reaction products), therefore the inhibition can not
be removed
 Vmax is decreased
Simplified cases: Uncompetitive inhibition
• Changing both Km and Vmax leads to double reciprocal plots, in
which intercepts on the vertical and horizontal axis are proportionately
changed
 Parallel lines in inhibited and uninhibited reactions
Vmax [S]
no EI formation  v =



Km + [S]  1 
• Reciprocal plot
[I ] 

' 
Ki 
Examples of uncompetitive inhibitors
• Lithium and the phosphoinositide cycle: an example
of uncompetitive inhibition and its pharmacological
consequences
Nahorski SR, et al Trends Pharmacol Sci. 1991 Aug;12(8):297303
– The ability of lithium to exert profound and selective
psychopharmacological effects to ameliorate manic-depressive
psychosis has been the focus of considerable research effort.
– There is increasing evidence that lithium exerts its therapeutic
action by interfering with polyphosphoinositide metabolism in
brain and prevention of inositol recycling by an uncompetitive
inhibition of inositol monophosphatase
Examples of uncompetitive inhibitors
• Many successful pesticides and drugs are tight-binding inhibitors,
but these are difficult to design because of the need to deliver and
maintain concentrations at least 1000-fold higher than the inhibition
constants
• A few pesticides are uncompetitive inhibitors, the best-known
example being the herbicide N-phosphonomethylglycine, commonly
known as glyphosate or Roundup, an uncompetitive inhibitor of 3phosphoshikimate 1-carboxyvinyltransferase.
Examples of uncompetitive inhibitors
Kamali and Rawlins, Biopharmaceutics & Drug Disposition, 13(6), 403-409
• The effects of probenecid on zidovudine (a potent HIV
inhibitor) glucuronidation were investigated, in vitro,
using human liver microsomal preparations
• The presence of probenecid:
– reduced the Vmax for zidovudine glucuronide formation by more
than 60 %
– reduced Km by 47 %
– uncompetitive inhibition
Determination of Ki
Secondary Plots
1. 1/Vmax vs [I]
– In the primary plot, each [I] gives a different Vmax
– The intercept on the inhibitor axis gives us the value for -Ki'
2. slope vs [I]
– This time the intercept on the inhibitor axis indicates -Ki
• Secondary plots with different inhibitor types
– Classical noncompetitive inhibitor
• The two plots should give identical results as Ki and Ki' are equal
– Competitive inhibitor
• The first secondary plot cannot be made as there is no change in Vmax
– Uncompetitive inhibitor
• This is the opposite of the competitive inhibitor. The second secondary plot
can't be made as there is no change in slope
Determination of Ki
Dixon plots (1953)
• It's a bit restricted as it can't be used to
calculate Km, Vmax, or even Ki’
– Measure vo as a function of [I] at two or more [S]
– Plot data as 1/v vs [I]
• A vertical line dropped from the point where the
lines intersect each other down to the inhibitor
axis gives -Ki
– For a classical noncompetitive inhibitor the
intersection would be on the axis so the constant
can be read off directly
– For competitive, or mixed inhibitors the
intersection will be some way above the axis
– With an uncompetitive inhibitor the lines would
be parallel - there's no intersection as Ki is
irrelevant to these inhibitors
For competitive and mixed
For noncompetitive
Assumptions vs reality???
• Although some examples exist of true uncompetitive and noncompetitive inhibitors, in most cases, the kinetics are not quite that
simple
– For uncompetitive inhibition: Inhibitor binding should only
occur if the active site is occupied by substrate. But in most
cases, the inhibitor will have some affinity for the unoccupied
enzyme as well
– For non-competitive inhibition: the inhibitor affinity should be
unchanged regardless of whether substrate is bound or not. The
affinity for the inhibitor usually changes when substrate is bound
in reality
• True competitive inhibition is common
 In reality, we have competitive and mixed inhibition
Dose-response curves
"The right dose differentiates a poison from a remedy"
Paracelsus
(Auroleus Phillipus Theostratus Bombastus von Hohenheim)
• Dose-response curves can be used to plot the results of many kinds
of experiments:
– The X-axis plots concentration of a drug or a hormone
– The Y-axis plots response, which could be almost anything: enzyme
activity, accumulation of an intracellular second messenger, membrane
potential, secretion of a hormone, heart rate or contraction of a muscle
• Assumptions in this relationship:
– There is almost always a dose below which no response occurs
– Once a maximum response is reached any further increases in the dose
will not result in any increased effect
 Dose-response curves can also be used to follow the effects of an
inhibitor on v0 at fixed [S]
Dose-response curves
• [I] required to achieve halfmaximal degree of inhibition:
IC50
•
vi
1

Fractional activity:
v0 1  [ I ]
IC50
• This strategy is very convenient when many compounds of unknown
and varying inhibitory potency are to be screened
• Effect of different inhibitors in a cellular assay: without [S] and
effective [I] info...
• IC50 can change with solution conditions so it is important to report
assay conditions...
Partial inhibitors
• In some situations, the enzyme can still work with the inhibitor
bound, but at a reduced rate
 Activity of the enzyme can not be driven to zero...
• To be sure if the compound is a partial inhibitor or not, one
should pay attention to experimental conditions
• Lineweaver-Burk plot would be linear, BUT secondary plots of
intercept or slope vs [I] will not be linear
• Dose-response curves (DRC) and Dixon plots are also different
for partial inhibitors
– At high [I]  there is a residual fractional activity in DRC
– Dixon plots are not linear but hyperbolic
Binding of two inhibitors
• Two structurally distict inhibitors, I and J, acting on the same NZ
– EIJ
– ESIJ or
– Mutually exclusive fashion: competitive with each other
• By labelling one of the inhibitors, ability of the second inhibitor to
interfere with this binding can be measured
• Effect of inhibitors on reaction rate:
1
1  [ I ] [ J ] [ I ][ J ] 

1



vij v0 
Ki K j Ki K j 
• What can be the value of :
– Mutually exclusive, completely indp, synergistic or antagonistic????
=
 =1
<1
>1
Tight binding inhibitors
• Many contemporary therapeutic enzyme inhibitors, like
anticancer drugs (for dihydrofolate reductase), anti-AIDS
drugs (for HIV aspartyl protease) are tight binders
• Many naturally occuring inhibitors playing role in
homeostasis are also tight binders
• Steady state approach is not valid for these inhibitors
– Classical double reciprocal plots fail  e.g. A tight binding
competitive inhibitor will look like a noncompetitive inhibitor. Only
at high [S] and high [I], the data curves downward....
– Misinterpretations of data is likely to occur  e.g. Natural
inhibitors of ribonuclease
Affinity labeling agents
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•
Active-site directed irreversible inhibitors
Recognized by the enzyme (reversible, specific binding) followed
by covalent bond formation
1. structurally similar to substrate, transition state or product allowing
for specific interaction between the compound and target enzyme
2. contains reactive functional group (e.g. a nucleophile, -COCH2Br)
allowing for covalent bond formation
•
•
Unwanted side reactions (non-specific alkylations) may still occur
due to the presence of the reactive group
It is a common and powerful tool to isolate and characterize the
enzymes
Mechanism-based enzyme inactivators
(Suicide Inhibitors)
• Active-site directed reagent (unreactive) binds to the enzyme active
site  transformed to a reactive form. Once activated, a covalent
bond between the inhibitor and the enzyme forms
• This approach minimizes side reactions (non specific covalent bond
formation) which may occur with an affinity reagent
• 1) inhibitor binds to active site
2) converted to reactive compound via enzyme's catalytic capabilities
3) covalently reacts with the enzyme
• Inactivation (covalent bond formation, k4) must occur prior to
dissociation (k3) otherwise the now reactive inhibitor is released into
the environment
Metabolically activated inactivator
• large partition ratio (k3/k4; favors dissocation) leading to nonspecific
reactions:
– MPTP is activated by an enzyme and then reacts elsewhere
• Neurotoxin, that causes Parkinsons disease related symptoms, is
activated from nontoxic agent via MAO-B
• Numerous carcinogenic compounds are initially nontoxic but are
activated by the cytochrome P450s
Structure-activity relationship and
inhibitor design
• Apart from physiochemical features, shape or topology of inhibitory
molecule is important
• Correlations of the structural changes with inhibitor potency are
referred as structure-activity relationship (SAR) studies
– SAR in the absence of structural info on target NZ
• Choose a lead compound: random screening of a compound library,
a known substrate or inhibitor
• Identify the pharmacophore (use as template)
– SAR that utilize structural info (rational or structure-based
inhibitor design)
• XRD and NMR structure of an enzyme with and without bound
inhibitor will provide structural details of enzyme-inhibitor interaction
Structure-activity relationship
An example: in the absence of structural info on target NZ
• Dihydrofolate reductase (DHFR) catalyse a key step in the biosynthesis of
deoxythymidine
• Its inhibition block the DNA synthesis
– In cancer therapy and as an antibiotic
•
Analogues of the substrate (dihydrofolate)
– Pteridines:
e.g. Methotrexate (human cancer)
- 5-R-2,4-Diaminopyrimidines:
e.g. Trimethoprim (bacterial infection)
A well-known example
Aspirin
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Hippocrates, use for childbirth
Reverend Edmund Stone, 1763 noted that
willow bark could be used as substitute for
Peruvian bark as a treatment for fever
– observation based on similar bitter taste
Salicin isolated in 1829
hydrolyzed to glucose and salicyclic alcohol  metabolized to salicylic acid
German chemist Felix Hoffmann for the Bayer Company: his father with
severe rheumatoid arthritis complained of irritation from salicylic acid: he
synthesized a number of analogs: acetyl analog was the best (1897)
Aspirin inhibits prostaglandin synthase (cylcooxygenase) via irreversible
inactivation by acetylating the enzyme