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.

2.

3.

4.

Always possible Not possible with 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 • 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

K app

• • • To obtain accurate data: At [S] = Km  check broad range of [I] Choose [I] that yield between 30 % to 75 % inhibition Measure v o 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 = k  2 [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 anti depressants: 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 no EI formation  V max [S] v = Km + [S]   1  [

I

K i ' ]   • Reciprocal plot

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):297 303 – 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 3 phosphoshikimate 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

K

m by 47 % – uncompetitive inhibition

Determination of K

i Secondary Plots 1. 1/V max vs [I]

– In the primary plot, each [I] gives a different V max – 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 V max

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

Dixon plots (1953)

Determination of K

i

• It's a bit restricted as it can't be used to calculate

Km

,

Vmax

, or even

Ki’

– Measure v o 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 non competitive 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 v 0 at fixed [S]

Dose-response curves

• • [I] required to achieve half maximal degree of inhibition: IC 50

v i

Fractional activity:

v

0  1  1 [

I

]

IC

50 • 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...

• IC 50 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

v ij

 1

v

0 1  [

I

] 

K i

[

J

] 

K j

[

I

][

J



K i 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

• • 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, -COCH 2 Br) 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 (k 3 /k 4 ; 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

• • • 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