Chapter IV Enzyme Inhibitor and Inhibitor Classification

8/10/2019 Chapter IV Enzyme Inhibitor and Inhibitor Classification

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Enzymes catalyze the chemical reactionsnecessary for life by factors of 10 10 10 15 . Thefirst step to initiate catalysis is binding thereactant molecules into the catalytic sites to formthe Michaelis complex.

In the second step , a conformational changeoccurs to enclose the reactants tightly in theenzyme and subsequently form products. The

enzyme then relaxes to open the catalytic siteand release roducts.


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Enzyme inhibition

Reaction velocity, V=k 3 [E-S] Rate of formation ES: k 1[E][S]Rate of decomposition ES: (k 1 + k 3)[E][S]

Assume steady state ([E- S] doesnt change)

k1[E][S] = (k 1 + k 3)[E][S]

[E-S] =[E] [S]

(k2 + k

3) / k

1

Michaelis constant: K M = (k 2 + k 3) / k 1

[E-S] =[E] [S]

KM

[E] = [E tot ] - [E-S]

[E-S] =([Etot ] - [E-S] [S]

KM

[E-S] =[S] + K M

[E tot ] [S]

E + S [ E- S ] [ E- P ] E + P

k3 E: EnzymeS: SubstrateP: Product

k1

k2


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[E-S] =

[S] + K M

[E tot ] [S]V=k

3 [E-S]

E + S [ E- S ] [ E- P ] E + P

k3 E: EnzymeS: SubstrateP: Product

k1

k2

V =[S] + KM

k3[Etot] [S]Vmax : All enzyme sites occupied by S

[S]>>K M, [S] + K M [S]

1Vmax =k 3 [E tot ]

V =

[S] + KM

Vmax [S]Michaelis Menten eq. V

[S]

Vmax

Vmax : 2

K M


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Enzymes frequently require coenzymes for optimumactivity.The coenzymes are vitamins and cofactors,such as mono and divalent metallic ions. Thesecoenzymes activate different enzymes bycomplexation and stereochemical interactions.

The ions may affect enzymes by:Direct interactions : induces changes in the

conformation or a charge on the enzyme or interactionof the cation with an enzyme-inhibiting substanceIndirect interactions : prevents or minimizes thedeactivation


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Cytochromes have heme iron

component wherein a Fe 2+ ion iscoordinated in a square planarstructure with four pyrrole ringnitrogen atoms.Peroxidases such as glutathioneperoxidases have selenocysteineamino acid presents in the enzymesPeptide-hydrolyzing enzymes suchas carboxypeptidase need Zn 2+ for

proper functioning.

Imidazole or pyrrole rings

-SH groups [cysteine]-COOH groups


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The active site of polyphenol oxidase shows that it uses sixamino acids to chelate two copper ions. These arehistidine which has a polar cyclic R-group with nitrogen

atoms with unshared electrons.


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10/27Catalytic site of thermolysin secreted by thebacterium Bacillus thermoproteolyticus [PDB ID 3DNZ ]
http://www.rcsb.org/pdb/explore/explore.do?structureId=3DNZhttp://www.rcsb.org/pdb/explore/explore.do?structureId=3DNZ


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Enzyme inhibitors prevent enzymes from their catalyticfunction by interfering with any step in this catalytic cycle.

Common types of enzyme inhibitors are:1. Reversible Inhibitors:

Competitive inhibitors are catalytic site inhibitors thatcompete with the substrate for formation of the Michaelis

complex;Noncompetitive inhibitors are inhibitors that alterformation of the Michaelis complex and full expression ofcatalytic potential;

2. Irreversible or covalent inhibitors that form a Michaeliscomplex followed by a chemical reaction with the enzymeto form a stable and inactive complex, often calledmechanism-based inhibitors or suicide inhibitors


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Drugs acting by a mechanism that involves enzyme inhibition

may involve definite groups on the protein portion, whichneed not necessarily be the site where normal substrateinteractions occur.

The reaction may be a weak nonspecific phenomenon ,

which may involve only a limited modification of the proteinconformation: e.g hydrogen bonding.

Enzyme inhibitors are chemical agents capable of modifyingan enzymes capacity to catalyze the reactions of its

normal substrate.

Inhibiting enzymes by chemical means offers manytherapeutic possibilities: reducing or increasing theproduction of a particular metabolite.


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Control of parasitic invasions has been successfullyachieved with compounds that inhibit key enzymesvital to the biosynthesis of nucleic acids in thispathogen microorganisms. This results in theirreproductive suppression or death.

An alternative mechanism: the inhibitor has a closechemical similarity to the enzymes normal substrate(metabolite). Such drugs are called antimetabolites , e.gantibacterial and anticancer agents.

The rationale for utilizing organic compounds structurally

similar to normal cellular metabolites as anticanceragents is: to interfere with the biosynthesis of thesesubstances within the cell and thus inhibit cellproliferation . The precursors that cells require fornormal growth are: pyrimidine and purine bases .


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THF is necessary for the biosynthesis ofpurine and thymine, which are theprecursors of nucleic acids.

Antimetabolite aminopterin, which has folatestructure, produces sustained remissionsin leukemia ( K i = 6x10 -10 ).

Methotrexate inhibits ( K i = 6x10 -10 ) theenzyme much more strongly than the

natural substrate.


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Certain factors must be considered in designing

drug molecules to function as antimetabolites.

Structural resemblance, in terms of both

dimensions and electronic factors , is usuallyessentials. Similarity has sometimes achievedby functional group subtitution, or even a singleatom. Similarity in electronegativity or

comparability of van der Waals radii, aresometimes important.


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Reversible inhibition may be subclassified as to itscompetitive and noncompetitivecharacteristics.

Competitive inhibition occurs when the inhibitorcompetes with the natural substrate at theenzymes active site [e.g accidental poisoningby ethylene glycol, methanol]Noncompetitive inhibition occurs when theinhibitor binds at other than the catalytic sitesof the enzyme and therefore does not directly

compete with the substrate at all.


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(a) Succinate binds to the enzyme succinate dehydrogenase. Adehydrogenation reaction occurs, and the product fumarate is released.

(b) Malonate also binds to the active site of succinate dehydrogenase. In thiscase, however, no subsequent reaction occurs.

Succinate dehydrogenase

Succinate dehydrogenase


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Irreversible inhibition is affected bycovalent bond formation with oneor more functional groups, primarilyat the active site of the enzyme.

Reactions very near the active site, asadditional anchoring points, arealso known to occur. Suchinhibition results in a catalyticallyinactive enzyme that is unable tointeract with its substrate.

Irreversible inhibitors are classifiedinto:1. Active-Site-Directed Inhibitors2. Mechanism-Based Inhibitors

(Sendovski, M., Kanteev,M., Shuster Ben-Yosef, V., Adir,N., Fishman, A., 2011,J.Mol.Biol. Volume 405, Issue 1 :227-237
http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=http://www.sciencedirect.com/science?_ob=PublicationURL&_tockey=


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B.R Baker (1960) postulated that it may be possible toinhibit reactive sites of enzymes irreversibly byincorporating alkylating or acylating moieties intomolecules closely resembling the enzymes substrate.

Effects of TPP + on oxidation of BZ byBPAO. BZ (200 M -48 mM) wasincubated with BPAO in the absence(controls; ) or presence of TPP + atconcentrations of 150 nM ( ), 300 nM( ), 700 nM ( ), 1.5 M ( ), 3 M ( ),and 10 M (). Initial velocities weremeasured by a peroxidase-coupledabsorbance assay.doi:10.1124/mol.107.040964

Molecular Pharmacology , 2008 vol. 73 no.

2: 525-538


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This type of irreversible inhibitor is often called suicidesubstrate . Essentially, it is designed to be an analogof a normal substrate, which once bound to theenzymes active site because of its structural analogy,is then modified to produce a highly active electrophilicgroup.

Mechanism-based inactivation is a unique type ofenzyme inhibition. By definition, a mechanism-basedinactivator itself does not inactivate the enzyme , butit is metabolized by the enzyme to a reactiveelectrophilic intermediate which, without prior releasefrom the active site, binds (most often covalently) tonucleophilic activesite amino acids resulting in acomplete or partial loss of activity ( Silverman, 1988 )

Th l i
http://dmd.aspetjournals.org/content/32/8/805.fullhttp://dmd.aspetjournals.org/content/32/8/805.full


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There are several importantclinical implications when adrug is a mechanism-based inactivator (suicidesubstrate) of P450:

The drug concentrationafter multiple dosing willbe much higher thanwhat is expected from

linear kinetics. Elevated drug

concentrations maylead to side effects andtoxicity.

(Okuda et al., 1997).Tacrine, a cholinesterase inhibitor, was approved for the treatment of Alzheimersdisease. Oxidative metabolism of tacrine occurs by CYP1A-catalyzedhydroxylation. In rats, it was observed that the AUC of the second oral dose wasconsistently higher than the AUC of the first oral dose, which was not due to the

accumulation of the drug in the plasma from the first dose. This finding suggestedinhibition of the enzyme during metabolism or inhibition by a metabolite.


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Proposed scheme for tacrine to form reactive intermediates that lead toenzyme inactivation

(doi: 10.1124/dmd.32.8.805)


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Unlike the active-site-directed inhibitors, the mechanism-based inhibitors have intrinsically unreactivefunctionalities that can not be activated by other

enzymes. This limitation of reactivity affords specificity and should result in low toxicity.

Designing such drug is not a simple task. The prerequisites

are:1. An understanding of target enzyme mechanism2. A Michael-type addition reaction [a conjugate addition

of a carbanion, a Michael donor, acting as nucleophile,

to the -carbon of an , unsaturated system].

To function successfully, this inhibitor must be able to bindto the enzymes reactive site with high affinity

Penicillins are cleaved by lactamase


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HN

SH

O

NH

R

O

OHN

SH

O

NH

R

O

O OH

-Lactamase

Penicillins are cleaved by -lactamase

N

OH

O

O OH

OH

Clavulanic acid irreversibly inhibits -lactamase

OH

HN

OH

O

O OH

OHHH

H HN

O

O

O OH

OH

O

Nu

Nu

O

Nu

H

HN

O

O

O OH

OH

O

HHNu


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Suicide substrates:VigabatrinNormal substrate for aminotransferase:

H 2 N CO 2

H R

Suicide substrate for aminotransferase:H 2N

H CO 2

ONE new electrophilic center

N

H

OH

NH

OP

O

HO

OH

H

B:

N

H

OH

NH

OP

O

HO

OH

BH

N

H

OH

NH

OP

O

HO

OH

BH

CO 2 CO 2 CO 2

N

H

OH

N

RO 2 C

H

OP

O

HO

OH

H

B:

N

H

OH

N

RO 2 C

H

OP

O

HO

OH

BH

N

H

OH

N

RO 2 C

H

OP

O

HO

OH

BH

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TWO new electrophilic centers!


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Reactivity of cationic intermediates: N+ is a good electron sink, making themolecule susceptible to nucleophilic attack. The nucleophile may be a groupon the enzyme, or another molecule

N

H

OH

N

RO 2C

H

OP

O

HO

OH

N

H

OH

NH

OPO

HO

OH

CO 2

NH N

H

Nu:Nu: Michael addition


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N

H

OH

N

RO 2 C

H

OP

O

HO

OH

H2

O

N

H

OH

NH 2

OP

O

HO

OH O

O 2 C R+

NH

OH

N

CO 2

H

OPO

HOOH

NH

OH

NH 2

OPO

HOOH

O

+

CO 2

H 2 O

Enz-Nu:

N

OH

N

CO 2

H

OPO

HO

OH

Enz-Nu:

Inactivateenzyme!

Active sitenucleophile i nreach of this electrophile

Normal substrate: final products. Enzyme unchanged and active

Suicide substrate: two pathways for products, one which inactivates theenzyme!

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Chapter IV Enzyme Inhibitor and Inhibitor Classification