Drug Discovery through Enzyme Inhibition and Inactivation

Learning outcomes

The student will be able to:

• List the types of enzyme inhibitors

• Describe the mechanism of enzyme catalysis and drug resistance

• Discuss and apply theory of drug synergism

• Discuss mechanism of action of reversible and irreversible enzyme inhibitors

• Describe rational design of enzyme inhibitors

Types of enzyme inhibitors

• Reversible inhibitors – involve reversible inhibition of enzyme via non-covalent interactions

• Irreversible inhibitor (Enzyme inactivator) - inhibits enzyme for an extended period of time via covalent interactions

Reversible Enzyme Inhibitors

1. Competitive inhibitors - compete with substrate for active site (structures similar to substrates or products)

a) Simple competitive inhibitors

b) Alternative substrate inhibitors

c) Transition state analogs

d) Slow, tight-binding inhibitors

2. Noncompetitive inhibitors - bind to a site other than active site (an allosteric site)

Mechanism of reversible inhibition

E + PE•PE•S

-S +S

E•I

koff

kon

E + I

• EI concentration depends on [I] and [S] and their dissociation constants (Ki and Km, respectively).

• Both S and I compete for E, (so a decrease in [I] or increase in [S], will decrease the [EI] and increases[ES]).

a) Simple Competitive Reversible Inhibitors

• E.g. antihypertensive drugs: ACE inhibitors - Captopril, enalapril, and lisinopril

• Hypertensive Effects of ACE

• Produces angiotensin II, a vasoconstrictor, also trigger release of a hormone, aldosterone that regulates the electrolyte balance by retention of Na+ and water.

• Angiotensin II is converted to angiotensin III, which also releases aldosterone.

• ACE also catalyzes hydrolysis of a vasodilator, bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg)

Hypertensive Effects of ACE

May be involved in memory retention & neuronal development

Lead Discovery

• 1965 - mixture of peptides in the venom of a South American pit viper shows inhibition of bradykininase activity and inhibits conversion of angiotensin I to angiotensin II Teprotide (Pyr-Trp-Pro-Arg-Pro –Gln-Ile-Pro-Pro)

– showed highest activity in vivo

Lead Modification

• Testing peptides inhibitors on ACE showed that:

– Pro is best at C-terminal, Ala at penultimate and an aromatic residue at antepenultimate position.

• ACE was found to contain Zn2+ that was believed to assist hydrolysis as a cofactor

ACE catalytic site was assumed to resemble carboxypeptidase A

• With (R)-2-benzylsuccinic acid as a model, a series of peptidomimetic, carboxyalkanoylproline derivatives (e.g. 1) were tested as inhibitors of ACE.

– All were only weak inhibitors of ACE.

• To increase potency, COO- was changed to SH that coordinate better to Zn(II).

Lead Modification

Hypothetical binding of inhibitors to ACE

Carboxylate analog

Thiol analog

Note: Active site of ACE has two additional binding site than carboxypeptidase

Effect of Structural Modifications

• Captopril had the best binding properties (Ki = 1.7 nM), It was also highly selective for ACE

• SAR studies showed that every part of captopril is important for binding.

(captopril)

Analog Relative Ki

1.0

12,500

10

12,000

120

120

1,100

CO2H

HS N

O

H3 C H

HH3 C

O

NHS

CO2H

HS N

O

HS

O

N

CO2H

CO2HHS

HN

O

CH3H

O

NHS

CO2H

CO2H

HOOC N

O

H3 C HCaptopril was the first ACE inhibitor on the drug market - hypertension and congestive heart failure.

• Captopril showed two reversible side effects (rashes and loss of taste) - Merck hypothesized side effects

from SH group.

A lipophilic group was added to reduce hydrophilicity and also to increase binding to S1 sub-site on ACE

Changed SH back to COO-

CH2 replaced by NH to increase interaction with enzyme,

– gave less potent molecule due to high hydrophilicity

enalaprilat: R = (S)-PhCH2CH2, R’ = H was the best candidate

enalaprilat was poorly absorbed orally - remedied by enalapril, an ethyl ester which is hydrolyzed in vivo by esterases to give enalaprilat

lisinopril5.29

CH2

CH2CO2H

H NH

N

H(CH2)4

CO2HO

NH2

Does not require prodrug activation.

b) Competitive Reversible Inhibitor that Acts as Substrate

• Examples are Sulfonamide Antibacterial Agents (Sulfa Drugs)

Lead discovery

• Bayer Co. tested azo dyes against streptococci. Prontosil was very effective in mice (1935).

• No activity in vitro unless a reducing agent is added.

• In vivo protonsil is metabolized by reduction to the active agent – it is a prodrug.

• the active agent is the bacteriostatic sulfanilamide

bacteriostatic - inhibits further growth of bacteria, but does not kill them.

Lead Modification

• Prontosil - beginning of modern chemotherapy

• Thousands of compounds synthesized and tested - the first SAR studies

• 1st example where new leads for other diseases discovered from side effects in clinical studies - antidiabetic and diuretic agents

Other developments from these studies:

• a simple assay for these compounds in body fluids

• showed antibacterial effect is proportional to the concentration in blood, which varied from patient to patient at a given dose - beginning of monitoring blood drug levels during chemotherapy

Mechanism of Action • In 1939 Stamp showed microorganisms contained a substance that

blocked the antibacterial action of sulfonamides

• In 1940 Woods hypothesized that this substance has structure similar to the enzyme substrate

• He also showed that it is p-aminobenzoic acid (PABA) and it compete with sulfanilamide for microbial growth

• Mid 1940s - Miller shows sulfanilamide inhibits folic acid biosynthesis

• Sulfonamides were shown to

be substrates for

Dihydropteroate synthase

• 1969 - Richey and Brown purified two enzymes involved in biosynthesis of dihydrofolate

(inhibiting formation of dihydrofolate needed for purines and DNA biosynthesis in bacteria)

Criteria for a good antimicrobial drug target

1. Target is essential to survival of microorganism

2. Target is unique to the microbe so humans are not harmed

3. Structure and function of the target is highly conserved across a variety of species of that microbe for broad spectrum

4. Resistance to inhibitors of target not easily acquired

• Dihydropteroate synthase satisfies the first 3 criteria – It generally takes 1-4 years for resistance to emerge;

resistance to sulfonamides took almost seven years.

Sulfonamides- Drug Resistance

• Overproduction of PABA

• Formation of a dihydropteroate synthase that binds PABA normally, but binds sulfonamides thousands of times less tightly

• Altered permeability of sulfonamides

c) Transition State Analogs

• Bernhard and Orgel – theorized that:

– inhibitor molecules resembling transition state structure are more tightly bound to enzyme than substrate

Pentostatin – an example of transition state analogs

2-deoxyinosine

2-deoxyadenosine

pentostatin5.58

HN

NN

N

HO H

OHO

OH

Antineoplastic agent isolated from bacterium Streptomyces antibioticus

A potent inhibitor of

adenosine deaminase Ki =

2.5 x 10-12 M (2.5

pM)(107 times lower than Km

for adenosine!)

pentostatin mimics this

Multisubstrate transition state analog

• When more than one substrate is involved, a single stable compound is made with a structure similar to the two or more substrates at the transition state - multisubstrate analog inhibitor.

‡

..

5.67

5.66

O

O

NH2

PO3=

NH2

COO-

H

COO-

COO-

H

COO-

NH2

O

PO3=

NH2

O

COO-

H

COO-

NH

NH2

O

NH

O

CH2

PO3=

COO-

H

COO-

5.65

N-Phosphonoacetyl-L-Asp (PALA) an example of Multisubstrate Analog

Aspartate transcarbamylase – catalyze de novo biosynthesis of pyrimidines from carbamoyl phosphate and L- Asp PALA resembles the transition state

carbamoyl phosphate

isostere - no longer a leaving group, mimics phosphate

N-carbamoyl-L-Asp

PALA

PALA was inffetctive due to tumor resistance: •tumor cells acquired ability to utilize preformed circulating pyrimidine nucleosides • increased carbamoyl phosphate • increased aspartate transcarbamylase

Slow, Tight-Binding Inhibitors

• Slow-binding inhibitors - equilibrium between enzyme and inhibitor is reached slowly (kon small); inhibition is therefore time-dependent

• Tight-binding inhibitors - substantial inhibition when [E] and [I] are comparable (koff small)

• Slow, tight-binding inhibitors – have both properties t1/2 for EI complex can be up to months

Slow, Tight-Binding Inhibitors – Examples • Examples of Slow, Tight-Binding Inhibitors the Antihypercholesterolemic

drugs Lovastatin and Simvastatin

• About 1/2 of body cholesterol is biosynthesized; the rest needs to be eaten. Some people biosynthesize much more than half of what they need.

• Rate-determining step in cholesterol biosynthesis is conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to mevalonic acid.

• Inhibition of HMG-CoA reductase should lower plasma cholesterol levels.

5.70

HMG-CoA reductase

2 NADP+2 NADPH

5.695.68

NADP+

NADPH-CoASH

NADP+

NADPH

O

H3 C

COOHHO

SCoA

OH

HO COOH

H3 C

O

H3 C

COOHHO

SCoA H

HO COOH

H3 C

OH

H:B

HMG-CoA

Mevalonic acid

O

CH2 C

CH3

CH3

CH

OH

C

O

NHCH2CH2 CNHCH2CH2 S

O

PO O-

O

PO O CH2

O- O

=O3PO OH

N

N

N

N

NH2

O

COOHHO

H3 C

CoA

HMG-CoA

mevastatin or compactin (R = H)

monacolin K, mevinolin, or lovastatin (R = CH3)5.71

CH3

O

O

H3C H

R

H

HO O

O

Lead Discovery • Endo and coworkers

(Sankyo Co. in Japan) found three compounds from fungus Penicillium citrinum that inhibited sterol biosynthesis – mevastatin was most potent.

• A more potent compound was isolated by Endo from a different fungus - called monacolin K.

• Mechanism of Action: Potent competitive reversible inhibitors of HMG-CoA reductase (Ki for lovastatin 6.4 x 10-10 M)

• The active form of lovastatin is the hydrolysis product.

• This mimics intermediate 5.70 in Scheme 5.10.

Active form of lovastatin

when H is replaced by CH3, it is simvastatin (2.5 times more potent than lovastatin)

5.70

O

H3C

COOHHO

SCoA

H

H:B

5.73

3

5

CH3

OH

H

COOH

O

H3C H

CH3

H

HOH

O

Irreversible Enzyme Inhibitors

• Two general types of irreversible inhibitors:

– Affinity labeling agents (reactive compounds)

– Mechanism-based enzyme inactivators (unreactive compounds)

Note: Irreversible inhibition does not mean permanent loss of the enzyme - gene encodes for other copies (may take hours or days).

Affinity Labeling Agents

• These are reactive compounds that have structure similar to that of substrate of the target enzyme

covalent complex

SN2 alkylation or acylation

reversible complex

Effective Affinity Labeling Agents have:

• low Ki for target enzyme for selectivity

• reactive group close to a nucleophile in active site of target enzyme

-lactam antibiotics – Examples affinity labeling agents

-lactam antibiotics inactivate Peptidoglycan transpeptidase which catalyze the cross-linking of peptidoglycan to form bacteria cell wall

OOHO

OH

NHAc

OO

OH

O

NHAcO

HH3 C

O

O

HO

OH

NHAc

O

GlcNAc MurNAc GlcNAc

-

L-Ala

D--Glu

Lys-NH2

NH

CHCH3

NH

CHCH3

COO

L

(D)

(D)

+

O

BH

H

B:

peptidoglycan transpeptidase

D-Ala

C O

GlcNAc MurNAc GlcNAc

OH

H3 CO

L-Ala

D--Glu

Lys-NH2

NH

CHCH3

O

L

(D)

C O

Ser

GlcNAc MurNAc GlcNAc

O

O

H

CH3

D-Ala

D-Ala

Lys

D--Glu

L-Ala

L NH2

GlcNAc MurNAc GlcNAc

OH

H3 CO

L-Ala

D--Glu

Lys-NH2

NH

CHCH3

L

(D)

C O

GlcNAc MurNAc GlcNAc

O

O

H

CH3

D-Ala

D-Ala

Lys

D--Glu

L-Ala

L NH

5.10

Penicillin inactivate the enzyme by acylating serine at active site

H

N b

N b

MeN a

Me

H

H

CO2H

O

Peptidoglycan

SN a

H H

CO2H

Me

Me

O

R

O

O

H

H

H

The structure of penicillin is comparable with D-Ala-D-Ala which is hydrolyzed by the enzyme

Penicillin Resistance: Occurs via 1. -Lactamase production - hydrolyzes -lactam ring 2. Also, transpeptidase becomes less susceptible to acylation. 3. Membrane permeability is modified.

Drug Synergism: combination therapy (penicillin + -lactamase inhibitor) e.g. • Augmentin (amoxicillin + clavulanate) • Unasyn (ampicillin + sulbactam)

clavulanate potassium5.92

N

O

O

OH

CO2-K+

sulbactam5.93

N

S

O

OO

CO2H

Mechanism-Based Enzyme Inactivators

• is an unreactive molecule with structure

similar to substrate or product

• once bound the target enzyme converts it

into a species that inactivates the enzyme

Their differences from affinity labeling agents are

• initially unreactive

• target enzyme activates them by catalysis

Mechanism-Based Inactivation Kinetics

E + I E • I E • I '

E + P

E - I ' '

k1

k-1

k2 k4

k3

partition ratio = k3/k4

ideally, partition ratio = 0

• Note: Inactivation occurs prior to release of the activated species

Vigabatrin – Examples of Mechanism-Based Inactivation

• Vigabatrin is an antiepileptic drug • Epilepsy - Can arise from imbalance in two neurotransmitters (L-Glu

and GABA)

• Thus an alternative to treat epilepsy could be introduce GABA into brain

• However, GABA does not cross blood-brain barrier

• Vigabatrin crosses blood-brain barrier, and inactivates GABA aminotransferase via mechanism-based inactivation.

- -

--+

-+GADPLP-CO2

GABA-AT

PLP PMP

-KGL-GluSSADH

NADP+

NADPH

H3N CO2

CO2

H3N CO2OHC

CO2

O2 CCO2

-

• Vigabatrin can cross the BBB because vinyl group: – increases lipophilicity of the compound – is electron withdrawing it will lower pKa of

the amino group thus increasing the nonzwitterionic form

Mechanism of action of Vigabatrin

1

2

3

4 5