Enzyme Inhibition and Inactivation

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Enzyme Inhibition and Inactivation

• Enzyme Inhibitor
• compound that slows or blocks enzyme catalysis
• 33 of the 97 drugs approved worldwide
  1998-2000 are enzyme inhibitors
• Why inhibit an enzyme?
• Enzyme substrate beneficial (essential), but
  depleted
• low levels of GABA lead to seizurestherefore
  inhibit GABA aminotransferase to prevent
  degradation of GABA
• Enzyme product harmful
• excess uric acid leads to goutinhibition of
  xanthine oxidase prevents conversion of xanthine
  to uric acid

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Chemotherapy

• The use of drugs to combat foreign organisms or
  aberrant cells.
• Enzyme inhibition is a promising approach for the
  rational discovery of new leads or drugs.
• Ideal enzyme inhibitors should be highly
  (totally) specific for target enzyme.
• For infectious diseases, enzyme inhibitors should
  target enzymes unique to the pathogen, or at
  least not essential to the host.

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Drug Resistance

• When a formerly effective drug dose is no longer
  effective.
• Resistance can be natural or acquired, and arises
  by natural selectionreplication of a resistant
  strain after drug has killed susceptible strains.
• Mutations
• Gene transfer
• Gene amplification

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Mechanisms of Drug Resistance

• Altered drug uptake
• Overproduction of target enzyme
• Altered target enzyme (site of action) structure
• Production of drug-destroying enzyme
• Deletion of prodrug activating enzyme
• Overproduction of target enzyme stubstrate
• New pathway for product formation
• Efflux pumps

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Altered target enzyme

• Minimize by designing a drug has very similar
  structure to natural substrate

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Altered site of action vancomycin
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Altered site of action vancomycin
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Antibiotics and Resistance

• Bacteria usually classified by the Gram stain
  test
• Gram-positive
• Thick cell wall (25 nm) up to 20 layers of
  peptidoglycan
• Gram-negative
• Thin cell wall (2-3 nm) outer lipid bilayer
  attached to peptidoglycan via peptide links
  across periplasmic space.
• Imprudent and indiscriminant use of antibiotics
  over the years has led to increasingly resistant
  bacteria several strains of bacteria are now
  resistant even to the antibiotics of last resort
  (e.g. vancomycin)
• Current research focus is on Gram-positive
  bacteria

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New Antibiotics

• Linezolid (Zyvox) Pharmacia
• Approved 4/2000
• First member of oxazolidinone class of
  antibiotics
• Blocks protein synthesis by peventing interaction
  of 30S ribosome, mRNA, and formyl methionine

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New Antibiotics

• Gemifloxicin (Factive) SmithKline Beecham
• NDA filed 2000
• Second-generation fluoroquinolone class of
  antibiotics
• Inhibits topoisomerase II

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New Antibacterial Targets

• Sortase
• Enzyme that attaches surface proteins in
  Gram-positive bacteria
• Inhibition of sortase prevents surface attachment
  of proteins, and bacteria cannot stick to other
  cells
• Deformylase
• Metalloenzyme that cleaves formyl group from
  N-terminus of bacterial proteinsnecessary for
  functional proteins
• Actinonin is naturally occurring formylase
  inhibitor
• Efflux pump inhibitors

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Drug Synergism

• Cases where therapeutic effect of two or more
  drugs used in combination is greater than the sum
  of the effects of the drugs administered
  individually
• Mechanisms of Synergism
• inhibition of drug-destroying enzyme
• sequential blocking
• inhibition of enzymes in different metabolic
  pathways
• efflux pump inhibitors
• multiple drugs for same target

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Reversible Enzyme Inhibitors

• Ki koff / kon therefore smaller Ki better
  inhibitor
• competitive inhibitor
• inhibitor binds at active site blocks substrate
  binding
• inhibitor may be metabolized
• easier to design
• non-competitive inhibitor
• binds at different (allosteric) site
• changes enzyme conformation to prevent binding or
  turnover
• difficult to design

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ACE Inhibitors

• Angiotensin converting enzyme (ACE) converts
  Angiotensin I to Angiotensin II
• Angiotensin II
• Vasoconstrictor
• Stimulates release of aldosterone
• Inactivates Bradykinin
• Triple play that increases blood pressure
• Therefore an ACE inhibitor should lower blood
  pressure
• Leads peptides from pit viper venom

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Renin-angiotensin system
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ACE Inhibitor Development Issues

• ACE is membrane bound and heavily glycosylated,
  making isolation and structural characterization
  difficult
• ACE is inhibited by EDTA, suggesting
  metalloenzyme (it has a Zn2 cofactor)
• ACE is similar to carboxypeptidase A, whose X-ray
  structure had been determined
• Knowledge of carboxypeptidase A applied to ACE
  problem
• Primary structure of ACE not determined until 1988

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ACE Inhibitors

• Captopril 1st ACE inhibitor marketed
• Side effects included rash and loss of taste
• Enalipril is a prodrug
• Ester group is hydrolyzed in vivo to carboxylate
• Enaliprilat is a slow-tight binding inhibitor and
  a transition state analog

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ACE Inhibitors

• Lisinopril is more slowly and less completely
  absorbed than enalipril, but has longer action of
  duration
• Cilazapril is conformationally restricted
• Cilazapril design based on 3D map of ACE active
  site from studies of the thiol inhibitors of ACE

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Antihypercholesterolemics

• Heart disease is leading cause of death in the
  U.S.
• One-half of all deaths attributable to
  atherosclerosis the deposition of plaque on
  artery walls
• Plaque is mostly cholesterol
• One-half of total cholesterol is synthesized in
  the liver
• Synthesis is gt20 enzymatic steps from acetyl-CoA
• Rate-limiting step is conversion of HMG-CoA to
  mevalonic acid, catalyzed by HMG-CoA reductase
• Inhibition of HMG-CoA reductase will lower
  cholesterol levels

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Early HMG-CoA Reductase Inhibitors

• Isolated from fungal cultures
• affinity for compactin 7,140 times that of
  natural substrate
• affinity for lovastatin 16,700 times that of
  natural substrate
• binding motifs hydroxyglutaryl and large
  hydrophobic groups

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HMG-CoA Reductase Inhibitors

• Simvastatin 2.5 times more potent than lovastatin
• Atorvastatin 1997 sales gt 1 billion 2000 sales
  5 billion

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Irreversible Enzyme Inhibitors

• Drug molecule (or a portion thereof) becomes
  irreversibly (covalently) bound to enzyme
• Affinity labeling agent
• Reactive compound similar to natural enzyme
  substrate
• Reacts with nucleophile in active site
  acylation, alkylation
• Enzyme selectivity
• Binding specificity

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b-lactam Antibiotics

• Inactivate peptidoglycan transpeptidase, a
  bacterial enzyme critical for cell wall synthesis
• Bacteria often resistant to b-lactam drugs due to
  production or overexpression of b-lactamase to
  destroy the drug

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b-lactamase Inhibitors

• Given in combination with b-lactam antibioticsan
  example of drug synergism
• Augmentin amoxicillin clavulanate
• Unasyn ampicillin sulbactam

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Pain and NSAIDs

• See Williams, M et al Emerging Molecular
  Approaches to Pain Therapy J. Med. Chem. 1999,
  42, 1481-1500.
• Pain Categories
• Acute
• Chronic
• Cancer pain
• Arthropathic pain
• AIDS pain
• Visceral
• Neuropathic
• NSAID Non-Steroidal Anti-Inflammatory Drug

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Traditional NSAIDs
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The Role of Cyclooxygenase

• Cyclooxygenase (COX) catalyzes the conversion of
  arachidonic acid to prostaglandin H2 (PGH2)
• Other enzymes convert PGH2 to a variety of
  prostaglandins

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Prostaglandin biosynthesis
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COX-1 vs. COX-2 Inhibition

• COX-1 is constitutive and necessary for
• Protecting GI tract mucosa, proper kidney
  function, platelet aggregation
• COX-2 (discovered in 1990) is inducible under
  inflammatory conditions and is responsible for
• Fever, pain, swelling
• COX-1 and COX-2 are 60 homologous
• Most traditional NSAIDs inhibit both COX-1 and
  COX-2 and thus have serious side effects
• GI bleeding
• Inhibited platelet aggregation
• Renal toxicity
• Liver toxicity

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Tricyclic COX-2 Inhibitors
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Methansulfonanilide COX-2 Inhibitors