Nucleotide metabolism Part 1 purine biosynthesis

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Nucleotide metabolism Part 1(purine
biosynthesis)

• By
• Henry Wormser, Ph.D
• Professor of Medicinal Chemistry

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Biological significance of nucleotide metabolism

• Nucleotides make up nucleic acids (DNA and RNA)
• Nucleotide triphosphates are the energy
  carriers in cells (primarily ATP)
• Many metabolic pathways are regulated by the
  level of the individual nucleotides
• Example cAMP regulation of glucose release
• Adenine nucleotides are components of many of the
  coenzymes
• Examples NAD, NADP, FAD, FMN, coenzyme A

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Dietary nucleotides

• do not contribute energy as do carbs, proteins
  and fats
• are not incorporated into RNA or DNA unless given
  I.V.
• normally metabolized to individual components
  (bases, sugar and phosphate)
• purines are converted to uric acid which is then
  excreted

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Medical significance of nucleotide metabolism

• Anticancer agents
• Rapidly dividing cells biosynthesize lots of
  purines and pyrimidines, but other cells reuse
  them. Cancer cells are rapidly dividing, so
  inhibitor of nucleotide metabolism kill them
• Antiviral agents
• Zidovudine (Retrovir)
• Lamivudine (Epivir)
• Valacyclovir (Valtrex)

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Structures of nucleotide building blocks and
nucleotides
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Structures of nucleotide building blocks and
nucleotides
guanine comes from guano thymine thymus gland
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Ribonucleotide phosphate ribonucleoside
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Biosynthesis of the purine nucleotide system
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Synthesis of Inosine Monophosphate

• Basic pathway for biosynthesis of purine
  ribonucleotides
• Starts from ribose-5-phosphate which is derived
  from the pentose phosphate pathway
• Requires 11 steps overall
• occurs primarily in the liver

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The big picture
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Steps 1 thru 3

• Step 1Activation of ribose-5-phosphate
• enzyme ribose phosphate pyrophosphokinase
• product 5-phosphoribosyl-a-pyrophosphate (PRPP)
• PRPP is also a precursor in the biosynthesis of
  pyrimidine nucleotides and the amino acids
  histidine and tryptophan

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Step 1 purine synthesis
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Steps 1 thru 3

• Step 2 acquisition of purine atom 9
• enzyme amidophosphoribosyl transferase
• displacement of pyrophosphate group by glutamine
  amide nitrogen (inversion of configuration a to
  b
• product b-5-phosphoribosylamine

Steps 1 and 2 are tightly regulated by feedback
inhibition
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Step 2 purine synthesiscommited step
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Steps 1 thru 3

• Step 3 acquisition of purine atoms C4, C5, and
  N7
• enzyme glycinamide synthetase
• b-phosphoribosylamine reacts with ATP and glycine
• product glycinamide ribotide (GAR)

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Step 3 purine synthesis
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Steps 4 thru 6

• Step 4 acquisition of purine atom C8
• formylation of free a-amino group of GAR
• enzyme GAR transformylase
• co-factor of enzyme is N10-formyl THF
• Step 5 acquisition of purine atom N3
• The amide amino group of a second glutamine is
  transferred to form formylglycinamidine ribotide
  (FGAM)
• Step 6 closing of the imidazole ring or
  formation of 5-aminoimidazole ribotide

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Step 6 purine synthesis
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Step 7

• Step 7 acquisition of C6
• C6 is introduced as HCO3-
• enzyme AIR carboxylase (aminoimidazole ribotide
  carboxylase)
• product CAIR (carboxyaminoimidazole ribotide)
• enzyme composed of 2 proteins PurE and PurK
  (synergistic proteins)

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Step 7 purine synthesis
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Steps 8 thru 11

• Step 8 acquisition of N1
• N1 is acquired from aspartate in an amide
  condensation reaction
• enzyme SAICAR synthetase
• product 5-aminoimidazole-4-(N-succinylocarboxamid
  e)ribotide (SAICAR)
• reaction is driven by hydrolysis of ATP

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Step 8 purine synthesis
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Steps 8 thru 11

• Step 9 elimination of fumarate
• Enzyme adenylosuccinate lyase
• Product 5-aminoimidazole-4-carboxamide ribotide
  (AICAR)
• Step 10 acquisition of C2
• Another formylation reaction catalyzed by AICAR
  transformylase
• Product 5-formaminoimidazole-4-carboxamide
  ribotide (FAICAR)

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Step 9 purine synthesis
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Step 10 purine synthesis
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Step 11

• cyclization or ring closure to form IMP
• water is eliminated
• in contrast to step 6 (closure of the imidazole
  ring), this reaction does not require ATP
  hydrolysis
• once formed, IMP is rapidly converted to AMP and
  GMP (it does not accumulate in cells

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Step 11 purine synthesis
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Synthesis of adenine and guanine nucleotides
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Purine nucleoside diphosphates and
triphosphates - to be incorporated into DNA
and RNA, nucleoside monophosphates (NMPs)
must be converted into nucleoside
triphosphates (NTPs) - nucleoside
monophosphate kinases (adenylate guanylate
kinases) - nucleoside diphosphate kinase
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Regulation of purine nucleotide biosynthesis
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The purine salvage pathway

• Purine bases created by degradation of RNA or DNA
  and intermediate of purine synthesis were costly
  for the cell to make, so there are pathways to
  recover these bases in the form of nucleotides
• Two phosphoribosyl transferases are involved
• APRT (adenine phosphoribosyl transferase) for
  adenine
• HGPRT (hypoxanthine guanine phosphoribosyl
  transferase) for guanine or hypoxanthine

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Salvage of purines
Adenine phosphoribosyltransferase (APRT)
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Salvage of purines

• Salvage is needed to maintain the purine pool
  (biosynthesis is not completely adequate,
  especially in neural tissue)
• Hypoxanthine-guanine phosphoribosyltransferase
  (HGPRT)
• Hypoxanthine PRPP IMP Ppi
• Guanine PRPP GMP Ppi
• Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack
  of enzyme leads to overproduction of purines
  which are metabolized to uric acid, which damages
  cells

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Lesch-Nyhan syndrome

• there is a defect or lack in the HGPRT enzyme
• the rate of purine synthesis is increased about
  200X
• uric acid level rises and there is gout
• in addition there are mental aberrations
• patients will self-mutilate by biting lips and
  fingers off

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Lesch-Nyhan syndrome
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Salvage of purine bases
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Next Part 2 - biosynthesis of pyrimidine
nucleotides
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Nucleotide metabolism Part 2(pyrimidine
biosynthesis)

• By
• Henry Wormser, Ph.D
• Professor of Medicinal Chemistry

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Synthesis of pyrimidine ribonucleotides

• shorter pathway than for purines
• base is made first, then attached to ribose-P
  (unlike purine biosynthesis)
• only 2 precursors (aspartate and glutamine, plus
  HCO3-) contribute to the 6-membered ring
• requires 6 steps (instead of 11 for purine)
• the product is UMP (uridine monophosphate)

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Origin of atoms in pyrimidine ring
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The big picture
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Step 1 synthesis of carbamoyl phosphate

• Condensation of glutamine, bicarbonate in the
  presence of ATP
• Carbamoyl phosphate synthetase exists in 2 types
  CPS-I which is a mitochondrial enzyme and is
  dedicated to the urea cycle and arginine
  biosynthesis) and CPS-II, a cytosolic enzyme used
  here

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Step 1 pyrimidine synthesis
CPS-II is the major site of regulation in
animals UDP and UTP inhibit the enzyme and ATP
and PRPP activate it It is the committed step in
animals
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Step 2 synthesis of carbamoyl aspartate

• enzyme is aspartate transcarbamoylase (ATCase)
• catalyzes the condensation of carbamoyl phosphate
  with aspartate with the release of Pi
• ATCase is the major site of regulation in
  bacteria it is activated by ATP and inhibited by
  CTP
• carbamoyl phosphate is an activated compound,
  so no energy input is needed at this step

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Step 2 pyrimidine synthesis
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Step 3 ring closure to form dihydroorotate

• enzyme dihydroorotase
• forms a pyrimidine from carbamoyl aspartate
• water is released in this process

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Step 3 pyrimidine synthesis
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• the first 3 enzymatic reactions are catalyzed by
  3 separate proteins/enzymes in E. coli
• in animals, all 3 steps are found in a
  multifunctional enzyme (210 kD). This allows
  channeling of the substrates and products
  between active sites without releasing them to
  the medium where they could be degraded.
• The acronym CAD is used as a name for the
  multienzyme carbamoyl phosphate synthetase,
  aspartate transcarbamoylase and dihydroorotase
• channeling also increases the overall rate of
  multistep processes

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Step 4 oxidation of dihydroorotate to orotate

• an irreversible reaction
• enzyme dihydroorotate dehydrogenase
• oxidizing power is derived from quinones (thru
  coenzyme Q)

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Step 4 pyrimidine synthesis
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Step 5 acquisition of ribose phosphate moiety

• enzyme orotate phosphoribosyl transferase
• ribose phosphate originates from PRPP
• product is orotidine-5-monophosphate (OMP)
• orotate phosphoribosyl transferase is also used
  in salvage of uracil and cytosine to their
  corresponding nucleotide

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Step 5 pyrimidine synthesis
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Step 6 decarboxylation of OMP

• enzyme OMP decarboxylase
• product uridine monophosphate (UMP)
• in animals, steps 5 and 6 are catalyzed by a
  single polypeptide with 2 active sites

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Step 6 pyrimidine synthesis
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The big picture again
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Orotic aciduria

• an inherited human disease caused by a deficiency
  in the multifunctional enzyme that catalyzes the
  last 2 steps in the pyrimidine synthesis
• large amounts of orotic acid in urine
• retarded growth and severe anemia
• treat by administration (injection) of uridine
  and/or cytidine

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Leflunomide (Arava)

• Leflunomide is an isoxazole immunomodulatory
  agent which inhibits dihydroorotate
  dehydrogenase) and has antiproliferative
  activity. Several in vivo and in vitro
  experimental models have demonstrated an
  anti-inflammatory effect.
• It is currently used as a DMARD in patients with
  serious rheumatoid arthritis

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Leflunomide (Arava)
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Activation of leflunomide
Opening of the isoxazole yields a reactive
compound which can then inhibit the enzyme
dihydroorotate dehydrogenase
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Synthesis of uridine and cytidine triphosphate
(in bacteria, ammonia donates the amino group)
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Regulation of pyrimidine nucleotide biosynthesis
UTP and CTP are feeback inhibitors of CPS II
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Formation of deoxyribonucleotides
All pathways shown previously led to synthesis of
ribonucleotides
dADP, dGDP, dUDP and dCDP are all synthesized by
the same enzyme Synthesized from nucleoside
diphosphate (not mono or triphosphate)
by ribonucleotide reductase
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Synthesis of dTMP

• Methylation of d-UMP via N5,N10-methylene THF
• Reaction inhibited by 5-fluorouracil (Efudex)

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Activation of 5-fluorouracil
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Dihydrofolate reductase
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Next - Part 3 catabolism
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Nucleotide metabolism Part 3(nucleotide
degradation)

• By
• Henry Wormser, Ph.D
• Professor of Medicinal Chemistry

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Nucleotide degradation
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Degradation of AMP
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PENTOSTATIN
previously called deoxycoformycin (DCF) a purine
analog with a 7-membered-ring potent inhibitor
of adenosine deaminase ADA is a key enzyme which
regulates adenosine levels in cells indicated
for refractory hairy cell leukemia other uses
chronic lymphocytic leukemia and lymphomas
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Adenosine deaminase
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ADA deficiency

• In the absence of ADA lymphocytes are destroyed
• deoxyadenosine is not destroyed, is converted to
  dAMP and then into dATP
• dATP is a potent feedback inhibitor of
  deoxynucleotide biosynthesis
• this leads to SCID (severe combined
  immunodeficiency disease)
• Infants with this deficiency have a high fatality
  rate due to infections

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ADA deficiency

• treatment consists of administering pegylated ADA
  which can remain in the blood for 1 2 weeks
• more efficient is gene therapy replacing the
  gene that is missing or defective
• gene therapy has been performed on selected
  patients

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Degradation of GMP and XMP
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CATABOLISM OF PURINES
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GOUT

• a disorder associated with abnormal amounts of
  urates in the body
• early stage recurring acute non-articular
  arthritis
• late stage chronic deforming polyarthritis and
  eventual renal complication
• disease with rich history dating back to ancient
  Greece

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GOUT

• once fashionable to associate gout with
  intelligence
• people with gout
• Isaac Newton
• Benjamin Frankin
• Martin Luther
• Charles Darwin
• Samuel Johnson

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Gout

• prevails mainly in adult males
• rarely encountered in premenopausal women
• symptoms are cause by deposition of crystals of
  monosodium urate monohydrate (can be seen under
  polarized light)
• usually affect joints in the lower extremities
  (the big toe is the classic site)

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Gout
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Four Stages of Gout

• 1. asymptomatic hyperuricemia
• 2. acute gouty arthritic attacks
• 3. asymptomatic intercritical period
• 4. tophaceous gout (characterized by the
  formation of tophi in joints)
• podagra (big toe)
• cheiagra (wrist) according to Hippocrates
• gonadra (knee)

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Diagnostic features

• usually affect joints in the lower extremities (
  95)
• onset is fast and sudden
• pain is usually severe joint may be swollen, red
  and hot
• attack may be accompanied by fever, leukocytosis
  and an elevated ESR

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Drugs which may induce hyperuricemia

• niacin
• thiazides and other diuretics
• low dose aspirin
• pyrazinamide
• ethambutol
• cyclosporine
• cytotoxic drugs

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Non-pharmacological approaches

• Avoid purine rich foods
• red meat and organ meat (liver, kidneys)
• shellfish, anchovies, mackerel, herring
• meat extracts and gravies
• peas and beans, aspargus, lentils
• beer, lager, other alcoholic beverages
• Weight loss
• Control alcohol (binge drinking)

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Pharmacological management of gout

• based on the premise that the hyperuricemia is
  due to both overproduction and underexcretion of
  uric acid
• symptomatic relief of pain is also achieved with
  analgesics (i.e. indomethacin)
• drugs used
• analgesics (NSAIDs)
• uricosuric agents
• xanthine oxidase inhibitors

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Therapy of acute gout

• treat with colchicine or NSAIDs
• avoid aspirin
• do not treat with allopurinol or uricosuric drugs
• uric acid lowering agents should never be started
  or stopped during acute attack
• pain resolution occurs within 48-72 hrs

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Colchicine
a non-basic alkaloid from the seeds and corms of
Colchicum autumnale (Meadow Safron)
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COLCHICINE

• used in the symptomatic treatment of acute
  attacks of gout
• decreases leukocyte motility, decreases
  phagocytosis and lactic acid production
• not used in other forms of arthritis
• a very potent drug
• can cause severe GI distress and abdominal pain

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Probenecid (Benemid)
A uricosuric agent
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Probenecid (Benemid)

• inhibits the tubular reabsorption of uric acid
• it can also inhibit the tubular excretion of
  certain organic acid via the transporter
• used in gout to promote the elimination of uric
  acid (not effective in acute attack)
• also used to enhance plasma concentration of
  certain antiinfectives (beta lactams)

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ALLOPURINOL (Zyloprim)

• prevention of attacks of gouty arthitis and
  nephropathy
• also used during chemotherapy of cancer and to
  prevent recurrent calcium oxalate calculi
• metabolized to oxypurinol (also an inhibitor of
  xanthine oxidase)
• inhibits the metabolism of certain anticancer
  drugs (6-MP, azathioprine)

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Allopurinol (Zyloprim)
An inhibitor of xanthine oxidase prevents the
formation of uric acid from precursorial purines
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Fate of uric acid

• in human and other primates uric acid is the
  final product of purine degradation and is
  excreted in the urine
• the same is true in bird, reptiles and many
  insects
• in other mammals uric acid is oxidized to
  allantoin (urate oxidase)
• teleost (bony) fish convert allantoin to
  allantoic acid
• cartilaginous fish and amphibian further degrade
  allantoic acid to urea
• and finally marine invertebrates decompose urea
  to ammonia

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Rasburicase (Elitek)
A recombinant form of uric acid oxidase. Used for
initial management of plasma uric acid levels in
pediatric patients with leukemia, lymphoma, and
solid tumor malignancies who are receiving
anticancer therapy expected to result in tumor
lysis and subsequent elevation of plasma uric
acid.
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Catabolism of a pyrimidine
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Formation of deoxyribonucleotides

• ribonucleotide reductase studied by JoAnne Stubbe
  (Wisconsin, then MIT)
• very complex enzyme contains
• Tyrosine radical
• 2 non-heme irons
• Two catalytically active cysteine residues
• Cys are reduced by other proteins thioredoxin
• Ribo. Reductase is the therapeutic target of the
  anticancer drug hydroxyurea

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Mechanism of ribonucleotide reductase

• Free radical mechanism involving tyrosyl residues
  and cysteine residues on the enzyme
• The enzyme is a dimer of dimers
• R1 a dimer of identical a subunits (85 kD each)
• R2 a dimer of identical b subunits (45 kD each)

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Reduction of the disulfide bond in ribonucleotide
reductase

• 2 proteins can perform this reductive reaction
• Thioredoxin (ubiquitous 12 kD monomer)
• Glutaredoxin which functions similarly to
  thioredoxin. Oxidized glutredoxin is reduced by
  glutathione (g-glutamylcysteinylglycine)

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Regeneration of thioredoxin and ribonucleotide
reductase
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HYDROXYUREA (Hydrea)

• inhibits the enzyme ribonucleotide reductase
• this enzyme causes ribonucleotides to be
  converted to deoxyribonucleotides
• DNA synthesis cannot occur
• cell are killed in the S phase
• drug holds other cells in the G1 phase
• primarily used to treat chronic myelogenous
  leukemia
• cancer cell develop resistance by
• increasing quantity of inhibited enzyme
• decreasing sensitivity of enzyme for inhibitor
• used orally
• major side effect is leukopenia

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GEMCITABINE (Gemzar)
Another inhibitor of ribonucleotide
reductaseindicated for non-small cell lung
cancer (usually with cisplatin) also first line
treatment for non-resectable pancreatic cancer
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The purine nucleotide cycle for anaplerotic
replenishment of citric acid cycle intermediates
in skeletal muscle
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The end