Lec 19- Antimetabolites

Question 1

Antimetabolites are

Answer 1

• Among the various compounds used as chemotherapeutic agents against cancer
• Chemicals that inhibit use of a metabolite where the metabolite is a naturally occurring chemical that is part of normal metabolism- this includes interfering with metabolism with healthy as well as cancer cells
• Often similar structure to what they interfere (folate= purine= pyrimidine)
• Cytotoxic properties halting cell growth/division
• Able to induce cell death during the S phase of cell growth when incorporated into RNA/DNA
• Antimetabolites are not restricted to the chemotherapy of cancer- recall that sulfonamide drugs inhibit folate biosynthesis in bacteria

Question 2

Folate biosynthesis

Answer 2

• Folic acid is converted to 7,8-dihydrofolic acid

1. GTP is the starting material for folic acid and phosphorylates the OH group and place 2 phosphate groups on
2. Black atoms are p-aminobenzoic acid
   
   • These are the nucleophile (NH2) which attacks electrophilic carbon to displace the phosphate group to attach the side chain
3. Glutamic acid is attached to the carboxyl group (of the benzoic acid) to form an amide bond
4. Imine bond between Nitrogen- carbon 5-6 is reduced

• For the antibacterial sulphonamide- the benzoic acid the red stuff attaches
• Instead of a carboxyl group, we will have an amino group- (we have nucleophilic N instead of electrophilic carbon) so can’t attach amino acid so can’t form folate

Question 3

Examples of antimetabolites

Just remember the important ones

Answer 3

• Purine analogues (end in ine)
  
  • Mercaptopurine
  • Tioguanine
• Pyrimidine analogues
  
  • 5-FU (flurouracil)
  • Tegafur
• Folic acid analogues- (trex)
  
  • MTX

Question 4

Fludarabine

Answer 4

• Purine antimetabolite gave orally or IV
• Interferes with ribonucleotide reductase and a DNA polymerase enzyme
• Active against dividing and resting cells
• Fluorinated analogue of adenine attached to an arabinose sugar has the BETA configuration (up) compared with ribose (down)

Question 5

Fludarabine

Answer 5

• Highly effective against chronic lymphocytic leukaemia, producing higher response rates than alkylating agents such as chlorambucil alone
• Fludarabine is used in the treatment of non-Hodgkin’s lymphomas in various combinations with
• Cyclophosphamide- DNA alkylator acting at the N7 of guanine
  
  • Mitoxantrone- Topoisomerase II inhibitor- interferes with DNA rep
  • Dexamethasone- steroid to counteract the side effect of chemotherapy- combat serious damage and inflammation caused by the harsh chemotherapy chemicals
  • Rituximab- mAb, destroys excessive, overactive B lymphocyte
    
    • This is given as a pre-chemotherapy treatment

Answer 6

• Highly effective against chronic lymphocytic leukaemia, producing higher response rates than alkylating agents such as chlorambucil alone
• Fludarbine is used in the treatment of non-hodgkinson lymphomas in various combinations with
  
  • Cyclophosphamide- DNA alkylator acting at N7 of guanine
  • Mitoxantrone- topoisomerase II inhibitor, interferes with DNA replication/repair
  • Dexamethasone- steroid counteracts side-effects of chemotherapy
  • Rituximab- mAb, destroy excessive, overactive B lymphocytes

Question 7

Ribonucleotide reductase- Learn this could be an exam question

Answer 7

• Accepts ribose structures and switches the hydroxyl groups to hydrogen via a complex mechanism
  
  • Tyrosine radical takes the hydrogen A-
  • Protonation of hydroxyl by the thiol
  • Water is then produced leaving behind a carbocation
  • Negative Sulphur forms a disulphide bond with other S neighbour, H moves off and binds to the carbo-cation
  • Ha is then placed back on to the structure
• Complex mechanism involving tyrosyl radical
• NADPH reduces disulphide S-S to S-H after catalysis
• Reduction NDP to dNDP occurs at diphosphate level (PP)

Question 8

Purine anticancer agents

Answer 8

• Purine bases that are pro-drugs that exploit purine salvage
• Ribonucleotide products cause feedback inhibition of purine biosynthesis
• Used against leukaemia, non-Hodgkin lymphoma
• Drug interaction with allopurinol, a xanthine oxidase inhibitor, that can exacerbate mercaptopurine toxicity
  
  • Allopurinol has a very similar structure (swap out S or O and on 5 membered ring make it a N-Ndouble bond)

Question 9

Antifolates

Answer 9

• Methotrexate- Black mimics folate, Red mimics- cytochain
  
  • Anti-cancer agents
  • Difference between MTX and FA- Instead of amino pointing Upwards FA has a carbonyl, MTX has methylated N
  • Most tight binding DHFR inhibitor- but no covalent bond
• Premetrexed
  
  • Lung cancer
  • Combined with cisplatin and dexamethasone
  • folate analogue
• Leucovorin (folinic acid)
  
  • Rescue therapy
  • Kickstart the folate pool- cancer need to make copies of themselves
  • Combats toxicity caused by use of MTX and purine antimetabolites

Question 10

Purine biosynthesis

Answer 10

• Folates act as one-carbon unit donors to supply C2 and C8
• 11 step de novo biosynthesis gives inosine monophosphate (IMP) from which ATP/dATP and GTP/dGTP are derived
• Need folate co-factors to work as carbon donors for purine synthesis

Question 11

Folate pool

Answer 11

• N⁵-methyltetrahydrofolate is the most reduced form of folate
• N¹⁰-formyltetrahydrofolate is the most oxidised form of folate- C2
• N5-N10 is an intermediate- C8
• Folate pool depletion is losing the ability to supply carbon units for purine synthesis
• Leucovorin we feed into the top of the pool
  
  • Isomerised to form N¹⁰ form and therefore all of the other folate co-factors

Question 12

Thymidylate synthase

Answer 12

• DHFR important for folate antimetabolites
• DHFR reduces dihydrofolate to tetrahydrofolate
• Serine supplies the missing carbon to regenerate
  
  • N⁵, N¹⁰- methylenetetrahydrofolate supplies the missing carbon to form 7,8-dihydrofolate =(DHFR)=> tetrahydrofolate with serine giving the missing carbon back

Question 13

Thymidylate synthase

Answer 13

• The drug works just like dUMP- just as a drug as the substrate
• SH (thiol part)-ENZ (enzyme)
• S is a nucleophile (much better than oxygen)
• In dUMP- there is an a,b- unsaturated ketone; b-carbon is electrophilic- nucleophile forms a covalent bond between substrate and enzyme (known as a binary complex)
• Folate co-factor then binds (co-enzyme) its job is to supply the missing carbon
  
  • we attach 2 pi electrons and attach them to the CH2 of the co-factor (known we have a ternary complex)
  • FU can be used in combination with folic acid- folic acid is converted into N⁵,N¹⁰MethyleneTHF helping to stabilise the ternary complex
• H⁺ attaches to the methylene group to break off the co-enzyme (left with a methyl group)
• H is lost and reform the C-C double bond to but also to reform the SH-ENZ

Question 14

5-FU

Answer 14

• A pro-drug that is converted to active 5-FdU by pyrimidine salvage that then inhibits the folate-dependent enzyme thymidylate synthase
• Removal of F⁺ is impossible so final step incomplete
• Leucovorin (folinate) is given with 5-FU to synergistic effect by stabilising the ternary complex
• Through the salvage pathway, a sugar is given to 5-FU which is the active form
• Enzyme accepts the active (sugar form)
  
  • b-carbon is electrophilic- Nucleophilic attack of sulphur- sulphur attaches
  • Same intermediate (accept with an F), stabilise negative charge ane use 2 pi electrons to form the bond with co-factor and use H⁺ (hydride to form CH³ losing co-factor
  • Hydrogen is normally lost to re-form SH- Due to F electronegativity it wants to form a negative ion, if the F were to undergo the same reaction it would have to accommodate a positive charge which it can’t do there the reaction can’t happen and the mechanism stalls