Lecture 9 - Antimetabolites Flashcards
What hallmark of cancer do antimetabolites target?
Limitless replicative potential
How do antimetabolites work as anticancer agents?
- usually via enzyme inhibition of enzymes involved in DNA biosynthesis
- inhibiting cellular processes
What are the 4 groups of antimetabolite drugs?
- Folate antagonists
- Pyrimidine antagonists
- Purine antagonists
- Sugar-modified nucleosides
What was Dr Sidney Farbers thinking process behind folates?
- Realised rapidly dividing cells needed folate, so treated them with a folate antagonist
What is the differences between folic acid, dihydrofolate and tetrahydrofolate?
- Folic acid –> reduced to dihydrofolate by addition of an H atom
- Tetrahydrofolate –> doubly reduced and contains two H’s
What is folic acid converted to in the body?
Folate
- which is vitamin B9, required for DNA synthesis
What are the 3 important enzymes in the folate cycle?
- Dihydrofolate reductase (DHFR)
- Serine hydroxymethyltransferase (SHMT)
- Thymidylate synthetase (TS)
Of the 3 enzymes of the folate cycle, which are rate limiting and which is not?
Rate limiting -
TS and DHFR
Not -
SHMT
3 examples of folate antagonists
- Analogues of Folate
- Lipophilic antifolates
- Inhibitors of thymidylate synthetase
Example of an analogues of folate
Methotrexate
Example of lipophilic antifolates
- Pyrimethamine
- Nolatrexed
- Piritrexim
- Methylbenzoprim
Inhibitors of thymidylate synthetases examples
- Pemetrexed
- Raltitrexed
What is the mode of action of methotrexate?
- Binds to DHFR at folate binding site
- Potent competitive inhibitor of DHFR
What are some of the issues with methotrexate?
- Too polar for passive diffusion into cell, taken up through reduced folate carrier
- Must be polyglutamylated to be retained in cells
What are the structure features of Methrotrexate?
- Analogue of Dihydrofolate
- Methyl and NH2 instead of carbonyl in dihydrofolate
- Polar
What are the mechanisms of resistance to methotrexate?
- Mutations to DHFR enzyme
- Mutations to RFC, reducing the uptake
- Active efflux of the drug
Difference between lipophilic antifolates and methotrexate
Lipophilic can enter cells by passive diffusion – don’t need RFC
What are inhibitors of thymidylate synthetase analogues of?
5,10-CH2-tetrahydrofolate
What are lipophilic antifolates analogues of?
Dihydrofolate
Example of pyrimidine antagonists
5FU - Mechanism based inhibitor of Thymidylate synthetase
FdURD
Azacytidine - Competitive binding inhibitor of TS
What is important to remember about Mechansism of Thymidylate synthetase?
- Ternary complex breaking apart is triggered by the loss of a proton
Overview of the Thymidylate Synthase reaction
- dUMP –> dTMP
- TS sulphur attacks dUMP in a 1,4 fashion
- forming dUMP-TS intermediate
- At same time 5,10-CH2-THF exists in an equilibrium
- Intermediate unwinds and attacks carbon of 5,10-CH2-THF forming ternary intermediate molecule
- BREAKDOWN of ternary intermediate by loss of proton liberating TS and binary complex
- Binary complex breaksdown –> dTMP and DHF
What happens to the TS mechanism with 5-FU and FdURD?
- 5-FU –> FdURD –> FdUMP
- FdUMP instead of dUMP which goes into TS mechanism
- In ternary complex there is fluorine instead of proton and TS cannot be eliminated
- Ternary complex gets stuck and process stops
What is the mechanism of action of Azacytidine?
- Gets phosphorylated to form azacytidine triphosphate, then incorporated into RNA
- causing RNA damage as is unstable
- inhibits methyltransferases
Is Azacytidine weak or strong inhibitor of TS?
Weak
Examples of purine antagonists
- 6-Mercaptopurine
- 6-thioguanine
- tiazofurin
Mechanism of action of tiazofuran
- Mimics NAD+ as it is converted to tiazofurin adenosine disphosphate (TAD)
- mimics with an azofurin ring
- TAD can inhibit IMP dehydrogenase in the NAD+ binding site –> no IMP –> no purines
How does TAD inhibit as a purine antagonist
TAD (from tiazofurin) inhibits the NAD+ binding site blocking IMP dehydrogenase
Examples of sugar-modified nucleosides
- Ara-C
- fludarabine
- gemcitabine
As a general, how do sugar-modified nucleosides work?
- They are converted to triphosphates and inhibit DNA polymerases making DNA non functional
Which is the most effective sugar modified nuceloside?
Gemcitabine
Overview of Gemcitabine mode of action
Gemcitabine (F2dc) to F2dCMP is rate limiting step –
Drug taken into cells by same molecular transporters as nucleosides
CTP synthase converts UTP to CTP
F2dCDP inhibits CTP synthase and ribonucleotide reductase = therefore less dCTP is produced.
Depletion of dCTP activates cCK, and thus MORE F2dCTP is made – FEEDBACK!!!!
SELF POTENTIATION
Applications of methotrexate in the clinic
- Widely used against many cancers
- Used in high dose regimen with leucovorin
What is the mode of action of Pyrimethamine?
- Inhibits DHFR as an analogue of dihydrofolate
What is the mode of action of Nolatrexed?
Inhibits DHFR and TS
What is the mode of action of Piritrexim?
Lipophilic inhibitor of DHFR
What is the mode of action of Methylbenzoprim?
Lipophilic inhibitor of DHFR
Example of cancer Nolatrexed used for
Liver carcinoma
What is the structure of Inhibitors of Thymidine Synthetase, drugs Pemetrexed and Raltitrexed?
- Analogues/similar to 5-10-Ch2-tetrahydrofolate
What is the mode of action of Inhibitors of Thymidine Synthetase, drugs Pemetrexed and Raltitrexed?
- Competitive inhibitors of TS
- Binding in the 5, 10-Ch2-tetrahydrofolate binding site
What are the structural features of 5-FU?
- Analogue of uracil
- Fluorine atom at the C-5 position in place of hydrogen
Resistance mechanisms against 5-FU
- DPD overexpression –> DPD mediated degradation of 5-FU
- Increased levels of TS
- LOF of p53 –> reduced sensitivity
What markers can indicate response to 5-FU?
- DPD and TS levels can indicate response to 5-FU
Structural features of Azacytidine
- Mimics C in RNA
- however it is unstable and causes RNA damage
Structural features of 6-mercaptopurine
- Hypoxanthine analogue
- competes with Hypoxanthine for HPRT
What is HPRT?
- Hypoxanthine phosphoribosyl transferase
- catalyses guanine –> guanosine monophosphate which is used as a nucleotide for DNA or RNA equivalent
Mechanism of action of 6-mecaptopurine
- 6-MP via HPRT –> Thio-IMP
-Thio-IMP –> thio-GMP (sometimes 6-MP end up as nucleotide derivatives of 6-TP) - Thio-GMP –> thio-GTP or thio-dGTP
- inhibits de novo purine synthesis
- Incorporated into RNA or DNA (respectively) mimic guanine
- Act as poisions for RNA and DNA via incorporation and inhibit GTP-binding protein
Mechanism of action of 6-thioguanine
- 6-TG via HPRT to Thio-GMP
- Thio-GMP –> Thio-GTP or ThiodGTP
- Incorporated into RNA or DNA (respectively) to mimic G
- Act as poisions for RNA and DNA via incorporation and inhibit GTP-binding protein
Structural features of 6-thioguanine
- Guanine analogue
Structure of Cytarabine (Ara-C)
- analogue of dCTP
- OH groups on carbon
Structure of Fludarabine
- Analogue of dATP
- OH group on carbon
What structural feature about gemcitabine makes it most efficient?
- fluorine’s on carbon
What does Gemcitabine need which the other two sugar-modified nucleosides don’t need
- Transporters to get into the cell
What ways can a cell become resistant to gemcitabine?
- Downregulation of rate limiting enzyme dCk
Clinical application of gemcitabine
- first line treatment for pancreatic cancer
- as a combo or monotherapy
- better to treat PDAC than 5-FU –> better overall survival
What is the importance of the products of gemcitabine in the mode of action of Gemcitabine?
- F2dCTP competes with dCTP as an inhibitor of DNA polymerase
- DNA polymerase usually uses energy from hydrolysis of dCTP for DNA synthesis
- F2dCDP is a potent inhibitor of ribonucleoside reductase, resulting in depletion of dCTP which inhibits dCK and is necessary for DNA synthesis
- SELF POTENTITATION
What do the purine antagonists 6-MP and 6-TG inhibit?
formation of AMP and GMP
- either encorpoorated into DNA or blocking synthesis of GMP and AMP
