MD8 - Anti-metabolites

Main groups of antimetabolites

folate antagonists e.g. methotrexate, pemetrexed and non-classical lipophillic antifolates

pyrimidine antagonists e.g. 5-fluorouracil, fluorodeoxyuridine and azacytidine

purine antagonists e.g. 6-mercaptopurine, thioguanine, tiazofurin

sugar-modified nucleotides e.g. cytarabine, fludarabine and gemcitibine

Folate

Vitamine B9

cannot be synthesised in the body

Folic acid is converted into a folate by the body

used as a dietary substance and is required by the body for the synthesis of DNA and RNA and to metabolise AA

Aminopterin

folate antagonist

Farber realised a folate deficiency helped patients with cancer get better

provided TEMPORARY remission in trials

Dihydrofolate vs Tetrahydrofolate

Dihydrofolate - saturated only one of the double bonds in the aromatic ring

Tetrahydrofolate: saturated all the bonds in the aromatic ring

Folate Cycle

L-Serine is converted to Glycine by Serine hydroxymethyltransferase by removing a CH2 and adds it to tetrahydrofolate

this forms 5,10-CH2- tetrahydrofolate (5-membered ring in the molecule between the two nitrogens and the carbon unit) and a conformational change from this

then 5,10-CH2-tetrahydrofolate is converted to dihydrofolate by thymidylate synthase via conversion of dUMP to dTMP by adding the CH2 from the L-serine to it (5,10-CH2tetrahydrofolate acted as a carrier for it)

we are left with dihydrofolate

to create tetrahydrofolate again it is converted by DHFR (reductase) with NADPH giving a H+ to create this

What are the best targets for this cycle and why?

DHFR and TS because they are the rate-limiting enzymes

What is methotrexate and why is it’s structure important?

Analogue of folate

N in the middle of the molecule is methylated which is very important because in the folate process, the 5,10-CH2 tetrahydrofolate has an N in the middle and it is part of a 5 membered ring. Because of the Me in methotraxate it cannot form that ring so it cannot be converted to something similar to this intermediate

Structural differences between dihydrofolate and methotrexate

methylated N in the middle

unsaturated aromatic ring (only one carbon saturated)

amine replacing the carboxyl in the dihydrofolate structure

Methotrexate as a folate inhibitor

Inhibits DHFR at the folate binding site

very potent competitive inhibitor

too polar for passive diffusion into cell so taken up via reduced folate carrier (RFC)

must be polyglutaylated to be retained in cells

often used in high dose regimen for folate rescue in normal cells and widely used for cancer

Mechnisms of resistance to methotrexate

• mutations to DHFR enzyme to modify the folate binding site

• MDR (multi-drug resistant) phenotype causing active efflux of drug

• mutations to RFC reducing drug uptake

Lipophillic antifolates

don’t need RFC to enter cells - can enter via diffusion

Pyrimethamine

Lipophillic antifolates

inhibits DHFR in many species

used atm as antibacterial

Methylbenzoprim

very potent experimental lipophilic inhibitor of DHFR

Piritrexim

Potent lipophillic inhibitor of DHFR and active in several tumour types

Nolatrexed

Inhibits DHFR and TS and is active against liver carcinoma

Pemetrexed and Raltitrexed

competitive inhibitors of TS and bind to 5,10-CH2-tetrahydrofolate binding site

MOA of TS

TS has a sulfur that attacks the dUMP in a 1,4 addition

TS is now covalently bound to dUMP

TS binds to the 5,10-CH2-THF complex to form a ternary complex (3 components)

TS is released via the loss of a proton on the uracil and a regeneration of a double bond

complex breaks down to give dTMP and DHFR

How does 5FU inhibit TS?

5FU is metabolised to FdURD

FdURD is metabolised to FdUMP

FdUMP binds to the TS and 5,10 CH2 complex but the F is in place of the proton

F is electronegative so cannot be released so TS is stuck and it acts as an inhibition

Azacytidine

weak inhibitor of TS

phosphorylated to form azacytidine TP the incorporated into RNA and mimics C in RNA but it’s unstable so it decomposes and causes damage to RNA

inhibits DNA methyltransferase

Metboli activation of purine analogues

parent compounds are hypoxanthine and guanine

hypoxanthine → 6-mercaptopurine (6-MP) → Thio-IMP (by HPRT) →Thio-GMP

guanine → 6-thioguanine (6-TG) → Thio-GMP (by HPRT) → Thio-GTP by TG-RNA → Thio-dGTP by TG-DNA

HPRT - hypoxanthine phosphoribosyltransferase

Tiazofurine

Experimental drug

converted to TAD

mimics NAD+

Inhibiting the biosynthesis of purine nucleotides

Thio-IMP nd Thio-GMP inhibit by binding at the purine binding site

Thio-IMP also inhibits IMP → AMP

Ara-C

Cytarabine - sugar modified nucleoside

can be converted to triphosphate which inhibits DNA polymerase as an analogue of dCTP

there is some incorporation into the DNA but all it does is make the DNA non-functional

Fludarabine

sugar modified nucleoside

converted to triphosphate

inhibits FNA polymerase as an analogue of dATP

Gemcitabine

sugar modified nucleoside

two fluorines on the sugar

converted to triphosphate very efficiently but also converted to diphosphate

inhibits DNA polymerase as analogue of dCTP but 100x more potent than Ara-C

Metabolism of Gemcitabine

Gemcitabine → F2dCMP by deoxycytidine kinase

f2dCMP → F2dCDP by UMP/CMP kinase

F2dCDP → F2dCTP by NDP kinase

How does Gemcitabine work?

blcoks CTP synthase and ribonucleotide reductase which are needed to convert UTP → CTP and CTP→ dCTP respectively

dCTP is a negative feedback loop inhibitor of dCK so less dCTP means mroe dCK which means more F2dCMP from gemcitabine