Antimetabolites

• What is the rationale for antimetabolites?

  • A key hallmark of cancer is its limitless replicative potential - requires limitless source of DNA for constant division and growth
  • Animetabolites kill (cancer) cells by inhibiting critical enzymes involved in the synthesis of DNA constituents (DNA biosynthesis) - essential for tumour proliferation
• How do antimetabolites work?

  • DNA biosynthesis requires: purines (A, G bases), pyrimidines (C, T bases in DNA and U in RNA), and essential vitamin and fuel folic acid (folate)
  • Antimetabolites trick cancer cells into using the antimetabolite — instead of the true molecules it needs (metabolite) to make genetic material
  • Essentially antimetabolites act as decoys that closely resemble the correct molecule
• What are the four main groups of antimetabolite drugs?

  • Folate "antagonists" - e.g. Methrotrexate, non-classical lipophilic antifolates, pemetrexed, raltitrexed
  • Pyrimidine "antagonists" - e.g. 5-Fluorouracil (5-FU), fluorodeoxyuridine (FdURD), azacytidine
  • Purine "antagonists" - e.g. 6-Mercaptopurine, thioguanine, tiazofurin
  • Sugar-modified nucleosides - e.g. , Gemcitibine, Cytarabine (Ara-C), fludarabine
• What is the story of folate?

  • Folate (natural) = folic acid (synthetic)= forms of vitamin B9, an essential B vitamin
  • Folic acid used as a dietary supplement - converted into folate by the body (folic acid not naturally synthesised)
  • Folate is required for the synthesis of DNA and RNA and to metabolise amino acids
  • Sidney Farber was the first to achieve temporary remission in cancer patients using the folate antagonist aminopterin to block the action of folate in rapidly dividing cells
  • There are different types/forms of folate, e.g. DHF, THF

  
• What is the folate cycle?

  • Also known as one carbon metabolism
  • Main compounds involved: dihydrofolate, tetrahydrofolate, and 5,10-CH2-tetrahydrofolate (5,10-CH2-THF)
  • Aim: convert deoxyuridine monophosphate (dUMP) via. thymidylate synthetase into deoxythymidine monophosphate (dTMP) to be used in DNA synthesis - this is mediated by folate derivatives
• What are the rate limiting steps of the folate cycle?

  • Thymidylate synthetase (TS) stage - TS catalyses the conversion of dUMP to dTMP
  • Dhihydrofolate reductase (DHFR) stage - DHFR regenerates the active form of folate (THF) required for one-carbon transfer reactions, including dTMP synthesis
  • Inhibition of these disrupts nucleotide metabolism, leading to impaired DNA synthesis and cell growth

  Serine hydroxymethyltransferase (SHMT) is not rate limiting - catalyses addition of one-carbon unit, but other alternative sources exist, thus inhibiting SHMT would not interfere too much with the cycle
• Folate antagonists (methotrexate) - DHFR

  • Very potent competitive inhibitor of DHFR - binds at DHFR folate-binding site and prevents normal DHFR function (reduction of DHF)
  • Too polar for passive diffusion into cells - taken up through reduced folate carrier (RFC)
  • Must be polyglutamylated to be retained in cells
  • Often used in high-dose regimens with leucovorin (folate) rescue/counterbalance of normal cells - as treatment with methotrexate alone would impede DNA synthesis in both cancer cells and healthy cells too
  • Widely used against many cancer types

  
• What are the mechanisms of resistance to methotexate?

  • Mutations to DHFR enzyme, modifying folate-binding site so methotrexate cannot competitively inhibit
  • Multi-drug resistance phenotype, causing active efflux of drug out of cancer cells
  • Mutations to reduced folate carrier (RFC), reducing uptake
• Folate antagonists (lipophilic antifolates) - DHFR

  Contain hydrophobic (lipophilic) groups or substituents that enhance their lipid solubility and membrane permeability - enter cells via. passive diffusion rather than reduced folate carrier (RFC)

  
  

  Pyrimethamine

  • Inhibit DHFR of many species
  • Mainly used as antibacterial agent

  Nolatrexed

  • Inhibits DHFR and indirectly TS (due to reduced levels of THF)
  • Active against liver carcinoma

  Piritrexim

  • Potent lipophilic inhibitor of DHFR
  • Active in several tumour types

  Methylbenzoprim

  • Very potent liphophilic inhibitor of DHFR
  • Not yet in clinic

  
• Analogues of folate (pemetrexed and raltitrexed) - TS

  • Pemetrexed and raltitrexed are competitive inhibitors of TS
  • Mimic the shape of 5,10-CH2-tetrahydrofolate - new CH2 carbon unit induces a new angle
  • Binding at the 5,10-CH2-tetrahydrofolate-binding site

  
• What is a Michael addition?

  • Also known as conjugate addition or 1,4-addition
  • Involves an unsaturated bond, carbonyl (COO), and amide (-NH2)
  • Nucleophile (e.g. RSH) with an electron-rich center (i.e. pair of electrons on sulfur) attacks an unsaturated carbonyl compound to form a new carbon-carbon bond
  • Forms covalent compound

  
• What is the mechanism of thmidylate synthetase (TS)?

  • dUMP and TS form a binary complex, covalently attaching dUMP to TS enzyme. Electrons on S- of TS attack the double bond next to amide of dUMP. This occurs in TS active site
  • 5,10-CH2-THF exists in equilibrium with 2 forms: RHS = closed ring while LHS = opened ring that forms an imine available to react with (dUMP+TS) complex and form ternary complex (crux of reaction)
  • Ternary complex broken - loss of H+ (proton) causes electrons between C and H to form a double bond and release TS enzyme. Reforms a binary complex (dUMP+5,10-CH2-THF)

  
• What is the most important step of thymidylate synthetase mechanism?

  Elimination of H+ (proton) - releases TS enzyme

  Essentially a suicide inhibitor effect
• Pyrimidine antagonists (5-FU and FdURD) - TS

  • 5-Fluorouracil (5-FU) is effectively a uracil with fluorine (F) attached
  • Attachment of deoxy sugar to 5-FU = fluorodeoxyuridine (FdURD)
  • Phosphorylation of FdURD = FdUMP
  • FdUMP equivalent to dUMP (but with F) - undergoes same process as dUMP with TS to make ternary complex
  • While dUMP ternary complex loses H+ to form a double bond, FdUMP has an F (very electronegative) that cannot be eliminated as F+ (too strong for electrons to pull)
  • Therefore, stuck at ternary complex stage - FdUMP stops the whole process = suicide inhibitor

  
• Pyrimidine antagonists (azacytidine) - TS

  • Weak inhibitor of TS
  • Phosphorylated to form azacytidine triphosphate, then incorporated into RNA
  • Mimics cytidine in RNA but with a nitrogen (N) in the aromatic ring
  • However this is unstable and decomposes, causing damage to RNA
  • Inhibits DNA methyltransferases (epigenetic effects)

  
• Purine antagonists (6-MP and 6-TG)

  • Sulfur (S) + purine analogues hypoxathine and guanine = 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) respectively
  • Competitive inhibitors for hypoxanthine phosphoribosyltransferase (HPRT)
  • HPRT puts analogues on sugar and monophosphorylates to get thio-IMP or thio-GMP respectively - can have conversion from thio-IMP into thio-GMP
  • Thio-GMP can be incorporated into RNA (thio-GTP) or DNA (thio-dGTP)
  • Not known which effects are responsible for cell death or what the major effect is

  
• Purine antagonists (tiazofuran)

  • Tiazofuran is an experimental drug converted intracellularly to its active form, tiazofurin adenine dinucleotide (TAD)
  • TAD mimics NAD+
  • TAD competitively inhibits IMP dehydrogenase by occupying NAD+ binding site - disrupts guanine nucleotide synthesis which needs IMPDH
  • Leads to reduced DNA and RNA synthesis and impaired cellular function, particularly in rapidly dividing cancer cells (which need correct nucleotides)

  
• How do the purine antagonists work together to inhibit the biosynthesis of purine nucleosides?

  • IMP uses a 2-step process to make either AMP or XMP
  • Both AMP and XMP are important - doubly phosphorylating these makes base pairs
  • TAD (from tiazofurin) inhibits IMPDH enzyme at NAD+ binding site
  • Additionally thio-IMP and thio-GMP are able to inhibit at purine binding site
  • IMPDH enzyme has NAD+ binding site but also for purine – both parts affected and suppress formation of GMP
  • Essentially some antagonsists target incorporation into DNA/RNA and others inhibit synthesis of AMP and GMP

  
• Sugar modified nucleosides (Ara-C, fludarabine, gemcitabine)

  Cytarabine (Ara-C)

  • Converted to triphosphate - inhibits DNA polymerases as analogue of dCTP
  • Incorporation induces DNA strand termination - makes DNA non-functional (-OH equatorial rather than axial)

  Fludarabine

  • F present
  • Converted to triphosphate - inhibits DNA polymerases as analogue of dATP

  Gemcitabine

  • Two F present
  • Converted efficiently to di-(F2dCDP) and tri-phosphate (F2dCTP)
  • F2dCDP inhibits ribonucleotide reductase - reduces deoxynucleotide pool
  • F2dCTP inhibits DNA polymerases as analogue of dCTP
  • 100x more potent than Ara-C

  
• What is the mechanism of metabolism/activation of gemcitabine?

  • dCK converts gemcitabine into F2dCMP (monophosphate)
  • UMP/CMP kinase converts F2dCMP to F2dCDP (diphosphate)
  • NDP kinase converts F2dCDP to F2dCTP (triphosphate)

  
• Interaction of Niraparib with gemcitabine mechanism

  
  

  • Niraparib inhibits deoxycytidine kinase (dCK) - reduces activation of gemcitabine, i.e. limit conversion to the active monophosphate (F2dCMP), diphosphate (F2dCDP), and triphosphate (F2dCTP) forms
  • However the DNA damage caused by gemcitabine that managed to be activated can be potentiated with Niraparib - as a PARP inhibitor, further impairs the repair of DNA damage

  
• What is the mechanism of self-potentiation in gemcitabine?

  • UTP is converted to CTP via. CTP synthase - carbonyl is converted to amide (red circle)
  • CTP is converted to dCTP via. ribonucleotide reductase - removes -OH (empty red circle)
  • F2dCDP inhibits both CTP synthase and ribonucleotide reductase - therefore less dCTP produced
  • dCTP = feedback inhibitor of dCK
  • Therefore, depletion of dCTP activates dCK = increased formation of F2dCMP from gemcitabine
  • Essentially, gemcitabine self-potentiates itself and is a good inhibitor of CTP synthase and ribonucleotide reductase

  
• Describe the folate cycle

  1. Reduction of oxidised-folate form, dihydrofolate (DHF), into active form, tetrahydrofolate (THF) via. dihydrofolate reductase (DHFR) - cofactor = NADPH converted to NADP+ to provide the hydrogens to produce THF
  2. THF + L-serine (providing a CH2 carbon unit) is converted into 5,10-CH2-THF and glycine via. serine hydroxymethyltransferase (SHMT)
  3. 5,10-CH2-THF + dUMP is converted into DHF and dTMP via. thymidylate synthetase (TS)

  
• What are the effects of 6-MP and 6-TG?

  • Incorporation into RNA/DNA - 6-MP and 6-TG conversion into Thio-GTP and Thio-dGTP can be incorporated into RNA/DNA, causing instability, DNA damage, and apoptosis
  • Inhibition of GTP-binding proteins - e.g. 6-TG nucleotides incorporated into GTP pools
  • Inhibition of de novo purine synthesis, - both drugs inhibit enzymes involved in purine synthesis, leading to reduced production of nucleotides necessary for DNA and RNA synthesis, e.g. thio-GMP + IMP dehydrogenase

  Major cytotoxic effect is unknown because 6-MP and 6-TG form multiple active metabolites