Mike Threadgill Flashcards
Antimetabolites
Kill (cancer) cells by inhibiting critical cellular processes, especially those involved in growth
Generally inhibit critical enzymes involved in DNA biosynthesis, because DNA biosynthesis is essential for tumour cell proliferation
Main groups of antimetabolite drugs
There are 4:
- Folate “antagonists” e.g. methotrexate, non-classical lipophilic antifolates, pemetrexed, raltitrexed
- Pyrimidine “antagonists” e.g. 5-fluorouracil, fluorodeoxyuridine, azacytidine
- Purine “antagonists” eg. 6-mercaptopurine, thioguanine, tiazofurin
- Sugar-modified nucleosides e.g. cytarabine, fludarabine, gemcitabine
Why “antagonists”?
The drugs are more like inhibitors than classical antagonists
Folate cycle enzymes
Dihydrofolate –> tetrahydrofolate = DHFR (dihydrofolate reductase)
Tetrahydrofolate –> 5,10-CH2-tetrahydrofolate = SHMT (serine hydroxymethyltransferase)
5,10-CH2-tetrahydrofolate –> dihydrofolate = TS (thymidylate synthetase)
Methotrexate
Analogue of dihydrofolate
Binds to DHFR at folate binding site
Very potent competitive inhibitor (Ki = 5 pM for human DHFR)
Too polar for passive diffusion into cells - needs to be taken up via RFC (reduced folate carrier) and must be polyglutamylated for intracellular retention
Widely used against many cancer types, often in combination with leucovorin (folinic acid) to rescue normal cells and reduce toxic effects of methotrexate
Problems associated with methotrexate
Cancer cells can develop several mechanisms of resistance to methotrexate e.g.
Mutations in DHFR that modify the folate binding site (so methotrexate can’t bind, but folate may still be able to bind)
Mutations in RFC therefore reducing the uptake of methotrexate into cancer cells
“Multi-drug resistance” phenotype, where the cancer cell actively pumps the drug out
Non-classical lipophilic antifolates
Also inhibit DHFR
Can enter cells by passive diffusion (RFC not required, circumvents potential resistance)
No glutamate side chain so not polyglutamylated, which means administration via continuous infusion is required due to lack of intracellular retention
Pyrimethamine
Mainly used as antibacterial
Piritrexim
Potent, active in several tumour types
Nolatrexed
Can inhibit DHFR and TS due to pyridine ring
(Non-competitive inhibitor of TS)
Use in liver carcinomas
Methylbenzoprim
Very potent experimental lipophilic inhibitor of DHFR
Ki ~ 10 pM
TS inhibitors
e.g. pemetrexed, raltitrexed
Analogues of the endogenous TS substrate
Competitive inhibitors of TS, bind at the same site as 5,10-CH2-THF
Require RFC for uptake
TS is over expressed in tumour cells so inhibition of TS is an important strategy in the development of chemotherapeutic drugs
TS mechanism
Draw
Reductive methylation, 5,10-CH2-THF serves as both methyl donor and reducing agent
Effect of inhibition of TS
Blocks DNA synthesis, eventually leading to cell death (“thymineless death”)
This reaction is the sole de novo source of thymidine, which is necessary for DNA replication and repair
Raltitrexed
Direct and specific inhibitor of TS
Water-soluble, non-nephrotoxic
Transported into cells by RFC and extensively polyglutamylated by folylpolyglutamate synthase (FPGS)
Polyglutamylation increases raltitrexed inhibitory activity by > 100-fold and is retained within cells for a prolonged period of time
Effective against metastatic colorectal cancer, but usage is now generally restricted to patients intolerant to FUra
Pemetrexed
New-generation antifolate - TS is its primary target by also inhibits other enzymes involved in pyrimidine synthesis e.g. DHFR
Can be taken up by RFC and PCFT (proton-coupled folate transporter), meaning its activity is preserved even if RFC is lost/impaired
Extensive polyglutamylation so persists in cells for a prolonged period of time
Inhibitory effect against TS is insensitive to dUMP accumulation because dUMP and pemetrexed bind to TS at different sites
Pemetrexed + cisplatin = first-line treatment for NSCLC
And shows activity in a number of other tumours
5-fluorouracil metabolism
5-Fu —> FdURD —> FdUMP
Mechanism of TS inhibition by 5-Fu
Fluorine substituent fails to dissociate from the pyrimidine ring, meaning elimination cannot occur to give loss of -S-TS
Therefore TS remains bound to FdUMP and is inhibited and unable to catalyse other processes
5-Fu also has other mechanisms of action e.g. metabolised to FdUTP and FUTP which can be misincorporated into DNA and RNA, respectively
Very insoluble
Major use in colorectal cancer
Azacytidine
Weak TS inhibitor (binds at substrate binding site but doesn’t inhibit mechanism)
Phosphorylated to form azacytidine triphosphate, that is then incorporated into RNA as a cytidine mimetic
However, azacytidine is unstable and decomposes, causing damage to RNA
Also inhibits DNA methyltransferases which has epigenetic effects and affects regulation
Limitations to clinical use of 5-Fu
Drug resistance
Inefficiently converted into FdUMP - some is catabolised to toxic metabolites
Dihydrofolate
Precursor to 5,10-CH2-THF
Required for pyrimidine biosynthesis
Mechanism of resistance to 5-Fu
5-Fu acutely induces TS expression due to inhibition of a negative feedback mechanism whereby ligand-free TS binds to and inhibits the translation of TS mRNA
When TS is bound by FdUMP, it is no longer able to bind to its mRNA and suppress its own translation, resulting in increased TS expression
This acute increase in TS levels would facilitate recovery of enzyme activity
HPRT
Hypoxanthine phosphoribosyltransferase
6-MP –> Thio-IMP
6-TG –> Thio-GMP
(I = inosine)
Metabolism of Thio-GMP
2 phosphate groups added to give Thio-GTP which is incorporated into RNA (TG-RNA)
Can also lose OH in 2 position on ribose to give Thio-dGTP which is incorporated into DNA (TG-DNA)
Mechanism of action of purine “antagonists” 6-MP/6-TG
The replacement of ) by S means that H-bonding can no longer occur because S is not electronegative enough
This breaks the H-bond pattern in DNA which damages the DNA and affects chromatin structure
Tiazofurin
Metabolised to TAD
= NAD+ mimetic
TAD doesn’t have cationic N (which is what gives NAD+ its oxidising ability because it can accept H-)
TAD can bind to the enzyme IMPDH (IMP dehydrogenase) but cannot carry out the oxidation of IMP to XMP
Inhibition of the biosynthesis of purine nucleosides
Draw
Cytarabine
Ara-C
Converted into the triphosphate
Inhibits DNA polymerase as an analogue of dCTP
Some is incorporated into DNA, making DNA non-functional
Fludarabine
Converted into the triphosphate
Inhibits DNA polymerase as an analogue of dATP
Gemcitabine
100x more potent than Ara-C
Converted to the di- and triphosphates F2dCDP and F2dCTP
Triphosphate F2dCTP inhibits DNA polymerase in a competitive, reversible fashion as an analogue of dCTP
Enzymes involved in activation of Gemcitabine
F2dC —> F2dCMP = deoxycytidine kinase (dCK)
F2dCMP —> F2dCDP = UMP/CMP kinase
F2dCDP —> F2dCTP = NDP kinase
Potential drug-drug interaction of Gemcitibine
With niraparib (PARP-1 inhibitor) Niraparib inhibits dCK
Self-potentiation of gemcitabine
dCTP is a feedback inhibitor of dCK
F2dCDP inhibits CTP synthase and ribonucleotide reductase which are involved in the conversion of UTP into dCTP
Therefore there is reduced formation of dCTP and less inhibitor of dCK
Activation of dCK increasing the formation of F2dCMP from gemcitabine
Reduced production of dCTP also means F2dCTP has reduced competition for DNA polymerase
Role of microtubules
Responsible for maintaining the structure of a cell and for separating the sets of chromosomes during mitosis
Interfering with the formation/remodelling of microtubules inhibits mitosis and therefore inhibits the proliferation of cancer cells0
Microtubule structure
Assemblies of tubulin heterodimers (a/b)
Microtubules can drink by losing dimers and grow by adding dimers
Microtubules are in a dynamic equilibrium with individual tubulin dimers
Mitotic spindle poisons interfere with this dynamic equilibrium
Vinca alkaloids
Bind strongly and reversibly to individual tubulin dimers, leading to a conformational change and preventing the binding of the tubulin dimers to microtubules
Individual complexes of vinca alkaloids and tubulin dimers condense into paracrystalline aggregates, taking the dimers out of the dynamic equilibrium and causing microtubules to shrink
Vinblastine
Vinca alkaloid
Binds to the (+) end of microtubules (acts as a cap), which prevents new tubulin dimers from adding
Treatment of lymphomas, testicular and ovarian carcinomas and Hodgkin’s lymphoma
Vincristine
Vinca alkaloid
Treatment of lymphomas, leukaemias, mammary carcinomas
Vindesine
Vinca alkaloid
Treatment of leukaemias, lymphomas, NSCLC
Vinrelbine
Vinca alkaloid
Treatment of NSCLC and mammary carcinomas
Taxols
Bind to taxol-binding sites on the inside surface of the microtubule, preventing their disassembly (“inappropriate microtubule”)
This leads to a decrease in the concentration of free tubulin dimers because there is no disassembly of old microtubules
Therefore new microtubules cannot be assembled
Paclitaxel
= taxol
Treatment of mammary and ovarian carcinomas
Docetaxel
= taxotere
Treatment of mammary, ovarian, prostate and lung carcinomas
Colchicine-like drugs
Bind to colchicine-binding sites on the beta subunits of the tubulin dimers and in the microtubules
When bound to dimers, this disfavours protofilament assembly
When bound to microtubules, this disfavours the disassembly of inappropriate microtubules
Colchicine
Not currently approved for treatment of cancer
Approved for treatment of gout
Combretastatin A4
Currently in clinical trials for cancer treatment, binds at colchicine binding site
