MDT - Antimetabolites Flashcards
What is an antimetabolite?
Kill cells by inhibiting cellular process which is critical. Normally some critical enzyme in the growth and replication of cells (DNA biosynthesis).
How many groups of antimetabolite drugs are there?
Four. Folate antagonists. Pyrimidine antagonists. Purine antagonists. Sugar-modified nucleosides.
What are some examples of folate antagonists?
Methotrexate, non-classical lipophillic antifolates, pemetrexed, raltitrexed.
Are folate antagonists really antagonists?
No they just inhibit enzymes, not true receptor antagonists.
What are some examples of pyrimidine antagonists?
5-Fluorouracil (5-FU), fluorodeoxyuridine (fdURD), azacytidine.
Pemetrexed
Folate antagonist
What is the folate cycle?
L-serine –> Thymidine
Thymidylate synthase (TS) ThyA is an enzyme crucial for
DNA synthesis
TS catalyses the conversion of ______ and _______ into ______ and ______ via _________
TS catalyses the conversion of dUMP and mTHF into dTMP and DHF via reductive methylation.
What is “thymineless death”?
The reaction catalysed by TS is a pivotal step in the de novo biosynthesis of dTTP (one of the four building blocks of DNA). Inhibition of TS, in the absence of preformed thymidine, blocks DNA synthesis -> cell death.
The mechanism which TS catalyses to form dTMP and DHF from dUMP and mTHF
Reductive methylation
What is the most widely used agent for targeting overexpression of TS?
5-FU - based on the observation that uracil may be preferentally used in DNA synthesis in tumours compared to normal cells.
Why is 5-FU effective at treatment of tumours?
5-FU is anablolised to a nucleotide analog of dUMP (FdUMP), which is a tight binding inhibitor of TS.
The reaction stops as the fluorine substituent fails to dissociate from the pyrimidine ring, resulting in inactivation of the enzyme.
Why is 5-FU not an optimal drug? [3]
It is inefficiently converted to FdUMP.
Part of it catabolised to toxic metabolites.
Also tumours can exhibit acquired or intrinsic resistance to FUra.
What is an important factor reducing the effectiveness of FdUMP as TS inhibitor?
Metabolic resistance, a phenomenon consisting of the protection of enzyme against drugs by accumulation of substrate to the level sufficient to compete efficiently with the inhibitor, leading to full activity of the metabolic pathway involving the target enzmye.
Why did researchers focus on analogues of the folate cofactor of TS? What did this lead to?
The metabolic resistance to FdUMP, this led to the development of inhibitors of the folate cofactor - the antifolates.
What is Raltitrexed?
Direct and specific inhibitor of TS.
On what was Raltitrexed modelled?
CB3717
How does the structure of Raltitrexed differ from that of CB3717?
CH2-C~CH group and benzene ring replaced with methyl group and thiophene ring.
What is metabolic resistance?
Metabolic resistance, a phenomenon consisting of the protection of enzyme against drugs by accumulation of substrate to the level sufficient to compete efficiently with the inhibitor, leading to full activity of the metabolic pathway involving the target enzmye.
RE: FdUMP effectiveness.
What was the rational behind the changes to the structure of CB3717 to make Raltitrexed
The CH2-C~CH group and benzene ring replaced with methyl group and thiophene ring was done to create a more water soluble, non-nephrotoxic and more potent drug.
Raltitrexed is an analogue of
mTHF and therefore cannot be incorporated into DNA.
How is Raltitrexed transported into the cell?
Reduced Folate Carrier (RFC)
What happens to Raltitrexed once it is in the cell?
It becomes polyglutamated via FPGS, this makes it 100-fold more inhibitory active and retains within cells for a much longer time.
The ability of Raltitrexed to compete with the mTHF cofactor at the enzyme site does not depend on what?
Does not depend on the formation of a covalent bond between the catalytic cysteine and the pyrimidine ring o the dUMP substrate - further drug study came from this.
Raltitrexed was first approved for treatment of
mCRC, metastatic colorectal cancer.
Who now uses Raltitrexed?
Only those who are intolerant to other therapies, such as 5-FU.
What causes Raltitrexed to become 100-fold more inhibitory?
Polyglutaminaton via FPGS.
What are pemetrexed and raltitrexed?
Antifolate metabolites
Inhibit TS
What is cytarabine?
How does it work?
Modified sugar nucleoside.
Converted into triphosphate inhibits DNA polymerase as it is an analogue of dCTP.
A sugar modified nucleoside that is converted to a triphosphate and inhibits DNA polymerase as it is an analogue of dCTP
Cytarabine (see also fludarabine)
_________ is a sugar modified ___________ that is converted to a __________ and inhibits ___ ____________ because it is an analogue of ________
Cytarabine is a sugar modified nucleoside that is converted to a triphosphate and inhibits DNA polymerase as it is an analogue of dCTP
What is fludarabine?
How does it work?
Fludarabine (Fludara ®) is a chemotherapy drug used to treat chronic lymphocytic leukaemia (CLL), acute myeloid leukaemia (AML) and some types of non-Hodgkin lymphoma.
Sugar modified nucleoside that is converted to a triphosphate and inhibits DNA polyermas as it is an analogue of dCTP.
What does 6-mercaptopurine look like?
Hypoxanthine.
Hypoxanthine is a naturally occurring purine derivative. It is occasionally found as a constituent of nucleic acids, where it is present in the anticodon of tRNA.
What are 4 lipophillic antifolate metabolites? why are they better?
They do not need reduced folate carriers.
Primethamime
Methylbenzoprim
Piritrexim
Nolatrexed
What are 3 antimetabolites that are classed as pyrimidine antagonists?
5-FU
FdURD
Azacytidine - chemical analogue of cytidine a nucleoside in DNA.
What are three antimetabolites that are sugar-modified nucleosides?
Cytarabine (ARa-C)
Fludarabine
Gemcitabine
Converted to triphosphates and inhibit DNA as analogues for dCTP.
What are pemetrexed and ralititrexed?
Antifoalte metabolites that inhibit TS
6-mercaptopurine inhibits which enzyme?
6-MP ribonucleotide inhibits purine nucleotide synthesis and metabolism by inhibiting an enzyme called Phosphoribosyl pyrophosphate amidotransferase (PRPP Amidotransferase). PRPP Amidotransferase is the rate limiting enzyme of purine synthesis. This alters the synthesis and function of RNA and DNA.
(The conversion of IMP to adenylic acid (AMP) via adenylosuccinate (SAMP).)
Thio-IMP is a metabolite of this.
What is nolatrexed? What is it licensed to treat?
How does it work?
Nolatrexed = lipophillic inhibitor of folate cycle.
Licensed: liver cancer.
Works: Inhibits DHF and TS
What are the order of products in the gemcitabine (f2dC) mechanism?
F2dC >dCK< F2dCMP >UMP/CMP kinase< F2dCDP >NDP kinase< F2dCTP
F2dCDP inhibits both CTP synthase and the next enzyme in the step ribonucleotide reducatase. Preventing formation of CTP and then dCTP.
Also, dCTP is a feedback inhibitor of the enzyme dCK, which turns F2dC into F2dCMP. Hence, depletion of dCTP activates dCK.
Activation of dCK increases the formation of F2dCMP from gemcitabine.
What is the relevance of the sulphur (instead of oxygen) in the structures of 6-MP and 6-TG?
Unable to form hydrogen bonds as it is not as electronegative as oxygen.
Both 6-MP and 6-TG bind where?
Purine binding site.
How does 6-MP inhibit DNA synthesis?
6-MP, used in the treatment of leukeamia, is an example of an allosteric inhibitor. It inhibits the first enzyme involved in the synthesis of purines and blocks their synthesis.
How does azacytidine work?
Pyrimidine antagonist.
Mimics cytidine, prodrug which is phosphorylated to azacytidine triphosphate and incorporated into RNA making it unstable and prone to decomposition.
What are the four main groups of antimetabolite drugs?
What are some examples?
Folate antagonists: methotrexate, non-classical lipophillic antifolates, pemetrexed, raltitrexed.
Pyrimidine antagonists: 5-FU, Fluorodeoxyuridine (FdURD) and azacytidine.
Purine antagonists: 6-Mercaptopurine, thioguanine, tiazofurin.
Sugar-modified nucleosides: Cytabarine, fludarabine, gemcitibine.
F2dCDP, the product of Gemcitabine, inhibits which two enzymes?
What is the outcome of this?
F2dCDP inhibits both CTP synthase and ribonucleotide reductase.
CTP synthase converts UTP into CTP.
Ribonucleotide reductase converts CTP into dCTP.
Gemicitabine self-potentiates: dCTP normally inhibits the activation of dCK, the first enzyme in the activation pathway of Gemcitabine that ultimately results in F2dCTP.
What is the role of microtubules?
Responsible for maintaining the structure of the cell and for separating the sets of chromsomes during mitosis.
What are three purine antagonists?
6-MP
Thioguanine
Tiazofurin
How are the purine antagonists activated?
6-MP —HPRT—> Thio-IMP —> Thio GMP.
Thio-GMP —–> Thio-GTP ——-> RNA
Thio-GMP ——> Thio-dGTP —-> DNA
6-TG —-HPRT—> Thio-GMP ——> As above.
How is GMP created?
At which stage in this process do the following work:
Thio-IMP (2 places)
TAD (from where?)
Thio-GMP
IMP is converted into AMP via the enzyme Adenylo-succinate synthetase.
Thio-IMP inhibits the Adenylo-succinate synthetase enzyme preventing this conversion. (1 place of action for Thio-IMP).
IMP is converted into GMP via IMP dehydrogenase into XMP and then via XMP-Gln amino-transferase into GMP.
Thio-IMP (second place of action) inhibits the enzyme IMP dehydrogenase by binding at the purine binding site. (Thio-GMP, from 6-TG, also works at this site).
TAD, from the now withdrawn drug tiazofurin, also inhibits the IMP dehydrogenase enzyme. It does this by binding at the NAD+ binding site.
What are the five stages of mitosis?
PPMAT
Prophase Prometaphase Metaphase Anaphase Telophase
What occurs during prophase? (3)
- Chromosomal material condenses to form a compact mitotic chromosome
- Cytoskeleton is disassembled and mitotic spindles assembled.
- Nuclear envelope disperses
What happens in prometaphase?
The microtubules attach to the centromeres of the chromosomes.
What happens in metaphase?
Chromosomes align along the metaphase plate.
What happens in anaphase?
Centromeres and chromosomes separate, spindle poles move further apart.
What happens in telophase?
Nuclear envelope assembles
What is a kinetochore?
Bit of microtubule that attaches to chromosome.
What is the spindle pole?
The position to which the chromosomes are to be pulled.
What is the kinetochore microtubule?
The bit that anchors the chromosomes to the centre of the spindle.
What is the astral microtubule?
They anchor the spindle poles to the membrane of the cell (one at each end)
What is a microtubule made up of?
Tubulin dimers
What are tubulin dimers made up of?
One alpha and one beta subunit.
Microtubules and individual tubulin dimers have what type of relationship?
They are in a dynamic equilibrium: Le Chateliers principle.
Mitotic spindle poisons ork principaly by interfering with the dynamic equilibrium between microtubules and individual tubulin dimers.
How do mitotic spindle poisons work primarily?
They interfere with the dynamic equilibrium that exists between microtubules and individual tubulin dimers.
What are 4 vinca alkaloids?
VinCRISTINE
VinBLASTINE
VinORELBINE
VinDESINE
Name 2 taxols
Paclitaxel (taxol)
Docetaxol (taxotere)
How does taxol and its derivatives work? [4]
- Taxols bind to taxol-binding sites on the inside surface of the microtubule, preventing disassembly.
- Inappropriate microtububles remain.
- Concentrations of free tubulin dimers decreases.
- The low concentration of free tubulin dimers means that new microtubules cannot be assembled.
What are the problems with taxol and its derivatives? [3]
- Very insoluble so have to be given IV.
- Hypersensitivity reactions due to the excipients it is formulated with.
- There is a correlation between increased solubility and decreased activity.
The better taxol is:
Docetaxel (semi-synthetic) has slightly better activity and a wider spectrum of activity.
Paclitaxel: mammary carcinoma, ovarian carcinoma.
Docetaxel: Prostate, mammary, ovarian and lung carcinoma.
Where does taxol come from and why is this a problem?
Pacific yew (Taxus brevifolia). It takes 3 trees to make one course of treatment for a patient.
How do the colchicine drugs work?
- Bind to colchicine binding sites on beta-tubulin subunits.
- This disfavours assembly of protofilaments.
- Also disfavours the DISASSEMBLY of inappropriate microtubules.
Two colchicine like drugs: Colchicine (gout)
Combrestatin a-4. (CT for cancer)
What is Combretastatin A-4?
Colchicine like drug in clinical trials for treatment of cancer.
Why were the lipophillic antifolates developed?
- Overcome the problems of methothrexate
2. Enter cell by passive diffusion - dont need RFC
Where do pemetrexed and ralitrexed bind?
The 5,10, CH2-tetrahydroglate binding site of TS.
Ralitrexed is transported into the cells by the RFC and then polyglutamated via ___________ becoming ____________. It binds _________.
FPGS (Folypolyglutamate synthase)
100 fold more inhibitory due to polyglutamated.
Binds non-covalently though.
What does pemetrexed bind to other than TS?
DHFR
AICARFT
GARFT
What is the major advantage that pemetrexed has?
IT binds with high affinity for several folate transporters in particular proton-coupled folate transporter (PCFT), avoiding RFC mutation resistance??
ALSO insensitive to DUMP accumulation.
Why was plevitrexed designed?
To be active without need for polyglutamation, so it can overcome resistance due to reduced FPGS enzyme expression.
Why is the TS reaction important? what type of reaction is it?
Reductive methylation.
dUMP -> dTMP
The sole de novo source of thymidylate, which is necessary for DNA replication and repair.
C-F bonds are equal to
C-H bond lengths, F wont form F+ as too electronegative.
How does the 5-FU metabolite work?
FdUMP binds to the NUCLEOTIDE-BINDING SITE of TS, forming a stable ternary complex with the enzyme and CH2 THF, thereby blocking the binding of the normal substrate dUMP and inhibiting dTMP synthesis.
5FU treats
BC and CRC
5FU is an analogue of ______ with what difference?
Uracil, F atom at pos 5 instead of H.
What are the resistance mechanisms to 5-FU?
TS can be salvaged from thymidine through the action of thmyidine kinase. This salvage pathway represents a resistance mechanism.
Also increased TS expression and increased DPD expression causes TS break down faster than normal.
What are the other mechanisms of 5-FU (other than TS irreversible inhibition)?
- Less dTMP leads to less dTTP which leads to a disruption of the ratios of all deoxynucleotides (especially the dATP/dTTP ratio) and leads to severe disruption of DNA synthesis and repair.
- Accumulation of dUMP leads to increased dUTP and both dUTP and the 5-FU metabolite FdUTP can become misincorporated into DNA. [Repair from UDG is futile in present of high (F)dUTP/dTTP].
- 5FU metabolite FUTP is extensively incorporated into RNA, disrupting normal RNA processing and function.
5-FU shows poor BA due to rapid degradation by to DHFU by DPD (the breakdown enzyme), how can this be alleviated?
Use DPD inhibitor, ENILURACIL in combination.
The loss of function of what molecule reduces cellular sensitivity to 5-FU?
p53
What are the anti-proliferative actions of azacytidine?
What is it?
Weak TS inhibitor.
Main action is to be incorporated into RNA instead of Cytidine.
Azacytidine is cytidine but with an extra N in the ring structure off the sugar. A triforce of ringbound Ns.
What is Hypoxanthine phosphoriboysltransferase?
HPRT: 6MP -> Thio-IMP
6TG -> Thio-GMP.
IT addds ribose and phosphate to both.
How does the structure of 6MP differ from that of hypoxathine?
IT has =S not =O at top of left ring.
hypoxathine is just guanine with no bottom left NH2 off the ring
Thio-GTP is incorporated into
RNA messing it up.
Thio-dGTP is incorporated into DNA messing it up.
How does the inhibition of the biosynthesis of purine nucleosides occur?
IMP —- adenylosuccinate synthase [inhibited by Thio-IMP] + adenylosuccinate lyase –> AMP
IMP –IMPDH [inhibited by TAD from tiazofurin, Thio-IMp and Thio-GMP]—–> XMP —— XMP-gln-aminotransferase—> GMP
What are the sugar modified nucleosides?
Cytabarine (Ara-C)
Fludabarine
Gemcitabine bae
What is ‘masked chain-termination’?
When dFdCTP is incorporated into DNA, a single deoxynucleotide is incorporated afterwards, preventing chain elongation.
Hence, this non-terminal position of gemcitabine makes DNA polymerase unable to proceed and is called masked chain termination.
Gemcitabine also activates p38 mitogen-activated protein kinase (MAPK) to trigger apoptosis in response to cellular stress in tumour cells, but not in normal cells.
Gemcitabine activates what causing apoptosis?
p38 Mitogen-activated-protein kinase (MAPK)
