W8 Anticancer drugs l (SF)

Question 1

Cytotoxic or chemotherapeutic drugs
Most traditional anticancer drugs working by disrupting the function of DNA.. How? (3)

Answer 1

1) Act on DNA directly
2) Act indirectly: inhibition enzymes involved in the synthesis of of DNA itself
or of nucleotide building blocks
3) Act on microtubule dynamics involved in mitosis

Question 2

What are the 3 main classes
of cytotoxic drugs?

Answer 2

1-Drugs targeting DNA structure & template activity
2-Antimetabolites: target enzymes involved in the synthesis of DNA itself or
of nucleotide building blocks
3-Mitotic arrest agents target microtubules

Question 3

Sites of actions of cytotoxic drugs:

Question 4

Cytotoxic drugs effects related to the cell cycle

Answer 4

Active at specific points in the cell cycle
* Antimetabolites: S phase
(synthesis of nucleosides)
* Intercalators and topoisomerases:
end of G1, during S and early G2
(DNA strands are being used for
synthesis of DNA)
* Mitotic inhibitors: M phase- vinca
early -taxoids late
* Alkylating agents: attack DNA,
single or double stranded throughout the whole cell cycle

Question 5

DNA Alkylating and Crosslinking Agents:
What are the features?

Answer 5

o Highly electrophilic compounds: react with nucleophilic groups on DNA (mainly the N-7 position of deoxyguanylates) → strong covalent bonds
* 2 alkylating groups → covalent cross-linking of adjacent strands of DNA
(inter-strand cross-linking) → disrupted replication/transcription → cell tries
unsuccessfully to repair → cell cycle arrest → apoptosis
o Organoplatinum: binding to adjacent guanine nucleotides on a single strand of
DNA → intra-strand DNA cross-linking
o Higher effect in tumour cells: divide more rapidly (high DNA synthesis)
o All phases of the cell cycle are susceptible (not cell cycle specific) but they are more toxic in late G1 or S phases (DNA is unwinding and exposing nucleotides)

Question 6

Issues with DNA Alkylating and Crosslinking Agents? (3)

Answer 6

o Most alkylating agents require chemical or enzymatic activation
o Poor selectivity: react with nucleophilic groups on proteins as well
o Can damage DNA of healthy cells: intrinsically mutagenic and carcinogenic!

Question 7

Five main groups that form DNA Alkylating and Crosslinking Agents? (for info?) he skipped

Answer 7

1. Nitrogen Mustards
2. Nitrosoureas
3. DNA methylators
4. Organoplatinum complexes (metallating agent)
5. Miscellaneous DNA alkylators
   All frequently include in combination therapy

Question 8

DNA Alkylating and Crosslinking Agents:
Effects of alkylating agents on DNA

Answer 8

• Crosslinked guanine bases =DNA replication impaired
• Guanine N7 alkylation can give an enol
  tautomer which can bind to thymidine = Mismatching of bases- leading to defective coding of proteins
• Guanine N7 alkylation can cause cleavage of imidazole ring = Excision of guanine residue leading to DNA breakage

Question 9

DNA Alkylating and Crosslinking Agents
1. Nitrogen Mustards
Structure:

Answer 9

R= can be either aliphatic or aromatic; prime determinant of chemical reactivity, oral bioavailability, nature/extent of adverse effects

Chlorine atoms: decrease N basic strength through a strong negative inductive effect → unionised drug predominates at physiologic pH → lone pair of electrons on N allows for the formation of the highly electrophilic aziridinium ion, which is
the reactive DNA-destroying intermediate

e.g. Chlormethine Chlorambucil Cyclophosphamide

Question 10

Nitrogen Mustards: MoA

Answer 10

1. Intramolecular nucleophilic displacement
2. Highly electrophilic azirdinium ion
3. Mono-alkylation
4. Repeat for the second Cl

Aziridinium ion (reactive 𝝱-carbon)
-strained three membered ring
-strong negative inductive effect of the cationic nitrogen

Mono-alkylated
lone pair on the mustard N is regenerated

Di-alkylation (bifunctional) (guanine N-7): cross-linking between DNA chains or within same chain → DNA strands cannot separate nor replicate, transcription of DNA to RNA is halted

Question 11

DNA Alkylating and Crosslinking Agents
Nitrogen Mustards: the R group is
important

Answer 11

• (Chlormethine) CH3 → electrons to the amine → enhances N nucleophilicity → very reactive (tumour and healthy cells) with unpaired DNA and other cell
  nucleophiles (SH, OH and NH of amino acids, and H2O body) → no tissue or cell specificity → increased risk of serious adverse effects and use-limiting toxicity Too reactive for oral route
• (Chlorambucil) Phenyl → stabilise the lone pair through resonance →
  significantly slows the rate of intramolecular nucleophilic
  attack, aziridinium ion formation, and DNA alkylation. Reactivity sufficiently controlled to permit oral administration, attenuate the severity of adverse effects, enhanced tissue selectivity

Question 12

Nitrogen Mustards: Cyclophosphamide
Is what type of drug

Answer 12

Chiral prodrug: requiring activation by metabolic and nonenzymatic processes

See mechanism on slide

Question 13

DNA Alkylating and Crosslinking Agents
Nitrogen Mustards: Cyclophosphamide
Toxic effects?

Answer 13

• Most common alkylating agent in cancer chemotherapy, oral administration (or i.v.), for leukaemias, limphomas, sarcoma, solid tumours
• Metabolic activation in the liver: lowered GI toxicity and less non-specific toxicity

Toxic effects:
* CYP3A4/CYP2B6: inactivate cyclophosphamide by N-dechloroethylation → chloroacetaldehyde formed → electrophilic alkylates Cys residues of critical cell proteins→ highly nephrotoxic and neurotoxic
* Acrolein: very electrophilic/highly reactive species→ generated in liver, readily conjugates with GSH→ delivered to the bladder for excretion → extensive
damage to cells of the kidney and bladder. If low GSH → acrolein will be attacked by the SH of bladder cell Cys residues → haemorrhagic cystitis

Question 14

Nitrogen Mustards: Cyclophosphamide
To minimise the risk of haemorrhagic cystitis what is given?

Answer 14

Mesna as adjuvant therapy
(active sylfhydryl reagent)

Question 15

DNA Alkylating and Crosslinking Agents
Nitrosoureas: MoA

Answer 15

Chloroethyl-containing nitrosoureas (CNUs)
Decompose spontaneously in the aqueous environment of the cell → active species…

Cross-linking of N1-Guanine-N3 Cytosine
Alkylatkion with O6 guanine
Carbamoylation of Lys residues

Highly lipophilic drugs → cross BBB → treat Brain tumour

See Mechanism on slide

Question 16

DNA Alkylating and Crosslinking Agents:
DNA methylators: triazenes (temozolomide and dacarbazine) and procarbazine

Why does alkylation disrupt DNA?

Answer 16

Prodrug: after metabolic oxidation (CYP450), generate a reactive methyldiazonium ion → DNA/RNA methylation (guanine N-7 or O6)

• Alkylation results in abnormal H-bonding
• O6-methyl or methyl N-7 guanosine, → alkyl group disrupts inter-strand hydrogen bonding, destabilizing DNA leading to cell death.

IV administration, used in combination therapies for melanomas and sarcomas

Question 18

Intercalating Agents
Examples?
Structure?
Features?

Answer 18

• Mainly antibiotics of microbial origin: doxorubicin, daunorubicin, bleomycin, etc
• Act in late G1, S and early G2 phases (DNA strands used for DNA synthesis)
• Planar aromatic or heteroaromatic ring system: insert into the space between successive nucleotide base pairs disrupting the double Helix of DNA → inhibit the enzymes involved in the replication and transcription processes,
  impairing cell division and triggering apoptosis

Binding reversible: stabilised by hydrophobic interactions (pi-stacking) between the opposing aromatic rings of the drug & adjacent DNA bases
Sugar amino group: essential for activity interacts with DNA (H-bond) May
interact/inhibit topoisomerase
enzymes (DNA unwinding/replication)

Question 18

Intercalating Agents
Synthetic drugs: rationally designed Doxorubicin analogues- Mitoxantrone

Answer 18

• Simplified anthracycline analogue
• No sugar ring: easy to synthetise (amino group still present)
• Less active than doxorubicin
• Less cardiotoxic than doxorubicin :sugar ring: thought to be responsible for cardiotoxicity
• For leukaemia, lymphomas, advanced breast cancer

Pharmacophoric features:
1. Overlapping planar region
(planar acridine system)
(intercalation)
2. Sugar amino (Dox) and NH (Mito) in
good alignment
3. OH groups in similar position
4. The scaffold in mitoxantrone contains the planar ring system and positions the OH and NH similarly to doxorubicin

Question 19

DNA Alkylating and Crosslinking Agents

4. Organoplatinum complexes (metallating agents)
   Examples?

Answer 19

Cisplatin, Carboplatin, Oxaliplatin

Central Pt with 2 EWGs and 2 ammonia
ligands: neutral and unreactive structure
Pt: electron-deficient metal reacts with
electron-rich DNA nucleophiles

-Bifunctional: accept electrons from two DNA nucleophiles
-Intra-strand cross-links AND Inter-strand cross-linking

1.(Cisplatin is) Neutral and unreactive
2. Inside cells: low [Cl-] → water is (abundant) displaces 1 or both Cl
3. Positively charged, reactive compounds
4. Covalent Pt-DNA links

Question 20

Topoisomerase inhibitors

Answer 20

Topoisomerase I (topI): cuts and re-ligates a single DNA strand
Topoisomerase II𝛼 (topII): cuts and re-ligates a double DNA strand
Cleavage (cut): transesterification reaction. Repair: reverse transesterification

Topoisomerase poisons: stimulate the DNA cleavage reaction BUT inhibit the DNA resealing activity of the enzymes, leaving the DNA irreversibly damaged and unable to replicate

• Act in late G1, S and early G2 phases (DNA strands used for DNA synthesis)
• Camptothecins
• Epipodophyllotoxins
• Anthracyclines and related anthracenediones (family of Doxorubicin)

Question 20

Topoisomerase inhibitors
What is the role of Topoisomerase in DNA replication?

Answer 20

1. Normal DNA
2. Supercoiled DNA: no longer accessible to the enzymes
3. Topoisomerase:
   * Binds
   * Cuts strand
   * Strand rotates
   * Reanneals strand
4. removal of torsional stress such as unknotting & relaxation
5. Normal DNA restored

Question 21

Topoisomerase I inhibitors: Camptothecins

Answer 21

o Water-insoluble natural conjugated pentacyclic lactones
o MoA: stabilise a covalent DNA–topoisomerase bond at the
point of single-strand breakage; sterically keep topI from catalysing
the DNA re-ligation reaction → NO resealing!!!

• Binding pocket: in the DNA strand→ revealed only after the normal DNA nicking has occurred→ preferentially bind to the enzyme–DNA complex

Question 22

Topoisomerase II𝛼 inhibitors: Epipodophyllotoxins

Answer 22

o Semisynthetic glycosidic derivatives of podophyllotoxin (mayapple plant)

Topoisomerase II principle function:
catalyse the separation of daughter
DNA strands just prior to mitosis

Drug: stabilizes enzyme-DNA complex inhibiting the annealing process → DNA double strand breaks not amenable to repair

Question 23

Antimetabolites

Answer 23

Suppress de novo DNA biosynthesis by: * inhibiting the enzymes involved in the synthesis of the nucleotides * inhibiting other enzymes required in DNA biosynthesis * arresting chain elongation: incorporation of false nucleotides into the growing DNA strand “Super attractive version” of the normal substrate (false substrates)→ * bind enzymes irreversibly or pseudoirreversibly * nucleotides cannot be synthesised, or DNA polymerisation is blocked * DNA synthesis is stopped→ apoptosis Work in S phase of the cell cycle only (synthesis DNA is active) * Purine antagonists and Pyrimidine antagonists * Antifolates * DNA polymerase and chain elongation inhibitors * Miscellaneous antimetabolites (DNA methyltransferase, adenosine deaminase inhibitors, ribonucleotide reductase inhibitors)

Question 24

Purine antagonists

Answer 24

Inhibit synthesis of adenosine monophosphate (AMP and dAMP) and guanosine monophosphate (GMP and dGMP) * inhibit **different enzymes involved in purine synthesis** (complex effect→ AMP and GMP biosynthesis halted) * incorporation of di- (ADP, dADP, GDP, dGDP) and triphosphate (ATP, dATP, GTP, dGTP) generated within the tumour cell into DNA and RNA→**inhibits further strand elongation →cleavage, cell-cycle arrest and apoptosis** Drugs: 6-thiol analogues of purine 6-Mercaptopurine 6-Thioguanine Two anticancer prodrugs: once converted to the nucleoside monophosphates (cellular enzymes):

Question 25

Purine antagonists

Answer 25

* Both drugs are administered orally and used for the treatment of leukaemias. Both are more effective in children than adults * Tox: myelosuppression, immunosuppression, GI distress. Methylated (-SCH3) metabolites induce potentially fatal hepatotoxicity requiring drug discontinuation * Xanthine oxidase inactivates 6-mercaptopurine (but not thioguanine) Allopurinol inhibits xanthine oxidase → increases levels of mercaptopurine nucleoside monophosphate→ co-administered with mercaptopurine increases duration of action and potency. Mercaptopurine dose must be cut to **25%-33% of the standard dose to avoid serious toxicity** **6-Mercaptopurine is usually given with Allopurinol**

Question 26

Pyrimidine antagonists:

Answer 26

Crucial step DNA synthesis: synthesis of **deoxythymidine monophosphate** (dTMP) from deoxyuridine monophosphate (dUMP). **Thymidylate synthase** catalyses the transfer of a methylene group from the essential cofactor N5,N10-methylene-tetrahydrofolate to dUMP, giving dTMP

Question 27

Pyrimidine antagonists

Answer 27

All dTMP synthesis inhibitors: inhibit thymidylate → “thymineless death” in actively dividing cells→ DNA will fragment, and the cell will die Most common- 5-Fluorouraclin (5-FU) * Fluorinated pyrimidine prodrug: must be converted to its deoxyribonucleotide (dFUMP) →binding thymidylate → presence of F (EWG)→ very stable ternary complex formed →cannot break down, no thymidine-based product formed, no folate cofactor released, thymidylate enzyme inhibited→ DNA irreversibly damaged (irreversible inhibitor) * Secondary MoA: incorporation of the false triphosphorylated ribonucleotide (5-F-UTP) into RNA, a chemical deal breaker for RNA viability

Question 28

Antifolates

Answer 28

* Purine and Pyrimidine biosynthesis requires a cofactor called folic acid * Folic acid derivatives are carriers of one carbon units

Question 29

Antifolates: DHFR

Answer 29

Dihydrofolate Reductase Inhibitors (DHFR) * DHFR: vital enzyme in the biosynthesis of the nucleoside thymidine * Only source of thymidine is the folate mediated addition of methyl to deoxyuridine monophosphate * Inhibition of this step deprives the cell of thymidine and leads to cell death Methotrexate

Question 30

Methotrexate

Answer 30

* Folic acid antagonist * Compete with 7,8-DHF for DHFR * DHFR direct inhibition → cellular levels of 7,8-DHF to rise, → **feedback (indirect) inhibition of thymidylate synthase** * NH2-makes difficult the reduction * Also inhibits glycinamide ribonucleotide (GAR) formyltransferase (synthesis of purine) =DNA synthesis is blocked

Question 31

Methotrexate: Used to treat??

Answer 31

Orally in the treatment of breast, head and neck, and various lung cancers as well as in non-Hodgkin lymphoma (NHL) * Tox: lung disease, severe GI adverse effects * Severe methotrexate toxicity occurs: reduced folate replacement therapy with 5-formyltetrahydrofolate (leucovorin) → generates the folate cofactors needed by DHFR and GAR formyltransferase →continued synthesis of pyrimidine/purine nucleotides in healthy cells * Cancer cells resistant to methotrexate over time: increased expression of DHFR and other enzyme targets, active cellular efflux by P-gp

Question 32

DNA polymerase and chain elongation inhibitors

Answer 32

DNA polymerases catalyse DNA synthesis from the 4 deoxyribonucleotide building blocks Six halogenated and/or ribose-modified DNA nucleoside analogues are marketed for the treatment of a wide variety of hematologic cancers and solid tumors Complex and multifaceted mechanisms; * Competitive inhibition of DNA polymerase * Chain Terminators * Prevent replication of modified DNA Nucleosides (not active) → actively transported into tumor cells → converted to triphosphorylated nucleotides by specific kinases →incorporated into the growing DNA chain→ arresting further elongation T Tumors deficient in the nucleoside transporter: resistant to these anticancers Secondary MoA: mono and diphosphorylated forms also inhibit the biosynthesis of essential deoxyribonucleotides

Question 33

DNA polymerase and chain elongation inhibitors Purine analogues

Answer 33

* All drugs (except one) administered IV; excreted predominantly via the kidneys * Tox: myelosuppression * Resistance: loss of functional nucleoside transporter proteins and deoxycytidine kinase enzymes → causes of acquired resistance to DNA polymerase inhibitors Purine analogues Fludabarine, Cladribine, Clofarabine\ Halogenated adenosine-based nucleosides→ transport into tumor cells→ conversion to the active triphosphate nucleotides→ competitive pol inhibitor, chain terminator + prevents replication. Used for leukaemias (IV)

Question 34

DNA polymerase and chain elongation inhibitors Pyrimidine analogues

Answer 34

Cytarabine (Ara C) Gemcitabine Trifluridine Pyrimidine-based anticancers: initial phosphorylation to the monophosphate required→MP→DP→TP (active form)→competitive pol inhibitor, chain terminator + prevents replication. Cytarabine: used for various leukemias (IV) Gemcitabine: analogue of cytarabine, fewer side effects. Used for pancreatic, lung, breast cancer (IV) Trifluridine: only orally administered DNA polymerase inhibitor

Question 35

Mitotic arrest agents

Answer 35

Mitotic process depends on the structural and functional viability of microtubules: polymeric heterodimers consisting of of α- and β-tubulin Tubulin subunits: lie adjacent to one another and roll up to form an open, pipe-like cylinder (microtubule). One end of microtubule is anchored to an organising center (aster); the other end is either growing or shrinking During cell division: tubulin alternatively polymerize (growth) and depolymerize (erosion) “dynamic instability.” Polymerization: addition of tubulin dimers to either end of the tubule→ tubular elongation; Depolymerization →structural shortening. Allow formation of the mitotic spindle, attachment of chromosomes →cell division

Question 36

Mitotic arrest agents What are the two general chemical classes of mitosis inhibitors?

Answer 36

* Taxanes: e.g. taxol. Bind with high affinity to polymerized microtubules of the mitotic spindle, the interaction is reversible. They bind to 𝝱-tubulin at specific amino acid residues→ inhibit disassembly of the spindle * Vinca alkaloids; e.g. vincristine. Bind to the dimers of 𝝰 and 𝝱 tubulin subunits preventing the build up of the polymerised tubulin -formation of spindle inhibited =ALL block M phase of cell cycle

Question 37

Molecular targeted therapeutics: What is the goal of targeted chemotherapy? Examples of new chemotherapy?

Answer 37

“Traditional” cancer drugs target DNA (cytotoxic drugs). Major problems: dose limiting side effects, resistance, poor selectivity, patient compliance, QoL In the last 20 years: better understanding of the molecular biology of cancer has greatly increased→ **targeting proteins rather than DNA**→ greater structural variety→ potentially more attractive for drug design Goal of targeted chemotherapy: sidestep the nonspecific toxicity inherent in cytotoxic chemotherapy by **directing drugs selectively to the genes or proteins within cancer cells** that mediate uncontrolled growth * Anti-hormonal therapy * Epigenetic inhibitors * Kinase inhibitors * PARP inhibitors (targeting repairing mechanisms) * Proteasome inhibitors * Many others