MITOTIC SPINDLE POISONS- PHARMACOLOGY {S1}

MITOTIC SPINDLE POISONS- PHARMACOLOGY {S1} BY RANJEET RAMAN

Spindles made of dynamic microtubules. Polymerization requires GTP. Positive ends more dynamic than negative ends. • Microtubules used for axonal transport, which is why these poisons have serious neurotoxicity.

Spindle poisons in vitro: Vinca drugs destabilize microtubules, Taxanes stabilize Spindle poisons in vivo: both Vinca and Taxanes slow polymerization dynamics

Vinca drugs • In vitro they depolymerize MTs but in vivo they decrease the dynamics of spindle MTs. Their serious toxicity is neurotoxicity which affects motor and sensory functions. • Vinblastine binds β tubulin and disturbs its polymerization. Treats lymphomas. • Vincristine treats acute leukemias and lymphomas. • Vinorelbine treats solid breast and lung tumors.

Taxanes • In vitro they polymerize MTs but in vivo they’re just like Vinca. Decrease spindle dynamics. Their serious toxicities are Myelosupression and peripheral neurotoxicity.

Mechanisms of Drug Resistance • 1. Multidrug resistance involves outward organic transporters such as MDR1 and drug sequestering proteins such as MRP1. • MDR1 is part of the ABC pump super family. • MPR1 is a glutathione/drug cotransporter found on both plasma and ER membranes. It is very good at pumping out Topo II poisons.

2. Breast cancer resistance protein (BCRP) gives huge resistance to Topo I and II poisons. • 3. Atypical drug resistance in which a mutation changes the drug binding site, preventing the drug’s effects. Many examples with Topo I and II drugs.

Topoisomerase-Targeting Drugs • Topoisomerase • A normal enzyme in the nucleus that catalyzes reversible substitutions of the DNA backbone using tyrosine as the nucleophile. The linkage preserves the energy of the original linkage. This process allows DNA to relieve supercoil strain.

All topoisomerase form covalent enzyme/DNA intermediates (keeps the DNA strands together). • Type I topoisomerase are monomeric, using a single tyrosine to cleave one DNA strand. Removes one supercoil at a time. These require no ATP. • Type II topoisomerase are dimeric, using two active site tyrosine's to cleave both DNA strands, forming a gate. Removes two supercoils at a time. These require ATP.

Topoisomerase-Targeting Drugs • Drugs against Type I and Type II Topoisomerase stabilize the enzyme’s covalent complex with DNA. Drugs against Type I intercalate and block ligation of the strands, and the enzyme gets stuck. • Type II poisons are sensitive to amount of ATP.

Stabilizing the covalent complex causes double strand break, inducing apoptosis. This occurs in the S phase for Type II poisons, and in G2 for Type I poisons • Both types, but especially Topo II poisons, eventually have resistance by cancer cells.

Topo I targeted drugs • Camptothecin, topotecan, irinotecan: These intercalate and stabilize the covalent complex. Used to treat solid tumors, such as in colorectal and ovarian cancer. Major toxicities include Myelosupression, alopecia. • Topotecan has a lactone ring that opens at acid pH, inactivating it. This is a limitation. • Irinotecan is a prodrug that must be metabolized.

Minor-Groove DNA binding drugs: Induce DNA bending in the minor groove because of their crescent shaped molecules. Bending stops Topo I. These drugs are S phase specific, and augment effect of other Topo I drugs. • Actinomycin D: Intercalates due to ring structure. Probably targets RNA polymerase, not Topo I. • Very potent against childhood malignancies such as Willm’s tumor.

Topo II targeted drugs • Anthracyclines such as Doxorubicin, Daunorubicin: Intercalate, stabilizing Topo II. Unusual toxicities include potent cardio toxicity and liver damage from free radicals generated during metabolism. • Doxorubicin for solid tumors such as breast cancer and lymphomas -Daunorubicin for acute leukemias -Mitoxantrone has somewhat less cardio toxicity • DNA Topoisomerase-Targeted Drugs and Mitotic Spindle Poisons.

TOPOISOMERASE • Enzyme in the nucleus that catalyzes reversible sub. of the DNA backbone using tyrosine as the nucleophile .The phosphoryl-tyrosyl linkage preserves the energy of the original linkage and prevents losing the DNA end This process allows DNA to relieve supercoil strain. • All topoisomerase form covalent enzyme/DNA intermediates (forms basis of Topo-targeted drug inhibition)

Type I = monomeric, uses one tyrosine to cleave one DNA strand. Removes one supercoil at a time. (no ATP) Type II = dimeric, uses 2 tyr to cleave both DNA strands, forms a gate. 2 supercoils removed. (ATP & Mg2++.) • TOPOISOMERASE-TARGETING DRUGS • Drugs against Type I and Type II Topo stabilize the enzyme’s covalent complex with DNA. • Type I topoisomerase drugs block ligation of strands and enzyme gets stuck by one of two ways

(1) DNA intercalation (ex: Camptothecin “CPT”) = Direct block of ligation • (2) DNA bending (ex: minor groove binding agents ) =Indirect thru DNA distortion Type II topoisomerase drugs not as understand but likely similar method but are sensitive to amount of ATP.

Mechanism of Killing by Drugs • (1)Stabilizing covalent complex DNA replication fork arrest double strand break apoptosis This occurs in the S phase for Type II poisons, and in G2 for Type I poisons! • (2) Arrest of RNA synthesis at elongation step also leads to apoptosis Both types, but especially Topo II poisons, eventually have resistance by cancer cells.

TOPO I TARGETED DRUGS • Camptothecin, topotecan, irinotecan (CPT-11): E2 derivative • Mech: intercalation mechanism and stabilize the covalent complex by direct block of ligation -Topotecan has a lactone ring that opens w/OH , inactivating it. pH dependent --Irinotecan is a prodrug that must be metabolized.

Uses: Treat solid tumors, such as in colorectal (irinotecan) and ovarian cancer (topotecan, but not that eff.). • Toxicity: Myelosupression (neutropenia), hair loss, fatigue, alopecia, diaherea • Irinotecan can also cause liver toxicity in patients w/Gilbert disease (↓ gluco conjugation]

Minor-Groove DNA binding drugs: • Mech: Induce DNA bending in the minor groove (AT rich) b/c of crescent shaped molecules and +ve charge Bending indirectly Topo I (↑complex of Topo w/DNA also effects on replication/transcription) Genomic targets unknown These drugs are S phase specific, and augment effect of other Topo I drugs. • Uses: Still in vitro stage

Actinomycin D: (2 cyclic peptide w/aromatic ring) • Mech: Intercalates due to ring . Probably targets RNA polymerase, not Topo I. Genomic targets unknown Phenoxazone intercalates at GpC and cyclic peptide binds minor grooves (H bonds to guanine base) • Uses: Against childhood malignancies such as Willm’s tumor, Ewings sarcoma, embryonal Rhabdosarcoma • Toxicity: The usual = Myelosupression, hair loss, oral and gastrointestinal ulceration

TOPO II TARGETED DRUGS • Epipodophylotoxin • Mech: No intercalative, indirect thru unknown structural perturbation • Uses: Many types of Cancer • Toxicity: Myelosupression, mucositis, nausea, anaphylaxis

**Anthracyclines such as Doxorubicin, Daunorubicin, Epirubicin: • Mech: Intercalate, stabilizing Topo II complex (direct or indirect) • Uses: -Doxorubicin for solid tumors such as breast cancer and lymphomas, Hodgkins, soft tissue sarcoma

Daunorubicin for acute leukemias • Toxicity: Potent cardio toxicity and liver damage from free radicals generated during metabolism. • Quinoids = redox active (ex: DNA damage and lipid and protein per oxidation) LIMITS DOSAGE • Also the usual Myelosupression, mucositis, nausea, anaphylaxis

Others: Aminoacridines,ellipticines, anthracences (≈ mech.& tox. as anthras but Mitoxantrone has ↓cardiac tox • BASIC MECHANISTIC PRINCIPLES OF MITOTIC SPINDLE POISONS -Spindles mediate chromosome segregation during mitosis and meiosis -Spindles made up of dynamic microtubules (w/ a and b tubulin) w/polarity -Polymerization and Depolymerization of MT involves GTP binding and hydrolysis -Since lots of tubulin in neurons for axonal transport, get neurologic toxicity of tubulin-binding agents -Spindle Poisons slow MT Polymerization Dynamics

1) Bind Intact Tubules2) Bind Free Tubulin Dimers • Vinca Alkaloids (from the periwinkle plants) “The three Vinnys” • Uses: Mostly blood disorders but also for some solid tumors Vincristine for acute lymphocytic leukemia (ALL), lymphomas, Hodgkins, childhood malignancy Vinblastine for germ cell tumors and Hodgkins Disease Vinorelbine for lung and breast cancers

Toxicity: Liver toxicity = significant metabolism in liver • Neurotoxicity = (esp w/vincristine) motor sensory, autonomic and cranial nerves affected (limits) • Myelosupression limits the dosage w/vinblastine and vinorelbine?Taxol mimics a loop absent in B-tubulin thus providing rigidity and dynamic equilibrium?

Taxanes (from Pacific yew tree) • Mech: Bind B-tubulin to stabilize lateral tubulin contacts • Use: Paclitaxel for ovarian and breast cancer: Docetaxel for metastatic breast cancer • Toxicity: Myelosupression and peripheral neuropathy (paclitaxel > docetaxel) • BIOCHEMICAL MECH OF ANTINEOPLASTIC DRUG RESISTANCE (Drug Efflux/Sequestration + Atypical Drug resistance (alter E or target site) + Metabolic Detox)

1) Multi-Drug Resistance (MDR) • through cellular drug efflux (MRP/Pgp) or IC redistribution of cytotoxic agents away from side of action (MRP) affected by ATP-dependent transporter proteins (ABC super family) • i) MDR1 (P-glycoprotein “P-gp”)for irinotecan, anthracyclines, vinnies etc

ii) MDR associated protein (MRP1)=ATP dependent organic ion transporter (pumps out –ve drugs) =Acts as glutathione drug cotransporter Found in PM and ER so it can kick drug out and sequester it away Usually works on Topo II inhibitors, Daunorubicin/etoposide

iii) Breast cancer resistance protein (BCRP) -great resistance to mitoxantrone, daunorubicin, doxorubicin -cross-resistance to topoisomerase I inhibitors (not taxanes, vinnies, or cisplatin) • iv) Lung Resistance-related protein (LRP)↓ N: C ratio of drugs or ↑ entrapment of drugs in vesicles -resistance to doxorubicin, vincristine, etoposide and paclitaxel

2) Atypical Drug Resistance • mutation of drug binding site, ↓gene expression (ex: topos), proteolysis of target protein, -↓ enzymatic activity needed for activation of prodrug (ex: carboxyl esterase activation of irinotecan)

MITOTIC SPINDLE POISONS- PHARMACOLOGY {S1}