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Pharmacology Dr. James T. Stivers, Ph.D.

Topoisomerase-Targeted Anticancer Drugs

You should concentrate on the following key points in this lecture (BOLD = KNOW THIS DRUG!) I. Topoisomerases and their Drugs

1. How type I and II topoisomerases work, and how they differ from each other. 2. How the enzymatic mechanism relates to the mechanism of drug action. 3. The major drug classes that target topoisomerases and their clinical uses and

common toxicities (topotecan, irinotecan, actinomycin D, etoposide, teniposide, doxorubicin, daunorubicin).

Outline I. Overview of Topoisomerase Mechanism and Cellular Function

A. Type I topos B. Type II topos

II. Mechanisms of DNA Topoisomerase-Targeted Anti-cancer Drugs III. Topoisomerase-Targeted Drugs Used to Treat Human Cancers

A. Topo I-Targeted Drugs i. camptothecin and its derivatives ii. Actinomycin D

B. Topoisomerase II-Targeted Drugs i. epipodophylotoxins ii. anthracyclines iii. others

I. Overview of Topoisomerase Mechanism and Cellular Function

All topoisomerases catalyze reversible nucleophilic substitution reaction at the phosphodiester backbone of DNA using an active site tyrosine as the nucleophile. The covalent phosphotyrosyl linkage preserves the energy of the initial phosphodiester linkage and prevents losing the end of the DNA.

All topoisomerases form covalent enzyme-DNA intermediates that form the basis for inhibition by many topo-targeted drugs.

Topoisomerases come in two general types: Type I: Monomeric enzymes that use a single tyrosine to cleave only one strand of the DNA without any ATP requirement (see Type IB mechanism below). Type II: Dyadic enzymes that use two active site tyrosines to cleave both strands of the duplex DNA, and require ATP and Mg2++ to do so.

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p-Tyr

linkage

O

HN

N

O

O

O

PO

O-DNA

DNA

O-

OH

O

HN

N

O

O

O

PO

HO-DNA

DNA

O-O

cleavage

ligation

5'

5'

cleavage

ligation

5'

5'

O

HN

N

O

O

OH

DNA5'

PO

O-O

O

DNA

3'

Type IB

Type II, IA

Topo

Topo

Topo

Mechanism of Type IB Topoisomerase (e.g. the human enzyme)

Mechanism of Type II DNA Topoisomerases

Dyadic enzyme with two active site tyrosines

Staggered double strand cleavage reactions to generate a DNA gate ATP hydrolysis is required and drives enzyme conformational changes

Two supercoils removed per cleavage event (duplex transport through gate)

General Chemical Mechanism

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Type II Topisomerase Mechanism

Cellular Function

Topoisomerases are required to remove superhelical strain that would otherwise accumulate during various essential DNA transactions such as replication, transcription, chromosome condensation and segregation and recombination.

The covalent linkage serves to keep the ends of the DNA together so that double strand breaks or DNA recombination reactions are minimized.

A DNA

duplex can

pass

through

this gate.

Action of Topo I on supercoiled plasmid DNA

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Example: Camptothecins Example: Minor groove binding agents

II. Mechanisms of DNA Topoisomerase-Targeted Anti-cancer Drugs

The primary mode of action of current Type I topoisomerase drugs is to stabilize the covalent complex by one of two general mechanisms:

The mechanism of type II topoisomerase drug action is less well understood, but likely involves stabilization of the covalent complex by similar mechanisms as that shown above.

Proposed Cell Killing Mechanism of Topo

Poisons

Stabilization of the covalent complex leads to DNA replication fork arrest and double strand breaks in the DNA that induces cell death or the apoptotic response (S phase specific).

Topo I drug complexes cause G2 cell cycle arrest

Arrest of RNA synthesis at the step of elongation also occurs and may also induce apoptosis

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III. Topoisomerase-Targeted Drugs Used to Treat Human Cancers A. Topo I Targeted Drugs

i. Camptothecin and its derivatives topotecan and irinotecan (CPT-11)

Mechanism: Acts by an intercalation mechanism to stabilize covalent complex by directly blocking ligation (see above). (Irinotecan is a prodrug that must be activated by carboxyesterase.) Clinical Uses: Generally effective treatment for solid tumors. First line treatment in colorectal cancer in U.S. and Europe. Currently, 5-FU/irinotecan combination therapy for advanced colorectal cancer is the state-of-the art. Also used for ovarian, lung and other adult malignancies.

General Resistance Mechanisms for Topo I Drugs

o Mutations in Topo gene, or down regulation of enzyme expression o Over expression of multidrug resistance efflux pump o Sequestration in non-target compartment

Specific Resistance Mechanisms

Topo adduct removal by proteolysis, then tyrosine phosphodiesterase

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Topo adduct removal by endonuclease action

General Toxicities: myelosuppression (neutropenia), nausea, hair loss, and fatigue. Specific Toxicities: Irinotecan may cause liver toxicity in patients deficient in gluconconjugation (Gilberts disease) which is the primary elimination route. Early onset and late onset diarrhea is also common. ii. Minor Groove DNA-Binding Drugs Actinomycin D A nautral product derived from Streptomyces that is composed of two cyclic peptides and a phenoxazone ring system: Mechanism: Likely increases the amount of covalent complex by inducing DNA bending or other structural perturbations (see above). Recent data suggests the major target of this drug in cells is likely to be RNA polymerase and not topo I. The precise genomic target is not known.

Binds with high affinity to 5 GpC sites in DNA. The phenoxazone ring intercalates at the GpC step and the cyclic peptides bind in the minor groove, forming specific hydrogen bonds to guanine bases.

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Clinical Uses: Very potent anticancer drug used in the treatment of a number of childood malignancies: Wilms tumor, Ewings sarcoma, embryonal rhabdosarcoma. Common Toxicities: The usual suspectsmyelosuppression, hair loss, oral and gastrointestinal ulceration. B. Topoisomerase II-Targeted Drugs i. Epipodophylotoxins (semisynthetic; parent molecule derived from mandrake plant) Examples: Mechanism: Nonintercalative. Increases the amount of covalent complex by an unknown structural perturbation of the enzyme and/or DNA. Clinical Uses: Used to treat many types of cancers in both the U.S. and Europe. Common Toxicities: myelosuppression, mucositis, nausea, anaphylaxis. ii. Anthracyclines

Mechanism: Intercalative. Stabilizes covalent complex of Topo II by direct or indirect interaction. Clinical Uses:

Examples:

Doxorubicin and Daunorubicin

both derived from Streptomyces.

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DoxorubicinTreatment of solid tumors (breast cancer, lymphomas, Hodgkins disease, and soft tissue sarcomas)

Daunorubicin--treatment of acute leukemias (AML and ALL) Common Toxicities:

Significant metabolism and toxicity occurs in liver.

cardiotoxicityacute and chronic. Serious problem that limits dosage. myelosuppression, mucositis, nausea, anaphylaxis.