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.
1

O
HN

General Chemical Mechanism

O
5'

DNA

Topo
O
O P OO

O
HN
O

Topo

5'

Type IB

5' HO-DNA

cleavage
N

ligation
DNA

OH O
O P

O
O-

O-DNA

5'

cleavage
HN
O

ligation
5'

Topo

DNA

O
OH 3'

Type II, IA

O
O P OO
DNA

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

p-Tyr
linkage

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)

Type II Topisomerase Mechanism

A DNA
duplex can
pass
through
this gate.

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.
Action of Topo I on
supercoiled plasmid
DNA

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:

Example: Camptothecins

Example: Minor groove binding agents

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

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

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.

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
Examples:
Doxorubicin and Daunorubicin
both derived from Streptomyces.

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

interaction.
Clinical Uses:

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.