Note: Topoisomerases as anticancer targets

Note: Topoisomerases as anticancer targets

(doi: 10.1042/BCJ20160583)

topoisomerase poisoning -- replication fork arrest and DSB formation.

Side effect of topo-II -- development of secondary cancers and cardiotoxicity.

There are two types of DNA topoisomerases, type I and type II. – Actually it should be 3?

Type I topoisomerases break one DNA strand of duplex DNA to allow either the passage of the other DNA strand through the break or the rotation of downstream DNA duplex about the break, and then reseal the broken strand.

Topo-I (each subtype has different mechanism);

1.TopoIA -- requires nick or single-stranded region to bind

2.TopoIB -- cleave one strand of duplex DNA

3.TopoIC -- cleave one strand of duplex DNA

Type II topo isomerases alter the linking number in steps of two by breaking both DNA strands of duplex DNA.

There are two subtypes of type II topoisomerases (working as homodimer), type IIA and type IIB.

Each subtype of topoisomerase is structurally and functionally conserved and forms a protein family.

Both human Top 1 (hTop 1) and the two isoforms of human topoisomerase II (hTop 2) are the cellular targets of clinically important anticancer drugs.

Manipulation of enzyme activities to achieve therapeutic effects has been accomplished by the following mechanisms:

(1) suppressing enzyme activity by catalytic inhibitors -- not success bc of cancer cells overexpressed topo-II which can overcome the efficacy of catalytic inhibitor.

(2) activating enzyme activity

toward the bona fide or a surrogate substrate; and

(3) turning the enzyme into a poisonous agent that is toxic to cells (many clinical successful inhibitor) -- through inhibition of religation.

These topoisomerase poisons convert a topoisomerase into a cellular poison by trapping a covalent topoisomerase–DNA catalytic intermediate as a topoisomerase–drug–DNA ternary complex.

Ternary complex formation triggers cytotoxic events, which include the inhibition of DNA replication, the generation of double-strand breaks (DSBs), and subsequent cell death.

human cells also have two isoforms of Top 3 (hTop 3α and hTop 3β). no inhibitor

of human Top 3 has been reported.

In this review;

focuses on recent structural studies of topoisomerase–drug–DNA ternary complexes that have advanced our understanding of the molecular basis of topoisomerase poisoning.

Top 1 is essential for removing DNA superhelical tension that arises during

both DNA replication and transcription. the topoisomerase activity of Top 2β is required for transcription.

mTop I (mitochondria) is the only topoisomerase exclusively targeted to mitochondria, plays critical roles in transcription and DNA replication of circular mitochondrial DNA. loss of mTop 1 severely affects mitochondrial integrity and energy metabolism.

eukaryotic Topo2A enzymes are homodimers.

These isoforms share roughly 70% amino acid sequence similarity and exhibit similar catalytic activities, but they have distinct cellular functions.

Top 2α is required for DNA replication and chromosome segregation, and its

expression is significantly elevated in proliferating and cancer cells -- therefore, it could be served as biomarker in some cancers.

Top 2β plays critical roles in transcription in normal mitotic and post-mitotic cells, as well as in cancer cells. required for development and the survival of some neural cells.

Structural analyses of hTop 1 in covalent and noncovalent complexes with DNA suggested that the postulated DNA rotation during the catalytic cycle is likely achieved via a ‘controlled rotation mechanism’.

Types IA, IB, and IIA topoisomerases have distinct requirements of Mg2+ for their catalytic activities.

hTop 1 is the cellular target of the quinoline alkaloid camptothecin and its clinically active derivatives, topotecan and irinotecan. these drugs trap the Top 1–DNA covalent complex and convert the enzyme into a cytotoxic covalently linked protein adduct on DNA. Causing the inhibition of DNA replication and the generation of a DSB.

Interestingly, no catalytic inhibitor of type IB topoisomerases has been reported thus far.

Camptothecin is a plant alkaloid that poisons hTop 1. The clinical development of camptothecin was discontinued because of its intolerable adverse effects and low therapeutic index.

Derivatives of camptothecin, topotecan and irinotecan -- their clinical use is limited due to their dose-limiting toxicity, especially neutropenia, myelosuppression, and diarrhea, as well as chemical instability due to the rapid opening of the E-ring (structural instability).

Topotecan (Hycamtin);

1. semi-synthetic water-soluble derivative of camptothecin.

2. first hTop 1 inhibitor approved for oral administration.

3. used to treat ovarian and small cell

lung cancer.

Irinotecan (CPT-11, Campostar);

1. water-soluble derivative of camptothecin

2. "prodrug" and is converted to a biologically active metabolite (ethyl-10-hydroxy-camptothecin (SN-38)) by a

carboxylesterase.

3. Irinotecan, together with fluorouracil, is often used for the treatment of advanced

colorectal cancer.

Both two drugs;

Good point

1.show effectiveness

Bad points

1.instability

2.severse side effects

3.drug resistance due to P-glycoprotein (drug exporter)

Therefore, there is the trend that ppl try to develop non-campthotecin derivative hTop1 inhibitors -- include;

1.indolocarbazoles (NB-506) -- synthetic analog isolated from several actinomycetes

2.indenoisoquinolines --

3.dibenzonaphathyridinones (ARC-111)

Several type IIA topoisomerase poisons are used successfully in the clinic as anticancer and antibacterial drugs.

It is worth noting that the co-crystallization approach had been applied successfully to the structural analyses of etoposide- and mitoxantrone-bound cleavage complexes of hTop 2α.

the crystal structure of doxorubicin-bound cleavage complex was only determined using a post-crystallization drug-replacement procedure, and therefore, the functional significance of this structure has remained to be elucidated.

the accumulation of hTop 2β-induced DNA breaks are associated with deleterious chromosome translocation and rearrangement events, resulting in frequent occurrence of therapy-related leukemia. Therefore, it would be clinically desirable to have an anticancer drug that preferentially targets hTop 2α.

I have not realized before that targeting the wrong target would cause another problem in particular cancer.

The development of isoform-selective drug may be achieved by targeting this hTop 2α-specific methionine.

The catalytic activities of type IIA topoisomerases are modulated through their interactions with various proteins.

Biochemical and structural studies suggest that these DNA-mimicking proteins may interact with the primary DNA-binding groove of DNA gyrase.

ATPase inhibitor works through disturbing the conformational change during the ATP hydrolysis.

ICRF-193, the most potent bisdioxopiperazine, contains two methyl groups and, thus, binds tighter to eukaryotic Top 2s.

hTop 2 poisons are successful anticancer drugs used in the treatments of various cancers. However, two serious side effects, therapy-related cancer and cardiotoxicity, associated with these drugs limit their use.

Etoposide, and other hTop 2 poisons cause the development of secondary malignancies, especially therapy-related acute myeloid leukemia (t-AML), therapy-related acute promyelocytic leukemia (t-APL).

Cardiomyocytes are particularly sensitive to oxidative stress. hTop 2β is the only topoisomerase expressed in myocytes.

Just learnt again that each cell type expressed different isoforms of hTop2.

Further studies are required to determine the exact mechanism(s) of the cardiotoxicity of doxorubicin, but the current strategy to prevent cardiotoxicity is to limit the cumulative dose of doxorubicin.

hTop2 poison;

1. Epipodophyllotoxins: etoposide and teniposide

2. Anthracyclines: doxorubicin, daunorubicin, and epirubicin

3. Mitoxantrone

Podophyllotoxin was originally isolated from the podophyllum plants, American Podophyllum peltatum and Podophyllum emodi.

To improve its water solubility, a prodrug etoposide phosphate (Etophos) was developed for intravenous use.

Doxorubicin (Adriamycin and Doxil) is an anthracycline antibiotic originally isolated from Streptomyces peucetius.

Anthracyclines act as Top 2 poisons. In addition, they can intercalate into DNA and may influence the functions of various proteins besides hTop 2 -- anthracyclines also generate ROS.

Just learnt that some anticancer drugs can generate ROS besides their hTop2 poisoning.

hTop2 catalytic inhibitor;

1. Dexrazoxane

Both hTop 1 and hTop 2s are targets of current anticancer drugs. New inhibitors of these enzymes are also in the pipeline. The expression of hTop 2α is often elevated in cancer cells, and the anticancer activity of these drugs is mainly mediated by the poisoning of hTop 2α. that both the development of therapy-related leukemia and cardiotoxicity are caused by the poisoning of hTop 2β. it seems reasonable to develop hTop 2α-specific poisons as anticancer drugs to prevent the therapy-related leukemia and cardiotoxicity.

topoisomerase catalytic inhibitors have not been successfully used in clinic.

hTop 3 inhibitors may be discovered and developed as new anticancer drugs in the future.

x