Human Tyrosyl-DNA Phosphodiesterase 1 : new activities and development of

inhibitors as anticancer drugs
Olga Lavrik

SB RAS Institute of Chemical Biology

repair (NER)
- X-rays
double strand
breaks
homologous
recombination (HR)
non-homologous
end joining (NHEJ)
- Replication
errors
base

mismatch
repair (MMR)

Most common DNA-damaging agents, lesions, and repair pathways

abasic sites
oxidized, deaminated,
alkylated bases
ssDNA breaks

base excision
repair (BER)

- Spontaneous reactions
- Oxygen radicals
- Alkylating agents
- X-rays

of antitumor drugs that have to reveal their cytotoxic effects

DNA repair systems are targets for development of anticancer drugs

1NOP).

Protein is represented in light pink, catalytic residues in

The crystal structure of Tyrosyl-DNA Phosphodiesterase 1 (TDP1)

Detailed contacts between substrate and TDP1 residues in the catalytic site.

Catalytic residues are represented as yellow sticks; residues involved in polar interactions are in cyan sticks; residues involved in hydrophobic interactions in magenta sticks.
All stick sare colored by element (N, blue; O, red; P, orange; Vanadate, grey). Dashed lines highlight polar interactions.

peripheral neuropathy typified by patients with SCAN1
Tdp1
Removal of the Top1

Prevention of the anticancer drug action

H493R mutation in TDP1 is responsible for the inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1)

Wild
type

SCAN1 mutation

+

The cerebellum is a common target for disorders with DNA repair defects

of supercoiling
Religation
Supercoiled duplex
Nicking
Supercoiled DNA
Relaxed DNA

derivatives
Topotecan
Irinotecan
Belotecan

of cancer to anticancer Top1- inhibitors
Tdp1 knockout mice and human

T.S. et al. (2008) Anticancer Agents Med Chem. 8, 381-389
Tdp1

10,000-50 000 per mammalian cell per day

Unrepaired AP sites are

NaBH4

АР site

АР site

Covalent binding of proteins to
AP sites in DNA

Spontaneous hydrolysis
(abasic site)

Reactive oxygen species
Methylation, deamination

Repair of apurinic/apyrimidinic sites (AP sites)

radicals
Abasic site (AP site)
Repair of AP sites initiated by

Base

Lebedeva NA, et al. DNA Repair. 2013, 12, 1037-1042.

the AP site cleavage activities of Tdp1 and APE1
Tdp1 is

Tdp1 generates breaks
with the 3'–P and 5'–P termini

APE1 generates DNA strand break from the AP site with the 3'-OH and
5’-dRP termini

APE1 activity depends on Mg2+ ions

APE1 is more efficient in the cleavage of AP sites in ds DNA

Tdp1 activity does not depend on Mg2+ ions

repair machinery
TDP1 is a component of the multiprotein complex formed

Moor NA, Vasil'eva IA, Anarbaev RO, Antson AA, Lavrik OI. Quantitative characterization of protein-protein complexes involved in base excision DNA repair. Nucleic Acids Res. 2015 May 26. pii: gkv569.

The X-ray structure of TDP1 (D148)
has been determined
(PDB code: 4DQY).

As a general 3’-DNA phosphodiesterase catalyzes hydrolysis of:
a) Top1-DNA covalent complexes
b) 3’-end DNA alterations (phosphoglycolate,
AP site,
a,b-unsaturated aldehyde,
nucleoside,
ribonucleoside);

Catalyzes cleavage of natural and synthetic AP sites.

peripheral neuropathy typified by patients with SCAN1
The cerebellum is

H493R mutation in Tdp1 is responsible for the inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1)

XMLR
Mental retardation

SCAN1
Cerebellar degeneration

MCSZ
Microcephaly and seizures

state of Tdp1. Cannot be used as pharmacological inhibitors because

Indenoisoquinolines

dual Topoisomerase I (Top1)− TDP1 Inhibitors.
Nguyen TX, et al. J. Med. Chem. 2015, 58, 3188−3208
Nguyen TX, et al. J. Med Chem. 2012 55, 4457–4478
Conda-Sheridan M, et al. J. Med. Chem. 2013, 56, 182−200

5‑Arylidenethioxothiazolidinones

submicromolar inhibitor of Tdp1 has not yet been tested for cytotoxicity
Sirivolu VR, et al. J. Med. Chem. 2012, 55, 8671−8684

JBIR-21,
compound from the culture of an anamorphic fungus, RF-13305 (IC50 value, 18 μM)
Takagi M, et al., J. Nat. Prod., 2012, 75 (4), pp 764–767

Tdp1 inhibitors

in the Laboratory of physiologically active substance, NIOCH SB RAS,

Lissoclinum vareau

varacin

This compound has a reactive amino group making it ideal for the development of new biologically active derivatives carrying various linkers.

varacin analog

Organic
synthesis

Chem., 2015, 23, 2044–2052.
5’-
-3’
We designed a new simple fluorophore-quencher coupled

The dependence of TDP1 residual activity as a function of the concentration of varacin analogs.

TDP1 using a new oligonucleotide-based fluorescence assay
0.22 ± 0.03
1.30

1.57 ±0.39

1.62 ± 0.40

3.70 ± 1.04

3.66 ± 2.88

6.03 ± 3.12

IC50, ϻМ

Zakharenko A., et al. Bioorg. Med. Chem., 2015, 23, 2044–2052,
Application for a patent No 2014139787, the priority date 30.09.2014.

of Chemical Sciences, University of Auckland, New Zealand)

According to the

The protein surface is rendered. The tertiary amine occupies a cleft exposed to the water environment. Red depicts a positive partial charge on the surface, blue depicts negative partial charge and grey shows neutral/lipophilic areas.

Hydrogen bonds are depicted as green lines between the ligand and the amino acids ASN516 and HIS263.

2-(Dibutylamino)-N-(8-(trifluoro-
methyl)benzo[f][1,2,3,4,5]
pentathiepin-6-yl) acetamide

IC50 = 0,22 µM.

caused apoptotic cell death in human mammary adenocarcinoma cell line

Cytotoxicity of varacin analogs

is the beneficial target to design the new classes of

- Tdp1 inhibitors caused apoptotic cell death in cancer cell lines

- The most potent TDP1 inhibitor has the greatest potential to be developed further as an anticancer drug in combination with the established Top1 inhibitors.

PARP1 is also a regulator of NHEJ, a mechanism of

DNA repair

BER, DSB, NHEJ

Transcriptional
regulation

Component of the TLE/Groucho corepressor complex involved in Wnt signalling implicated in transcriptional regulation of androgen receptor expression

Chromatin
modification

Mitosis

Cell
death

Maintenance of telomere length and chromosomal stability

Involved in mitotic- spindle formation

Component of pathways mediating apoptosis

Function of PARP1

repair

acid derivatives –
selective PARP1 inhibitors
Enhancement of the effect of

Protective effect

PARP1 is multifunctional protein involved in DNA damage response and a number of inflammatory processes. New antitumor drugs (PARP1 inhibitors) are based on modified natural compounds.

PARP1 inhibition (inactivation)

Hyperactivation

Moderate activation

DNA repair

Piperazine derivatives of betulonic acid

the universal inhibitors of DNA repair enzymes

Necrosis

of betulonic acid
Varacine analogs
the inhibitors of Tdp1
Glycirritinic acid derivatives –

Usnic acid derivatives – the selective inhibitors of PARP1 and Tdp1

DNA repair inhibitors that are potential medical products:

The inhibition of DNA repair systems is essential for the stable chemo- and radiotherapy

The activity of DNA repair proteins such as apurinic/apyrimidinic endonuclease 1 (АРЕ1), DNA polymerase β (Pol β), tyrosyl-DNA phosphodiesterase 1 (Tdp1) and poly(ADP-ribose) polymerase 1 (PARP1) levels the effect of antitumore drugs.

the universal inhibitors of DNA repair enzymes

SB RAS
Benzopentathiepine derivatives
K.P. Volcho, T.M. Khomenko
Usnic acid derivatives
O.A. Luzina, D.V.

Betulinic acid derivatives
I.Ya. Mainagashev

Glycyrretinic acid derivatives
O.A. Salomatina

O.I. Lavrik
Head of Laboratory of Bioorganic Chemistry of Enzymes of ICBFM SB RAS

Design of TDP1 sensor
R.O. Anarbaev, N.A. Lebedeva

In vitro screening of inhibitors
A.L. Zakharenko

TDP1 intractions with AP-sites
R.O. Anarbaev, N.A. Lebedeva, N.I. Rechkunova

J. Reynisson
School of Chemical Sciences, University of Auckland, New Zealand
(computer modelling)

Protein-protein interactions
N.A. Moor, I. Vasil’eva

This work was supported by
the Ministry of Education and Science of RF, grants of Russian Science Foundation
and Russian Foundation for Basic Research

fluorescence in MCF-7 cells treated with the pentathiepines for 24

*not determined

The study of cytotoxicity of these compounds revealed that all compounds cause an apoptotic cell death in human mammary adenocarcinoma cell line MCF-7 or human liver cell carcinoma Hep G2

The compound 3d demonstrated concentration-dependence as evaluated by the MTT cytotoxicity assay. The 50% cytotoxic concentration (CC50) was defined as the concentration required to reduce the cell number by 50% compared to that for the untreated controls. The CC50 values of Tdp1-active compounds after 72 hours treatment applying the MTT cell based assay.

The detection of apoptotic DNA fragments after 72 hours treatment of MCF-7 cells with 100 μM of the derivatives. Electrophoretic analysis revealed the appearance of DNA fragments corresponded to mono- (170-200) and oligonucleosoms, confirming an apoptotic mechanism of cell death for the all pentathiepines tested.

1 - designates DNA markers 4 - empty;
2 - control cells; 5 - 3d;
3 - 1%DMSO; 6 - varacin analog.

750

1000

2000

3000

180-200

360-400

540-600

superfamily, inhibit Tdp1 but require high concentrations (in the low

Furamidine,

antiparasitic preparation in phase III clinical trials, inhibits Tdp1 at low micromolar concentrations
Antony S, et al. Nucleic Acids Res. 2007; 35(13):4474–84.
Pommier, Y., et al. USA patent. 60/786,604. 2006.

Tetracyclines

The preliminary results on tetracyclines showed micromolar inhibitory effect against Tdp1 but lacked apparent structure-activity relationship.
Pommier, Y. et al. USA patent. US 12/241,011. 2006.

Phosphotyrosine Mimetics

inhibit Tdp1 at sub-micromolar concentrations
Pommier, Y. et al. USA patent. 60/921,980. 2007.
Marchand C, et al. Mol Cancer Ther. Jan; 2009 8(1):240–8.
Pommier, Y. et al. USA patent. 61/042,706. 2008.
Dexheimer TS, et al. J Med Chem. 2009 2(22):7122–31.

methyl-3,4-dephostatin

steroid compound NSC88915