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Brettrager et al. Cancer Drug Resist 2019;2:1153-63 I http://dx.doi.org/10.20517/cdr.2019.91 Page 1155

Table 1. Therapeutic and endogenous agents generating Tdp1 substrates
Agent Substrate(s) Ref.
Reactive oxygen species 3’ abasic site [8-14]
Chain terminating nucleoside analogs 1 3’ nucleoside adducts [10]
Irradiation/Bleomycin 3’ phospho-glycolate, 3’ abasic sites [15-19]
MMS, TMZ 2 Methylated bases [9,20]
PARP1 inhibitors 3 PARP1-DNA Schiff base [21-23]
Camptothecins 4 3’ phospho-tyrosine [2,3]
Etoposide, doxorubicin 5 5’ phospho-tyrosine [20,24,25]
Tdp1 mutants 3’ phospho-histidine [1,16,26-29]
2
1 Arcyclovir (ACV), cytarabine (Ara-C), zidovudine (AZT), zalcitabine (ddC), and sapacitabine; methyl methanesulfonate (MMS) and
3
4
temozolomide (TMZ); BMN673, Olaparib, and Rucaparip; U.S. Food and Drug Administration (FDA) approved analogs: topotecan and
5
irinotecan; epipodophyllotoxins such as etoposide and anthracyclins, with Doxorubicin as an example, are Topo2-DNA stabilizing agents
with different mechanisms of action [30] . Tdp1: Tyrosyl-DNA phosphodiesterase I
CATALYTIC INHIBITORS
To date, many catalytic Tdp1 inhibitors have been identified, yet only a few were tested in cell or cancer
models. Interestingly, many of these Tdp1 catalytic inhibitors are based on natural products isolated from
fungi, plants, nucleoside analogs, and bile acids showing inhibition in the low micromolar to the high
nanomolar range. The first reported catalytic Tdp1 inhibitors were the non-specific transition metals
3-
vanadate (VO ) and tungstate at millimolar concentrations and were used to resolve the crystal structure of
4
the transition state complex of hTdp1 catalytic core domain with VO -DNA-hTopo1 peptide fragments [42-44] .
4
[45]
Vanadate and tungstate are known general inhibitors for protein phosphotyrosyl phosphatases . Yves
Pommier and collaborators advanced the search for catalytic inhibitors by exploring existing compounds
and developing in vitro/alpha-screen based high-throughput screens (HTSs) using oligonucleotides with
a 3’ phospho-linked fluorescent adduct to mimic the 3’ phospho-tyrosyl bond. They exploited antibiotic
ribosomal inhibitors and identified as the first pharmacological active Tdp1 inhibitors Neomycin, an
aminoglycoside analog, and non-aminoglycosides such as puromycin and thiostrepton with IC ranging
50
[46]
2-30 mmol/L . They further identified rolitetracycline and potentially other tetracycline analogs to
[48]
[47]
inhibit Tdp1 catalysis at micromolar levels . Huang et al. successfully translated this observation by
repurposing minocycline in combination with irinotecan to treat high grade ovarian cancer cell lines
and an orthotopic xenograft mouse model of human ovarian carcinomatosis. Intriguingly, minocycline
decreased Tdp1 expression levels that enhanced irinotecan toxicity in platinum resistant cell lines, while in
vivo this combination reduced micro-metastases to improve overall survival. Why minocycline specifically
reduces Tdp1 protein levels and/or inhibits Tdp1 activity as suggested for another tetracycline analog -
rolitetracycline - is currently unknown [47,48] . How tetracyclines inhibit eukaryotic protein synthesis is still
[49]
questionable - the most popular explanation is ribosome inhibition , but the reduction of one protein
(Tdp1) by minocycline remains an enigma.

The diamidine analogue furamidine [2,5-bis (4-amidinophenyl) furan] was the first inhibitor identified
[50]
in a HTS at National Institutes of Health (NIH) . Furamidine is used to treat Trypanosomiasis and
[51]
leishmaniasis, which express Tdp1, but it is unclear if Tdp1 inhibition is part of the antiparasitic activity .
[52]
An interesting twist is that furamidine combined with irinotecan suppresses murine lupus nephritis .
Additional HTSs at NIH identified additional micromolar to high nanomolar Tdp1 inhibitors: progesterone
derivative NSC88915 (3,20-dioxopregn-4-en-21-yl 4-bromobenzenesulfonate) and phospho-tyrosyl
mimetics such as suramin and dephostatin derivative methyl-3,4-dephostatin (3,4-Dihydroxy-N-methyl-N-
nitrosoaniline) [53,54] . The Pommier and Wang collaboration identified arylidene thioazolidinone derivatives
[55]
as high nanomolar inhibitors of Tdp1 catalysis .
The identification of non-hydrolysable phospho-tyrosyl mimetics that perfectly docked in the catalytic
pocket led to the first in silico-docking screen to classify 46 additional potential Tdp1 inhibitors that dock