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Page 1154 Brettrager et al. Cancer Drug Resist 2019;2:1153-63 I http://dx.doi.org/10.20517/cdr.2019.91

INTRODUCTION
Tyrosyl-DNA phosphodiesterase I (Tdp1) is a eukaryotic DNA repair enzyme which is a member of the
phospholipase D superfamily and hydrolyzes the phosphodiester bond that links an adduct to the end
[1-3]
of a nicked DNA strand . Tdp1 was discovered as an enzyme activity able to hydrolyze a 3’ phospho-
tyrosyl linkage, which is the chemical bond between the active site tyrosine of Tyrosine-recombinases and
[3]
eukaryotic DNA Topoisomerase I (Topo1), and the 3’ phosphoryl-end of a DNA strand . Over the last
two decades, a broad spectrum of phosphodiester linked 3’ and 5’ DNA-adducts were identified as Tdp1
[4]
substrates . Tdp1 natural substrates can be divided into two groups: small adducts consisting of damaged
nucleotides, DNA inserted ribonucleotides, and non-canonical nucleotide/nucleoside analogs, and large
adducts including covalent protein-DNA adducts that are generated as a transient reaction intermediate
by, for example, DNA topoisomerases and Tdp1, or protein fragments (peptides) as a result of failed Schiff
base linked proteins such as proteolytically processed poly (ADP-ribose) polymerase 1 (PARP1)-DNA
adducts [Table 1]. These transient protein-DNA adducts can be stabilized by chemotherapeutics including
camptothecins (CPT), epipodophyllotoxins (e.g., etoposide), and local DNA perturbations introduced by,
for example, irradiation and endogenously generated reactive oxygen species [Table 1]. Moreover, Tdp1 is
localized in both the nuclear and mitochondrial compartments to catalyze the hydrolysis of phosphodiester
[4-7]
linked DNA adducts . This reveals the general role of Tdp1 in maintaining nuclear and mitochondrial
genome stability and chemotherapeutic-resistance.

Tdp1 utilizes two highly conserved histidine-lysine-aspartate (HxKx D; x being any amino acid) motifs
4
in two coordinated S 2 nucleophilic attacks to hydrolyze the phosphodiester linkage. First, His263
N
nucleophilically attacks the phosphodiester bond to release the adduct/protein by forming a transient
Tdp1-DNA adduct, which is broken by the general acid/base His493 mediated hydrolysis via activation of
water releasing Tdp1 from the DNA-end [Figure 1] [2,3,11,16,26,28,31,32] . The intriguing fact is that cells risk the
formation of second potentially toxic enzyme-DNA reaction intermediate to resolve the primary toxic
insult. This potential danger to the cell is highlighted by the catalytic human Tdp1 (H493R) mutant, which
is the molecular basis for the rare autosomal recessive neurodegenerative disease spinocerebellar ataxia
with axonal neuropathy (SCAN1) [29,33] . Tdp1 is expressed in all human tissues at a low level, probably due to
its potential danger. However, elevated levels of Tdp1 are detected in a heterologous distribution in virtually
[35]
all tumors [7,34] . Elevated Tdp1 levels stimulate chromosome instability and increase cell sensitivity to DNA
damaging agents [24,26,35-37] . Thought-provoking is that deletion of Tdp1 in yeast, DT40 chicken cells, HEK293
cells, and mice also results in enhanced cell sensitivity to DNA damaging agents [2,5,16,20,27,31,38-40] . Overall, this
supports the two different therapeutic strategies hypothesized: (1) no Tdp1 activity or catalytic inhibition
of Tdp1 that prevents repair of DNA-adducts leads to cytotoxicity; and (2) accumulation of Tdp1-DNA
adducts via poisoning/stabilization of the protein-DNA complex, or increase of Tdp1 levels, results in
cytotoxicity [Figure 1]. This review focuses on the current development of Tdp1 as a therapeutic target to
[30]
improve treatment response with FDA approved chemotherapeutics - a topoisomerase targeting drug .

TDP1 AS THERAPEUTIC TARGET
The potential of Tdp1 as a therapeutic target for catalytic inhibitors was directly proposed by Nash and co-
[3]
workers in its discovery paper . Over the last five years, the search for Tdp1 inhibitors has been rapidly
growing with the therapeutic focus to combine catalytic Tdp1 inhibiting agents with the current FDA-
approved Topo1 targeting chemotherapeutics. Besides catalytic inhibition, we champion a second strategy
to poison Tdp1 or to pharmacologically stabilize the enzyme-DNA adduct turning Tdp1 into a cellular
toxin, similar to topoisomerase inhibitors [4,30] . The principle of this strategy is supported by the SCAN1
His Arg-mutant and other Tdp1 catalytic mutants tested [1,16,26,28,29,36,37,41] . Currently, no small molecules for
gab
this strategy have been reported and, herein, we focus only on catalytic Tdp1 inhibitors.