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Soren et al. Cancer Drug Resist 2020;3:18-25 I http://dx.doi.org/10.20517/cdr.2019.106 Page 23
[49]
[48]
the -1 position of the scissile strand for binding of CPT . Chrencik et al. suggested that six residues
(Phe361, Gly363, Arg364, Glu418, Gly503, and Asp533) present in the Lip1-Lip2 region harbor a CPT
[49]
resistant mutations cluster . Other studies on the Gly363 mutation show the role of this residue in CPT
sensitivity [50,51] . Even though the linker domain is located far away from the active site of Tyr723, it can
deeply affect the drug resistance or sensitivity of hTopIB. The main reason for this long-distance interaction
is its flexibility. Evidence of these phenomena comes from several works where key residues involved in the
mobility of the domain were mutated. The mutation of Ala653Pro has demonstrated that a large flexibility
[52]
of the linker domain is associated to CPT resistance . This correlation has been shown also in the
[30]
Glu710Gly mutation . This paper demonstrated that a lower degree of linker motion leads to an increase
of religation rate and consequently to CPT resistance. On the other hand, in the case of Asp677Gly-
[53]
Val703Ile double mutant, the reduction of the linker flexibility confers a hypersensitivity to CPT . Other
evidence of linker involvement in enzyme religation rate comes from the construction of a chimeric
enzyme, in which the human enzyme contains a linker from Plasmodium falciparum TopI (PfTopI). In the
chimeric enzyme, the PfTopI linker shows a great flexibility and confers the chimeric enzyme to be CPT
[54]
resistant . Moreover, inserting a long yeast linker domain in hTopIB drastically altered enzyme function in
vivo. Expression of this chimera was toxic in yeast even in the absence of CPT, with no particular changes
[55]
in enzyme catalysis . Altering the linker structure, either through changing its flexibility by the mutation
of specific residues or by deleting the entire domain, affects the enzyme rate and consequently the CPT
reactivity. An additional residue that was shown to impact CPT reactivity is Thr729, which is part of the
[56]
hydrophobic pocket present in the C-terminal domain. Losasso et al. studied this residue extensively by
producing different mutations, namely Thr729Ala, Thr729Glu, Thr729Lys, and Thr729Pro, and showed the
[56]
importance of this residue in modulation of hTopIB DNA binding and drug resistance . Residue Asn722,
which is present near the Thr729 residue, was also shown to impart CPT resistance to the protein [57,58] .

CONCLUSION
HTopIB is still a fascinating enzyme that is worth studying. However, some considerations should be given
to try to focus the attention of the scientific community. Many inhibitors of the binding and cleavage phases
have never entered into clinical protocols. The most important step for inhibition remains the religation
and compounds that reversibly inhibit this phase of the enzyme catalysis. The only derivatives of CPT that
are now in clinical use targeting hTopIB are still Topotecan and Irinotecan. It is probably time to look for
other natural compounds that could inhibit this enzyme. Our group is focusing on a metabolite from an
Antarctic sponge that was found to be a potent inhibitor of hTopIB (unpublished results). Perhaps it is time
to study and search different ecosystems to obtain promising drugs for cancer therapy, as sometimes it is
from nature that we have the best solution.

DECLARATIONS

Acknowledgments
We thank Prof. A. Desideri and Dr. B. Morozzo Della Rocca for proof reading of the review.

Authors’ contributions
Wrote the review: Soren BC, Dasari JB
Made all the pictures together with the writing part: Ottaviani A, Iacovelli F
Supervised both parts graphic and writing part: Fiorani P

Availability of data and materials
Not applicable.

Financial support and sponsorship
None.