Identification of Natural Products That Inhibit the Catalytic Function of Human Tyrosyl-DNA Phosphodiesterase (TDP1)

Abstract

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an enzyme crucial for cleavage of the covalent topoisomerase 1-DNA complex, an intermediate in DNA repair. TDP1 plays a role in reversing inhibition of topoisomerase I by camptothecins, a series of potent and effective inhibitors used in the treatment of colorectal, ovarian, and small-cell lung cancers. It is hypothesized that inhibition of TDP1 activity may enhance camptothecin sensitivity in tumors. Here, we describe the design, development, and execution of a novel assay to identify inhibitors of TDP1 present in natural product extracts. The assay was designed to address issues with fluorescent "nuisance" molecules and to minimize the detection of false-positives caused by polyphenolic molecules known to nonspecifically inhibit enzyme activity. A total of 227,905 purified molecules, prefractionated extracts, and crude natural product extracts were screened. This yielded 534 initial positives (0.23%). Secondary prioritization reduced this number to 117 (0.05% final hit rate). Several novel inhibitors have been identified showing micromolar affinity for human TDP1, including halenaquinol sulfate, a pentacyclic hydroquinone from the sponge Xestospongia sp.

Dual DNA topoisomerase 1 and tyrosyl‐DNA phosphodiesterase 1 inhibition for improved anticancer activity

“…Thus, inhibiting Tdp1 can increase the efficacy of Top1 poisons in tumor cells and thus improve the efficacy of anticancer therapy. In addition, Tdp1 is gathering interest in cancer treatment because of its role in repairing various other DNA adducts induced by chemotherapeutics . Recently, new classes of Tdp1 inhibitors have been developed that are active in the micromolar to nanomolar range and have low intrinsic toxicity …”

“…In addition, Tdp1 is gathering interest in cancer treatment because of its role in repairing various other DNA adducts induced by chemotherapeutics. 38,39,44 Recently, new classes of Tdp1 inhibitors have been developed that are active in the micromolar to nanomolar range and have low intrinsic toxicity. 39,43,52,60,[64][65][66][67][71][72][73]84 Using the same compounds to inhibit Tdp1 and Top1 is a prospective approach to develop anticancer drugs as both Tdp1 and Tdp2 inhibition could hypothetically potentiate the cytotoxicities of topoisomerase poisons.…”

“…18,38,39 Many other synthesized compounds, including naturally occurring plant and fungal metabolites and their derivatives or analogs, have been tested as Tdp1 inhibitors. [40][41][42][43][44] However, none of these studies used an animal model, and have not proceeded to trials. Such compounds can simultaneously cause damage to DNA and prevent its repair.…”

“…The search for Tdp1 inhibitors commenced in 2002 by testing weakly effective nonspecific inhibitors including transition metals, such as vanadate and tungstate; aminoglycoside antibiotics; diamidines, and phosphotyrosine mimetics . Many other synthesized compounds, including naturally occurring plant and fungal metabolites and their derivatives or analogs, have been tested as Tdp1 inhibitors . However, none of these studies used an animal model, and have not proceeded to trials.…”

Creating and screening natural product libraries

“…As discussed in Section 2 of this review, partial purication, or prefractionation, of crude natural product extracts can be highly useful in removing or sequestering problematic compounds while providing increased test concentrations of potentially active compounds (ideally in different fractions) as well as providing less complex mixtures which can result in higher conrmation rates. 107 The transition from crude to prefractionated extracts for cell-based HTS has been validated in HTS campaigns at the NCI. 106,107 Fig.…”