5-Arylidenethioxothiazolidinones as inhibitors of tyrosyl-DNA phosphodiesterase I

Abstract

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a cellular enzyme that repairs the irreversible topoisomerase I (Top1)-DNA complexes and confers chemotherapeutic resistance to Top1 inhibitors. Inhibiting Tdp1 provides an attractive approach to potentiating clinically used Top1 inhibitors. However, despite recent efforts in studying Tdp1 as a therapeutic target, its inhibition remains poorly understood and largely underexplored. We describe herein the discovery of arylidene thioxothiazolidinone as a scaffold for potent Tdp1 inhibitors based on an initial tyrphostin lead compound 8. Through structure-activity relationship (SAR) studies we demonstrated that arylidene thioxothiazolidinones inhibit Tdp1 and identified compound 50 as a submicromolar inhibitor of Tdp1 (IC₅₀ = 0.87 μM). Molecular modeling provided insight into key interactions essential for observed activities. Some derivatives were also active against endogenous Tdp1 in whole cell extracts. These findings contribute to advancing the understanding on Tdp1 inhibition.

Figure 1.

Tdp1 in the repair of the stalled Top1–DNA complex: (A) noncovalent Top1–DNA complex; (B) transient Top1–DNA complex, which is trapped in the presence of a Top1 inhibitor; (C) the stalled Top1–DNA complex (cleavage complex) is repaired by Tdp1 via a transient Tdp1–DNA covalent intermediate; (D) Top1 is released and the Tdp1–DNA covalent intermediate resolved by hydrolysis; (E) 3′-phospho–DNA–end product.

Figure 2.

Known inhibitors of Tdp1.

Figure 3.

Inhibition of Tdp1 by tyrphostin derivatives. (A) Schematic representation of the Tdp1 biochemical gel-based assay. Tdp1 hydrolyzes the 3′-phosphotyrosine bond and converts the N14Y DNA substrate to a 3′-phosphate oligonucleotide product (N14P); (B) representative gel demonstrating concentration-dependent inhibition of Tdp1 by compounds 8 and 9; (C) representative compounds. The pharmacophore of lead compound 8 comprises two domains: the functional head domain and the aromatic moiety.

Figure 4.

Design of heterocyclic arylidene scaffolds 19–22 based on lead compound 8 and PTP inhibitors 18.

Figure 5.

(A) Representative gel demonstrating concentration-dependent inhibition of Tdp1 by compounds 48, 50, and 51. (C) Concentration–response curves. Each point represents the mean value ± standard deviation of three independent experiments.

Figure 6.

Binding of 50 to Tdp1 was examined using SPR spectroscopy. A concentration series of 50 (50, 25, 6.25, 3.12, 1.56. 0.78, and 0.39 μM) were injected over immobilized Tdp1 (A) or a tyrosyl-labeled oligonucleotide (GATCTAAAAGACTT-Tyr) (B). The theoretical maximal binding response to Tdp1 was 45 RU and to the oligonucleotide was 140 RU.

Figure 7.

Modeled structure of Human Tdp1 (PDB: 1NOP) with (A) Top1–DNA substrate, (B) 9, (C) 31, and (D) 50 docked within the active site. The model shows three specific regions important for substrate binding—the hydrophobic (Y204, P461, W590, labeled in white), the catalytic (K265, H263, N283, H493, K495, N516, labeled in blue), and an adjacent hydrophilic region (S400, S518, S536, E538, labeled in red). Effective binding of the hydrolytic tyrosyl–nucleosidic group of the Top1–DNA substrate involves the hydrophobic and the catalytic regions (boxed in yellow).

Scheme 1.. Synthesis of Scaffolds 19–22^a

^aReagents and conditions: (a) EtOAc, reflux, 48 h, 92%; (b) EtOH, piperidine, reflux, 16 h, 41–78%; (c) 140 °C, 4 h, 72%; (d) Lawesson’s reagent, toluene, reflux, 2 h, 98%; (e) NaOAc, AcOH, reflux, 2 h, 58–90%.