Discovery of new ATP-competitive inhibitors of human DNA topoisomerase IIα through screening of bacterial topoisomerase inhibitors

Highlights

• Discovery of novel chemotype of DNA topoisomerase II inhibitors.

• Bacterial topoisomerase inhibitors were an excellent starting point in search of new topo II inhibitors.

• The most potent inhibitor had an IC₅₀ of 3.2 µM on topo II.

• Two compounds showed very potent activity on MCF-7 cell line (IC₅₀ < 10 µM).

Abstract

Human DNA topoisomerase II is one of the major targets in anticancer therapy, however ATP-competitive inhibitors of this target have not yet reached their full potential. ATPase domain of human DNA topoisomerase II belongs to the GHKL ATPase superfamily and shares a very high 3D structural similarity with other superfamily members, including bacterial topoisomerases. In this work we report the discovery of a new chemotype of ATP-competitive inhibitors of human DNA topoisomerase IIα that were discovered through screening of in-house library of ATP-competitive inhibitors of bacterial DNA gyrase and topoisomerase IV. Systematic screening of this library provided us with 20 hit compounds. 1,2,4-Substituted N-phenylpyrrolamides were selected for a further exploration which resulted in 13 new analogues, including 52 with potent activity in relaxation assay (IC₅₀ = 3.2 µM) and ATPase assay (IC₅₀ = 0.43 µM). Cytotoxic activity of all hits was determined in MCF-7 cancer cell line and the most potent compounds, 16 and 20, showed an IC₅₀ value of 8.7 and 8.2 µM, respectively.

Introduction

Topoisomerases are a family of enzymes with ability to change the topology of DNA molecule. Based on their structure and mechanism of action, topoisomerases can be divided into two types. Type I topoisomerases are monomeric enzymes that catalyse single-strand breaks in DNA double helix, while multimeric type II topoisomerases catalyse double-strand breaks. Based on structural and functional distinctions, both types of topoisomerases are further divided into two subfamilies: IA and IB, and IIA and IIB. Topoisomerases have important roles in processes such as replication and transcription and are essential for cell survival; hence their inhibition leads to cell death. Bacterial type IIA topoisomerases (DNA gyrase and DNA topoisomerase IV) are well known targets of antibacterial drugs, while human DNA topoisomerase IIA (topo II) is targeted by anticancer agents such as etoposide, doxorubicin, daunorubicin and mitoxantrone [1], [2].

Prokaryotic and eukaryotic type IIA topoisomerases share many structural and functional similarities. They all require Mg(II) ions as cofactors and the energy from ATP hydrolysis to perform their function. Prokaryotic topoisomerase IIA enzymes have heterotetrameric structure, while eukaryotic enzymes are homodimeric. All type IIA topoisomerases have a three-domain structure, which was determined based on their homology with Escherichia coli DNA gyrase. Subunit B of E. coli DNA gyrase (GyrB) is homologous to the N-terminal part of topo II, subunit A of E. coli DNA gyrase (GyrA) is homologous to the central domain of topo II, and the C-terminal tail of both enzymes is responsible for their nuclear localization and for their interactions with other proteins. The ATP binding sites lie within the N-terminal domains of type IIA topoisomerases. The ATPase domain belongs to the GHKL superfamily (gyrase, Hsp90, histidine kinase and MutL) whose members contain a unique Bergerat fold. Despite low sequence homology between GHKL proteins, the 3D structures of their ATP binding domains are almost superimposable [2], [3], [4].

There are many known crystal structures of E. coli GyrB with bound inhibitors (for example PDB: 4ZVI [5], 1KZN [6], 6F86 [7], 6F8J [7], 6F94 [7], 6F96 [7], 5MMN [8], 5MMO [8], 5MMP [8], 4DUH [9], 3G7E [10]) and recently a full structure of E. coli gyrase was resolved with cryo-EM (PDB: 6RKU [11]). On the other hand, the crystal structure of ATPase domain of topo II with bound inhibitor is not yet known, however, crystal structures with ATP analogues are available (PDB: 1ZXM [12], 1ZXN [12]). Fig. 1 shows the comparison of the binding poses of ATP analogues adenosine diphosphate (ADP) and adenylyl-imidodiphosphate (AMP-PNP) in the ATP binding site of E. coli GyrB and topo II. The main interactions in both enzymes are hydrogen bonds of the adenine ring with Asp73 of E. coli GyrB or Asn120 of topo II and interactions of the phosphate moiety with Mg(II) ions and multiple amino acid residues in the phosphate binding pocket.

Topo II exists in two isoforms, α and β. The expression of topo IIα is dependent on the cell cycle step and is distinctive for the proliferating cells, and essential for their survival [13]. The main function of the α isoform is to release the DNA topology strains that are created during DNA replication and mitosis [14]. Topo IIβ is expressed in all post-mitotic cells independent of the cell cycle, and while it shows similar function to α isoform in vitro, its in vivo role is poorly understood. Topo IIβ seems not to be essential in the proliferating cells, however, the current findings support its important function in local regulation of chromatin architecture. In mice, the genetic deletion of topo IIβ resulted in perinatal defects and death [14], [15].

Topo II inhibitors can be divided into two large groups, topo II poisons and catalytic inhibitors. Clinically successful topo II inhibitors belong in the first group, which act through stabilising the covalent enzyme–DNA complex, either through slowing down the religation step or speeding up the complex formation. As a consequence, increased levels of damaged DNA are present in the cells, which – if not repaired – leads to cell death. Catalytic inhibitors act through various mechanisms, such as blocking the ATP binding site, preventing the binding of topo II on DNA, or inhibiting the formation of covalent cleavable complex. The only approved drug from this group of inhibitors is dexrazoxane, which inhibits topo II cleavage reaction, but is only used in the clinic in combination with doxorubicin to antagonise its cardiotoxicity [1], [16].

Topo II poisons used in the clinic were discovered in the 1960s and 1970s and have remained a clinically important class of anticancer drugs. There are some issues connected to the use of topo II poisons in therapy, such as toxicity (eg. secondary leukaemia, cardiotoxicity and myelosuppression) and development of resistance [1], [17]. Because of these liabilities, catalytic inhibitors of topo II represent an interesting alternative to topo II poisons with potentially lower toxicity. Among catalytic inhibitors, ATP-competitive inhibitors show good promise, especially considering the recent clinical success of some other ATP-competitive inhibitors, such as kinase inhibitors [18], [19]. Additionally, the unique GHKL ATP binding site architecture makes the non-specific binding of topo II inhibitors to non-GHKL kinases unlikely [4], [20]. Several ATP-competitive inhibitors of topo II have been reported, most of which are purine analogues, including the most promising inhibitor described so far, QAP1, which was developed by Novartis. The inhibitory activity of QAP1 on topo II was determined with ATPase assay (IC₅₀ = 128 nM) and its cytotoxicity was evaluated with MTT assay on SK-BR-3 and MCF-7 cancer cell lines (IC₅₀ values 10 µM and 32 µM respectively) [21], [22]. Other structural types of topo II ATP-competitive inhibitors include thiosemicarbazones [23], N-fused imidazoles [24], urothilins [25], xanthones [26] and triazinones [27]. Novobiocin, a known ATP-competitive inhibitor of bacterial topoisomerases, also shows very weak inhibitory activity on topo II (IC₅₀ = 650 µM), however, it is highly selective for bacterial topoisomerases (E. coli DNA gyrase IC₅₀ = 98 nM) [28].