The overview of development of novel bacterial topoisomerase inhibitors effective against multidrug-resistant bacteria in an academic environment: From early hits to in vivo active antibacterials.

Antimicrobial resistance caused by the excessive and inappropriate use of antibacterial drugs is a global health concern. Currently, we are walking a fine line between the fact that most bacterial infections can still be cured with the antibiotics known so far, and the emergence of infections with bacteria resistant to several drugs at the same time, against which we no longer have an effective drug. Therefore, new antibacterial drugs are urgently needed to curb the hard-to-treat infections. Our group has developed new antibacterials from the class of novel bacterial topoisomerase inhibitors (NBTIs) that exhibit broad-spectrum antibacterial activity. This article reviews our efforts in developing highly potent NBTIs over the past decade. Following the discovery of an initial hit with potent enzyme inhibitory and broad-spectrum antibacterial activity, an extensive hit-to-lead campaign was conducted with the goal of optimizing physicochemical properties, reducing hERG inhibition, and maintaining antibacterial activity against both Gram-positive and Gram-negative bacteria, with a focus on methicillin-resistant Staphylococcus aureus (MRSA). This optimization strategy resulted in an amide-containing, focused NBTI library with compounds exhibiting potent antibacterial activity against Gram-positive bacteria, reduced hERG inhibition, no cardiotoxicity in in vivo zebrafish model, and favorable in vivo efficacy in a neutropenic murine thigh infection model for MRSA infections.

Study of nitrogen heterocycles as DNA/HSA binder, topoisomerase inhibitors and toxicological safety.

Four new nitrogen-containing heterocyclic derivatives (acridine, quinoline, indole, pyridine) were synthesized and their biological properties were evaluated. The compounds showed affinity for DNA and HSA, with CAIC and CAAC displaying higher binding constants (Kb) of 9.54 × 104 and 1.06 × 106, respectively. The fluorescence quenching assay (Ksv) revealed suppression values ranging from 0.34 to 0.64 × 103 M-1 for ethidium bromide (EB) and 0.1 to 0.34 × 103 M-1 for acridine orange (AO). Molecular docking confirmed the competition of the derivatives with intercalation probes at the same binding site. At 10 µM concentrations, the derivatives inhibited topoisomerase IIα activity. In the antiproliferative assays, the compounds demonstrated activity against MCF-7 and T47-D tumor cells and nonhemolytic profile. Regarding toxicity, no acute effects were observed in the embryos. However, some compounds caused enzymatic and cardiac changes, particularly the CAIC, which increased SOD activity and altered heart rate compared to the control. These findings suggest potential antitumor action of the derivatives and indicate that substituting the acridine core with different cores does not interfere with their interaction and topoisomerase inhibition. Further investigations are required to assess possible toxicological effects, including reactive oxygen species generation.

Demystifying the potential of inhibitors targeting DNA topoisomerases in unicellular protozoan parasites.

DNA topoisomerases are a group of enzymes omnipresent in all organisms. They maintain the DNA topology during replication, repair, recombination, and transcription. However, the structure of topoisomerase in protozoan parasites differs significantly from that of human topoisomerases; thus, this enzyme acts as a crucial target in drug development against parasitic diseases. Although the therapeutic potential of drugs targeting the parasitic topoisomerase is well known, to manage the shortcomings of currently available therapeutics and the emergence of drug resistance, the discovery of novel antiparasitic molecules is an urgent need. In this review, we describe various investigational and repurposed topoisomerase inhibitors developed against protozoan parasites over the past few years.

Structure activity relationship of N-1 substituted 1,5-naphthyrid-2-one analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-9).

Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 µg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 µg/mL).

The Structural Features of Novel Bacterial Topoisomerase Inhibitors That Define Their Activity on Topoisomerase IV.

The continued emergence of bacterial resistance has created an urgent need for new and effective antibacterial agents. Bacterial type II topoisomerases, such as DNA gyrase and topoisomerase IV (topoIV), are well-validated targets for antibacterial chemotherapy. The novel bacterial topoisomerase inhibitors (NBTIs) represent one of the new promising classes of antibacterial agents. They can inhibit both of these bacterial targets; however, their potencies differ on the targets among species, making topoIV probably a primary target of NBTIs in Gram-negative bacteria. Therefore, it is important to gain an insight into the NBTIs key structural features that govern the topoIV inhibition. However, in Gram-positive bacteria, topoIV is also a significant target for achieving dual-targeting, which in turn contributes to avoiding bacterial resistance caused by single-target mutations. In this perspective, we address the structure-activity relationship guidelines for NBTIs that target the topoIV enzyme in Gram-positive and Gram-negative bacteria.

Design, Synthesis and Cytotoxicity of Thiazole-Based Stilbene Analogs as Novel DNA Topoisomerase IB Inhibitors.

A series of new thiazole-based stilbene analogs were designed, synthesized and evaluated for DNA topoisomerase IB (Top1) inhibitory activity. Top1-mediated relaxation assays showed that the synthesized compounds possessed variable Top1 inhibitory activity. Among them, (E)-2-(3-methylstyryl)-4-(4-fluorophenyl)thiazole (8) acted as a potent Top1 inhibitor with high Top1 inhibition of ++++ which is comparable to that of CPT. A possible binding mode of compound 8 with Top1-DNA complex was further provided by molecular docking. An MTT assay against human breast cancer (MCF-7) and human colon cancer (HCT116) cell lines revealed that the majority of these compounds showed high cytotoxicity, with IC50 values at micromolar concentrations. Compounds 8 and (E)-2-(4-tert-butylstyryl)-4-(4-fluorophenyl)thiazole (11) exhibited the most potent cytotoxicity with IC50 values of 0.78 and 0.62 µM against MCF-7 and HCT116, respectively. Moreover, the preliminary structure-activity relationships of thiazole-based stilbene analogs was also discussed.

Identification of new halogen-containing 2,4-diphenyl indenopyridin-5-one derivative as a boosting agent for the anticancer responses of clinically available topoisomerase inhibitors.

Based on previous reports on the significance of halogen moieties and the indenopyridin-5-one skeleton, we designed and synthesized a novel series of halogen (F-, Cl-, Br-, CF3- and OCF3-)-containing 2,4-diphenyl indenopyridin-5-ones and their corresponding -5-ols. Unlike indenopyridin-5-ols, most of the prepared indenopyridin-5-ones with Cl-, Br-, and CF3- groups at the 2-phenyl ring conferred a strong dual topoisomerase I/IIα inhibitory effect. Among the series, para-bromophenyl substituted compound 9 exhibited the most potent topoisomerase inhibition and antiproliferative effects, which showed dependency upon the topoisomerase gene expression level of diverse cancer cells. In particular, as a DNA minor groove-binding non-intercalative topoisomerase I/IIα catalytic inhibitor, compound 9 synergistically promoted the anticancer efficacy of clinically applied topoisomerase I/IIα poisons both in vitro and in vivo, having the great advantage of alleviating poison-related toxicities.

Design and synthesis of ciprofloxacin-sulfonamide hybrids to manipulate ciprofloxacin pharmacological qualities: Potency and side effects.

Fluoroquinolones are a class of antibacterial agents used clinically to treat a wide array of bacterial infections. Although being potent, susceptibility to CNS side effects limits their use. It was observed that improvements in absorption, activity and side effects were achieved via modifications at the N atom of the C7 of the side chain. To meet the increasing demand for development of new antibacterial agents, nineteen novel ciprofloxacin-sulfonamide hybrid molecules were designed, synthesized and characterized by IR, 1H NMR and 13C NMR as potential antibacterial agents with dual DNA gyrase/topoisomerase IV inhibitory activity. Most of the synthesized compounds showed significant antibacterial activity that was revealed by testing their inhibitory activity against DNA gyrase, DNA topoisomerase IV as well as their minimum inhibitory concentration against Staphylococcus aureus. Six ciprofloxacin-sulfonamide hybrids (3f, 5d, 7a, 7d, 7e and 9b) showed potent inhibitory activity against DNA topoisomerase IV, compared to ciprofloxacin (IC50: 0.55 µM), with IC50 range: 0.23-0.44 µM. DNA gyrase was also efficiently inhibited by five ciprofloxacin-sulfonamide hybrids (3f, 5d, 5e, 7a and 7d) with IC50 range: 0.43-1.1 µM (IC50 of ciprofloxacin: 0.83 µM). Compounds 3a and 3b showed a marked improvement in the antibacterial activity over ciprofloxacin against both Gram-positive and Gram-negative pathogens, namely, Staphylococcus aureus Newman and Escherichia coli ATCC8739, with MIC = 0.324 and 0.422 µM, respectively, that is 4.2-fold and 3.2-fold lower than ciprofloxacin (MIC = 1.359 µM) against the Gram-positive Staphylococcus aureus, and MIC = 0.025 and 0.013 µM, respectively, that is 10.2-fold and 19.6-fold lower than ciprofloxacin (MIC = 0.255 µM) against the Gram-negative Escherichia coli ATCC8739. Also, the most active compounds showed lower CNS and convulsive side effects compared to ciprofloxacin with a concomitant decrease in GABA expression.

N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents: Synthesis and biological evaluation.

Glioblastoma is one of the most lethal brain tumors. The crucial chemotherapy is mainly alkylating agents with modest clinical success. Given this desperate need and inspired by the encouraging results of a phase II trial via concomitant Topo I inhibitor plus COX-2 inhibitor, we designed a series of N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents based on structure modification on 1,5-naphthyridine derivatives (Topo I inhibitors). Notably, the target compounds I-1 (33.61 ± 1.15 µM) and I-8 (45.01 ± 2.37 µM) were confirmed to inhibit COX-2, while a previous reported compound (1,5-naphthyridine derivative) resulted nearly inactive towards COX-2 (IC50 > 150 µM). Besides, I-1 and I-8 exhibited higher anti-proliferation, anti-migration, anti-invasion effects than the parent compound 1,5-naphthyridine derivative, suggesting the success of modification based on the parent. Moreover, I-1 obviously repressed tumor growth in the C6 glioma orthotopic model (TGI = 66.7%) and U87MG xenograft model (TGI = 69.4%). Besides, I-1 downregulated PGE2, VEGF, MMP-9, and STAT3 activation, upregulated E-cadherin in the orthotopic model. More importantly, I-1 showed higher safety than temozolomide and different mechanism from temozolomide in the C6 glioma orthotopic model. All the evidence demonstrated that N-2-(phenylamino) benzamide derivatives as novel anti-glioblastoma agents could be promising for the glioma management.

Iridium(iii) complexes as mitochondrial topoisomerase inhibitors against cisplatin-resistant cancer cells.

Herein, we developed the first metal-based mitochondrial topoisomerase inhibitors to achieve an effective therapeutic outcome for the therapy of cisplatin-resistant tumour cells.

Thiosemicarbazide Derivatives Decrease the ATPase Activity of Staphylococcus aureus Topoisomerase IV, Inhibit Mycobacterial Growth, and Affect Replication in Mycobacterium smegmatis.

Compounds targeting bacterial topoisomerases are of interest for the development of antibacterial agents. Our previous studies culminated in the synthesis and characterization of small-molecular weight thiosemicarbazides as the initial prototypes of a novel class of gyrase and topoisomerase IV inhibitors. To expand these findings with further details on the mode of action of the most potent compounds, enzymatic studies combined with a molecular docking approach were carried out, the results of which are presented herein. The biochemical assay for 1-(indol-2-oyl)-4-(4-nitrophenyl) thiosemicarbazide (4) and 4-benzoyl-1-(indol-2-oyl) thiosemicarbazide (7), showing strong inhibitory activity against Staphylococcus aureus topoisomerase IV, confirmed that these compounds reduce the ability of the ParE subunit to hydrolyze ATP rather than act by stabilizing the cleavage complex. Compound 7 showed better antibacterial activity than compound 4 against clinical strains of S. aureus and representatives of the Mycobacterium genus. In vivo studies using time-lapse microfluidic microscopy, which allowed for the monitoring of fluorescently labelled replisomes, revealed that compound 7 caused an extension of the replication process duration in Mycobacterium smegmatis, as well as the growth arrest of bacterial cells. Despite some similarities to the mechanism of action of novobiocin, these compounds show additional, unique properties, and can thus be considered a novel group of inhibitors of the ATPase activity of bacterial type IIA topoisomerases.

Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L.

Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections.

Oxocrebanine: A Novel Dual Topoisomerase inhibitor, Suppressed the Proliferation of Breast Cancer Cells MCF-7 by Inducing DNA Damage and Mitotic Arrest.

BACKGROUND: DNA topoisomerase (Topo) inhibition plays key role in breast cancer treatment. Stephania hainanensis H. S. Lo et Y. Tsoong (S. hainanensis), a Li nationality plant that has abundant aporphine alkaloids, can inhibit Topo. PURPOSE: To identify a dual Topo inhibitor, a deep and systematic study of active aporphine alkaloids in S. hainanensis and their mechanisms of inhibiting breast cancer proliferation and Topo activity are essential. STUDY DESIGN: This study aimed to assess the anti-breast cancer and Topo inhibitory activities of oxocrebanine and explore the underlying mechanisms. METHODS: The growth inhibitory activities of 12 compounds in S. hainanensis were screened by MTT assay in MCF-7, SGC-7901, HepG-2 cells, and compared with the effects on human normal mammary epithelial MCF-10A cells as non cancer control cells. The Topo inhibitory activity was assessed by DNA relaxation and unwinding assays, kDNA decatenation assay and western blot. Cell cycle and autophagy analyses were carried out with flow cytometry and staining. Acridine orange staining and α-tubulin morphology were observed by fluorescence microscopy. Western blot was used to examine microtubule assembly dynamics and the expression levels of key proteins associated with DNA damage, autophagy and mitotic arrest. RESULTS: Oxocrebanine was the anti-breast cancer active alkaloid in S. hainanensis. It exhibited the best inhibitory effect on MCF-7 cells with an IC50 of 16.66 µmol/l, and had only weak effect on the proliferation of MCF-10A cells. Oxocrebanine inhibited Topo I and II α in a cell-free system and in MCF-7 cells. The DNA unwinding assay suggested that oxocrebanine intercalated with DNA as a catalytic inhibitor. Oxocrebanine regulated the levels of Topo I and IIα and DNA damage-related proteins. Oxocrebanine led to the mitotic arrest, and these effects occurred through both p53-dependent and p53-independent pathways. Oxocrebanine induced autophagy, abnormal α-tubulin morphology and stimulated enhanced microtubule dynamics. CONCLUSION: Oxocrebanine was the anti-breast cancer active aporphine alkaloid in S. hainanensis. Oxocrebanine was a Topo I/IIα dual inhibitor, catalytic inhibitor and DNA intercalator. Oxocrebanine caused DNA damage, autophagy, and mitotic arrest in MCF-7 cells. Oxocrebanine also disrupted tubulin polymerization. Accordingly, oxocrebanine held a great potential for development as a novel dual Topo inhibitor for effective breast cancer treatment.

DNA topoisomerases as additional targets for anticancer monofunctional platinum(ii) complexes.

Topoisomerases are ubiquitous enzymes and important targets for DNA-oriented anticancer drugs. Two mitochondrion-targeted monofunctional platinum(ii) complexes, [Pt(ortho-PPh3CH2Py)(NH3)2Cl](NO3)2 (OPT) and [Pt(para-PPh3CH2Py)(NH3)2Cl](NO3)2 (PPT; PPh3 = triphenylphosphonium, Py = pyridine), show significant inhibition towards the activity of DNA topoisomerases in addition to their DNA binding and mitochondrial targeting capabilities. OPT exhibits strong cytotoxicity toward the human renal clear cell carcinoma 786-O and the murine prostate cancer RM-1 cell lines. The complex could bind to the minor groove of DNA, as well as DNA topoisomerases I and IIα, thereby acting as an inhibitor of topoisomerase I/IIα and causing DNA damage. The damage was evidenced by the enhanced expression of γ-H2AX, Chk1/2 phosphorylation, p53 and cell cycle arrest in the G2/M phase. In contrast, the inhibitory effect of PPT on DNA topoisomerases was largely limited to the isolated enzymes. The results demonstrate that the cellular inhibition of the complex towards the DNA topoisomerases positively correlated with its mitochondrial accumulation. Molecular docking provided more detailed structural insights into the interactions of OPT or PPT with DNA and topoisomerase I/IIα. The binding sites of OPT and PPT in topoisomerase-DNA complexes are different from each other. Aside from previously revealed DNA and mitochondrial targets, this study discovered new evidence that DNA topoisomerases may also serve as targets of monofunctional platinum(ii) complexes. For a multispecific platinum complex, strong DNA binding ability does not necessarily lead to potent cytotoxicity as other factors including the cell types, mitochondrial accumulation, and activity of DNA topoisomerases also affect the outcome of DNA damage.

DNA topoisomerases as molecular targets for anticancer drugs.

The significant role of topoisomerases in the control of DNA chain topology has been confirmed in numerous research conducted worldwide. The prevalence of these enzymes, as well as the key importance of topoisomerase in the proper functioning of cells, have made them the target of many scientific studies conducted all over the world. This article is a comprehensive review of knowledge about topoisomerases and their inhibitors collected over the years. Studies on the structure-activity relationship and molecular docking are one of the key elements driving drug development. In addition to information on molecular targets, this article contains details on the structure-activity relationship of described classes of compounds. Moreover, the work also includes details about the structure of the compounds that drive the mode of action of topoisomerase inhibitors. Finally, selected topoisomerases inhibitors at the stage of clinical trials and their potential application in the chemotherapy of various cancers are described.

Necroptosis Induced by Ruthenium(II) Complexes as Dual Catalytic Inhibitors of Topoisomerase I/II.

Inducing necroptosis in cancer cells is an effective approach to circumvent drug-resistance. Metal-based triggers have, however, rarely been reported. Ruthenium(II) complexes containing 1,1-(pyrazin-2-yl)pyreno[4,5-e][1,2,4]triazine were developed with a series of different ancillary ligands (Ru1-7). The combination of the main ligand with bipyridyl and phenylpyridyl ligands endows Ru7 with superior nucleus-targeting properties. As a rare dual catalytic inhibitor, Ru7 effectively inhibits the endogenous activities of topoisomerase (topo) I and II and kills cancer cells by necroptosis. The cell signaling pathway from topo inhibition to necroptosis was elucidated. Furthermore, Ru7 displays significant antitumor activity against drug-resistant cancer cells in vivo. To the best of our knowledge, Ru7 is the first Ru-based necroptosis-inducing chemotherapeutic agent.

Novel bacterial topoisomerase inhibitors derived from isomannide.

A series of Novel Bacterial Topoisomerase Inhibitors (NBTIs) employing a linker derived from isomannide were synthesized and evaluated. Reduced hERG inhibition was observed compared to structure-matched analogues with different linkers, and compound 6 showed minimal proarrhythmic potential using an in vitro panel of cardiac ion channels. Compound 6 also displayed excellent activity against fluoroquinolone-resistant MRSA (MIC90 = 2 µg/mL) and other Gram-positive pathogens.

Dual-Target Inhibitors Based on HDACs: Novel Antitumor Agents for Cancer Therapy.

Histone deacetylases (HDACs) play an important role in regulating target gene expression. They have been highlighted as a novel category of anticancer targets, and their inhibition can induce apoptosis, differentiation, and growth arrest in cancer cells. In view of the fact that HDAC inhibitors and other antitumor agents, such as BET inhibitors, topoisomerase inhibitors, and RTK pathway inhibitors, exert a synergistic effect on cellular processes in cancer cells, the combined inhibition of two targets is regarded as a rational strategy to improve the effectiveness of these single-target drugs for cancer treatment. In this review, we discuss the theoretical basis for designing HDAC-involved dual-target drugs and provide insight into the structure-activity relationships of these dual-target agents.

Synthesis, anticancer evaluation and molecular docking studies of new heterocycles linked to sulfonamide moiety as novel human topoisomerase types I and II poisons.

A series of heterocyclic compounds with a sulfonamide moiety were synthesized from reaction of enaminone 4 with active methylene compounds, glycine derivatives, 1,4-benzoquinone, hydroxylamine hydrochloride, hydrazonyl halides and dimethylacetylenedicarboxylate. The newly synthesized sulfonamide derivatives were characterized by FT-IR, 1H NMR, 13C NMR, mass spectroscopy, elemental analysis and alternative synthetic routes. The reactions products were evaluated for their antiproliferative activity against a panel of three different human cancerous cell lines, MCF-7 (breast), HepG-2 (liver) and HCT-116 (colon) and the results were deployed to derive the structure-activity relationships (SAR). Various test compounds were potent antiproliferative to cancerous cells; reaching very low micromolar levels, as in case of 21 which showed IC50 value of 6.2 µM against HepG-2 cell. In addition, treatment of cancerous cells with the synthesized compounds induced cell apoptosis and G2/M phase arrest evidenced by flow cytometric analysis. Furthermore, the activity of the synthesized compounds against TOP I and II were documented by DNA relaxation assays. Data revealed that compound 24 significantly interfered with TOP I- and II-mediated DNA relaxation, nicking and decatenation, with IC50 values 27.8 and 33.6 µM, respectively. Moreover, the molecular docking studies supported the results from enzymatic assays, where compound 24 was intercalated between nucleotides flanking the DNA cleavage site via pi-pi stacking and hydrophobic interactions. In conclusion, aromatic heterocycles linked to sulfonamides are excellent molecular frameworks amenable for optimization as dual TOP I and II poisons to control various human malignancies.