Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents.

Bacterial topoisomerase functions are required for regulation of DNA supercoiling and overcoming the DNA topological barriers that are encountered during many vital cellular processes. DNA gyrase and topoisomerase IV of the type IIA bacterial topoisomerase family are important clinical targets for antibacterial therapy. Topoisomerase I, belonging to the type IA topoisomerase family, has recently been validated as a potential antitubercular target. The topoisomerase I activity has been shown to be essential for bacterial viability and infection in a murine model of tuberculosis. Mixture-based combinatorial libraries were screened in this study to identify novel bacterial topoisomerase I inhibitors. Using positional-scanning deconvolution, selective small-molecule inhibitors of bacterial topoisomerase I were identified starting from a polyamine scaffold. Antibacterial assays demonstrated that four of these small-molecule inhibitors of bacterial topoisomerase I are bactericidal against Mycobacterium smegmatis and Mycobacterium tuberculosis The MICs for growth inhibition of M. smegmatis increased with overexpression of recombinant M. tuberculosis topoisomerase I, consistent with inhibition of intracellular topoisomerase I activity being involved in the antimycobacterial mode of action.

A novel copper(II) complex identified as a potent drug against colorectal and breast cancer cells and as a poison inhibitor for human topoisomerase IIα.

A novel complex, [Cu(acetylethTSC)Cl]Cl•0.25C2H5OH 1 (where acetylethTSC = (E)-N-ethyl-2-[1-(thiazol-2-yl)ethylidene]hydrazinecarbothioamide), was shown to have anti-proliferative activity against various colon and aggressive breast cancer cell lines. In vitro studies showed that complex 1 acted as a poison inhibitor of human topoisomerase IIα, which may account for the observed anti-cancer effects.

Specialized proresolving mediators in infection and lung injury.

Specialized proresolving mediators (SPMs) are endogenous lipid metabolites of long-chain polyunsaturated fatty acids that are involved in promoting the resolution of inflammation. Many disease conditions characterized by excessive inflammation have impaired or altered SPM biosynthesis, which may lead to chronic, unresolved inflammation. Exogenous administration of SPMs in infectious conditions has been shown to be effective at improving infection clearance and survival in preclinical models. SPMs have also shown tremendous promise in the context of inflammatory lung conditions, such as acute respiratory distress syndrome and chronic obstructive pulmonary disease, mostly in preclinical settings. To date, SPMs have not been studied in the context of the novel Coronavirus, severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), however their preclinical efficacy in combatting infections and improving acute respiratory distress suggest they may be a valuable resource in the fight against Coronavirus disease-19 (COVID-19). Overall, while the research on SPMs is still evolving, they may offer a novel therapeutic option for inflammatory conditions.

Synthesis, characterization, DNA binding, topoisomerase inhibition, and apoptosis induction studies of a novel cobalt(III) complex with a thiosemicarbazone ligand.

In this study, 9-anthraldehyde-N(4)-methylthiosemicarbazone (MeATSC) 1 and [Co(phen)2(O2CO)]Cl·6H2O 2 (where phen = 1,10-phenanthroline) were synthesized. [Co(phen)2(O2CO)]Cl·6H2O 2 was used to produce anhydrous [Co(phen)2(H2O)2](NO3)33. Subsequently, anhydrous [Co(phen)2(H2O)2](NO3)33 was reacted with MeATSC 1 to produce [Co(phen)2(MeATSC)](NO3)3·1.5H2O·C2H5OH 4. The ligand, MeATSC 1 and all complexes were characterized by elemental analysis, FT IR, UV-visible, and multinuclear NMR (1H, 13C, and 59Co) spectroscopy, along with HRMS, and conductivity measurements, where appropriate. Interactions of MeATSC 1 and complex 4 with calf thymus DNA (ctDNA) were investigated by carrying out UV-visible spectrophotometric studies. UV-visible spectrophotometric studies revealed weak interactions between ctDNA and the analytes, MeATSC 1 and complex 4 (Kb = 8.1 × 105 and 1.6 × 104 M-1, respectively). Topoisomerase inhibition assays and cleavage studies proved that complex 4 was an efficient catalytic inhibitor of human topoisomerases I and IIα. Based upon the results obtained from the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay on 4T1-luc metastatic mammary breast cancer cells (IC50 = 34.4 ±â€¯5.2 µM when compared to IC50 = 13.75 ±â€¯1.08 µM for the control, cisplatin), further investigations into the molecular events initiated by exposure to complex 4 were investigated. Studies have shown that complex 4 activated both the apoptotic and autophagic signaling pathways in addition to causing dissipation of the mitochondrial membrane potential (ΔΨm). Furthermore, activation of cysteine-aspartic proteases3 (caspase 3) in a time- and concentration-dependent manner coupled with the ΔΨm, studies implicated the intrinsic apoptotic pathway as the major regulator of cell death mechanism.

Discovery of novel bacterial topoisomerase I inhibitors by use of in silico docking and in vitro assays.

Topoisomerases are important targets for antibacterial and anticancer therapies. Bacterial topoisomerase I remains to be exploited for antibiotics that can be used in the clinic. Inhibitors of bacterial topoisomerase I may provide leads for novel antibacterial drugs against pathogens resistant to current antibiotics. TB is the leading infectious cause of death worldwide, and new TB drugs against an alternative target are urgently needed to overcome multi-drug resistance. Mycobacterium tuberculosis topoisomerase I (MtbTopI) has been validated genetically and chemically as a TB drug target. Here we conducted in silico screening targeting an active site pocket of MtbTopI. The top hits were assayed for inhibition of MtbTopI activity. The shared structural motif found in the active hits was utilized in a second round of in silico screening and in vitro assays, yielding selective inhibitors of MtbTopI with IC50s as low as 2 µM. Growth inhibition of Mycobacterium smegmatis by these compounds in combination with an efflux pump inhibitor was diminished by the overexpression of recombinant MtbTopI. This work demonstrates that in silico screening can be utilized to discover new bacterial topoisomerase I inhibitors, and identifies a novel structural motif which could be explored further for finding selective bacterial topoisomerase I inhibitors.

Inhibition of Zn(II) binding type IA topoisomerases by organomercury compounds and Hg(II).

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3ß may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.

Synthesis, structure-activity relationship studies, and antibacterial evaluation of 4-chromanones and chalcones, as well as olympicin A and derivatives.

On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 µg/mL. SAR analysis revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2',4'-dihydroxylated A ring and the lipophilic substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting complex mechanisms of actions for compounds in this series.