Amsacrine Derivatives Selectively Inhibit Mycobacterial Topoisomerase I (TopA), Impair M. smegmatis Growth and Disturb Chromosome Replication.

Amsacrine, which inhibits eukaryotic type II topoisomerase via DNA intercalation and stabilization of the cleavable topoisomerase-DNA complex, promotes DNA damage and eventually cell death. Amsacrine has also been shown to inhibit structurally distinct bacterial type I topoisomerases (TopAs), including mycobacterial TopA, the only and essential topoisomerase I in Mycobacterium tuberculosis. Here, we describe the modifications of an amsacrine sulfonamide moiety that presumably interacts with mycobacterial TopA, which notably increased the enzyme inhibition and drug selectivity in vivo. To analyse the effects of amsacrine and its derivatives treatment on cell cycle, we used time-lapse fluorescence microscopy (TLMM) and fusion of the ß-subunit of DNA polymerase III with enhanced green fluorescence protein (DnaN-EGFP). We determined that treatment with amsacrine and its derivatives increased the number of DnaN-EGFP complexes and/or prolonged the time of chromosome replication and cell cycle notably. The analysis of TopA depletion strain confirmed that lowering TopA level results in similar disturbances of chromosome replication. In summary, since TopA is crucial for mycobacterial cell viability, the compounds targeting the enzyme disturbed the cell cycle and thus may constitute a new class of anti-tuberculosis drugs.

A quinoline compound inhibits the replication of dengue virus serotypes 1-4 in Vero cells.

BACKGROUND: The global occurrence of dengue, a mosquito-​borne viral disease caused by four distinct dengue viruses (DENV-1, -2, -3 and -4), is reported to have increased approximately 30-fold in the last 50 years, causing approximately 400 million infections a year. A limited use, sub-optimal live attenuated dengue vaccine has become available recently. It is becoming apparent that antibodies to DENVs can promote infection by Zika virus (ZIKV), a related mosquito-borne flavivirus. A drug to treat these flaviviral infections continues to be an unmet public health need. METHODS: We screened an 'in-house' library of approximately 2,000 small molecules for inhibitors of cloned DENV-2 protease. Putative inhibitor binding to DENV-2 protease was analysed by in silico docking. Anti-DENV activity was analysed by monitoring viral antigen synthesis by ELISA, viral RNA synthesis by reverse-transcription​ coupled to real-time polymerase chain reaction and infectious virus production by plaque assay, in DENV-infected Vero cells. RESULTS: A quinoline derivative, BT24, was identified for the first time as a potent inhibitor of the cloned DENV-2 protease (half maximal inhibitory concentration [IC50]=0.5 µM). In silico analysis revealed that BT24 binds to an allosteric site in the vicinity of the active site of DENV-2 protease. Cell-based assays demonstrated that BT24 can inhibit all four DENVs in infected Vero cells. CONCLUSIONS: BT24 is a DENV-2 protease inhibitor which manifests the capacity to inhibit the replication of all four DENVs in cultured cells. It may provide a lead for a pan-DENV inhibitory drug.

Pharmacological evaluation of novel alagebrium analogs as methylglyoxal scavengers in vitro in cardiac myocytes and in vivo in SD rats.

BACKGROUND: Methylglyoxal (MG) is a byproduct of glucose metabolism and an inducer of advanced glycation end products (AGEs). AGEs are implicated in the pathogenesis of diabetes as well as hypertension. Most of the currently available MG scavengers are non-specific and have other effects as well. Alagebrium (ALA), developed by Alteon Corporation is a MG scavenger. Thus the aim of the present study was to investigate the potential of novel ALA analogs as possible MG scavengers and whether they could prevent any deleterious effects of MG. METHODS AND RESULTS: MG levels were measured by HPLC. The different biochemical and molecular parameters were measured by assay kits, RT-PCR and immunocytochemistry. Out of the 15 ALA analogs tested in vitro, compound no. 13 was found to be an effective inhibitor of MG in a concentration and time dependent manner. Compound no. 13 significantly attenuated the MG levels in vitro in MG treated cultured H9C2 cardiomyocytes as well as in vivo in MG treated SD rats. MG induced oxidative stress and apoptosis were attenuated by pretreatment of H9C2 cardiac myocytes with compound no. 13. MG induced cardiac hypertrophy and apoptosis were also attenuated by treating MG treated SD rats with compound no. 13. CONCLUSION: Our results indicate compound 13 as an effective inhibitor of MG in vitro in cultured cardiomyocytes and in vivo in SD rats and thus it may prove very useful in blocking the multiple deleterious effects of MG, including AGEs and vascular complications of diabetes.

Development of acridine derivatives as selective Mycobacterium tuberculosis DNA gyrase inhibitors.

In this study we have designed p-phenylene diamine linked acridine derivative from our earlier reported quinoline-aminopiperidine hybrid MTB DNA gyrase inhibitors with aiming more potency and less cardiotoxicity. We synthesized thirty six compounds using four step synthesis from 2-chloro benzoic acid. Among them compound 4-chloro-N-(4-((2-methylacridin-9-yl)amino)phenyl)benzenesulphonamide (6) was found to be more potent with MTB DNA gyrase super coiling IC50 of 5.21±0.51µM; MTB MIC of 6.59µM and no zHERG cardiotoxicity at 30µM and 11.78% inhibition at 50µM against mouse macrophage cell line RAW 264.7.

The Small Molecule Indirubin-3'-Oxime Inhibits Protein Kinase R: Antiapoptotic and Antioxidant Effect in Rat Cardiac Myocytes.

Double-stranded, RNA-dependent protein kinase R (PKR) is a serine/threonine protein kinase activated by various stress signals. It plays an important role in inflammation, insulin sensitivity and glucose homeostasis. Increased PKR activity has been observed in obese humans as well as in obese diabetic mice. Indirubin-3'-oxime (I3O) is an effective inhibitor of cyclin-dependent kinases and glycogen synthase kinase 3-beta. However, the effects of I3O on PKR activity/expression in cultured rat cardiomyocytes have not been reported. We investigated whether I3O attenuates the effects of high glucose on PKR, oxidative stress and apoptotic gene markers. Quantitative PCR and western blotting were used to measure protein and mRNA, respectively. High glucose treatment caused significant increase in the PKR protein/mRNA expression, which was attenuated by co-treatment with I3O. High glucose-treated, cultured cardiomyocytes developed a significant increase in mRNA expression for c-Jun-N-terminal kinase, caspase-3 and NF-ĸB, which were all attenuated by pretreatment with I3O. There was also a significant increase in reactive oxygen species generation in high glucose-treated, cultured cardiomyocytes, which was attenuated by pretreatment with I3O. In conclusion, I3O may have a preventive role against the deleterious effects of high glucose in the heart.

4-Aminoquinoline derivatives as novel Mycobacterium tuberculosis GyrB inhibitors: Structural optimization, synthesis and biological evaluation.

Mycobacterial DNA gyrase B subunit has been identified to be one of the potentially underexploited drug targets in the field of antitubercular drug discovery. In the present study, we employed structural optimization of the reported GyrB inhibitor resulting in synthesis of a series of 46 novel quinoline derivatives. The compounds were evaluated for their in vitro Mycobacterium smegmatis GyrB inhibitory ability and Mycobacterium tuberculosis DNA supercoiling inhibitory activity. The antitubercular activity of these compounds was tested over Mtb H37Rv strain and their safety profile was checked against mouse macrophage RAW 264.7 cell line. Among all, three compounds (23, 28, and 53) emerged to be active displaying IC50 values below 1 µM against Msm GyrB and were found to be non-cytotoxic at 50 µM concentration. Compound 53 was identified to be potent GyrB inhibitor with 0.86 ± 0.16 µM and an MIC (minimum inhibitory concentration) of 3.3 µM. The binding affinity of this compound towards GyrB protein was analysed by differential scanning fluorimetry which resulted in a positive shift of 3.3 °C in melting temperature (Tm) when compared to the native protein thereby reacertaining the stabilization effect of the compound over protein.

Design and synthesis of novel quinoline-aminopiperidine hybrid analogues as Mycobacterium tuberculosis DNA gyraseB inhibitors.

Antibiotics with good therapeutic value and novel mechanism of action are becoming increasingly important in today's battle against bacterial resistance. One of the popular targets being DNA gyrase, is currently becoming well-established and clinically validated for the development of novel antibacterials. In the present work, a series of forty eight quinoline-aminopiperidine based urea and thiourea derivatives were synthesized as pharmacophoric hybrids and evaluated for their biological activity. Compound, 1-(4-chlorophenyl)-3-(1-(6-methoxy-2-methylquinolin-4-yl)piperidin-4-yl)thiourea (45) was found to exhibit promising in vitro Mycobacterium smegmatis GyrB IC50 of 0.95 ± 0.12 µM and a well correlated Mycobacterium tuberculosis (MTB) DNA gyrase supercoiling IC50 of 0.62 ± 0.16 µM. Further, compound 45 also exhibited commendable MTB MIC, safe eukaryotic cytotoxic profile with no signs of cardiotoxicity in zebrafish ether-a-go-go-related gene (zERG).

Enabling the (3 + 2) cycloaddition reaction in assembling newer anti-tubercular lead acting through the inhibition of the gyrase ATPase domain: lead optimization and structure activity profiling.

DNA gyrase, the sole type II topoisomerase present in Mycobacterium tuberculosis, is absent in humans and is a well validated target for anti-tubercular drug discovery. In this study, a moderately active inhibitor of Mycobacterium tuberculosis GyrB, the pharmaceutically unexploited domain of DNA gyrase, was reengineered using a combination of molecular docking and medicinal chemistry strategies to obtain a lead series displaying considerable in vitro enzyme efficacy and bacterial kill against the Mycobacterium tuberculosis H37Rv strain. Biophysical investigations using differential scanning fluorimetry experiments re-ascertained the affinity of these molecules towards the GyrB domain. Furthermore, the molecules were completely devoid of hERG toxicity up to 30 µM, as evaluated in a zebra fish model with a good selectivity index, and from a pharmaceutical point of view, turned out as potential candidates against TB.