Nucleoside hydrolase immobilized on magnetic particles as a tool for onflow screening and characterization of inhibitors.

Nucleoside Hydrolases (NH) are considered a target for the development of new antiprotozoal agents. The development of new and automated screening assays for the identification of NH inhibitors can accelerate the first stages of the drug discovery process. In this work, NH from Leishmania donovani (LdNH) was covalently immobilized onto magnetic particles (LdNH-MPs) and trapped by magnets into a TFE tube to yield an immobilized enzyme reactor (IMER). For an automated assay, the LdNH-MP-IMER was connected in-line to an analytical column in an HPLC-DAD system to monitor the enzyme activity through quantification of the product hypoxanthine. Kinetic studies provided a KM value of 2079 ± 87 µmol.L-1 for the inosine substrate. Validation of the LdNH-MP-IMER for onflow screening purposes was performed with a library containing 12 quinolone ribonucleosides. Among them, three were identified as new competitive LdNH inhibitors, with Ki values between 83.5 and 169.4 µmol.L-1. This novel in-line screening assay has proven to be reliable, fast, low cost, and applicable to large libraries of compounds.

Chloroquinolone Carboxamide Derivatives as New Anti-HSV-1 Promising Drugs.

BACKGROUND: Since the emergence of HSV resistant strains, new antiviral agents have emerged and still are urgently needed, especially those with alternative targets. OBJECTIVE: In this work, we evaluated new quinolone derivatives as anti-HSV. METHODS: For this study, cells were infected and treated with different components to evaluate the profile of HSV replication in vitro. In addition, studies were performed to determine the pharmacokinetic toxicity and profile of the compound. RESULTS: Indeed the EC50 values of these promising molecules ranged between 8 µM and 32 µM. We have also showed that all compounds inhibited the expression of ICP27 viral proteins, which gives new insights in the search for new target for antiherpetic therapy. Chlorine in positions C6 and phosphonate in position C1 have shown to be important for viral inhibition. The chloroquinolone carboxamide derivatives fulfilled "Lipinsky Rule of Five" for good oral bioavailability and showed higher intestinal absorption and blood brain barrier penetration, as well as lower toxicity profile. CONCLUSION: Although the inhibition activities of chloroquinolone carboxamide derivatives were lower than acyclovir, they showed different modes of action in comparison to the drugs currently available. These findings encourage us to continue pre-clinical studies for the development of new anti-HSV-1 agents.

Aminoquinolones and Their Benzoquinone Dimer Hybrids as Modulators of Prion Protein Conversion.

Prion Diseases or Transmissible Spongiform Encephalopathies are neurodegenerative conditions associated with a long incubation period and progressive clinical evolution, leading to death. Their pathogenesis is characterized by conformational changes of the cellular prion protein-PrPC-in its infectious isoform-PrPSc-which can form polymeric aggregates that precipitate in brain tissues. Currently, there are no effective treatments for these diseases. The 2,5-diamino-1,4-benzoquinone structure is associated with an anti-prion profile and, considering the biodynamic properties associated with 4-quinolones, in this work, 6-amino-4-quinolones derivatives and their respective benzoquinone dimeric hybrids were synthesized and had their bioactive profile evaluated through their ability to prevent prion conversion. Two hybrids, namely, 2,5-dichloro-3,6-bis((3-carboxy-1-pentyl-4-quinolone-6-yl)amino)-1,4-benzoquinone (8e) and 2,5-dichloro-3,6-bis((1-benzyl-3-carboxy-4-quinolone-6-yl)amino)-1,4-benzoquinone (8f), stood out for their prion conversion inhibition ability, affecting the fibrillation process in both the kinetics-with a shortening of the lag phase-and thermodynamics and their ability to inhibit the formation of protein aggregates without significant cytotoxicity at ten micromolar.

Selective AMPK activator leads to unfolded protein response downregulation and induces breast cancer cell death and autophagy.

AIMS: AMPK plays a critical role regulating cell metabolism, growth and survival. Interfering with this enzyme activity has been extensively studied as putative mechanism for cancer therapy. The present work aims to identify a specific AMPK activator for cancer cells among a series of novel heterocyclic compounds. MATERIALS AND METHODS: A series of novel hybrid heterocyclic compounds, namely naphtoquinone-4-oxoquinoline and isoquinoline-5,8-quinone-4-oxoquinoline derivatives, were synthesized via Michael reaction and their structures confirmed by spectral data: infrared; 1H and 13C NMR spectroscopy (COSY, HSQC, HMBC); and high-resolution mass spectrometry (HRMS). The novel compounds were screened and tested for antitumoral activity and have part of their mechanism of action scrutinized. KEY FINDINGS: Here, we identified a selective AMPK activator among the new hybrid heterocyclic compounds. This new compound presents selective cytotoxicity on breast cancer cells but not on non-cancer counterparts. We identified that by specifically activating AMPK in cancer cells, the drug downregulates unfolded protein response pathway, as well as inhibits mTOR signaling. SIGNIFICANCE: These effects, that are selective for cancer cells, lead to activation of autophagy and, ultimately, to cancer cells death. Taken together, our data support the promising anticancer activity of this novel compound which is a strong modulator of metabolism.

1,2:5,6-Di-O-iso-propyl-idene-3-C-methyl-α-d-allo-furan-ose.

The title carbohydrate, C13H22O6, is a derivative of d-glycose, in which the furan-osidic and iso-propyl-idene rings are in twisted conformations. The mean plane of the furan-osidic ring makes a dihedral angle of 70.32 (18)° with the mean plane of the fused iso-propyl-idene ring. The methyl groups in the other iso-propyl-idene ring are disordered over two sets of sites, with an occupancy ratio of 0.74 (6):0.26 (6). In the crystal, mol-ecules are linked by O-H⋯O hydrogen bonds into chains with graph-set notation C(5) along [100]. Weak C-H⋯O interactions also occur.

Characterization of HIV-1 enzyme reverse transcriptase inhibition by the compound 6-chloro-1,4-dihydro-4-oxo-1-(beta-D-ribofuranosyl) quinoline-3-carboxylic acid through kinetic and in silico studies.

We recently described that the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(beta-D-ribofuranosyl) quinoline-3-carboxylic acid (compound A) inhibits the human immunodeficiency virus type 1 (HIV-1) enzyme reverse transcriptase (RT), and its replication in primary cells. Based on these findings, we performed kinetic studies to investigate the mode of inhibition of compound A and its aglycan analog (compound B). We found that both molecules inhibited RT activity independently of the template/primer used. Nevertheless, compound A was 10-fold more potent than compound B. Compound A inhibited the RNA-dependent DNA polymerase (RDDP) activity of RT with an uncompetitive and a noncompetitive mode of action with respect to dTTP incorporation and to template/primer (TP) uptake, respectively. The kinetic pattern of the inhibition displayed by compound A was probably due to its greater affinity for the ternary complex (RT-TP-dNTP) than the enzyme alone or the binary complex (RT-TP). Besides, by means of molecular modeling, we show that compound A bound on the NNRTI binding pocket of RT. However, our molecule targets such a site by making novel interactions with the enzyme RT, when compared to NNRTIs. These include a hydrogen bridge between the 2'-OH of our compound and the Tyr675 of the enzyme RT's chain B. Therefore, compound A is able to synergize with both a NRTI (AZT-TP) and a NNRTI (efavirenz). Taken together, our results suggest that compound A displays a novel mechanism of action, which may be different from classical NRTIs and NNRTIs.

The compound 6-chloro-1,4-dihydro-4-oxo-1-(beta-D-ribofuranosyl) quinoline-3-carboxylic acid inhibits HIV-1 replication by targeting the enzyme reverse transcriptase.

We describe in this paper that the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(beta-D-ribofuranosyl)-quinoline-3-carboxylic acid (compound A) inhibits the HIV-1 replication in human primary cells. We initially observed that compound A inhibited HIV-1 infection in peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner, resulting in an EC(50) of 1.5 +/- 0.5 microM and in a selective index of 1134. Likewise, compound A blocked HIV-1(BA-L) replication in macrophages in a dose-dependent manner, with an EC(50) equal to 4.98 +/- 0.9 microM. The replication of HIV-1 isolates from subtypes C and F was also inhibited by compound A with the same efficiency. Compound A inhibited an early event of the HIV-1 replicative cycle, since it prevented viral DNA synthesis in PBMCs exposed to HIV-1. Kinetic assays demonstrated that compound A inhibits the HIV-1 enzyme reverse transcriptase (RT) in dose-dependent manner, with a K(I) equal to 0.5 +/- 0.04 microM. Using a panel of HIV-1 isolates harboring NNRTI resistance mutations, we found a low degree of cross-resistance between compound A and clinical available NNRTIs. In addition, compound A exhibited additive effects with the RT inhibitors AZT and nevirapine, and synergized with the protease inhibitor atazanavir. Our results encourage continuous studies about the kinetic impact of compound A towards different catalytic forms of RT enzyme, and suggest that our nucleoside represents a promising molecule for future antiretroviral drug design.