Effect of α-Methoxy Substitution on the Anti-HIV Activity of Dihydropyrimidin-4(3 H)-ones.

Conformational restriction applied to dihydrobenzylpyrimidin-4-(3 H)-ones (DABOs) by the intoduction of a methyl group at the α-benzylic position is known to massively improve the anti-HIV-1 activity of these compounds. Here, we report the effects of methoxy substitution at the α-benzylic position in S-, NH-, and N, N-DABOs carrying 2,6-difluoro, 2-chloro-6-fluoro, or 2,6-dichloro substituted benzyl moieties. The various α-methoxy DABO series (12-14) present different SAR at the dihalo benzyl substitution, with the most potent compounds (12d,e and 13c) showing similar (picomolar/nanomolar) anti-HIV-1 potency as the corresponding α-methyl analogues against wt HIV-1, and 10-100-fold increased potency (up to low nanomolar) against clinically relevant K103N, Y181C, Y188L, IRLL98, and K103N+Y181C HIV-1 mutant strains, highlighting the importance of the α-methoxy substitution to provide highly efficient DABOs as "second generation" NNRTIs. HPLC enantioseparation of three of the most potent derivatives (12d, 13c, and 14c) provided single enantiomers with significant enantioselectivity in HIV-1 inhibition. Computational studies allowed to correlate the best antiviral activity with the ( R) absolute configuration at the α-methoxy stereogenic center.

Disruptor of telomeric silencing 1-like (DOT1L): disclosing a new class of non-nucleoside inhibitors by means of ligand-based and structure-based approaches.

Chemical inhibition of chromatin-mediated signaling involved proteins is an established strategy to drive expression networks and alter disease progression. Protein methyltransferases are among the most studied proteins in epigenetics and, in particular, disruptor of telomeric silencing 1-like (DOT1L) lysine methyltransferase plays a key role in MLL-rearranged acute leukemia Selective inhibition of DOT1L is an established attractive strategy to breakdown aberrant H3K79 methylation and thus overexpression of leukemia genes, and leukemogenesis. Although numerous DOT1L inhibitors have been several structural data published no pronounced computational efforts have been yet reported. In these studies a first tentative of multi-stage and LB/SB combined approach is reported in order to maximize the use of available data. Using co-crystallized ligand/DOT1L complexes, predictive 3-D QSAR and COMBINE models were built through a python implementation of previously reported methodologies. The models, validated by either modeled or experimental external test sets, proved to have good predictive abilities. The application of these models to an internal library led to the selection of two unreported compounds that were found able to inhibit DOT1L at micromolar level. To the best of our knowledge this is the first report of quantitative LB and SB DOT1L inhibitors models and their application to disclose new potential epigenetic modulators.

Development of 1,2,4-Oxadiazoles as Potent and Selective Inhibitors of the Human Deacetylase Sirtuin 2: Structure-Activity Relationship, X-ray Crystal Structure, and Anticancer Activity.

Sirt2 is a target for the treatment of neurological, metabolic, and age-related diseases including cancer. Here we report a series of Sirt2 inhibitors based on the 1,2,4-oxadiazole scaffold. These compounds are potent Sirt2 inhibitors active at single-digit µM level by using the Sirt2 substrate α-tubulin-acetylLys40 peptide and inactive up to 100 µM against Sirt1, -3, and -5 (deacetylase and desuccinylase activities). Their mechanism of inhibition is uncompetitive toward both the peptide substrate and NAD+, and the crystal structure of a 1,2,4-oxadiazole analog in complex with Sirt2 and ADP-ribose reveals its orientation in a still unexplored subcavity useful for further inhibitor development. Tested in leukemia cell lines, 35 and 39 induced apoptosis and/or showed antiproliferative effects at 10 or 25 µM after 48 h. Western blot analyses confirmed the involvement of Sirt2 inhibition for their effects in NB4 and in U937 cells. Our results provide novel Sirt2 inhibitors with a compact scaffold and structural insights for further inhibitor improvement.

Lysine Deacetylase Inhibitors in Parasites: Past, Present, and Future Perspectives.

Current therapies for human parasite infections rely on a few drugs, most of which have severe side effects, and their helpfulness is being seriously compromised by the drug resistance problem. Globally, this is pushing discovery research of antiparasitic drugs toward new agents endowed with new mechanisms of action. By using a "drug repurposing" strategy, histone deacetylase inhibitors (HDACi), which are presently clinically approved for cancer use, are now under investigation for various parasite infections. Because parasitic Zn2+- and NAD+-dependent HDACs play crucial roles in the modulation of parasite gene expression and many of them are pro-survival for several parasites under various conditions, they are now emerging as novel potential antiparasitic targets. This Perspective summarizes the state of knowledge of HDACi (both class I/II HDACi and sirtuin inhibitors) targeted to the main human parasitic diseases (schistosomiasis, malaria, trypanosomiasis, leishmaniasis, and toxoplasmosis) and provides visions into the main issues that challenge their development as antiparasitic agents.

A new nitrobenzoxadiazole-based GSTP1-1 inhibitor with a previously unheard of mechanism of action and high stability.

CONTEXT: The nitrobezoxadiazole derivative NBDHEX is a potent inhibitor of glutathione transferase P1-1 (GSTP1-1) endowed with outstanding anticancer activity in different tumor models. OBJECTIVE: To characterize by in vitro biochemical and in silico studies the NBDHEX analogues named MC2752 and MC2753. MATERIALS AND METHODS: Synthesis of MC2752 and MC2753, biochemical assays and in silico docking and normal-mode analyses. RESULTS: The presence of a hydrophobic moiety in the side chain of MC2753 confers unique features to this molecule. Unlike its parent drug NBDHEX, MC2753 does not require GSH to trigger the dissociation of the complex between GSTP1-1 and TRAF2, and displays high stability towards the nucleophilic attack of the tripeptide under physiological conditions. DISCUSSION AND CONCLUSION: MC2753 may represent a lead compound for the development of novel GSTP1-1 inhibitors not affected in their anticancer action by fluctuations of cellular GSH levels, and characterized by an increased half-life in vivo.

Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features.

Because of its involvement in the progression of several malignant tumors, the histone lysine-specific demethylase 1 (LSD1) has become a prominent drug target in modern medicinal chemistry research. We report on the discovery of two classes of noncovalent inhibitors displaying unique structural features. The antibiotics polymyxins bind at the entrance of the substrate cleft, where their highly charged cyclic moiety interacts with a cluster of positively charged amino acids. The same site is occupied by quinazoline-based compounds, which were found to inhibit the enzyme through a most peculiar mode because they form a pile of five to seven molecules that obstruct access to the active center. These data significantly indicate unpredictable strategies for the development of epigenetic inhibitors.

Sirtuin functions and modulation: from chemistry to the clinic.

Sirtuins are NAD(+)-dependent histone deacetylases regulating important metabolic pathways in prokaryotes and eukaryotes and are involved in many biological processes such as cell survival, senescence, proliferation, apoptosis, DNA repair, cell metabolism, and caloric restriction. The seven members of this family of enzymes are considered potential targets for the treatment of human pathologies including neurodegenerative diseases, cardiovascular diseases, and cancer. Furthermore, recent interest focusing on sirtuin modulators as epigenetic players in the regulation of fundamental biological pathways has prompted increased efforts to discover new small molecules able to modify sirtuin activity. Here, we review the role, mechanism of action, and biological function of the seven sirtuins, as well as their inhibitors and activators.

c-Jun N-terminal kinase activation by nitrobenzoxadiazoles leads to late-stage autophagy inhibition.

BACKGROUND: Nitrobenzoxadiazole derivatives (NBDs), including NBDHEX and the recently developed MC3181, are promising anticancer agents able to target glutathione transferase and inhibit both its catalytic activity and ability to sequester TNF-receptor associated factor 2 (TRAF2) and c-Jun N-terminal kinase (JNK). NBDs have been shown to impair the growth and survival of a broad-spectrum of tumor types, in vitro and in vivo. Herein, we evaluated the effects of the new compound MC3181 on U-2OS osteosarcoma cells and investigated the impact of both NBDHEX and MC3181 on autophagy. METHODS: Cell viability was evaluated by sulforhodamine B assay. The dissociation of the TRAF2-GSTP1-1 complex was detected by proximity ligation assay, while the phospho-activation of JNK was assessed by western blotting. The effects of NBDs on autophagy were evaluated by GFP-LC3 puncta formation, western blotting for LC3-II and p62, and LC3 turnover assay in the presence of bafilomycin A1. The role of JNK in the reduction of autophagic flux caused by NBDs was investigated using JNK1 shRNA-transfected cells. Fluorogenic caspase activity assay and flow cytometric analysis of DNA content were used to determine the cytotoxic effects of NBDs on JNK1-silenced cells. RESULTS: Similar to NBDHEX, MC3181 reduced viability and activated TRAF2/JNK signaling in U-2OS cells. Moreover, NBDs induced the accumulation of autophagic vesicles and LC3-II while reducing both basal and nutritional stress-induced autophagic flux. Furthermore, increased levels of both LC3-II and the autophagy selective substrate p62 were observed in different tumor cell lines treated with NBDs, the concurrent increase of these markers being consistent with an impairment of autophagosome clearance. Autophagy inhibition by NBDs required JNK activity: NBDs caused autophagy inhibition and caspase-3 activation in JNK-positive U-2OS, but no autophagic flux inhibition or caspase-3 activation in JNK-silenced cells. CONCLUSIONS: Our demonstration that NBDs can act as late-phase autophagy inhibitors opens new opportunities to fully exploit their therapeutic potential. This may not rely solely on their effectiveness in inducing cell cycle arrest and apoptosis, but also on their ability to weaken the capacity of tumor cells to endure stress conditions via autophagy. In addition, this study provides evidence that JNK can participate in impairing autophagy.

Synthesis and structure--activity relationship of new cytotoxic agents targeting human glutathione-S-transferases.

The 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX, 1), a "suicide inhibitor" of the glutathione-S-transferase GSTP1-1, showed pro-apoptotic properties in tumor cells, but in vivo studies were limited by poor bioavailability and high affinity towards GSTM2-2, expressed in many non-cancerous tissues. Here we describe the synthesis and biological characterization of new 1 analogs (2-40), in which the hydroxyhexyl portion at the C4-sulfur atom has been replaced with phenyl-containing moieties as well as substituted alkyl chains. Some of the new compounds displayed 10-100 times increased water-solubility (8, 11, 17, 26-28, 34, 35), and most of them showed higher GSTP1-1 selectivity (2-20, 23-26, 31-33, 35) than 1. The presence of a phenyl ring with polar substituents is in general associated, with some exceptions (23, 24) to low cytotoxicity in osteosarcoma U-2OS cells. Differently, some alkyl derivatives possess cytotoxicity comparable (26, 34, 35) or higher (30, 32) than 1. Among the novel compounds, selected ones (26, 27, 34, and 35) deserve further investigation for their anticancer potential.