Ciro Milite

p300/CBP-associated factor selectively regulates the extinction of conditioned fear

It is well established that the activity of chromatin-modifying enzymes is crucial for regulating gene expression associated with hippocampal-dependent memories. However, very little is known about how these epigenetic mechanisms influence the formation of cortically dependent memory, particularly when there is competition between opposing memory traces, such as that which occurs during the acquisition and extinction of conditioned fear. Here we demonstrate, in C57BL/6 mice, that the activity of p300/CBP-associated factor (PCAF) within the infralimbic prefrontal cortex is required for long-term potentiation and is necessary for the formation of memory associated with fear extinction, but not for fear acquisition. Further, systemic administration of the PCAF activator SPV106 enhances memory for fear extinction and prevents fear renewal. The selective influence of PCAF on fear extinction is mediated, in part, by a transient recruitment of the repressive transcription factor ATF4 to the promoter of the immediate early gene zif268, which competitively inhibits its expression. Thus, within the context of fear extinction, PCAF functions as a transcriptional coactivator, which may facilitate the formation of memory for fear extinction by interfering with reconsolidation of the original memory trace.

Design, Synthesis and Biological Evaluation of Carboxy Analogues of Arginine Methyltransferase Inhibitor 1 (AMI-1)

Here we report the synthesis of a number of compounds structurally related to arginine methyltransferase inhibitor 1 (AMI‐1). The structural alterations that we made included: 1) the substitution of the sulfonic groups with the bioisosteric carboxylic groups; 2) the replacement of the ureidic function with a bis‐amidic moiety; 3) the introduction of a N‐containing basic moiety; and 4) the positional isomerization of the aminohydroxynaphthoic moiety. We have assessed the biological activity of these compounds against a panel of arginine methyltransferases (fungal RmtA, hPRMT1, hCARM1, hPRMT3, hPRMT6) and a lysine methyltransferase (SET7/9) using histone and nonhistone proteins as substrates. Molecular modeling studies for a deep binding‐mode analysis of test compounds were also performed. The bis‐carboxylic acid derivatives 1 b and 7 b emerged as the most effective PRMT inhibitors, both in vitro and in vivo, being comparable or even better than the reference compound (AMI‐1) and practically inactive against the lysine methyltransferase SET7/9.

Nutraceutical properties and polyphenolic profile of berry skin and wine of Vitis vinifera L. (cv. Aglianico)

Red grapes are rich in phenolics, flavonoids, anthocyanins and resveratrol, all substances which have been suggested as having nutraceutical and health benefits. The berry skin and wine of grape cultivar Vitis vinifera L. (cv. Aglianico), grown in Basilicata (Southern Italy) were examined to determinate the presence of the above mentioned compounds as well as to establish the inorganic cation profile. HPLC analysis coupled with LC-ESI/MS/MS detected high contents of total flavonols and anthocyanins in berry skin and wine. The wine made with the same grape used for berry skin assays showed a notable presence of quercetin-3-O-glucoside (39.4% of total flavonols), and malvidin and petunidin derivatives (63.9% and 10.8% of total anthocyanins, respectively). The strong antioxidant ROS-scavenging activity, determined by both DPPH and FRAP assays, and the high resveratrol content confer high sensory characteristics resulted to be associated with positive nutraceutical properties of these grapes and wine. The level of cis-resveratrol was lower than trans-resveratrol in both berry skin and wine reaching 44.1mg/kg and 0.3mg/l, respectively. The cation profile presents low levels of Ca, Cu, K, Fe, Zn and Cd compared to numerous, important red wines, such as Monastrell and Tempranillo.

Synthesis and cytotoxic activity evaluation of 2,3-thiazolidin-4-one derivatives on human breast cancer cell lines

It is well known that resveratrol (RSV) displayed cancer-preventing and anticancer properties but its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. Aim of this work was to synthesize pharmacologically active resveratrol analogs with an enhanced structural rigidity and bioavailability. In particular, we have synthesized a library of 2,3-thiazolidin-4-one derivatives in which a thiazolidinone nucleus connects two aromatic rings. Some of these compounds showed strong inhibitory effects on breast cancer cell growth. Our results indicate that some of thiazolidin-based resveratrol derivatives may become a new potent alternative tool for the treatment of human breast cancer.

Modulation of the activity of histone acetyltransferases by long chain alkylidenemalonates (LoCAMs).

A novel class of KAT modulators (long chain alkylidenemalonates, LoCAMs) has been identified. Variations of the alkyl chain length can change the activity profile from inhibition of both KAT3A/KAT2B (as derivative 2a) to the peculiar profile of pentadecylidenemalonate 1b, the first activator/inhibitor of histone acetyltransferases. Together with the powerful apoptotic effect (particularly notable if considering that anacardic acid and other KAT inhibitors are not cell permeable) appoint them as valuable biological tools to understand the mechanisms of lysine acetyltransferases.

A Novel Cell-Permeable, Selective, and Noncompetitive Inhibitor of KAT3 Histone Acetyltransferases from a Combined Molecular Pruning/Classical Isosterism Approach

Selective inhibitors of the two paralogue KAT3 acetyltransferases (CBP and p300) may serve not only as precious chemical tools to investigate the role of these enzymes in physiopathological mechanisms but also as lead structures for the development of further antitumor agents. After the application of a molecular pruning approach to the hardly optimizable and not very cell-permeable garcinol core structure, we prepared many analogues that were screened for their inhibitory effects using biochemical and biophysical (SPR) assays. Further optimization led to the discovery of the benzylidenebarbituric acid derivative 7h (EML425) as a potent and selective reversible inhibitor of CBP/p300, noncompetitive versus both acetyl-CoA and a histone H3 peptide, and endowed with good cell permeability. Furthermore, in human leukemia U937 cells, it induced a marked and time-dependent reduction in the acetylation of lysine H4K5 and H3K9, a marked arrest in the G0/G1 phase and a significant increase in the hypodiploid nuclei percentage.

Synthesis and Biological Evaluation of 4-Phenylquinazoline- 2-carboxamides Designed as a Novel Class of Potent Ligands of the Translocator Protein

A series of novel 4-phenylquinazoline-2-carboxamides (1-58) were designed as aza-isosters of PK11195, the well-known 18 kDa translocator protein (TSPO) reference ligand, and synthesized by means of a very simple and efficient procedure. A number of these derivatives bind to the TSPO with K(i) values in the nanomolar/subnanomolar range, show selectivity toward the central benzodiazepine receptor (BzR) and exhibit structure-affinity relationships consistent with a previously published pharmacophore/topological model of ligand-TSPO interaction.

Antifungal activity of azole compounds CPA18 and CPA109 against azole-susceptible and -resistant strains of Candida albicans

OBJECTIVES In this study we investigated the in vitro fungistatic and fungicidal activities of CPA18 and CPA109, two azole compounds with original structural features, alone and in combination with fluconazole against fluconazole-susceptible and -resistant Candida albicans strains. METHODS Antifungal activities were measured by MIC evaluation and time-kill studies. Azole binding analysis was performed by UV-Vis spectroscopy. Hyphal growth inhibition and filipin and propidium iodide staining assays were used for morphological analysis. An analysis of membrane lipids was also performed to gauge alterations in membrane composition and integrity. Synergism was calculated using fractional inhibitory concentration indices (FICIs). Evaluation of cytotoxicity towards murine macrophages was performed to verify selective antifungal activity. RESULTS Even though their binding affinity to C. albicans Erg11p is comparable to that of fluconazole, CPA compounds are active against resistant strains of C. albicans with a mutation in ERG11 sequences and/or overexpressing the ABC transporter genes CDR1 and CDR2, which encode ATP-dependent efflux pumps. Moreover, CPA18 is fungistatic, even against the two resistant strains, and was found to be synergistic with fluconazole. Differently from fluconazole and other related azoles, CPA compounds induced marked changes in membrane permeability and dramatic alterations in membrane lipid composition. CONCLUSIONS Our outcomes suggest that CPA compounds are able to overcome major mechanisms of resistance in C. albicans. Also, they are promising candidates for combination treatment that could reduce the toxicity caused by high fluconazole doses, particularly in immunocompromised patients.

A continuous-flow synthesis of 1,4-benzodiazepin-5-ones, privileged scaffolds for drug discovery

An efficient and gram-scale continuous-flow protocol for the synthesis of the privileged structure 3,4-dihydro-5H-benzo[e][1,4]diazepin-5-one is reported. If compared to the traditional metal mediated non-catalytic reduction procedure, this approach is high yielding and does not require purification steps and therefore could be conveniently used for the generation of compound libraries for drug discovery.

Identification of Structural Features of 2-Alkylidene-1,3-Dicarbonyl Derivatives that Induce Inhibition and/or Activation of Histone Acetyltransferases KAT3B/p300 and KAT2B/PCAF

Dysregulation of the activity of lysine acetyltransferases (KATs) is related to a variety of diseases and/or pathological cellular states; however, their role remains unclear. Therefore, the development of selective modulators of these enzymes is of paramount importance, because these molecules could be invaluable tools for assessing the importance of KATs in several pathologies. We recently found that diethyl pentadecylidenemalonate (SPV106) possesses a previously unobserved inhibitor/activator activity profile against protein acetyltransferases. Herein, we report that manipulation of the carbonyl functions of a series of analogues of SPV106 yielded different activity profiles against KAT2B and KAT3B (pure KAT2B activator, pan‐inhibitor, or mixed KAT2B activator/KAT3B inhibitor). Among the novel compounds, a few derivatives may be useful chemical tools for studying the mechanism of lysine acetylation and its implications in physiological and/or pathological processes.