Paolo Mellini

Sirtuin modulators: an updated patent review (2012 – 2014)

Introduction: Since 2000 sirtuins (SIRT1–7) have gained growing attention for their connections with many biological processes such as cellular metabolism regulation, neuroprotection, apoptosis, inflammation, and cancer progression. In particular, SIRT1 has been the most studied isoform, not only for its role during caloric restriction but also as target in prevention of aging-related diseases. SIRT inhibition can be useful for treating cancer, HIV infection or muscular diseases, SIRT activation can exert positive effects in aging-related disorders such as metabolism, cardiovascular, and neurodegenerative diseases. Areas covered: This review includes the patents about sirtuin modulation released during the 2012 – 2014 period, and covers the potential therapeutic uses of known sirtuin modulators as well as new related small molecules in various disease contexts. Expert opinion: The effective role of sirtuins in cancer is still controversial, because some of them seem to have tumor-promoter as well as tumor-suppressor properties. Thus, few patents describing SIRT inhibitors have been found in 2012 – 2014 period. Despite the still active debate on their role as direct or indirect activators of SIRT1, sirtuin-activating compounds are actually subjected to intense research for the ability to treat neurodegenerative diseases, metabolic disorders, inflammation, vascular system injuries, wound healing and endothelial dysfunctions. A great number of clinical trials are reported with either SIRT inhibitors or activators, thus it is possible that in the foreseeable future one or more of them will enter in the clinical arena.

Screen of Pseudopeptidic Inhibitors of Human Sirtuins 1–3: Two Lead Compounds with Antiproliferative Effects in Cancer Cells

In the past few years sirtuins have gained growing attention for their involvement in many biological processes such as cellular metabolism, apoptosis, aging and inflammation. In this contribution, we report the synthesis of a library of thioacetylated pseudopeptides that were screened against human sirtuins 1-3 to reveal their in vitro inhibition activities. Molecular modeling studies were performed to acquire data about the binding modes of the inhibitors. Three sirtuin inhibitors were subjected to cellular studies, and all of them showed an increase in acetylation of Lys382 of p53 after DNA damage. Furthermore, two of the compounds were able to inhibit both A549 lung carcinoma and MCF-7 breast carcinoma cell growth in micromolar concentration with the ability to arrest cancer cell cycle in the G1 phase.

Synthesis and antifungal activity of a new series of 2-(1H-imidazol-1-yl)- 1-phenylethanol derivatives

A new series of aromatic ester and carbamate derivatives of 2-(1H-imidazol-1-yl)-1-phenylethanol were synthesized and evaluated for their antifungal activity towards Candida albicans and non-albicans Candida species strains. The aromatic biphenyl ester derivatives 6a-c were more active than the reference compound fluconazole. 6c possesses a MIC mean values of 1.7 ± 1.4 μg mL(-1)vs C. albicans and 1.9 ± 2.0 μg mL(-1)vs non-albicans Candida species strains. The racemic mixtures of 6a, b were purified to afford the pure enantiomers. The (-) isomers were up to 500 times more active than (+) isomers. (-)-6a and (-)-6b were thirty and ninety times more active than fluconazole towards C. krusei strain respectively. The racemates of 6a-c showed low cytotoxicity against human monocytic cell line (U937) with 6a demonstrating a CC(50) greater than 128 μg mL(-1).

Studying SIRT6 regulation using H3K56 based substrate and small molecules

SIRT6 is a modulator of chromatin structure having an important role in healthy ageing, and there is a crucial need to find specific modulators for it. Therefore, the activity of SIRT6 should be studied using a variety of methods. We examined the capability of SIRT6 to deacetylate a set of five fluorogenic substrates based on p53 and histone H3 sequences. The substrate designed around H3K56 deacetylation site exhibited the best signal-to-background ratio and was chosen for further studies. Nicotinamide is a known inhibitor for sirtuins, and it was found to be less potent inhibitor for SIRT6 than it is for SIRT1. In addition, we studied 15 other small molecule sirtuin modulators using the H3K56 based substrate. EX-527, quercetin and three pseudopeptidic compounds were found to be the most potent SIRT6 inhibitors, exhibiting over 50% deacetylation inhibition. These findings describe the first modulators of SIRT6 activity at the physiologically important H3K56 deacetylation site.

1,4-Dihydropyridines Active on the SIRT1/AMPK Pathway Ameliorate Skin Repair and Mitochondrial Function and Exhibit Inhibition of Proliferation in Cancer Cells

Modulators of sirtuins are considered promising therapeutic targets for the treatment of cancer, cardiovascular, metabolic, inflammatory, and neurodegenerative diseases. Here we prepared new 1,4-dihydropyridines (DHPs) bearing changes at the C2/C6, C3/C5, C4, or N1 position. Tested with the SIRTainty procedure, some of them displayed increased SIRT1 activation with respect to the prototype 3a, high NO release in HaCat cells, and ameliorated skin repair in a mouse model of wound healing. In C2C12 myoblasts, two of them improved mitochondrial density and functions. All the effects were reverted by coadministration of compound C (9), an AMPK inhibitor, or of EX-527 (10), a SIRT1 inhibitor, highlighting the involvement of the SIRT1/AMPK pathway in the action of DHPs. Finally, tested in a panel of cancer cells, the water-soluble form of 3a, compound 8, displayed antiproliferative effects in the range of 8-35 μM and increased H4K16 deacetylation, suggesting a possible role for SIRT1 activators in cancer therapy.

SIRT2 inhibition modulate glutamate and serotonin systems in the prefrontal cortex and induces antidepressant-like action

&NA; Growing evidence suggests that changes in histone acetylation in specific sites of the chromatin modulate neuronal plasticity and contribute to antidepressant‐like action. Sirtuin 2 (SIRT2) is a class III NAD+‐dependent histone deacetylase involved in transcriptional repression of genes regulating synaptic plasticity. Importantly, a key role for the glutamate system in prefrontal cortex (PFC) synaptic plasticity changes induced by antidepressants has been suggested. Here, we asked whether SIRT2 could be a pharmacological target for depression therapy. The compound 2‐{3‐(3‐fluorophenethyloxy)phenylamino}benzamide (33i), a selective SIRT2 inhibitor in vitro, was studied in mice (C57Bl6). Firstly, the inhibitory effect of subchronic 33i (5–15 mg/kg, 10 days) on SIRT2 activity in the PFC was evaluated. Moreover, the effect of SIRT2 inhibition on the expression of synaptic plasticity markers linked to glutamate neurotransmission (VGLUT1, synaptophysin, mGluR4, GluA1, GluN2B, GluN2A) and on serotonin levels was studied. Further, neurochemical and behavioral effects of chronic (5 weeks) 33i (15 mg/kg) on the chronic mild stress (CMS) model were analyzed. Subchronic 33i inhibited SIRT2, increased GluN2A, GluN2B and serotonin levels in the PFC. Moreover, chronic 33i reverted CMS‐induced anhedonia and social avoidance. Moreover, 33i upregulated postsynaptic GluN2B and phosphorylated form of GluA1 (p‐GluA1), suggesting that SIRT2 inhibition enhance synaptic strength. Yet, CMS also increased both GluN2A and GluN2B in the postsynaptic fraction. These results suggest that Sirt2 inhibition induce antidepressant‐like action and this effect could be mediated by modulation of glutamate and serotonin system in the PFC. Moreover, it highlights the therapeutic potential of SIRT2 inhibitors as new antidepressant agents. HighlightsThe therapeutic potential of SIRT2 inhibitors as new antidepressants is proposed.The compound 33i shows in vivo Sirt2 inhibitory activity in the prefrontal cortex.33i increases serotonin levels and glutamate receptor subunits.33i induces antidepressant‐like action in the CMS model of depression.Glutamate and serotonin targets could be involved in antidepressant action of 33i.

Identification of SNAIL1 Peptide-Based Irreversible Lysine-Specific Demethylase 1-Selective Inactivators

Inhibition of lysine-specific demethylase 1 (LSD1), a flavin-dependent histone demethylase, has recently emerged as a new strategy for treating cancer and other diseases. LSD1 interacts physically with SNAIL1, a member of the SNAIL/SCRATCH family of transcription factors. This study describes the discovery of SNAIL1 peptide-based inactivators of LSD1. We designed and prepared SNAIL1 peptides bearing a propargyl amine, hydrazine, or phenylcyclopropane moiety. Among them, peptide 3, bearing hydrazine, displayed the most potent LSD1-inhibitory activity in enzyme assays. Kinetic study and mass spectrometric analysis indicated that peptide 3 is a mechanism-based LSD1 inhibitor. Furthermore, peptides 37 and 38, which consist of cell-membrane-permeable oligoarginine conjugated with peptide 3, induced a dose-dependent increase of dimethylated Lys4 of histone H3 in HeLa cells, suggesting that they are likely to exhibit LSD1-inhibitory activity intracellularly. In addition, peptide 37 decreased the viability of HeLa cells. We believe this new approach for targeting LSD1 provides a basis for development of potent selective inhibitors and biological probes for LSD1.

The histone methyltransferase EZH2 as a druggable target in SHH medulloblastoma cancer stem cells

The histone methyltransferase EZH2 plays a role in maintenance of the stem component of cancer, and its overexpression and/or mutation typically drives tumor aggressiveness, drug resistance and patients’ poor prognosis. In this study, we use mouse and human medulloblastoma stem-like cells belonging to the Sonic Hedgehog subgroup (SHH MB-SLCs) and demonstrate that genetic suppression of EZH2 reduces the level of its histone mark H3K27me3 and lowers proliferation and self-renewal. We designed an EZH2 inhibitor (EZH2i) as a simplified analog of EPZ005687 and GSK2816126, MC3629, and we tested its biological activity in SHH MB-SLCs. Pharmacological inhibition of EZH2 impairs SHH MB cells proliferation and self-renewal, and induces apoptosis in vitro. Finally, we generated xenograft MB-SLCs orthotopic tumors in nude mice to test MC3629 in vivo. In treated mice, we observed impairment of tumor growth, together with induction of apoptosis and reduction of proliferation and stemness. Overall, these findings describe EZH2 as a druggable target in MB and provide insight into the biological activity of MC3629 as an EZH2i.

Carprofen Analogues as Sirtuin Inhibitors: Enzyme and Cellular Studies

The best of both: SIRT1/2 inhibitors were developed by combining chemical features of selisistat (SIRT1-selective inhibitor; blue) and carprofen (anti-inflammatory drug; red). The most potent compound (shown) increased acetyl-p53 and acetyl-α-tubulin levels, and induced slight apoptosis at 50 μM in U937 cells, differently from selisistat and carprofen.

Activity of Drugs Against Dormant Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is the etiologic agent of tuberculosis (TB) which causes about 1.8 million deaths every year 1. Furthermore, 2 billion people are estimated to be latently infected with Mtb, with 10% of them reactivating to active TB during their lifetime 2. in persons with latent TB, Mtb is thought to live in a nonreplicating (dormant) stage in closed cavities of the lungs with caseous necrosis and little access to oxygen 3. Dormant Mtb is insensitive to the first-line anti-TB drug isoniazid, which is used for the chemoprophylaxis of latent TB 4-5. However, this treatment is very long (9 months), thus the search for drugs more effective against nonreplicating Mtb is an urgent need. several in vitro models have been developed to obtain dormant Mtb including the Wayne model by which is possible to obtain nonreplicating tubercle bacilli by adaptation of replicating cultures to hypoxia through the self-generated formation of an oxygen gradient 4. Using this model we showed that the activity of drugs is related to the growth stage of Mtb 6-7. The purpose of the present investigation was to study the activity of clinically used drugs and newly synthesized agents against dormant cultures obtained as previously described 4, 6-7. Briefly, Mtb strain H37Rv was grown in tubes containing Dubos Tween-Albumin (DTA, Difco Laboratories, Detroit, Mich) broth. Aerobic (A), replicating populations were generated by incubation of the tubes at 37°C with loosened screw-caps for 5 days (A5). For preparation of hypoxic (H), nonreplicating, bacilli, tight-fitting rubber caps were put under the screw-caps to allow addition of drugs by syringe, and the tubes were incubated for 5, 12 and 19 days (H5, H12, H19 cells, respectively). Control tubes with 1.5 μg/ml methylene blue as an indicator of oxygen depletion were added in each experiment. The following drugs known to be active against replicating Mtb 8 were tested: rifampin (RMP), which inhibits RnA transcription; moxifloxacin (Mx), an inhibitor of DnA replication; linezolid (LZ), an inhibitor of protein synthesis. some compounds active against anaerobic bacteria and protozoa 9 were also tested, including metronidazole (MZ) and four MZ-analogs, namely the clinically used ornidazole (ORn) 10 and three newly synthesized derivatives: MZ-A, MZ-B and MZ-C (Table 1). Briefly, MZ was obtained by extraction with dichloromethane after shattering commercial tablets (Ecobi Farmaceutici, Genoa, italy) in warm water. MZ-A was prepared by reaction of MZ with pirazinic acid ethyl anhydride, previously obtained by condensation with triethylamine and ethylchloroformate, in dry acetonitrile. MZ-B was obtained, in dry acetonitrile, by condensation of MZ with 3-trifluoromethybenzoyl chloride in the presence of triethylamine. Finally, MZ-C was obtained in dichloromethane from 3-fluoro-4-(morpholin-4-yl) aniline previously activated as isocianate by treatment with trifosgene and triethylamine 11. All synthesized compounds were purified by silica-gel column chromatography and analytically characterized by FT-iR, 1H and 13C nMR spectroscopy, mass spectrometry, elemental analysis (Table 1). To measure drug activity, the compounds were added to A5 cultures and, by syringe, to H5, H12 and H19 cultures, at the maximum serum concentration (Cmax) for RMP, MZ, ORn (sigma Chemical, st. Louis, Mo), Mx (Bayer, Berlin, Germany), LZ (Pfizer, sandwich, Kent, UK) (8, 8, 10, 4, 8 μg/ml, respectively), and at a concentration equimolar to that of MZ or ORn (46 μM) for MZ-A, MZ-B, MZ-C. After 0, 7 and 14 days of exposure, 1 ml of A or H cultures were washed and resuspended in 1 ml of DTA broth, and 0.2 ml were inoculated in Middlebrook 7H10 agar plates (Difco) for Colony Forming Unit (CFU) determination. The numbers of CFU/ml are shown in Figure 1. Overall, a differential activity of drugs against A5, H5, H12 and H19 cells was observed. Among antibiotics with known anti-TB activity, Mx was more active against A5 cells (6.2 and 6.5 log10 CFU decrease on day 7 and 14, respectively, in comparison with day 0) and H5 cells (3.6 and 3.2 log10 CFU decrease on day 7 and 14, respectively), and less active against H12 and H19 cells. LZ was active against A5 cells (2.7 and 4.7 log10 CFU decrease on day 7 and 14, respectively) and low or inactive against H5, H12 and H19 cells. RMP was active against all four populations on day 7 (6.0, 5.7, 4.2 and 4.8 log10 CFU decrease against A5, H5, H12 and H19 cells, respectively), then the activity increased on day 14 against H12 and H19 cells (5.9 and 6.0 log10 CFU decrease), but not against A5 and H5 cells. MZ, ORn and MZ-A, MZ-B and MZ-C were uniformly ineffective against A5 cells, however, their activity progressively increased with dormancy from H5 to H19 cells. ORn and MZ-C were usually less active than MZ, while MZ-A and MZ-B were significantly more active than MZ against H19 cells (P value of <0.05; student’s t test) on day 7 or 14. MZ-A was significantly more active than MZ (P = 0.031) against H12 cells on day 7. MZ is a drug used for treatment of anaerobic bacteria and protozoa 9 and has been shown to be highly effective at reducing total lung bacterial burdens in Mtb-infected rabbits 12. A trial was performed in south Korea to evaluate the effect of adding MZ to second-line therapy in patients who have multi-drug resistant TB of the lungs 13. ORn may be preferable to MZ for treatment of infections caused by anaerobic bacteria and protozoa because of its longer half-life and fewer side-effects 10, but no information is known for TB. We found that ORn and MZ-C were no more active than MZ while MZ-A and MZ-B were more active than MZ against H19 cells. The lipophilicity of our MZ-derived compounds as calculated by the 1-octanol/water partition (ALOGPs algorithm 14, Virtual Computational Chemistry LAB; http://www.vcclab.org/lab/alogps/start. html) was: MZ-B=2.49 >MZ-C=2.15 >ORn=0.51 >MZ-A=0.28 >MZ=-0.02. Other investigators reported that the addition of a lipophilic chain in position 1 of MZ did not confer aerobic activity and decreased anaerobic activity 15. A similar pattern was shown by MZ-C, but not MZ-A and MZ-B, which were more active than MZ in spite of having different lipophilicities. Most existing anti-TB drugs were developed for activity against logarithmic-phase Mtb; the result is that non-replicating bacilli are not killed by the majority of current drugs, with the ex1 Dipartimento di Malattie infettive, Parassitarie e immunomediate, istituto superiore di sanità, Via Regina Elena 299, 00161 Rome, italy. 2 Dipartimento di Chimica e Tecnologie del Farmaco, “sapienza”, Università di Roma, Piazzale Aldo Moro 5, 00185 Rome, italy.