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The aim of the present study was to investigate the feasibility of targeting Leishmania transporters via appropriately designed chemical probes. Leishmania donovani, the parasite that causes visceral leishmaniasis, is auxotrophic for arginine and lysine and has specific transporters (LdAAP3 and LdAAP7) to import these nutrients. Probes 1–15 were originated by conjugating cytotoxic quinone fragments (II and III) with amino acids (i.e. arginine and lysine) by means of an amide linkage. The toxicity of the synthesized conjugates against Leishmania extracellular (promastigotes) and intracellular (amastigotes) forms was investigated, as well their inhibition of the relevant amino acid transporters. We observed that some conjugates indeed displayed toxicity against the parasites; in particular, 7 was identified as the most potent derivative (at concentrations of 1 µg/mL and 2.5 µg/mL residual cell viability was reduced to 15% and 48% in promastigotes and amastigotes, respectively). Notably, 6, while retaining the cytotoxic activity of quinone II, displayed no toxicity against mammalian THP1 cells. Transport assays indicated that the novel conjugates inhibited transport activity of lysine, arginine and proline transporters. Furthermore, our analyses suggested that the toxic conjugates might be translocated by the transporters into the cells. The non-toxic probes that inhibited transport competed with the natural substrates for binding to the transporters without being translocated. Thus, it is likely that 6, by exploiting amino acid transporters, can selectively deliver its toxic effects to Leishmania cells. This work provides the first evidence that amino acid transporters of the human pathogen Leishmania might be modulated by small molecules, and warrants their further investigation from drug discovery and chemical biology perspectives.

26 p.-8 fig.-2 tab.-1 graph. abst.-+9 p. mat. supl.-10 fig.-2 tab., Common copathogenic factors, including oxidative stress and neuroinflammation, are found to play a vital role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Nowadays, owing to the multifactorial character of the diseases, no effective therapies are available, thus underlying the need for new strategies. Overexpression of the enzyme GSK-3β and downregulation of the Nrf2/ARE pathway are responsible for a decrease in antioxidant defense effects. These pieces of evidence underline the usefulness of dual GSK-3β inhibitors/Nrf2 inducers. In this regard, to design a dual modulator, the structures of a curcumin-based analogue, as GSK-3β inhibitor, and a diethyl fumarate fragment, as Nrf2 inducer, were combined. Among the hybrids, 5 and 6 proved to effectively inhibit GSK-3β, while 4 and 5 showed a marked ability to activate Nrf2 together to increase the neuronal resistance to oxidative stress. These last pieces of evidence translated into specific neuroprotective effects of 4 and 5 against PD pathological events including neurotoxicity elicited by α-synuclein aggregates and 6-hydroxydopamine. Hybrid 5 also showed neuroprotective effects in a C. elegans model of PD where the activation of GSK-3β is intimately involved in Nrf2 regulation. In summary, 5 emerged as an interesting multitarget derivative, valuable to be exploited in a multitarget PD perspective., This work was supported by the University of Bologna (RFO funds), MCIU (grant no. SAF2016-76693-R), and ISCiii CIBERNED (CB18/05/00040) and EDS, partially funded by the CNR project NUTR-AGE (FOE-2019, DSB.AD004.271).

Osteosarcoma (OS) is a malignant disease characterized by poor prognosis due to a high incidence of metastasis and chemoresistance. Recently, Licochalcone A (Lic-A) has been reported as a promising agent against OS. Starting from chalcones selected from a wide in-house library, a new series was designed and synthetized. The antitumor activity of the compounds was tested on the MG63 OS cell line through the innovative Quantitative Phase Imaging technique and MTT assay. To further investigate the biological profile of active derivatives, cell cycle progression and apoptosis induction were evaluated. An earlier and more consistent arrest in the G2-M phase with respect to Lic-A was observed. Moreover, apoptosis was assessed by Annexin V staining as well as by the detection of typical morphological features of apoptotic cells. Among the selected compounds, 1e, 1q, and 1r proved to be the most promising antitumor molecules. This study pointed out that an integrated methodological approach may constitute a valuable platform for the rapid screening of large series of compounds.

Despite the significant outcomes attained by scientific research, breast cancer (BC) still represents the second leading cause of death in women. Estrogen receptor-positive (ER+) BC accounts for the majority of diagnosed BCs, highlighting the disruption of estrogenic signalling as target for first-line treatment. This goal is presently pursued by inhibiting aromatase (AR) enzyme or by modulating Estrogen Receptor (ER) α. An appealing strategy for fighting BC and reducing side effects and resistance issues may lie in the design of multifunctional compounds able to simultaneously target AR and ER. In this paper, previously reported flavonoid-related potent AR inhibitors were suitably modified with the aim of also targeting ERα. As a result, homoisoflavone derivatives 3b and 4a emerged as well-balanced submicromolar dual acting compounds. An extensive computational study was then performed to gain insights into the interactions the best compounds established with the two targets. This study highlighted the feasibility of switching from single-target compounds to balanced dual-acting agents, confirming that a multi-target approach may represent a valid therapeutic option to counteract ER+ BC. The homoisoflavone core emerged as a valuable natural-inspired scaffold for the design of multifunctional compounds.

The unmet need for the development of effective drugs to treat Alzheimer's disease has been steadily growing, representing a major challenge in drug discovery. In this context, drug repurposing, namely the identification of novel therapeutic indications for approved or investigational compounds, can be seen as an attractive attempt to obtain new medications reducing both the time and the economic burden usually required for research and development programs. In the last years, several classes of drugs have evidenced promising beneficial effects in neurodegenerative diseases, and for some of them, preliminary clinical trials have been started. This review aims to illustrate some of the most recent examples of drugs reprofiled for Alzheimer’s disease, considering not only the finding of new uses for existing drugs but also the new hypotheses on disease pathogenesis that could promote previously unconsidered therapeutic regimens. Moreover, some examples of structural modifications performed on existing drugs in order to obtain multifunctional compounds will also be described.

Among the psychiatric diseases, bipolar disorder (BD) is the sixth leading cause of disability with a prevalence up to 4 % worldwide. BD is a complex neuropsychiatric condition which alternates episodes of mania with symptoms of depression. Although the neurobiological pathways are not completely clarified, the dopamine (DA) hypothesis, recognized as the leading theory explaining the pathophysiology of the malady, states that the dramatically compromised homeostatic regulation of dopaminergic circuits leads to alternated changes in DA neurotransmission. Modulation of D2 and D3 receptors (D2/3R) through partial agonists represents the first-line therapeutic strategy for psychiatric diseases. Moreover, a deregulation of the enzyme glycogen synthase kinase-3β (GSK-3β) has been reported as peculiar feature of BD. In this scenario, the concomitant modulation of D3R and GSK-3β, by employing multitarget compounds, could offer promises to achieve an effective cure of this illness. In the light of these findings, we rationally envisaged the pharmacophoric model at the basis of the design of several D3R partial agonists, suitable to be exploited for the dual D3R/GSK-3β ligand design. Thus, synthetic efforts were addressed to develop a first set of hybrid molecules able to concurrently modulate the selected targets. For a chemical structure point of view, we employed different spacers to combine a substituted aryl-piperazine moiety, reported in previously discovered D3R modulators, with a pyrazole-based fragment, already identified in GSK-3β inhibitors. A fluorescent and a cellular functional assays were carried out to assess the activity of all synthetized compounds against GSK-3β and on D3R, respectively. Most of the derivatives proved to effectively modulate both GSK-3β and D3R with potencies in the low-µM and low-nM range, respectively. The consistent biological data allowed us to identify some lead candidates worth to be further modified with the aim to optimize their biological profile and to perform a structure-activity relationship (SAR) study.

An abnormal increase in glucocorticoid levels is responsible for pathological disorders affecting different organs and systems, and the selective inhibition of appropriate steroidogenic enzymes represents a validated strategy to restore their physiological levels. In continuing our studies on CYP11B inhibitors, in this paper a small series of 6-substituted 3-imidazolylmethylxanthones was designed and synthesized, according to the data acquired from previously reported series of derivatives and from a purposely-performed docking study. The new compounds proved to be potent inhibitors of CYP11B isoforms, being effective on CYP11B1 in the low nanomolar range and improving selectivity with respect to CYP11B2, compared to previously reported related compounds. These data further confirmed that a suitable mutual arrangement of the imidazolylmethyl pharmacophore and a properly selected substituent on the xanthone core allows a fine tuning of the activity towards the different CYPs and further corroborate the role of the xanthone scaffold as a privileged structure in this field.

Aim: Alzheimer’s disease is a still untreatable multifaceted pathology, and drugs able to stop or reverse its progression are urgently needed. In this picture, the recent reformulation of the cholinergic hypothesis renewed the interest for acetylcholinesterase inhibitors. In this paper, a series of naturally inspired chalcone-based carbamates was designed to target cholinesterase enzymes and possibly generate fragments endowed with neuroprotective activity in situ. Results & methodology: All compounds presented in this study showed nanomolar potency for cholinesterase inhibition. Notably, fragment 11d also displayed an interesting neuroprotective profile. Conclusion: These new derivatives are able to simultaneously modulate different key targets involved in Alzheimer’s disease, and could be regarded as promising starting points for the development of disease-modifying drug candidates. [Formula: see text]

Current healthcare has significantly increased the average life expectancy, leading to a consequently greater incidence of age-related diseases, such as Alzheimer's disease. Following a multitarget approach, in this paper a series of polycyclic maleimide-based derivatives were designed and synthesized aimed at simultaneously modulate neuronal calcium channels and glycogen synthase kinase 3-beta (GSK-3β), validated targets to combat Alzheimer’ disease. Different structural modifications were performed on the polycyclic scaffold in order to investigate the structure-activity relationships and compound 10 emerged as a promising non-toxic lead compound, endowed with calcium modulating brain-addressed properties and significant GSK-3β inhibitory activity. Moreover, the easily affordable polycyclic core appears as a new appealing privileged structure in medicinal chemistry.

The modulation of the endocannabinoid system is emerging as a viable avenue for the treatment of neurodegeneration, being involved in neuroprotective and anti-inflammatory processes. In particular, indirectly enhancing endocannabinoid signaling to therapeutic levels through FAAH inhibition might be beneficial for neurodegenerative disorders such as Alzheimer's disease, effectively preventing or slowing the progression of the disease. Hence, in the search for a more effective treatment for Alzheimer's disease, in this paper, the multitarget-directed ligand paradigm was applied to the design of carbamates able to simultaneously target the recently proposed endocannabinoid system and the classic cholinesterase system, and achieve effective dual FAAH/cholinesterase inhibitors. Among the two series of synthesized compounds, while some derivatives proved to be extremely potent on a single target, compounds 9 and 19 were identified as effective dual FAAH/ChE inhibitors, with well-balanced nanomolar activities. Thus, 9 and 19 might be considered as new promising candidates for Alzheimer's disease treatment.

Alzheimer’s disease still represents an untreated multifaceted pathology, and drugs able to stop or reverse its progression are urgently needed. In this paper, a series of naturally inspired chalcone-based derivatives were designed as structural simplification of our previously reported benzofuran lead compound, aiming at targeting both acetyl (AChE)- and butyryl (BuChE) cholinesterases that, despite having been studied for years, still deserve considerable attention. In addition, the new compounds could also modulate different pathways involved in disease progression, due to the peculiar trans-α,β-unsaturated ketone in the chalcone framework. All molecules presented in this study were evaluated for cholinesterase inhibition on the human enzymes and for antioxidant and neuroprotective activities on a SH-SY5Y cell line. The results proved that almost all the new compounds were low micromolar inhibitors, showing different selectivity depending on the appended substituent, some of them were also effective antioxidant and neuroprotective agents. In particular, compound 4, endowed with dual AChE/BuChE inhibitory activity, was able to decrease ROS formation and increase GSH levels, resulting in enhanced antioxidant endogenous defense. Moreover, this compound also proved to counteract the neurotoxicity elicited by Aβ1–42 oligomers, showing a promising neuroprotective potential.

All currently used first-line and second-line drugs for the treatment of leishmaniasis exhibit several drawbacks including toxicity, high costs and route of administration. Furthermore, some drugs are associated with the emergence of drug resistance. Thus, the development of new treatments for leishmaniasis is a priority in the field of neglected tropical diseases. The present work highlights the use of natural derived products, i.e. chalcones, as potential source of antileishmanial agents. Thirty-one novel chalcone compounds have been synthesized and their activity has been evaluated against promastigotes of Leishmania donovani; 16 compounds resulted active against L. donovani in a range from 3.0 to 21.5 μM, showing low toxicity against mammalian cells. Among these molecules, 6 and 16 showed good inhibitory activity on both promastigotes and intracellular amastigotes, coupled with an high selectivity index. Furthermore, compounds 6 and 16 inhibited the promastigote growth of other leishmanial species, including L. tropica, L. major and L. infantum. Finally, 6 and 16 interacted with high affinity with trypanothione reductase (TR), an essential enzyme for the leishmanial parasite and compound 6 inhibited TR with sub-micromolar potency. Thus, the effective inhibitory activity against Leishmania, the lack of toxicity on mammalian cells and the ability to block a crucial parasite's enzyme, highlight the potential for compound 6 to be optimized as novel drug candidate against leishmaniasis.

open 11 no Several studies suggest that curcumin and related compounds possess antioxidant and anti-inflammatory properties including modulation of lipopolysaccharide- (LPS-) mediated signalling in macrophage cell models. We here investigated the effects of curcumin and the two structurally unrelated analogues GG6 and GG9 in primary human blood-derived macrophages as well as the signalling pathways involved. Macrophages differentiated from peripheral blood monocytes for 7 days were activated with LPS or selective Toll-like receptor agonists for 24 h. The effects of test compounds on cytokine production and immunophenotypes evaluated as CD80+/CCR2+and CD206+/CD163+subsets were examined by ELISA and flow cytometry. Signalling pathways were probed by Western blot. Curcumin (2.5-10 μM) failed to suppress LPS-induced inflammatory responses. While GG6 reduced LPS-induced IκB-α degradation and showed a trend towards reduced interleukin-1β release, GG9 prevented the increase in proinflammatory CD80+macrophage subset, downregulation of the anti-inflammatory CD206+/CD163+subset, increase in p38 phosphorylation, and increase in cell-bound and secreted interleukin-1β stimulated by LPS, at least in part through signalling pathways not involving Toll-like receptor 4 and nuclear factor-κB. Thus, the curcumin analogue GG9 attenuated the LPS-induced inflammatory response in human blood-derived macrophages and may therefore represent an attractive chemical template for macrophage pharmacological targeting. open Tedesco, Serena; Zusso, Morena; Facci, Laura; Trenti, Annalisa; Boscaro, Carlotta; Belluti, Federica; Fadini, Gian Paolo; Skaper, Stephen D.; Giusti, Pietro; Bolego, Chiara*; Cignarella, Andrea Tedesco, Serena; Zusso, Morena; Facci, Laura; Trenti, Annalisa; Boscaro, Carlotta; Belluti, Federica; Fadini, Gian Paolo; Skaper, Stephen D.; Giusti, Pietro; Bolego, Chiara*; Cignarella, Andrea

Protozoan infections caused by Plasmodium, Leishmania, and Trypanosoma spp. contribute significantly to the burden of infectious diseases worldwide, causing severe morbidity and mortality. The inadequacy of available treatments calls for cost- and time-effective drug discovery endeavors. To this end, we envisaged the triazole linkage of privileged structures as an effective drug design strategy to generate a focused library of high-quality compounds. The versatility of this approach was combined with the feasibility of a phenotypic assay, integrated with early ADME-tox profiling. Thus, an 18-membered library was efficiently assembled via Huisgen cycloaddition of phenothiazine, biphenyl, and phenylpiperazine scaffolds. The resulting 18 compounds were then tested against seven parasite strains, and counter-screened for selectivity against two mammalian cell lines. In parallel, hERG and cytochrome P450 (CYP) inhibition, and mitochondrial toxicity were assessed. Remarkably, 10-((1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-5-yl)methyl)-10H-phenothiazine (7) and 10-(3-(1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-4-yl)propyl)-10H-phenothiazine (12) showed respective IC50 values of 1.8 and 1.9 μg mL-1 against T. cruzi, together with optimal selectivity. In particular, compound 7 showed a promising ADME-tox profile. Thus, hit 7 might be progressed as an antichagasic lead.

Alzheimer's disease (AD) is a major public health challenge that faces an aging global population. Current drug treatment has demonstrated only symptomatic efficacy, leaving an unmet medical need for a new generation of disease-modifying therapies. Following the multitarget-directed ligand approach, a small library of coumarin-based derivatives was designed and synthesized as a follow-up to our studies on AP2238, aimed at expanding its biological profile. The coumarin substitution pattern at the 6- or 7-position was modified by introducing alkyl chains of variable lengths and with different terminal amino functional groups. 3-(4-{[Benzyl(ethyl)amino]methyl}phenyl)-6-({5-[(7-methoxy-6H-indeno[2,1-b]quinolin-11-yl)amino]pentyl}oxy)-2H-chromen-2-one, bearing the bulkiest amine, emerged as a non-neurotoxic dual acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) inhibitor, potentially suitable for the treatment of the middle stage of AD. Furthermore, the introduction of a diethylamino spacer, as in 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-6-{[5-(diethylamino)pentyl]oxy}-2H-chromen-2-one and 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-7-[4-(diethylamino)butoxy]-2H-chromen-2-one, led to nanomolar human AChE inhibitors endowed with significant inhibitory activity toward Aβ42 self-aggregation, whereas the reference compound was completely ineffective. Furthermore, 3-(4-{[benzyl(ethyl)amino]methyl}phenyl)-7-[4-(diethylamino)butoxy]-2H-chromen-2-one also showed promising neuroprotective behavior, which makes it a potential candidate for development into a disease-modifying agent.

Background and Purpose: Toll-like receptor 4 (TLR4) plays a key role in the induction of inflammatory responses both in peripheral organs and the CNS. Curcumin exerts anti-inflammatory functions by interfering with LPS-induced dimerization of TLR4–myeloid differentiation protein-2 (MD-2) complex and suppressing pro-inflammatory mediator release. However, the inhibitory mechanism of curcumin remains to be defined. Experimental Approach: Binding of bis-demethoxycurcumin (GG6) and its cyclized pyrazole analogue (GG9), lacking the 1,3-dicarbonyl function, to TLR4–MD-2 was determined using molecular docking simulations. The effects of these compounds on cytokine release and NF-κB activation were examined by ELISA and fluorescence staining in LPS-stimulated primary microglia. Interference with TLR4 dimerization was assessed by immunoprecipitation in Ba/F3 cells. Key Results: Both curcumin analogues bound to the hydrophobic region of the MD-2 pocket. However, only curcumin and GG6, both possessing the 1,3-diketone moiety, inhibited LPS-induced TLR4 dimerization, activation of NF-κB and secretion of pro-inflammatory cytokines in primary microglia. Consistent with the ability of 1,3-diketones to coordinate divalent metal ions, LPS stimulation in a low magnesium environment decreased pro-inflammatory cytokine release and NF-κB p65 nuclear translocation in microglia and decreased TLR4–MD-2 dimerization in Ba/F3 cells. Curcumin and GG6 also significantly reduced cytokine output in contrast to the pyrazole analogue GG9. Conclusions and Implications: These results indicate that phenolic 1,3-diketones, with a structural motif able to coordinate magnesium ions, can modulate LPS-mediated TLR4–MD-2 signalling. Taken together, these studies identify a previously uncharacterized mechanism involving magnesium, underlying the inflammatory responses to LPS.

As a follow-up to our previous studies on differently substituted 1,4-dihydropyridines endowed with a peculiar cardiac selectivity, in this paper, a small series of hybrid compounds bearing the pharmacophore fragment of lidoflazine in position 2 or 3 on a 4-(xanthen-9-one)-dihydropyridine core was reported. Lidoflazine was selected due to our promising previously reported data, and the xanthen-9-one substituent was introduced in position 4 of the dihydropyridine scaffold based on the cardiac selectivity observed in several of our studies. The new hybrid compounds were tested to assess cardiac and vascular activities, and the data were evaluated in comparison with those previously obtained for 4-(xanthen-9-one)-dihydropyridines and lidoflazine&ndash, nifedipine hybrid compounds. The functional studies indicated an interesting peculiar selectivity for the cardiac parameter inotropy, in particular when the lidoflazine fragment was introduced in position 2 of the dihydropyridine scaffold (4a&ndash, e), and thus a possible preferential binding with the Cav 1.2 isoform of l-type calcium channels, which are mainly involved in cardiac contractility.

Alzheimer's disease (AD) has been defined as a multi-factorial disorder resulting from a complex array of networked cellular and molecular mechanisms. In particular, elevated levels of Aβ protein and its aggregation products in the presence of metal ions proved to be highly neurotoxic and therapeutic strategies aimed at preventing Aβ generation and oxidative stress may represent an effective approach for AD treatment. A recent paradigm for the treatment of complex diseases such as AD suggests the employment of multifunctional compounds, single chemical entities capable of simultaneously modulating different targets involved in the pathology. In this paper, the "pharmacophores combination" strategy was applied, connecting the main scaffold of the BACE-1 ligand 1 to that of the chalcone 2, as metal chelating pharmacophore, to obtain a small library of compounds. Conjugate 5 emerged as the most interesting derivative, proving to inhibit BACE-1 with low-micromolar potency, and showing neuroprotective effects. In particular, 5 proved to be able to protect from metal-associated oxidative stress by hampering intracellular Cu(2+)-induced ROS formation without any direct neurotoxic effect.

The aim of this study was to develop chitosan-based nanoparticles that could encapsulate lipophilic molecules and deliver them to cancer cells. Nanoparticles were prepared with different molar ratios of chitosan, hyaluronic acid and sulphobutyl-ether-?-cyclodextrin and with or without curcumin. The nanosystems were characterized in terms of their size, zeta potential, morphology, encapsulation efficiency and stability in different media. Intestinal epithelial and colorectal cancer cells were treated with unloaded nanoparticles in order to study their effect on cellular membrane organization and ROS production. Finally, in vitro assays on both cellular lines were performed in order to evaluate the ability of nanoparticles to promote curcumin internalization and to study their effect on cell proliferation and cell cycle. Results show that nanoparticles were positively charged and their size increased with the increasing amounts of the anionic excipient. Nanoparticles showed good encapsulation efficiency and stability in water. Unloaded nanoparticles led to a change in lipid organization in the cellular membrane of both cell lines, without inducing ROS generation. Confocal microscopy, cell proliferation and cell cycle studies allowed the selection of the best formulation to limit curcumin cytotoxicity in normal intestinal epithelial cells and to reduce cancer cell proliferation. The latter was the result of the increase of expression for genes involved in apoptosis.

The multitarget approach has gained increasing acceptance as a useful tool to address complex and multifactorial maladies such as Alzheimer's disease (AD). The concurrent inhibition of the validated AD targets β-secretase (BACE-1) and glycogen synthase kinase-3β (GSK-3β) by attacking both β-amyloid and tau protein cascades has been identified as a promising AD therapeutic strategy. In our study, curcumin was identified as a lead compound for the simultaneous inhibition of both targets; therefore, synthetic efforts were dedicated to obtaining a small library of novel curcumin-based analogues, and a number of potent and balanced dual-target inhibitors were obtained. In particular, 2, 6, and 7 emerged as promising drug candidates endowed with neuroprotective potential and brain permeability. Notably, for some new compounds the symmetrical diketo and the β-keto-enol tautomeric forms were purposely isolated and tested in vitro, allowing us to gain insight into the key requirements for BACE-1 and GSK-3β inhibition.