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Sigma-1 receptor (S1R) is involved in a large array of biological functions due to its ability to interact with various proteins and ion channels. Crystal structures of human S1R revealed the trimeric organization for which each protomer comprises the ligand binding pocket. This study applied a multistep computational procedure to develop a pharmacophore model obtained from molecular dynamics simulations of available cocrystal structures of well-known S1R ligands. Apart from the well-established positive ionizable and hydrophobic features, the obtained model included an additional specific hydrophobic feature and different excluded volumes, thus increasing the selectivity of the model as well as a more detailed determination of the distance between two essential features. The obtained pharmacophore model passed the validation test by receiver operating characteristic (ROC) curve analysis of active and inactive S1R ligands. Finally, the pharmacophoric performance was experimentally investigated through the synthesis and binding assay of new 4-phenylpiperazine-based compounds. The most active new ligand 2-(3-methyl-1-piperidyl)-1-(4-phenylpiperazin-1-yl)ethanone ( 3 ) showed an S1R affinity close to the reference compound haloperidol ( K i values of 4.8 and 2.6 nM, respectively). The proposed pharmacophore model can represent a useful tool to design and discover new potent S1R ligands.

Copper, an essential metal for various cellular processes, requires tight regulation to prevent cytotoxicity. Intracellular pathways crucial for maintaining optimal copper levels involve soluble and membrane transporters, namely, metallochaperones and P -type ATPases, respectively. In this study, we used a simulation workflow based on free-energy perturbation (FEP) theory and parallel bias metadynamics (PBMetaD) to predict the Cu(I) exchange mechanism between the human Cu(I) chaperone, Atox1, and one of its two physiological partners, ATP7A. ATP7A, also known as the Menkes disease protein, is a transmembrane protein and one of the main copper-transporting ATPases. It pumps copper into the trans-Golgi network for the maturation of cuproenzymes and is also essential for the efflux of excess copper across the plasma membrane. In this analysis, we utilized the nuclear magnetic resonance (NMR) structure of the Cu(I)-mediated complex between Atox1 and the first soluble domain of the Menkes protein (Mnk1) as a starting point. Independent free-energy simulations were conducted to investigate the dissociation of both Atox1 and Mnk1. The calculations revealed that the two dissociations require free energy values of 6.3 and 6.2 kcal/mol, respectively, following a stepwise dissociation mechanism.

The design and synthesis of a series of 2,7-diazaspiro[4.4]nonane derivatives as potent sigma receptor (SR) ligands, associated with analgesic activity, are the focus of this work. In this study, affinities at S1R and S2R were measured, and molecular modeling studies were performed to investigate the binding pose characteristics. The most promising compounds were subjected to in vitro toxicity testing and subsequently screened for in vivo analgesic properties. Compound 9d ( AD258 ) exhibited negligible in vitro cellular toxicity and a high binding affinity to both SRs ( K i S1R = 3.5 nM, K i S2R = 2.6 nM), but not for other pain-related targets, and exerted high potency in a model of capsaicin-induced allodynia, reaching the maximum antiallodynic effect at very low doses (0.6-1.25 mg/kg). Functional activity experiments showed that S1R antagonism is needed for the effects of 9d and that it did not induce motor impairment. In addition, 9d exhibited a favorable pharmacokinetic profile.

The development of diazabicyclo[4.3.0]nonane and 2,7-diazaspiro[3.5]nonane derivatives as sigma receptors (SRs) ligands is reported. The compounds were evaluated in S1R and S2R binding assays, and modeling studies were carried out to analyze the binding mode. The most notable compounds, 4b (AD186, K i S1R = 2.7 nM, K i S2R = 27 nM), 5b (AB21, K i S1R = 13 nM, K i S2R = 102 nM), and 8f (AB10, K i S1R = 10 nM, K i S2R = 165 nM), have been screened for analgesic effects in vivo , and their functional profile was determined through in vivo and in vitro models. Compounds 5b and 8f reached the maximum antiallodynic effect at 20 mg/kg. The selective S1R agonist PRE-084 completely reversed their action, indicating that the effects are entirely dependent on the S1R antagonism. Conversely, compound 4b sharing the 2,7-diazaspiro[3.5]nonane core as 5b was completely devoid of antiallodynic effect. Interestingly, compound 4b fully reversed the antiallodynic effect of BD-1063, indicating that 4b induces an S1R agonistic in vivo effect. The functional profiles were confirmed by the phenytoin assay. Our study might establish the importance of 2,7-diazaspiro[3.5]nonane core for the development of S1R compounds with specific agonist or antagonist profile and the role of the diazabicyclo[4.3.0]nonane in the development of novel SR ligands.

Polypharmacology is a new trend in amyotrophic lateral sclerosis (ALS) therapy and an effective way of addressing a multifactorial etiology involving excitotoxicity, mitochondrial dysfunction, oxidative stress, and microglial activation. Inspired by a reported clinical trial, we converted a riluzole ( 1 )-rasagiline ( 2 ) combination into single-molecule multi-target-directed ligands. By a ligand-based approach, the highly structurally integrated hybrids 3 - 8 were designed and synthesized. Through a target- and phenotypic-based screening pipeline, we identified hit compound 6 . It showed monoamine oxidase A (MAO-A) inhibitory activity (IC 50 = 6.9 μM) rationalized by in silico studies as well as in vitro brain permeability. By using neuronal and non-neuronal cell models, including ALS-patient-derived cells, we disclosed for 6 a neuroprotective/neuroinflammatory profile similar to that of the parent compounds and their combination. Furthermore, the unexpected MAO inhibitory activity of 1 (IC 50 = 8.7 μM) might add a piece to the puzzle of its anti-ALS molecular profile.

A new series of pyrimidine and pyridine diamines was designed as dual binding site inhibitors of cholinesterases (ChEs), characterized by two small aromatic moieties separated by a diaminoalkyl flexible linker. Many compounds are mixed or uncompetitive acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE) nanomolar inhibitors, with compound 9 being the most active on Electrophorus electricus AChE ( Ee AChE) ( K i = 0.312 μM) and compound 22 on equine BChE ( eq BChE) ( K i = 0.099 μM). Molecular docking and molecular dynamic studies confirmed the interaction mode of our compounds with the enzymatic active site. UV-vis spectroscopic studies showed that these compounds can form complexes with Cu 2+ and Fe 3+ and that compounds 18 , 20 , and 30 have antioxidant properties. Interestingly, some compounds were also able to reduce Aβ 42 and tau aggregation, with compound 28 being the most potent (22.3 and 17.0% inhibition at 100 μM on Aβ 42 and tau, respectively). Moreover, the most active compounds showed low cytotoxicity on a human brain cell line and they were predicted as BBB-permeable.

Chromone-3-phenylcarboxamides ( Crom-1 and Crom-2 ) were identified as potent, selective, and reversible inhibitors of human monoamine oxidase B ( h MAO-B). Since they exhibit some absorption, distribution, metabolism, and excretion (ADME)-toxicity liabilities, new derivatives were synthesized to map the chemical structural features that compose the pharmacophore, a process vital for lead optimization. Structure-activity relationship data, supported by molecular docking studies, provided a rationale for the contribution of the heterocycle's rigidity, the carbonyl group, and the benzopyran heteroatom for h MAO-B inhibitory activity. From the study, N -(3-chlorophenyl)-4 H -thiochromone-3-carboxamide ( 31 ) ( h MAO-B IC 50 = 1.52 ± 0.15 nM) emerged as a reversible tight binding inhibitor with an improved pharmacological profile. In in vitro ADME-toxicity studies, compound 31 showed a safe cytotoxicity profile in Caco-2, SH-SY5Y, HUVEC, HEK-293, and MCF-7 cells, did not present cardiotoxic effects, and did not affect P-gp transport activity. Compound 31 also protected SH-SY5Y cells from iron(III)-induced damage. Collectively, these studies highlighted compound 31 as the first-in-class and a suitable candidate for in vivo preclinical investigation.

ELAV-like (ELAVL) RNA-binding proteins play a pivotal role in post-transcriptional processes, and their dysregulation is involved in several pathologies. This work was focused on HuD (ELAVL4), which is specifically expressed in nervous tissues, and involved in differentiation and synaptic plasticity mechanisms. HuD represents a new, albeit unexplored, candidate target for the treatment of several relevant neurodegenerative diseases. The aim of this pioneering work was the identification of new molecules able to recognize and bind HuD, thus interfering with its activity. We combined virtual screening, molecular dynamics (MD), and STD-NMR techniques. Starting from around 51 000 compounds, four promising hits eventually provided experimental evidence of their ability to bind HuD. Among the selected best hits, folic acid was found to be the most interesting one, being able to well recognize the HuD binding site. Our results provide a basis for the identification of new HuD interfering compounds which may be useful against neurodegenerative syndromes.

A new class of pyrrolo[2',3':3,4]cyclohepta[1,2- d ][1,2]oxazoles was synthesized for the treatment of hyperproliferative pathologies, including neoplasms. The new compounds were screened in the 60 human cancer cell lines of the NCI drug screen and showed potent activity with GI 50 values reaching the nanomolar level, with mean graph midpoints of 0.08-0.41 μM. All compounds were further tested on six lymphoma cell lines, and eight showed potent growth inhibitory effects with IC 50 values lower than 500 nM. Mechanism of action studies showed the ability of the new [1,2]oxazoles to arrest cells in the G2/M phase in a concentration dependent manner and to induce apoptosis through the mitochondrial pathway. The most active compounds inhibited tubulin polymerization, with IC 50 values of 1.9-8.2 μM, and appeared to bind to the colchicine site. The G2/M arrest was accompanied by apoptosis, mitochondrial depolarization, generation of reactive oxygen species, and PARP cleavage.

l-Asparaginase (EC 3.5.1.1) was first used as a component of combination drug therapies to treat acute lymphoblastic leukemia (ALL), a cancer of the blood and bone marrow, almost 50 years ago. Administering this enzyme to reduce asparagine levels in the blood is a cornerstone of modern clinical protocols for ALL; indeed, this remains the only successful example of a therapy targeted against a specific metabolic weakness in any form of cancer. Three problems, however, constrain the clinical use of l-asparaginase. First, a type II bacterial variant of l-asparaginase is administered to patients, the majority of whom are children, which produces an immune response thereby limiting the time over which the enzyme can be tolerated. Second, l-asparaginase is subject to proteolytic degradation in the blood. Third, toxic side effects are observed, which may be correlated with the l-glutaminase activity of the enzyme. This Perspective will outline how asparagine depletion negatively impacts the growth of leukemic blasts, discuss the structure and mechanism of l-asparaginase, and briefly describe the clinical use of chemically modified forms of clinically useful l-asparaginases, such as Asparlas, which was recently given FDA approval for use in children (babies to young adults) as part of multidrug treatments for ALL. Finally, we review ongoing efforts to engineer l-asparaginase variants with improved therapeutic properties and briefly detail emerging, alternate strategies for the treatment of forms of ALL that are resistant to asparagine depletion.

In the present study we investigated the structure-activity relationships of a new series of 4-[(3-ethyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides ( EMAC10101a - m ). All synthesized compounds, with the exception of compound EMAC10101k , preferentially inhibit off-target hCA II isoform. Within the series, compound EMAC10101d , bearing a 2,4-dichorophenyl substituent in position 4 of the dihydrothiazole ring, was the most potent and selective toward hCA II with an inhibitory activity in the low nanomolar range., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

C-6α and C-7α androstanes were studied to disclose which position among them is more convenient to functionalize to reach superior aromatase inhibition. In the first series, the study of C-6 versus C-7 methyl derivatives led to the very active compound 9 with IC 50 of 0.06 μM and K i = 0.025 μM (competitive inhibition). In the second series, the study of C-6 versus C-7 allyl derivatives led to the best aromatase inhibitor 13 of this work with IC 50 of 0.055 μM and K i = 0.0225 μM (irreversible inhibition). Beyond these findings, it was concluded that position C-6α is better to functionalize than C-7α, except when there is a C-4 substituent simultaneously. In addition, the methyl group was the best substituent, followed by the allyl group and next by the hydroxyl group. To rationalize the structure-activity relationship of the best inhibitor 13, molecular modeling studies were carried out.

A novel series of of 4-[(3-phenyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides ( EMAC10111a-g ) was synthesized and assayed toward both human carbonic anhydrase isozymes I, II, IX, and XII and cyclooxygenase isoforms. The majority of these derivatives preferentially inhibit hCA isoforms II and XII and hCOX-2 isozyme, indicating that 2,3,4-trisubstituted 2,3-dihydrothiazoles are a promising scaffold for the inhibition of hCA isozymes and of hCOX-2 enzyme. The nature of the substituent at the dihydrothiazole ring position 4 influenced the activity and selectivity toward both enzyme families. EMAC10111g resulted as the best performing compound toward both enzyme families and exhibited preferential activity toward hCA XII and hCOX-2 isozymes., Competing Interests: The authors declare no competing financial interest.

In the last years, it has been shown that the DNA secondary structure known as G-quadruplex is also involved in the regulation of oncogenes transcription, such as c-myc , c-Kit , KRAS , Bcl-2 , VEGF , and PDGF . DNA G-quadruplexes, formed in the promoter region of these proto-oncogenes, are considered alternative anticancer targets since their stabilization causes a reduction of the related oncoprotein overexpression. In this study, a structure-based virtual screening toward the experimental DNA G-quadruplex structures of c-myc and c-Kit was performed by using Glide for the docking analysis of a commercial library of approximately 693 000 compounds. The best hits were submitted to thermodynamic and biophysical studies, highlighting the effective stabilization of both G-quadruplex oncogene promoter structures for three N -(4-piperidinylmethyl)amine derivatives, thus proposed as a new class of dual G-quadruplex binders., Competing Interests: The authors declare no competing financial interest.

A small library of psoralen carboxylic acids and their corresponding benzenesulfonamide derivatives were designed and synthesized to evaluate their activity and selectivity toward tumor associated human carbonic anhydrase (hCA) isoforms IX and XII. Both psoralen acids and sulfonamides exhibited potent inhibition of IX and XII isozymes in the nanomolar concentration range. However, psoralen acids resulted as the most selective in comparison with the corresponding benzenesulfonamide derivatives. Our data indicate that the psoralen scaffold is a promising starting point for the design of highly selective tumor associated hCA inhibitors., Competing Interests: The authors declare no competing financial interest.

A series of N -acylbenzenesulfonamide dihydro-1,3,4-oxadiazole hybrids ( EMAC8000a-m ) was designed and synthesized with the aim to target tumor associated carbonic anhydrase (hCA) isoforms IX and XII. Most of the compounds were selective inhibitors of the tumor associated hCA XII. Moreover, resolution of EMAC8000d racemic mixture led to the isolation of the levorotatory eutomer exhibiting an increase of hCA XII inhibition potency and selectivity with respect to hCA II. Computational studies corroborated these data. Overall our data indicate that both substitution pattern and stereochemistry of dihydro-1,3,4-oxadiazole could be considered as key factors to determine activity and selectivity toward hCA isozymes. These results can provide further indication for the design and optimization of selective hCA inhibitors.

In this study, for the first time, we have considered essential oils (EOs) as possible resources of carbonic anhydrase inhibitors (CAIs), in particular against the mitochondrial isoform VA that, actually, represents an innovative target for the obesity treatment. In silico structure-based virtual screening was performed in order to speed up the identification of promising antiobesity agents. The potential hit compounds were submitted to in vitro assays and experimental results, corroborated by molecular modeling studies, showed EOs components as a new class of CAIs with a competitive mechanism of action due to the zinc ion coordination within the active sites of these metallo-enzymes.

G-quadruplexes, alternative DNA secondary structures present in telomeres, emerge as promising targets for the treatment of cancer, because they prevent telomere elongation and accordingly cell proliferation. Within this study, theoretically validated pharmacophore- and shape-based models as well as a theoretically validated docking protocol were generated and applied in parallel for virtual screening and the identification of novel G-quadruplex ligands. Top-ranked hits retrieved with all methods independently and in addition in a consensus approach were selected for biological testing. Of the 32 tested virtual hits seven selectively stabilized G-quadruplexes over duplex DNA in the fluorescence melting assay. For the five most active compounds, chemically closely related analogues were collected and subjected to in vitro analysis. Thereby, seven further novel G-quadruplex ligands could be identified. These molecules do not only represent novel scaffolds, but some of them are in addition even more potent G-quadruplex stabilizers than the established reference compound berberine. This study proposes an optimized in silico workflow for the identification of novel G-quadruplex stabilizers, which can also be applied in future studies. In addition, structurally novel and promising lead candidates with strong and selective G-quadruplex stabilizing properties are reported.

The purpose of this work was to determine the kaempferol content in three red wines of Calabria, a southern Italian region with a great number of certified food products. Considering that wine cultivar, climate, and soil influence the qualitative and quantitative composition in flavonoids of Vitis vinifera L. berries, the three analyzed samples were taken from the 2013 vintage. Moreover, the Gaglioppo samples, with assigned Controlled Origin Denomination (DOC), were also investigated in the production of years 2008, 2010, and 2011. In addition to the analysis of kaempferol, which is present in higher concentration than in other Italian wines, in vitro assays were performed to evaluate, for the first time, the inhibition of the human monoamine oxidases (hMAO-A and hMAO-B). Molecular recognition studies were also carried out to provide insight into the binding mode of kaempferol and selectivity of inhibition of the hMAO-A isoform.

PTPRJ is a receptor protein tyrosine phosphatase involved in both physiological and oncogenic pathways. We previously reported that its expression is strongly reduced in the majority of explored cancer cell lines and tumor samples; moreover, its restoration blocks in vitro cancer cell proliferation and in vivo tumor formation. By means of a phage display library screening, we recently identified two peptides able to bind and activate PTPRJ, resulting in cell growth inhibition and apoptosis of both cancer and endothelial cells. Here, on a previously discovered PTPRJ agonist peptide, PTPRJ-pep19, we synthesized and assayed a panel of nonapeptide analogues with the aim to identify specific amino acid residues responsible for peptide activity. These second-generation nonapeptides were tested on both cancer and primary endothelial cells (HeLa and HUVEC, respectively); interestingly, one of them (PTPRJ-19.4) was able to both dramatically reduce cell proliferation and effectively trigger apoptosis of both HeLa and HUVECs compared to its first-generation counterpart. Moreover, PTPRJ-pep19.4 significantly inhibited in vitro tube formation on Matrigel. Intriguingly, while ERK1/2 phosphorylation and cell proliferation were both inhibited by PTPRJ-pep19.4 in breast cancer cells (MCF-7 and SKBr3), no effects were observed on primary normal human mammary endothelial cells (HMEC). We further characterized these peptides by molecular modeling and NMR experiments reporting, for the most active peptide, the possibility of self-aggregation states and highlighting new hints of structure-activity relationship. Thus, our results indicate that this nonapeptide might represent a great potential lead for the development of novel targeted anticancer drugs.

PTPRJ is a receptor-type protein tyrosine phosphatase whose expression is strongly reduced in the majority of investigated cancer cell lines and tumor specimens. PTPRJ negatively interferes with mitogenic signals originating from several oncogenic receptor tyrosine kinases, including HGFR, PDGFR, RET, and VEGFR-2. Here we report the isolation and characterization of peptides from a random peptide phage display library that bind and activate PTPRJ. These agonist peptides, which are able to both circularize and form dimers in acqueous solution, were assayed for their biochemical and biological activity on both human cancer cells and primary endothelial cells (HeLa and HUVEC, respectively). Our results demonstrate that binding of PTPRJ-interacting peptides to cell cultures dramatically reduces the extent of both MAPK phosphorylation and total phosphotyrosine levels; conversely, they induce a significant increase of the cell cycle inhibitor p27(Kip1). Moreover, PTPRJ agonist peptides both reduce proliferation and trigger apoptosis of treated cells. Our data indicate that peptide agonists of PTPRJ positively modulate the PTPRJ activity and may lead to novel targeted anticancer therapies.

A computer-aided design of novel asymmetric pyrazoles with improved enantioselective properties was performed by docking experiments starting from a model of Chiralcel OJ chiral in the stationary phase. Synthesis and HPLC experiments confirmed the theoretical prediction and led to a detailed investigation of the enantioselective recognition process. For the first time, looking at the time spent by each enantiomer in contact with the CSP during long molecular dynamic simulations, the experimental analytical trend has been reproduced., (© 2012 American Chemical Society)

Oleuropein, the main phenolic compound in virgin olive oil, and several of its derivatives such as oleuropein aglycone, hydroxytyrosol, and their respective acetylated lipophilic forms were obtained by simple and environmentally friendly semisynthetic protocols. The same molecules were then tested in vitro and in vivo, comparing their intriguing anti-COX-1 and anti-COX-2 properties to those of well-known anti-inflammatory drugs such as ibuprofen and celecoxib. Finally, molecular modeling experiments displaying the most probable binding modes within the classical binding clefts of the enzymes suggest the heme moiety as a potential alternative target.

The current strategy to improve the quality of life of Human Immunodeficiency Virus (HIV) infected individuals through suppressing viral replication and maintaining the virus at low to undetectable levels is based on highly active antiretroviral therapy (HAART). Protease inhibitors are essential components of most HAART protocols and are often used as the first line of treatment. However, a considerable percentage of new HIV-1 infections are caused by viruses carrying antiretroviral drug-resistant mutations. In this paper molecular dynamics, docking simulations, and free energy analysis of mutated HIV protease complexes were used to estimate the influence of different drug resistance-associated mutations in lopinavir, amprenavir, saquinavir, and atazanavir protease recognition. In agreement with virological and clinical data, the structural analysis showed that the single mutations V82A, I84V, and M46I are associated with higher energetic values for all analyzed complexes with respect to wild-type, indicating their decreased stability. Interestingly, in atazanavir complexes, in the presence of the L76V substitution, the drug revealed a more productive binding affinity, in agreement with hypersusceptibility data.

In this work, we present molecular modeling studies carried out using six DNA sequences and six azinomycin analogues, including the naturally occurring compound azinomycin B, selected on the basis of known cell cytotoxicity and structural analogies (epoxide and aziridine alkylating moieties). Among several computational methods the Stochastic Dynamics with Energy Minimization (SDEM) approach yielded results superior to the others with the natural compound (r(2) > 0.9) and was adopted for studying other DNA adducts, obtaining good correlation between the average theoretical cross-linking properties and the antitumor activity scale. As a result, some interesting SAR considerations have been highlighted and a cross-linking conformation different from that of the azinomycin was identified in a less potent, simplified analogue.