Discovery of a new potent inhibitor of mushroom tyrosinase (Agaricus bisporus) containing 4-(4-hydroxyphenyl)piperazin-1-yl moiety.

Tyrosinase (TYR, EC 1.14.18.1) plays a pivotal role in mammalian melanogenesis and enzymatic browning of plant-derived food. Therefore, tyrosinase inhibitors (TYRIs) can be of interest in cosmetics and pharmaceutical industries as depigmentation compounds as well as anti-browning agents. Starting from 4-benzylpiperidine derivatives that showed good inhibitory properties toward tyrosinase from Agaricus bisporus (TyM), we synthesized a new series of TYRIs named 3-(4-benzyl-1-piperidyl)-1-(4-phenylpiperazin-1-yl)propan-1-one and 2-(4-benzyl-1-piperidyl)-1-(4-phenylpiperazin-1-yl)ethanone derivatives. Among them, compound 4b proved to be the most potent inhibitor (IC50 = 3.80 µM) and it also showed a good antioxidant activity. These new data furnished additional information about the SAR for this class of TYRIs.

Inhibition of HIV-1 RT activity by a new series of 3-(1,3,4-thiadiazol-2-yl)thiazolidin-4-one derivatives.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent potent anti-HIV agents targeting HIV-1 reverse transcriptase (RT), a crucial enzyme for the viral life cycle. We have previously identified a series of NNRTIs bearing a 2,3-diaryl-1,3-thiazolidin-4-one core and some compounds proved to be effective in inhibiting HIV-1 replication at micromolar concentration. As a continuation in this research work we report the design, the synthesis and the structure-activity relationship studies of a further series of 3-(1,3,4-thiadiazol-2-yl)thiazolidin-4-one derivatives containing an arylthioacetamide group as pharmacophoric structural requirement for binding to the RT catalytic area. The new compounds proved to be effective to inhibit RT activity at micromolar concentrations. Finally, docking studies were carried out in order to rationalize the biological results of the new synthesized inhibitors.

Exploring structural properties of potent human carbonic anhydrase inhibitors bearing a 4-(cycloalkylamino-1-carbonyl)benzenesulfonamide moiety.

Guided by the crystal structure of 4-(3,4-dihydroquinolin-1(2H)-ylcarbonyl)benzenesulfonamide 3 in complex with hCA II (PDB code 4Z0Q), a novel series of cycloalkylamino-1-carbonylbenzenesulfonamides was designed and synthesized. Thus, we replaced the quinoline ring with an azepine/piperidine/piperazine nucleus and introduced further modifications on cycloalkylamine nucleus by means the installation of hydrophobic/hydrophilic functionalities able to establish additional contacts in the middle area of the enzyme cavity. Among the synthesized compounds, the derivatives 7a, 7b, 8b exhibited a remarkable inhibition for hCA II and the brain-expressed hCA VII in subnanomolar range. The binding of these molecules to the target enzymes was characterized by means of a crystallographic analysis, providing a clear snapshot of the most important interactions established by this class of inhibitors into the hCA II and hCA VII catalytic site. Notably, our results showed that the benzylpiperazine tail of compound 8b is oriented both in hCA II and in hCA VII toward a poorly explored region of the active site. These features should be further investigated for the design of new isoform selective CA inhibitors.

Identification of influenza PA-Nter endonuclease inhibitors using pharmacophore- and docking-based virtual screening.

Searching for new antiviral agents, we focused our interest on the influenza PA-Nter endonuclease. Therefore, we developed a three-dimensional pharmacophore model which contains the binding features addressed to the metal-chelating active site. The obtained hypothesis has been fruitfully employed to select three "hit compounds" through an in silico screening campaign on our in-house database of small molecules. We studied the binding poses of these hit compounds using molecular docking, and subjected them to an enzymatic assay with recombinant PA-Nter endonuclease. Compound 20 proved the most active inhibitor of the endonucleolytic cleavage reaction, with an IC50 value of 12 µM.

Graphene Quantum Dots Based Systems As HIV Inhibitors.

Graphene quantum dots (GQD) are the next generation of nanomaterials with great potential in drug delivery and target-specific HIV inhibition. In this study we investigated the antiviral activity of graphene based nanomaterials by using water-soluble GQD synthesized from multiwalled carbon nanotubes (MWCNT) through prolonged acidic oxidation and exfoliation and compared their anti-HIV activity with that exerted by reverse transcriptase inhibitors (RTI) conjugated with the same nanomaterial. The antiretroviral agents chosen in this study, CHI499 and CDF119, belong to the class of non-nucleoside reverse transcriptase inhibitors (NNRTI). From this study emerged the RTI-conjugated compound GQD-CHI499 as a good potential candidate for HIV treatment, showing an IC50 of 0.09 µg/mL and an EC50 value in cell of 0.066 µg/mL. The target of action in the replicative cycle of HIV of the drug conjugated samples GQD-CHI499 and GQD-CDF119 was also investigated by a time of addition (TOA) method, showing for both conjugated samples a mechanism of action similar to that exerted by NNRTI drugs.

Discovery of benzimidazole-based Leishmania mexicana cysteine protease CPB2.8ΔCTE inhibitors as potential therapeutics for leishmaniasis.

Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However, its effectiveness is severely limited due to high toxicity, long treatment length, drug resistance, or inadequate mode of administration. As a consequence, there is a need to identify new molecular scaffolds and targets as potential therapeutics for the treatment of this disease. We report a small series of 1,2-substituted-1H-benzo[d]imidazole derivatives (9a-d) showing affinity in the submicromolar range (Ki  = 0.15-0.69 µM) toward Leishmania mexicanaCPB2.8ΔCTE, one of the more promising targets for antileishmanial drug design. The compounds confirmed activity in vitro against intracellular amastigotes of Leishmania infantum with the best result being obtained with derivative 9d (IC50  = 6.8 µM), although with some degree of cytotoxicity (CC50  = 8.0 µM on PMM and CC50  = 32.0 µM on MCR-5). In silico molecular docking studies and ADME-Tox properties prediction were performed to validate the hypothesis of the interaction with the intended target and to assess the drug-likeness of these derivatives.

Targeting Tyrosinase: Development and Structural Insights of Novel Inhibitors Bearing Arylpiperidine and Arylpiperazine Fragments.

The inhibition of tyrosinase (Ty, EC 1.14.18.1) represents an efficient strategy of decreasing melanogenesis and skin hyperpigmentation. A combination of crystallographic and docking studies on two different tyrosinases, that from Bacillus megaterium (TyBm) and that from a mushroom (TyM), has contributed to increasing our knowledge about their structural information and translating that information to the most druggable human Ty (TyH) isozyme. In particular, we designed and synthesized a series of 1-(4-fluorobenzyl)piperazine and 1-(4-fluorobenzyl)piperidine derivatives showing inhibitory activities on TyM at micromolar ranges and more potency than that of the reference compound, kojic acid. The crystal structures of TyBm with inhibitor 3 (IC50 value of 25.11 µM) and 16 (IC50 value of 5.25 µM) were solved, confirming the binding poses hypothesized by in silico studies and revealing the main molecular determinants for the binding recognition of the inhibitors.

Structural optimization of N1-aryl-benzimidazoles for the discovery of new non-nucleoside reverse transcriptase inhibitors active against wild-type and mutant HIV-1 strains.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of human immunodeficiency virus (HIV) infection. These regimens are extremely effective in suppressing virus replication. Recently, our research group identified some N1-aryl-2-arylthioacetamido-benzimidazoles as a novel class of NNRTIs. In this research work we report the design, the synthesis and the structure-activity relationship studies of new compounds (20-34) in which some structural modifications have been introduced in order to investigate their effects on reverse transcriptase (RT) inhibition and to better define the features needed to increase the antiviral activity. Most of the new compounds proved to be highly effective in inhibiting both RT enzyme at nanomolar concentrations and HIV-1 replication in MT4 cells with minimal cytotoxicity. Among them, the most promising N1-aryl-2-arylthioacetamido-benzimidazoles and N1-aryl-2-aryloxyacetamido-benzimidazoles were also tested toward a panel of single- and double-mutants strain responsible for resistance to NNRTIs, showing in vitro antiviral activity toward single mutants L100I, K103N, Y181C, Y188L and E138K. The best results were observed for derivatives 29 and 33 active also against the double mutants F227L and V106A. Computational approaches were applied in order to rationalize the potency of the new synthesized inhibitors.

Inhibitory effects and structural insights for a novel series of coumarin-based compounds that selectively target human CA IX and CA XII carbonic anhydrases.

Coumarin derivatives are a peculiar class of inhibitors of the family of metalloenzymes carbonic anhydrases (CA, EC 4.2.1.1). Several coumarins display higher affinity and selectivity toward most relevant and druggable CA isoforms. By decorating the natural compound umbelliferone (1) we have identified a new series of coumarin-based compounds demonstrating high CA inhibitory effects with nanomolar affinity for hCA IX and hCA XII isoforms that were considered a target amenable to develop antitumor agents. The most active tested compounds proved to be potent inhibitors with Ki values equal to that of the well-known inhibitor acetazolamide (AAZ), that lacks selectivity over ubiquitous hCA I and hCA II. As suggested by docking studies the coumarins, that are lacking of the canonical metal binding groups, do not interact with Zinc ion within the catalytic site as found for classical sulfonamide type inhibitors of CAs. Thus, the studied inhibitors might possess a non-classical inhibitory mode of action preventing the carbon dioxide to entry into catalytic cavity and its conversion into bicarbonate ion. Specifically, the most active inhibitor of hCA XII compound 18i (Ki value of 5.5 nM) and its supposed hydrolytic products could establish a web of H-bond interactions within the enzymatic cavity.

Searching for novel N1-substituted benzimidazol-2-ones as non-nucleoside HIV-1 RT inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent an integral part of the currently available combination antiretroviral therapy (cART) contributing to reduce the AIDS-mortality and turned the disease from lethal to chronic. In this context we recently reported a series of 6-chloro-1-(3-methylphenylsulfonyl)-1,3-dihydro-2H-benzimidazol-2-ones as potent non-nucleoside HIV-1 reverse transcriptase inhibitors. In this paper, we describe the design and the synthesis of two novel series of benzimidazolone analogues in which the linker moiety between the phenyl ring and the sulfonyl group was modified and new small lipophilic groups on the benzyl sulfonyl pendant were introduced. All the new obtained compounds were evaluated as RT inhibitors and were also tested against RTs containing single amino acid mutations. Finally, molecular docking studies were performed in order to rationalize the observed activity of the most promising compound.

Probing Molecular Interactions between Human Carbonic Anhydrases (hCAs) and a Novel Class of Benzenesulfonamides.

On the basis of X-ray crystallographic studies of the complex of hCA II with 4-(3,4-dihydro-1H-isoquinoline-2-carbonyl)benzenesulfonamide (3) (PDB code 4Z1J ), a novel series of 4-(1-aryl-3,4-dihydro-1H-isoquinolin-2-carbonyl)benzenesulfonamides (23-33) was designed. Specifically, our idea was to improve the selectivity toward druggable isoforms through the introduction of additional hydrophobic/hydrophilic functionalities. Among the synthesized and tested compounds, the (R,S)-4-(6,7-dihydroxy-1-phenyl-3,4-tetrahydroisoquinoline-1H-2-carbonyl)benzenesulfonamide (30) exhibited a remarkable inhibition for the brain-expressed hCA VII (Ki = 0.20 nM) and selectivity over wider distributed hCA I and hCA II isoforms. By enantioselective HPLC, we solved the racemic mixture and ascertained that the two enantiomers (30a and 30b) are equiactive inhibitors for hCA VII. Crystallographic and docking studies revealed the main interactions of these inhibitors into the carbonic anhydrase (CA) catalytic site, thus highlighting the relevant role of nonpolar contacts for this class of hCA inhibitors.

Chemical exploration of 4-(4-fluorobenzyl)piperidine fragment for the development of new tyrosinase inhibitors.

Tyrosinase is involved in the production of melanin through the hydroxylation of monophenols to o-diphenols. The role of this enzyme was extensively studied in order to identify new therapeutics preventing skin pigmentation and melanoma. In this work we initially identified the 3-(4-benzylpiperidin-1-yl)-1-(1H-indol-3-yl)propan-1-one (1a) as promising mushroom tyrosinase inhibitor (IC50 = 252 µM). Then, several chemical modifications were performed and new analogues related to compound 1a were synthesized. Biochemical assays demonstrated that several obtained compounds proved to be effective inhibitors showing IC50 values lower both than "lead compound" 1a and reference inhibitor kojic acid, as a well-known tyrosinase inhibitor. The inhibition kinetics analyzed by Lineweaver-Burk plots revealed that compounds 2 a-c and 10b act as non-competitive inhibitors while the most active inhibitor 2d (IC50 = 7.56 µM) is a mixed-type inhibitor. Furthermore, experimental and computational structural studies were performed in order to clarify the binding mode of the derivative 2d.

Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase.

Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors.

Rational Design, Synthesis and Evaluation of Coumarin Derivatives as Protein-protein Interaction Inhibitors.

Herein we describe the design and synthesis of a new series of coumarin derivatives searching for novel HIV-1 integrase (IN) allosteric inhibitors. All new obtained compounds were tested in order to evaluate their ability to inhibit the interaction between the HIV-1 IN enzyme and the nuclear protein lens epithelium growth factor LEDGF/p75. A combined approach of docking and molecular dynamic simulations has been applied to clarify the activity of the new compounds. Specifically, the binding free energies by using the method of molecular mechanics-generalized Born surface area (MM-GBSA) was calculated, whereas hydrogen bond occupancies were monitored throughout simulations methods.

Structure-guided design of new indoles as negative allosteric modulators (NAMs) of N-methyl-D-aspartate receptor (NMDAR) containing GluN2B subunit.

Negative allosteric modulators (NAMs) of GluN2B-containing NMDARs provide pharmacological tools for the treatment of chronic neurodegenerative diseases. Novel NAMs have been designed on the basis of computational studies focused on the 'hit compound' 3. This series of indoles has been tested in competition assay. Compounds 16 and 17 were the most active ligands (IC50 values of 83 nM and 71 nM, respectively) and they showed a potency close to that of reference compounds ifenprodil (1, IC50=47 nM) and 3 (IC50=25 nM). Furthermore, docking studies have been performed for active ligand 16 and the results were in a good agreement with biological data.

Carbonic anhydrase inhibitors: Design, synthesis and structural characterization of new heteroaryl-N-carbonylbenzenesulfonamides targeting druggable human carbonic anhydrase isoforms.

A set of heteroaryl-N-carbonylbenzenesulfonamides has been designed, synthesized, and screened as inhibitors of human carbonic anhydrases (hCAs). The new sulfonamide derivatives were tested against hCA I, hCA II, hCA VII, hCA IX, and hCA XII isoforms using acetazolamide (AAZ, 1) and topiramate (TPM, 2) as reference compounds. Six compounds were low nanomolar inhibitors of tumor-associated hCA IX isoform (Ki values < 10 nM); among them we identified three arylsulfonamides showing unexpected inefficacy over brain distributed hCA VII isoform (hCA IX/hCA VII selectivity ratio > 1500 for compound 5c). Thus, these compounds can offer the opportunity to highlight the interactions preventing the inhibition of hCA VII mainly expressed in central nervous system. Thereby, we used structural and computational techniques to study in depth the interaction with hCAs. In an effort to confirm the inhibitory action we determined crystal structures of five selected heteroaryl-N-carbonylbenzenesulfonamides (4a, 4b, 4e, 5c, and 5e) in complex with hCA II. Moreover, to explore the lack of inhibitory effects of selected compounds (e.g.4b and 5c) we also performed docking studies into hCA VII catalytic site.

Targeting GluN2B-containing N-Methyl-D-aspartate receptors: design, synthesis, and binding affinity evaluation of novel 3-substituted indoles.

In an effort to improve our knowledge about structure-affinity relationships (SARs) for a class of 3-substituted-indole derivatives as GluN2B-containing N-methyl-D-aspartate-type receptor (NMDAR) ligands, we herein describe the design, synthesis, and preliminary screening of a new series of molecules. The in vitro determination of binding affinities suggested that 5-hydroxy- and 6-hydroxyindole derivatives 12 and 13 were active ligands. Generally, the novel compounds proved to be less potent than their homologs previously reported as promising neuroprotective agents. In fact, our lead compound 3-(4-benzylpiperidin-1-yl)-1-(5-hydroxy-1H-indol-3-yl)ethan-1-one (2) was about 10-fold more active than the new propan-1-one derivative (12). To rationalize the low potency of the new analog 12, docking studies were also performed and the in silico results were consistent with the in vitro data.

Synthesis, modelling and biological characterization of 3-substituted-1H-indoles as ligands of GluN2B-containing N-methyl-d-aspartate receptors.

A three-step synthetic pathway has been employed to synthesize a small library of 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethanone and 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethane-1,2-dione derivatives that have been screened in [(3)H]ifenprodil competition binding assay. Some compounds exhibited significant binding affinity at nanomolar concentration, the most active being ligand 35 (IC50=5.5nM). Docking experiments suggested the main interactions between 35 and GluN2B-containing NMDA receptors. Notably, the compound 35 reduced NMDA-mediated excitatory post-synaptic currents recorded in mouse hippocampal slices indicating antagonistic effects (50nM). Moreover, the compound 35 has shown antioxidant effects in a preliminary screening, thus suggesting that it might be considered prototype for future drug development of novel 'dual target' neuroprotective agents.

From NMDA receptor antagonists to discovery of selective σ2 receptor ligands.

Following previous studies focused on the search for new molecules targeting GluN2B-containing NMDA, a small series of 1-(1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone derivatives has been synthesized by using Microwave Assisted Organic Synthesis (MAOS). Given that GluN2B ligands frequently exert off-target effects we also tested their affinity towards sigma receptors. Binding assay revealed that only the 1-(5-hydroxy-1H-indol-3-yl)-2-(4-phenylpiperidin-1-yl)ethanone (7a) retained GluN2B affinity. Interestingly, the 5-methoxyindoles 5a and 6a were efficient and selective ligands toward σ2 receptor (Ki values of 10nM and 20 nM, respectively). Thus, in this case the discovery of new σ2 receptor selective ligands was an unexpected result emerging from the screening of cross-activity against other CNS receptors.