Exploring Structural Requirements for Sigma-1 Receptor Linear Ligands: Experimental and Computational Approaches.

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 (Ki 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.

Structure-based design of a phosphotyrosine-masked covalent ligand targeting the E3 ligase SOCS2.

The Src homology 2 (SH2) domain recognizes phosphotyrosine (pY) post translational modifications in partner proteins to trigger downstream signaling. Drug discovery efforts targeting the SH2 domains have long been stymied by the poor drug-like properties of phosphate and its mimetics. Here, we use structure-based design to target the SH2 domain of the E3 ligase suppressor of cytokine signaling 2 (SOCS2). Starting from the highly ligand-efficient pY amino acid, a fragment growing approach reveals covalent modification of Cys111 in a co-crystal structure, which we leverage to rationally design a cysteine-directed electrophilic covalent inhibitor MN551. We report the prodrug MN714 containing a pivaloyloxymethyl (POM) protecting group and evidence its cell permeability and capping group unmasking using cellular target engagement and in-cell 19F NMR spectroscopy. Covalent engagement at Cys111 competitively blocks recruitment of cellular SOCS2 protein to its native substrate. The qualified inhibitors of SOCS2 could find attractive applications as chemical probes to understand the biology of SOCS2 and its CRL5 complex, and as E3 ligase handles in proteolysis targeting chimera (PROTACs) to induce targeted protein degradation.

Computational methods to analyze and predict the binding mode of inhibitors targeting both human and mushroom tyrosinase.

Tyrosinase, a copper-containing enzyme critical in melanin biosynthesis, is a key drug target for hyperpigmentation and melanoma in humans. Testing the inhibitory effects of compounds using tyrosinase from Agaricus bisporus (AbTYR) has been a common practice to identify potential therapeutics from synthetic and natural sources. However, structural diversity among human tyrosinase (hTYR) and AbTYR presents a challenge in developing drugs that are therapeutically effective. In this study, we combined retrospective and computational analyses with experimental data to provide insights into the development of new inhibitors targeting both hTYR and AbTYR. We observed contrasting effects of Thiamidol™ and our 4-(4-hydroxyphenyl)piperazin-1-yl-derivative (6) on both enzymes; based on this finding, we aimed to investigate their binding modes in hTYR and AbTYR to identify residues that significantly improve affinity. All the information led to the discovery of compound [4-(4-hydroxyphenyl)piperazin-1-yl](2-methoxyphenyl)methanone (MehT-3, 7), which showed comparable activity on AbTYR (IC50 = 3.52 µM) and hTYR (IC50 = 5.4 µM). Based on these achievements we propose the exploitation of our computational results to provide relevant structural information for the development of newer dual-targeting molecules, which could be preliminarily tested on AbTYR as a rapid and inexpensive screening procedure before being tested on hTYR.

Discovery and computational studies of piperidine/piperazine-based compounds endowed with sigma receptor affinity.

Herein, we describe our efforts to identify sigma receptor 1 (S1R) ligands through a screening campaign on our in-house collection of piperidine/piperazine-based compounds. Our investigations led to the discovery of the potent compound 2-[4-(benzyl)-1-piperidin-1-yl]-1-4-(4-phenylpiperazin-1-yl)ethanone (1) with high affinity toward S1R (Ki value of 3.2 nM) that was comparable to reference compound haloperidol (Ki value of 2.5 nM). Functional assay revealed that compound 1 acted as S1R agonist. To decipher the binding mode of this promising S1R ligand as a starting point for further structure-based optimization, we analysed the docking pose by using a S1R-structure derived from cocrystal structures of potent ligands in complex with target protein. The computational study was enriched with molecular dynamic simulations that revealed the crucial amino acid residues that interacted with the most interesting compound 1.

Screening Campaign and Docking Investigations in Identifying New Hit Compounds as Inhibitors of Human Carbonic Anhydrases Expressed In Tumour Cells.

The tumor-expressed human carbonic anhydrase (hCA) isoforms hCA IX and hCA XII have been extensively studied to develop anticancer agents targeting solid tumors in combined therapy. These CA  isoforms are considered key factors in controlling tumor microenvironment (TME) of cancer lines that develop high metastatic activity. Herein, we report the discovery of potent hCA IX/hCA XII inhibitors that were disclosed through a screening campaign on an in-house collection of arylsulfonamides preliminary tested toward other hCAs. Among them, the N-(4-sulfamoylphenyl)naphthalene-2-carboxamide (12) and N-(4-sulfamoylphenyl)-3,4-dihydroisoquinoline-2(1H)-carbothioamide (15) proved to be the most intriguing hCA IX/hCA XII inhibitors displaying favourable selectivity ratios over widespread hCA I and hCA II isoforms. To explore their binding mode, we conducted docking studies that described the poses of the best inhibitors in the catalytic site of hCA IX and hCA XII, thus suggesting the privileged pattern of interactions. These structural findings might further improve the knowledge for a successful identification of new sulfonamides as adjuvant agents in cancer management.

Evaluation of the In Vitro Antifungal Activity of Novel Arylsulfonamides against Candida spp.

The antifungal activity of molecules belonging to the arylsulfonamide chemotype has previously been demonstrated. Here, we screened arylsulfonamide-type compounds against a range of Candida spp. and further established the structure-activity relationship based on a "hit compound". A series of four sulfonamide-based compounds, N-(4-sulfamoylbenzyl) biphenyl-4-carboxamide (3), 2,2-diphenyl-N-(4-sulfamoylbenzyl) acetamide (4), N-(4-sulfamoylphenethyl) biphenyl-4-carboxamide (5) and 2,2-diphenyl-N-(4-sulfamoylphenethyl) acetamide (6), were tested against the American Type Culture Collection (ATCC) and clinical strains of C. albicans, C. parapsilosis and C. glabrata. Based on the fungistatic potential of prototype 3, a further subset of compounds, structurally related to hit compound 3, was synthesized and tested: two benzamides (10-11), the related amine 4-[[(4-4-((biphenyl-4-ylmethylamino)methyl) benzenesulfonamide (13) and the corresponding hydrochloride, 13.HCl. Both amine 13 and its hydrochloride salt had fungicidal effects against Candida glabrata strain 33 (MFC of 1.000 mg/mL). An indifferent effect was detected in the association of the compounds with amphotericin B and fluconazole. The cytotoxicity of the active compounds was also evaluated. This data could be useful to develop novel therapeutics for topical use against fungal infections.

Leveraging the 3-Chloro-4-fluorophenyl Motif to Identify Inhibitors of Tyrosinase from Agaricus bisporus.

Tyrosinase (EC 1.14.18.1) is implicated in melanin production in various organisms. There is a growing body of evidence suggesting that the overproduction of melanin might be related to several skin pigmentation disorders as well as neurodegenerative processes in Parkinson's disease. Based on this consideration, the development of tyrosinase inhibitors represents a new challenge to identify new agents in pharmaceutical and cosmetic applications. With the goal of identifying tyrosinase inhibitors from a synthetic source, we employed a cheap and facile preliminary assay using tyrosinase from Agaricus bisporus (AbTYR). We have previously demonstrated that the 4-fluorobenzyl moiety might be effective in interactions with the catalytic site of AbTYR; moreover, the additional chlorine atom exerted beneficial effects in enhancing inhibitory activity. Therefore, we planned the synthesis of new small compounds in which we incorporated the 3-chloro-4-fluorophenyl fragment into distinct chemotypes that revealed the ability to establish profitable contact with the AbTYR catalytic site. Our results confirmed that the presence of this fragment is an important structural feature to improve the AbTYR inhibition in these new chemotypes as well. Furthermore, docking analysis supported the best activity of the selected studied compounds, possessing higher potency when compared with reference compounds.

Structure-guided identification of a selective sulfonamide-based inhibitor targeting the human carbonic anhydrase VA isoform.

In recent years, multistep hybrid computational protocols have attracted attention for their application in the drug discovery of enzyme inhibitors. So far, there are large collections of human carbonic anhydrase (hCA) inhibitors, but only a few of them selectively inhibit the mitochondrial isoforms hCA VA and VB as potential therapeutics in obesity treatment. Most sulfonamide-based inhibitors show poor selectivity for inhibiting isoforms of therapeutic interest over ubiquitous hCA I and hCA II. Herein, we propose a combination of ligand- and structure-based approaches to generate pharmacophore models for hCA VA inhibitors. Then, we performed a virtual screening (VS) campaign on a database of commercially available sulfonamides. Finally, the in silico screening followed by docking studies suggested several "hit compounds" that demonstrated to inhibit hCA VA at a low nanomolar concentration in a stopped-flow CO2 hydrase assay. Notably, the best candidate, 2-(3,4-dihydro-2H-quinolin-1-yl)-N-(4-sulfamoylphenyl)acetamide (code name VAME-28) proved to be a potent hCA VA inhibitor (Ki value of 54.8 nM) and a more selective agent over hCA II when compared to the reference compound topiramate.

Ligand-Based Discovery of a Small Molecule as Inhibitor of α-Synuclein Amyloid Formation.

α-Synuclein (α-Syn) aggregates are implicated in Parkinson's disease (PD), so inhibitors of α-Syn aggregation have been intensively explored. It has been demonstrated that small molecules might be able to reduce α-Syn aggregation in fibrils, thus exerting neuroprotective effects in models of PD. To expand our knowledge about the structural requirements for blocking the recognition process into the oligomeric assembly of α-Syn aggregates, we performed a ligand-based virtual screening procedure using two well-known α-Syn aggregation inhibitors, SynuClean-D and ZPD-2, as query compounds. A collection of thirty-four compounds bearing distinct chemical functionalities and mutual chemical features were studied in a Th-T fluorescence test, thus identifying 5-(2,6-dinitro-4-(trifluoromethyl)benzyl)-1-methyl-1H-tetrazole (named MeSC-04) as a potent α-Syn amyloid formation inhibitor that demonstrated similar behavior when compared to SynuClean-D in the thioflavin-T-monitored kinetic assays, with both molecules reducing the number and size of amyloid fibrils, as evidenced by electron microscopy. Molecular modeling studies suggested the binding mode of MeSC-04 through the identification of putative druggable pockets on α-syn fibrils and a subsequent consensus docking methodology. Overall, this work could furnish new insights in the development of α-Syn amyloid inhibitors from synthetic sources.

The SIRT2 Pathway Is Involved in the Antiproliferative Effect of Flavanones in Human Leukemia Monocytic THP-1 Cells.

Acute myeloid leukemia (AML) represents the most alarming hematological disease for adults. Several genetic modifications are known to be pivotal in AML; however, SIRT2 over-expression has attracted the scientific community's attention as an unfavorable prognostic marker. The plant kingdom is a treasure trove of bioactive principles, with flavonoids standing out among the others. On this line, the aim of this study was to investigate the anti-leukemic properties of the main flavanones of Citrus spp., exploring the potential implication of SIRT2. Naringenin (NAR), hesperetin (HSP), naringin (NRG), and neohesperidin (NHP) inhibited SIRT2 activity in the isolated recombinant enzyme, and more, the combination between NAR and HSP. In monocytic leukemic THP-1 cells, only NAR and HSP induced antiproliferative effects, altering the cell cycle. These effects may be ascribed to SIRT2 inhibition since these flavonoids reduced its gene expression and hampered the deacetylation of p53, known sirtuin substrate, and contextually modulated the expression of the downstream cell cycle regulators p21 and cyclin E1. Additionally, these two flavanones proved to interact with the SIRT2 inhibitory site, as shown by docking simulations. Our results suggest that both NAR and HSP may act as anti-leukemic agents, alone and in combination, via targeting the SIRT2/p53/p21/cyclin E1 pathway, thus encouraging deeper investigations.

Design, Synthesis, and in Vitro Evaluation of 4-(4-Hydroxyphenyl)piperazine-Based Compounds Targeting Tyrosinase.

Melanin biosynthesis is enzymatically regulated by tyrosinase (TYR, EC 1.14.18.1), which is efficiently inhibited by natural and synthetic phenols, demonstrating potential therapeutic application for the treatment of several human diseases. Herein we report the inhibitory effects of a series of (4-(4-hydroxyphenyl)piperazin-1-yl)arylmethanone derivatives, that were designed, synthesised and assayed against TYR from Agaricus bisporus (AbTYR). The best inhibitory activity was predominantly found for compounds bearing selected hydrophobic ortho-substituents on the aroyl moiety (IC50 values in the range of 1.5-4.6 µM). They proved to be more potent than the reference compound kojic acid (IC50 =17.8 µM) and displayed competitive mechanism of inhibition of diphenolase activity of AbTYR. Docking simulation predicted their binding mode into the catalytic cavities of AbTYR and the modelled human TYR. In addition, these compounds displayed antioxidant activity combined with no cytotoxicity in MTT tests. Notably, the best inhibitor affected tyrosinase activity in α-MSH-stimulated B16F10 cells, thus demonstrating anti-melanogenic activity.

Synthesis and biological evaluation of sulfonamide-based compounds as inhibitors of carbonic anhydrase from Vibrio cholerae.

This study reports our continued efforts to identify inhibitors capable of targeting carbonic anhydrases (CAs) expressed in bacteria. Based on previously identified chemotypes, we designed and synthesized new analogs that were screened toward the α, ß, and γ classes encoded in Vibrio cholerae (Vch). The Ki values measured in the stopped-flow hydrase assay revealed that very simple structural modifications might induce a relevant impact on the inhibitory effects as well as the selectivity profile over ubiquitous human isozymes (hCA I/II). Unfortunately, the best active VchCA inhibitors demonstrated a dramatic loss of hCA II selectivity when compared to previously reported compounds. Among the new series of sulfonamides, several molecules proved to be about sevenfold more potent against VchCAγ than the reference compound acetazolamide, thus furnishing new insights for further development of inhibitors targeting CAs expressed in bacteria.

In Silico Insights towards the Identification of SARS-CoV-2 NSP13 Helicase Druggable Pockets.

The merging of distinct computational approaches has become a powerful strategy for discovering new biologically active compounds. By using molecular modeling, significant efforts have recently resulted in the development of new molecules, demonstrating high efficiency in reducing the replication of severe acute respiratory coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic. We have focused our interest on non-structural protein Nsp13 (NTPase/helicase), as a crucial protein, embedded in the replication-transcription complex (RTC), that controls the virus life cycle. To assist in the identification of the most druggable surfaces of Nsps13, we applied a combination of four computational tools: FTMap, SiteMap, Fpocket and LigandScout. These software packages explored the binding sites for different three-dimensional structures of RTC complexes (PDB codes: 6XEZ, 7CXM, 7CXN), thus, detecting several hot spots, that were clustered to obtain ensemble consensus sites, through a combination of four different approaches. The comparison of data provided new insights about putative druggable sites that might be employed for further docking simulations on druggable surfaces of Nsps13, in a scenario of repurposing drugs.

Discovery of Neuroprotective Agents Based on a 5-(4-Pyridinyl)-1,2,4-triazole Scaffold.

Parkinson's disease (PD) is characterized by the death of dopaminergic neurons. The common histopathological hallmark in PD patients is the formation of intracellular proteinaceous accumulations. The main constituent of these inclusions is alpha-synuclein (α-syn), an intrinsically disordered protein that in pathological conditions creates amyloid aggregates that lead to neurotoxicity and neurodegeneration. The main goal of our study was to optimize our previously identified α-syn aggregation inhibitors of 5-(4-pyridinyl)-1,2,4-triazole chemotype in terms of in vivo efficacy. Our efforts resulted in the identification of ethyl 2-((4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetate (15), which displayed the ability to prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridine-induced bradykinesia as well as to affect the levels of PD markers after the administration of the same neurotoxin. In addition to the in vivo evaluation, for the 5-(4-pyridinyl)-1,2,4-triazole-based compounds, we measured the prevention of the fibrillization process using light scattering and a ThT binding assay; these compounds have been shown to slightly reduce the α-syn aggregation.

4-Sulfamoylphenylalkylamides as Inhibitors of Carbonic Anhydrases Expressed in Vibrio cholerae.

A current issue of antimicrobial therapy is the resistance to treatment with worldwide consequences. Thus, the identification of innovative targets is an intriguing challenge in the drug and development process aimed at newer antimicrobial agents. The state-of-art of anticholera therapy might comprise the reduction of the expression of cholera toxin, which could be reached through the inhibition of carbonic anhydrases expressed in Vibrio cholerae (VchCAα, VchCAß, and VchCAγ). Therefore, we focused our interest on the exploitation of sulfonamides as VchCA inhibitors. We planned to design and synthesize new benzenesulfonamides based on our knowledge of the VchCA catalytic site. The synthesized compounds were tested thus collecting useful SAR information. From our investigation, we identified new potent VchCA inhibitors, some of them displayed high affinity toward VchCAγ class, for which few inhibitors are currently reported in literature. The best interesting VchCAγ inhibitor (S)-N-(1-oxo-1-((4-sulfamoylbenzyl)amino)propan-2-yl)furan-2-carboxamide (40) resulted more active and selective inhibitor when compared with acetazolamide (AAZ) as well as previously reported VchCA inhibitors.

Evaluation of 4-(4-Fluorobenzyl)piperazin-1-yl]-Based Compounds as Competitive Tyrosinase Inhibitors Endowed with Antimelanogenic Effects.

There is a considerable attention for the development of inhibitors of tyrosinase (TYR) as therapeutic strategy for the treatment of hyperpigmentation disorders in humans. Continuing in our efforts to identify TYR inhibitors, we describe the design, synthesis and pharmacophore exploration of new small molecules structurally characterized by the presence of the 4-fluorobenzylpiperazine moiety as key pharmacophoric feature for the inhibition of TYR from Agaricus bisporus (AbTYR). Our investigations resulted in the discovery of the competitive inhibitor [4-(4-fluorobenzyl)piperazin-1-yl]-(3-chloro-2-nitro-phenyl)methanone 26 (IC50 =0.18 µM) that proved to be ∼100-fold more active than reference compound kojic acid (IC50 =17.76 µM). Notably, compound 26 exerted antimelanogenic effect on B16F10 cells in absence of cytotoxicity. Docking analysis suggested its binding mode into AbTYR and into modelled human TYR.

Design, synthesis and biochemical evaluation of novel carbonic anhydrase inhibitors triggered by structural knowledge on hCA VII.

To tackle the challenge of isoform selectivity, we explored the entrance of the cavity for selected druggable human Carbonic Anhydrases (hCAs). Based on X-ray crystallographic studies on the 4-(4-(2-chlorobenzoyl)piperazine-1-carbonyl)benzenesulfonamide in complex with the brain expressed hCA VII (PDB code: 7NC4), a series of 4-(4(hetero)aroylpiperazine-1-carbonyl)benzene-1-sulfonamides has been developed. To evaluate their capability to fit the hCA VII catalytic cavity, the newer benzenesulfonamides were preliminary investigated by means of docking simulations. Then, this series of thirteen benzenesulfonamides was synthesized and tested against selected druggable hCAs. Among them, the 4-(4-(furan-2-carbonyl)piperazine-1-carbonyl)benzenesulfonamide showed remarkable affinity towards hCA VII (Ki: 4.3 nM) and good selectivity over the physiologically widespread hCA I when compared to Topiramate (TPM).

Exploring Molecular Contacts of MUC1 at CIN85 Binding Interface to Address Future Drug Design Efforts.

The modulation of protein-protein interactions (PPIs) by small molecules represents a valuable strategy for pharmacological intervention in several human diseases. In this context, computer-aided drug discovery techniques offer useful resources to predict the network of interactions governing the recognition process between protein partners, thus furnishing relevant information for the design of novel PPI modulators. In this work, we focused our attention on the MUC1-CIN85 complex as a crucial PPI controlling cancer progression and metastasis. MUC1 is a transmembrane glycoprotein whose extracellular domain contains a variable number of tandem repeats (VNTRs) regions that are highly glycosylated in normal cells and under-glycosylated in cancer. The hypo-glycosylation fosters the exposure of the backbone to new interactions with other proteins, such as CIN85, that alter the intracellular signalling in tumour cells. Herein, different computational approaches were combined to investigate the molecular recognition pattern of MUC1-CIN85 PPI thus unveiling new structural information useful for the design of MUC1-CIN85 PPI inhibitors as potential anti-metastatic agents.

In Silico Strategy for Targeting the mTOR Kinase at Rapamycin Binding Site by Small Molecules.

Computer aided drug-design methods proved to be powerful tools for the identification of new therapeutic agents. We employed a structure-based workflow to identify new inhibitors targeting mTOR kinase at rapamycin binding site. By combining molecular dynamics (MD) simulation and pharmacophore modelling, a simplified structure-based pharmacophore hypothesis was built starting from the FKBP12-rapamycin-FRB ternary complex retrieved from RCSB Protein Data Bank (PDB code 1FAP). Then, the obtained model was used as filter to screen the ZINC biogenic compounds library, containing molecules derived from natural sources or natural-inspired compounds. The resulting hits were clustered according to their similarity; moreover, compounds showing the highest pharmacophore fit-score were chosen from each cluster. The selected molecules were subjected to docking studies to clarify their putative binding mode. The binding free energy of the obtained complexes was calculated by MM/GBSA method and the hits characterized by the lowest ΔGbind values were identified as potential mTOR inhibitors. Furthermore, the stability of the resulting complexes was studied by means of MD simulation which revealed that the selected compounds were able to form a stable ternary complex with FKBP12 and FRB domain, thus underlining their potential ability to inhibit mTOR with a rapamycin-like mechanism.

Taking advantage of lithium monohalocarbenoid intrinsic α-elimination in 2-MeTHF: controlled epoxide ring-opening en route to halohydrins.

The intrinsic degradative α-elimination of Li carbenoids somehow complicates their use in synthesis as C1-synthons. Nevertheless, we herein report how boosting such an α-elimination is a straightforward strategy for accomplishing controlled ring-opening of epoxides to furnish the corresponding ß-halohydrins. Crucial for the development of the method is the use of the eco-friendly solvent 2-MeTHF, which forces the degradation of the incipient monohalolithium, due to the very limited stabilizing effect of this solvent on the chemical integrity of the carbenoid. With this approach, high yields of the targeted compounds are consistently obtained under very high regiocontrol and, despite the basic nature of the reagents, no racemization of enantiopure materials is observed.