A new series of dithiocarbamates (DTCs) was prepared from primary/secondary amines incorporating amino/hydroxyl-alkyl, mono- and bicyclic aliphatic ring systems based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, and carbon disulfide. The compounds were investigated for the inhibition of four mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, that is, the human (h) hCA I, II, IX and XII, drug targets for antiglaucoma (hCA II and XII) or antitumor (hCA IX/XII) agents. The compounds were moderate or inefficient hCA I inhibitors (off-target isoform for both applications), efficiently inhibited hCA II, whereas some of them were low nanomolar/subnanomolar hCA IX/XII inhibitors. One DTC showed excellent intraocular pressure (IOP) lowering properties in an animal model of glaucoma, with a two times better efficiency compared to the clinically used sulfonamide dorzolamide.

Cannabinoid (CB) and opioid systems are both involved in analgesia, food intake, mood and behavior. Due to the co-localization of µ-opioid (MOR) and CB1 receptors in various regions of the central nervous system (CNS) and their ability to form heterodimers, bivalent ligands targeting to both these systems may be good candidates to investigate the existence of possible cross-talking or synergistic effects, also at sub-effective doses. In this work, we selected from a small series of new Rimonabant analogs one CB1R reverse agonist to be conjugated to the opioid fragment Tyr-D-Ala-Gly-Phe-NH2. The bivalent compound (9) has been used for in vitro binding assays, for in vivo antinociception models and in vitro hypothalamic perfusion test, to evaluate the neurotransmitters release.

A hierarchical in silico screening procedure using the crystal structure of an agonist bound chimeric α7/Ls-AChBP protein was successfully applied to both proprietary and commercial databases containing drug-like molecules. An overall hit rate of 26% (pK(i) ≥5.0) was obtained, with an even better hit rate of 35% for the commercial compound collection. Structurally novel and diverse ligands were identified. Binding studies with [(3)H]epibatidine on chimeric α7/5-HT(3) receptors yielded submicromolar inhibition constants for identified hits. Compared to a previous screening procedure that utilized the wild type Ls-AChBP crystal structure, the current study shows that the recently obtained α7/Ls-AChBP chimeric protein crystal structure is a better template for the identification of novel α7 receptor ligands.

Novel sulfonamidoindole-based hydrazones with a 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide scaffold were synthesized and tested in enzyme inhibition assays against the tumor-associated carbonic anhydrase isoforms, hCA IX and XII, and the off-targets, hCA I and II. The compounds showed selectivity against hCA IX and XII over hCA I and II. Six compounds showed KI values lower than 10 nM against hCA IX or XII. Molecular modeling studies were performed to suggest binding interactions between the ligand and the hCA active sites.

A large library of derivatives based on the scaffold of 2-(benzylsulfinyl)benzoic acid were synthesised and tested as atypical inhibitors against four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). The exploration of the chemical space around the main functional groups led to the discovery of selective hCA IX inhibitors in the micromolar/nanomolar range, thus establishing robust structure-activity relationships within this versatile scaffold. HPLC separation of some selected chiral compounds and biological evaluation of the corresponding enantiomers was performed along with molecular modelling studies on the most active derivatives.

In this study, sixty novel coumarin-monoterpene compounds were synthesized in two series [thirty-two compounds (12-43) bearing a triazole ring in the first series, and twenty-eight compounds (44-71) bearing an alkyl chain in the second one]. Their inhibitory effects on the human carbonic anhydrase (hCA) isoforms I, II, IX, and XII and anticancer potentials were determined. All synthesized molecules selectively inhibited CA IX and XII. 23 and 42 were found to be the strongest inhibitors, with K i values of 1.9 nM against hCA IX. Also, 70 showed the highest inhibitory activity with a K i value of 4.9 nM against hCA XII. Moreover, their cytotoxic effects on colon adenocarcinoma (HT-29), prostate adenocarcinoma (PC-3), and breast adenocarcinoma (MCF-7) cell lines were evaluated. According to the cytotoxicity results, 14 (IC50 = 2.48 μM) and 63 (IC50 = 3.91 μM) exhibited the highest cytotoxicity on the MCF-7 cells, while 23 showed the strongest cytotoxic effect on both PC-3 (IC50 = 9.40 μM) and HT-29 (IC50 = 12.10 μM) cell lines. 14, 23, and 66 decreased CA IX and CA XII protein expression in HT-29 cells, while 23 and 66 showed the strongest reduction of both CA IX and CA XII in MCF-7 cells. All of the selected compounds increased total apoptosis in a concentration-dependent manner in HT-29 and MCF-7 cells. 14 has the strongest apoptotic effect in MCF-7 cells. 23 increased early apoptosis primarily, while 14 and 66 increased total apoptosis in HT-29. In addition, PI/Hoechst staining proves that apoptotic cells are increased in HT-29 with an effect of 14, 23, and 66. As a result of the modeling studies, it has been shown that only the open coumarin form of the compounds can interact directly with the active-site Zn2+ ion. It has been shown that coumarin-monoterpene structures with different alkyl and monoterpene groups both specifically inhibit CA IX and XII and exhibit specific cytotoxicity in different cell lines.

Carbonic anhydrases isoforms CA IX, and XII are known to be highly expressed in various human tissues and malignancies. CA IX is a prominent target for especially colorectal cancers, because it is overexpressed in colorectal cancer and this overexpression leads poor prognosis. Inhibition of CA IX activity by small molecule CA inhibitors like sulfonamides, sulfonamide derivative or coumarins leads to inhibition of tumorigenesis. Novel twenty-seven compounds in three series (sulfonamide-based imines (6a-6i), coumarin-based aldehydes (7a-7i), and coumarin-sulfonamide-based target molecules (8a-8i)) were synthesized and characterized by means of IR, NMR, and mass spectra. All compounds were tested for their ability to inhibit CA I, CA II, CA IX, and CA XII isoforms. 4-((((2-((1-(3-((2-oxo-2H-chromen-7-yl)oxy)propyl)-1H-1,2,3-triazol-4-yl)methoxy)naphthalen-1-yl)-methylene)amino)methyl)benzenesulfonamide (8i) exhibited the highest hCA IX inhibition with the Ki of 45.5 nM. In addition, 8i was found to be potent in inhibiting cancer cell proliferation as selective (IC50 = 17.01 ± 1.35 μM for HT-29, IC50 = 118.73 ± 1.19 μM for HEK293T). This novel compound inhibited the CA IX and CA XII protein expression in HT-29 cells. These findings indicate that 8i can inhibit cellular proliferation in human colon cancer cells by specifically targeting the CA IX and CA XII expression.

Hierarchical in silico screening protocols against the agonist bound acetylcholine binding protein (AChBP) crystal structure were efficient in identifying novel chemotypes for AChBP and the human α7 receptor. Two hit structures were cocrystallized with AChBP revealing intermolecular cation-π interactions with loop C but lacking intermolecular hydrogen bonding. The compounds act as competitive α7 receptor antagonists and as non-competitive α4β2 receptor inhibitors. These results underline the usability of AChBP in structure-based in silico screening strategies in finding novel scaffolds for the α7 receptor, but also illustrates some limitations of using AChBP as bait to find competitive α4β2 receptor ligands and α7 receptor agonists.

A large library of fibrate-based N-acylsulphonamides was designed, synthesised, and fully characterised in order to propose them as zinc binders for the inhibition of human carbonic anhydrase (hCA) enzymatic activity. Synthesised compounds were tested against four hCAs (I, II, IX, and XII) revealing a promising submicromolar inhibitory activity characterised by an isozyme selectivity pattern. Structural modifications explored within this scaffold are: presence of an aryl ring on the sulphonamide, p-substitution of this aryl ring, benzothiazole or benzophenone as core nuclei, and an n-propyl chain or a geminal dimethyl at Cα carbon. Biological results fitted well with molecular modelling analyses, revealing a putative direct interaction with the zinc ion in the active site of hCA I, II and IX. These findings supported the exploration of less investigated secondary sulphonamides as potential hCA inhibitors.

In our efforts to find new and selective thiazolidinone-based anti-Candida agents, we synthesized and tested 26 thiazolidinones against several Candida spp. and Gram-positive and Gram-negative bacteria. The compounds showed selective antifungal activity with potency similar to fluconazole and clotrimazole, while lacking strong antibacterial activity. Molecular docking and molecular dynamics studies were performed on Candida CYP51a1 and carbonic anhydrase (CA) enzymes to further suggest putative targets that could mediate the antifungal effects of these compounds. Finally, the compounds were tested in enzyme inhibition assays to assess their putative mechanism of action and showed promising KI values in the 0.1-10 µM range against the Candida glabrata β-CA enzyme CgNce103.

Inhibition of the β-carbonic anhydrase (CA, EC 4.2.1.1) from pathogenic Candida glabrata (CgNce103) by 1H-indole-2,3-dione 3-[N-(4-sulfamoylphenyl)thiosemicarbazones] 4a-m was investigated. All the compounds were found to be potent inhibitors of CgNce103, with inhibition constants in the range of 6.4-63.9 nM. The 5,7-dichloro substituted derivative 4l showed the most effective inhibition (KI of 6.4 nM) as well as the highest selectivity for inhibiting CgNce103 over the cytosolic human (h) isoforms hCA I and II. A possible binding interaction of compound 4l within the active site of CgNce103 has been proposed based on docking studies.

Human carbonic anhydrases IX and XII are upregulated in many tumors and form a novel target for new generation anticancer drugs. Here we report the synthesis of novel 2-indolinone derivatives with the sulfonamide group as a zinc binding moiety. Enzyme inhibition assays confirmed that the compounds showed selectivity against hCA IX and XII over the widely distributed off-targets hCA I and II. Molecular modelling studies were performed to suggest modes of binding for these compounds.

In order to obtain novel pharmacological tools and to investigate a multitargeting analgesic strategy, the CB1 and CB2 cannabinoid receptor agonist JWH-018 was conjugated with the opiate analgesic oxycodone or with an enkephalin related tetrapeptide. The opioid and cannabinoid pharmacophores were coupled via spacers of different length and chemical structure. In vitro radioligand binding experiments confirmed that the resulting bivalent compounds bound both to the opioid and to the cannabinoid receptors with moderate to high affinity. The highest affinity bivalent derivatives 11 and 19 exhibited agonist properties in [35S]GTPγS binding assays. These compounds activated MOR and CB (11 mainly CB2, whereas 19 mainly CB1) receptor-mediated signaling, as it was revealed by experiments using receptor specific antagonists. In rats both 11 and 19 exhibited antiallodynic effect similar to the parent drugs in 20 μg dose at spinal level. These results support the strategy of multitargeting G-protein coupled receptors to develop lead compounds with antinociceptive properties.

Enzyme inhibition data of structurally novel isatin-containing sulfonamides were determined for two carbonic anhydrases (CAs, EC 4.2.1.1) from pathogenic Candida species (CaNce103 from C. albicans and CgNce103 from C. glabrata). The compounds show KI values in the low nanomolar range for the fungal CAs, while they have significantly higher KI values for the human CAs. Homology models were constructed for the CaNce103 and CgNce103 and subsequently the ligands were docked into these models to rationalize their enzyme inhibitory properties.

A small collection of 26 structurally novel thiazolidinone-containing compounds, without the well-known sulphonamide zinc-binding group, were synthesised and tested in enzyme inhibition assays against the tumour-associated hCA IX enzyme. Inhibition constants in the lower micromolar region (KI < 25 μM) have been measured for 17 of the 26 compounds. Even though the KI values are relatively weak, the fact that they do not contain a sulphonamide moiety suggests that these compounds do not interact with the active site zinc ion. Therefore, docking studies and molecular dynamics simulations have been performed to suggest binding poses for these structurally novel inhibitors.

It has been proven that specific isoforms of human carbonic anhydrase (hCA) are able to fine-tune physiological pathways connected to signal processing, and that decreased CAs expression negatively influences cognition, leading to mental retardation, Alzheimer's disease, and aging-related cognitive dysfunctions. For this reason, a small library of natural and synthetic nitrogen containing cyclic derivatives was assayed as activators of four human isoforms of carbonic anhydrase (hCA I, II, IV and VII). Most of the compounds activated hCA I, IV and VII in the micromolar range, with KAs ranging between 3.46 and 80.5 μM, whereas they were not active towards hCA II (KAs > 100 μM). Two natural compounds, namely l-(+)-ergothioneine (1) and melatonin (2), displayed KAs towards hCA VII in the nanomolar range after evaluation by a CO₂ hydration method in vitro, showing a rather efficient and selective activation profile with respect to histamine, used as a reference compound. Corroborated with the above in vitro findings, a molecular modelling in silico approach has been performed to correlate these biological data, and to elucidate the binding interaction of these activators within the enzyme active site.

Due to the increasing resistance of currently used antimicrobial drugs, there is an urgent problem for the treatment of cholera disease, selective inhibition of the α-class carbonic anhydrases (CA, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae (VcCA) presents an alternative therapeutic target. In this study, a series of hydrazone derivatives, carrying the 2-(hydrazinocarbonyl)-3-phenyl-1H-indole-5-sulfonamide scaffold, have been evaluated as inhibitors of the VcCA with molecular modeling studies. The results suggest that these compounds may bind to the active site of VcCA. To verify this, VcCA enzyme inhibition studies were performed and as predicted most of the tested compounds displayed potent inhibitory activities against VcCA with three compounds showing KI values lower than 30 nM. In addition, all these compounds showed selectivity for VcCA and the off-targets hCA I and II.

Nonsteroidal aromatase inhibitors (NSAIs) are well-established drugs for the therapy of breast cancer. However, they display some serious side effects, and their efficacy can be compromised by the development of chemoresistance. Previously, we have reported different indazole-based carbamates and piperidine-sulphonamides as potent aromatase inhibitors. Starting from the most promising compounds, here we have synthesised new indazole and triazole derivatives and evaluated their biological activity as potential dual agents, targeting both the aromatase and the inducible nitric oxide synthase, being this last dysregulated in breast cancer. Furthermore, selected compounds were evaluated as antiproliferative and cytotoxic agents in the MCF-7 cell line. Moreover, considering the therapeutic diversity of azole-based compounds, all the synthesized compounds were also evaluated as antifungals on different Candida strains. A docking study, as well as molecular dynamics simulation, were carried out to shed light on the binding mode of the most interesting compound into the different target enzymes catalytic sites.

Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the enzyme (main protease) inhibition-based assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T/hACE2+TMPRSS2, and virus neutralization assay using xCELLigence MP real-time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of montelukast both on the main protease enzyme inhibition and virus entry into the host cell (spike/ACE2). The virus neutralization assay results showed that SARS-CoV-2 virus activity was delayed with montelukast for 20 h on the infected cells. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and, if its effect is proved in clinical phase studies, it should be used against coronavirus disease 2019 (COVID-19).

Caulerpa cylindracea Sonder is a green alga belonging to the Caulerpaceae family. This is the first chemical investigation of C. cylindracea in the Dardanelles which resulted in the isolation of four compounds, caulerpin (1), monomethyl caulerpinate (2), beta-sitosterol (3), and palmitic acid (4). Their structures were elucidated by spectroscopic analyses including 1D- and 2D NMR and mass. The isolated compounds 1 and 2 were tested against the SARS-CoV-2 viral targets spike protein and main protease (3CL) enzyme, and both compounds significantly inhibit the interaction of spike protein and ACE2, while the main protease activity was not significantly reduced. Docking studies suggested that compounds 1 and 2 may bind to the ACE2 binding pocket on spike, and compound 2 may also bind to an allosteric site on spike. As such, these compounds may inhibit the spike-ACE2 complex formation competitively and/or allosterically and have the potential to be used against SARS-CoV-2 virus infection. In addition, compounds 1 and 2 showed at least two-fold higher cytotoxicity against breast cancer cell lines MCF7 and MDA-MB-231 compared to the CCD fibroblast control cell line.