Synthesis of N-oxyamide analogues of protein kinase B (Akt) targeting anionic glycoglycerolipids and their antiproliferative activity on human ovarian carcinoma cells.

N-Oxyamides of bioactive anionic glycoglycerolipids based on 2-O-ß-D-glucosylglycerol were efficiently prepared. However, the oxidation step of the primary hydroxyl group of the glucose moiety in the presence of the N-oxyamide function appeared to be a difficult task that was nevertheless conveniently achieved for the first time by employing a chemoenzymatic laccase/TEMPO procedure. The obtained N-oxyamides exhibited a higher inhibition of proliferation of ovarian carcinoma IGROV-1 cells in serum-free medium than in complete medium, similarly to the corresponding bioactive esters. Stability and serum binding studies indicated that the observed reduced activity of the compounds in complete medium could be mainly due to a binding effect of serum proteins rather than the hydrolytic degradation of glycoglycerolipid acyl chains. Furthermore, the results of the cellular studies under serum-free conditions suggested that the N-oxyamide group could increase the antiproliferative activity of a glycoglycerolipid independently of the presence of the anionic carboxylic group. Cellular studies in other cell lines besides IGROV-1 also support a certain degree of selectivity of this series of compounds for tumor cells with Akt hyperactivation.

Elagolix Sodium Salt and Its Synthetic Intermediates: A Spectroscopic, Crystallographic, and Conformational Study.

Elagolix sodium salt is the first marketed orally active non-peptide gonadotropin-releasing hormone receptor antagonist (GnRHR-ant) for the management of hormone dependent diseases, such as endometriosis and uterine fibroids. Despite its presence on the market since 2018, a thorough NMR analysis of this drug, together with its synthetic intermediates, is still lacking. Hence, with the aim of filling this literature gap, we here performed a detailed NMR investigation, which allowed the complete assignment of the 1H, 13C, and 15N NMR signals. These data allowed, with the support of the conformational analysis, the determination of the stereochemical profile of the two atropisomers, detectable in solution. Moreover, these latter were also detected by means of cellulose-based chiral HPLC, starting from a sample prepared through an implemented synthetic procedure with respect to the reported ones. Overall, these results contribute to further understanding of the topic of atropisomerism in drug discovery and could be applied in the design of safe and stable analogs, endowed with improved target selectivity.

An Outline of the Latest Crystallographic Studies on Inhibitor-Enzyme Complexes for the Design and Development of New Therapeutics against Tuberculosis.

The elucidation of the structure of enzymes and their complexes with ligands continues to provide invaluable insights for the development of drugs against many diseases, including bacterial infections. After nearly three decades since the World Health Organization's (WHO) declaration of tuberculosis (TB) as a global health emergency, Mycobacterium tuberculosis (Mtb) continues to claim millions of lives, remaining among the leading causes of death worldwide. In the last years, several efforts have been devoted to shortening and improving treatment outcomes, and to overcoming the increasing resistance phenomenon. The structural elucidation of enzyme-ligand complexes is fundamental to identify hot-spots, define possible interaction sites, and elaborate strategies to develop optimized molecules with high affinity. This review offers a critical and comprehensive overview of the most recent structural information on traditional and emerging mycobacterial enzymatic targets. A selection of more than twenty enzymes is here discussed, with a special emphasis on the analysis of their binding sites, the definition of the structure-activity relationships (SARs) of their inhibitors, and the study of their main intermolecular interactions. This work corroborates the potential of structural studies, substantiating their relevance in future anti-mycobacterial drug discovery and development efforts.

Towards the Inhibition of Protein-Protein Interactions (PPIs) in STAT3: Insights into a New Class of Benzothiadiazole Derivatives.

Signal transducer and activator of transcription 3 (STAT3) is a validated anticancer target due to the relationship between its constitutive activation and malignant tumors. Through a virtual screening approach on the STAT3-SH2 domain, 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide (1) was identified as a potential STAT3 inhibitor. Some benzothiadiazole derivatives were synthesized by employing a versatile methodology, and they were tested by an AlphaScreen-based assay. Among them, benzosulfamide 1 showed a significant activity with an IC50 = 15.8 ± 0.6 µM as a direct STAT3 inhibitor. Notably, we discovered that compound 1 was also able to interact with cysteine residues located around the SH2 domain. By applying mass spectrometry, liquid chromatography, NMR, and UV spectroscopy, an in-depth investigation was carried out, shedding light on its intriguing and unexpected mechanism of interaction.

Structure-based drug design, synthesis and biological assays of P. falciparum Atg3-Atg8 protein-protein interaction inhibitors.

The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein-protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3-Atg8 reciprocal protein-protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds.

Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a ß-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-ß-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

Anionic glycolipids related to glucuronosyldiacylglycerol inhibit protein kinase Akt.

New glucuronosyldiacylglycerol (GlcADG) analogues based on a 2-O-ß-D-glucopyranosyl-sn-glycerol scaffold and carrying one or two acyl chains of different lengths have been synthesized as phosphatidylinositol 3-phosphate (PI3P) mimics targeting the protein kinase Akt. The Akt inhibitory effect of the prepared compounds was assayed using an in vitro kinase assay. The antiproliferative activity of the compounds was tested in the human ovarian carcinoma IGROV-1 cell line in which we found that two of them could inhibit proliferation, in keeping with the target inhibitory effect.

Automated radiosynthesis and preclinical evaluation of two new PSMA-617 derivatives radiolabelled via [18F]AlF2+ method.

BACKGROUND: In the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research. The most promising derivative in terms of interaction with the antigen and clinical properties has been found to be "PSMA-617", and its lutetium-177 radiolabelled version has recently been approved by EU and USA regulatory agencies for therapeutic purposes. For the above reasons, the development of new derivatives of PSMA-617 radiolabelled with fluorine-18 may still be of great interest. This paper proposes the comparison of two different PSMA-617 derivatives functionalized with NODA and RESCA chelators, respectively, radiolabelled via [18F]AlF2+ complexation. RESULTS: The organic synthesis of two PSMA-617 derivatives and their radiolabelling via [18F]AlF2+ complexation resulted to proceed efficiently and successfully. Moreover, stability in solution and in plasma has been evaluated. The whole radiosynthesis procedure has been fully automated, and the final products have been obtained with radiochemical yield and purity potentially suitable for clinical studies. The biodistribution of the two derivatives was performed both in prostate cancer and glioma tumour models. Compared with the reference [18F]F-PSMA-1007 and [18F]F-PSMA-617-RESCA, [18F]F-PSMA-617-NODA derivative showed a higher uptake in both tumors, faster clearance in non-target organs, and lower uptake in salivary glands. CONCLUSION: PSMA-617 NODA and RESCA derivatives were radiolabelled successfully via [18F]AlF2+ chelation, the former being more stable in solution and human plasma. Moreover, preclinical biodistribution studies showed that [18F]F-PSMA-617-NODA might be of potential interest for clinical applications.

Crystallographic, spectroscopic, and theoretical investigation of the efficiently separated 21R and 21S-diastereoisomers of argatroban.

Argatroban (I), a potent noncovalent reversible thrombin inhibitor, is used as an anticoagulant for the parenteral treatment of heparin-induced thrombocytopenia (HIT) patients. By virtue of its pharmacological properties and the well-balanced risks and benefits, argatroban is now emerging as a clinically relevant antithrombotic agent. The availability of this drug as a mixture of 21R and 21S-diastereoisomers, in a ratio of roughly 64:36, prompted us to design an efficient separation setup of the two epimers. We pursued our efforts on their detailed structural analysis with the aim of understanding their different activity and aqueous solubility. These investigations were accompanied by a modeling study of the two diastereoisomers, with particular attention on the easy interconverting half-chair of the tetrahydroquinoline system and its preferred conformation, which is determined by the configuration at C21. These results, together with the analysis of their physicochemical profiles, provide new useful information for the development of the individual diastereoisomers.

Curcumin and Related Compounds in Cancer Cells: New Avenues for Old Molecules.

Curcumin and related compounds are known for the large spectrum of activities. The chemical features of these compounds are important for their biological effects with a key role for the thiol-reactive α-ß unsaturated carbonyl groups. Curcumin derivatives may overcome the limitation of the bioavailability of the parent compound, while maintaining the key chemical features responsible for biological activities. Curcumin and related compounds show anti-viral, anti-fungal, anti-microbial and anti-tumor activities. The therapeutic effects of curcumin, used as a supplement in cancer therapy, have been documented in various cancer types, in which inhibition of cell growth and survival pathways, induction of apoptosis and other cell death pathways have been reported. Curcumin-induced apoptosis has been linked both to the intrinsic and extrinsic apoptotic pathways. Necroptosis has also been involved in curcumin-induced toxicity. Among curcumin-induced effects, ferroptosis has also been described. The mechanism of curcumin toxicity can be triggered by reactive oxygen species-mediated endoplasmic reticulum stress. Curcumin targets have been identified in the context of the ubiquitin-proteasome system with evidence of inhibition of the proteasome proteolytic activities and cellular deubiquitinases. Curcumin has recently been shown to act on the tumor microenvironment with effects on cancer-associated fibroblasts and immune cells. The related product caffeic acid phenethyl ester has shown promising preclinical results with an effect on the inflammatory microenvironment. Here, we review the mechanisms underlying curcumin and derivatives toxicity towards cancer cells with particular emphasis on cell death pathways and the ubiquitin-proteasome system.

Recent Progresses in Conjugation with Bioactive Ligands to Improve the Anticancer Activity of Platinum Compounds.

Platinum (Pt) drugs, including cisplatin, are widely used for the treatment of solid tumors. Despite the clinical success, side effects and occurrence of resistance represent major limitations to the use of clinically available Pt drugs. To overcome these problems, a variety of derivatives have been designed and synthetized. Here, we summarize the recent progress in the development of Pt(II) and Pt(IV) complexes with bioactive ligands. The development of Pt(II) and Pt(IV) complexes with targeting molecules, clinically available agents, and other bioactive molecules is an active field of research. Even if none of the reported Pt derivatives has been yet approved for clinical use, many of these compounds exhibit promising anticancer activities with an improved pharmacological profile. Thus, planning hybrid compounds can be considered as a promising approach to improve the available Pt-based anticancer agents and to obtain new molecular tools to deepen the knowledge of cancer progression and drug resistance mechanisms.

The deubiquitinase USP8 regulates ovarian cancer cell response to cisplatin by suppressing apoptosis.

The identification of therapeutic approaches to improve response to platinum-based therapies is an urgent need for ovarian carcinoma. Deubiquitinases are a large family of ubiquitin proteases implicated in a variety of cellular functions and may contribute to tumor aggressive features through regulation of processes such as proliferation and cell death. Among the subfamily of ubiquitin-specific peptidases, USP8 appears to be involved in modulation of cancer cell survival by still poorly understood mechanisms. Thus, we used ovarian carcinoma cells of different histotypes, including cisplatin-resistant variants with increased survival features to evaluate the efficacy of molecular targeting of USP8 as a strategy to overcome drug resistance/modulate cisplatin response. We performed biochemical analysis of USP8 activity in pairs of cisplatin-sensitive and -resistant cells and found increased USP8 activity in resistant cells. Silencing of USP8 resulted in decreased activation of receptor tyrosine kinases and increased sensitivity to cisplatin in IGROV-1/Pt1 resistant cells as shown by colony forming assay. Increased cisplatin sensitivity was associated with enhanced cisplatin-induced caspase 3/7 activation and apoptosis, a phenotype also observed in cisplatin sensitive cells. Increased apoptosis was linked to FLIPL decrease and cisplatin induction of caspase 3 in IGROV-1/Pt1 cells, cisplatin-induced claspin and survivin down-regulation in IGROV-1 cells, thereby showing a decrease of anti-apoptotic proteins. Immunohistochemical staining on 65 clinical specimens from advanced stage ovarian carcinoma indicated that 40% of tumors were USP8 positive suggesting that USP8 is an independent prognostic factor for adverse outcome when considering progression free survival as a clinical end-point. Taken together, our results support that USP8 may be of diagnostic value and may provide a therapeutic target to improve the efficacy of platinum-based therapy in ovarian carcinoma.

Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.

Hybrid Lipid/Polymer Nanoparticles to Tackle the Cystic Fibrosis Mucus Barrier in siRNA Delivery to the Lungs: Does PEGylation Make the Difference?

Inhaled siRNA therapy has a unique potential for treatment of severe lung diseases, such as cystic fibrosis (CF). Nevertheless, a drug delivery system tackling lung barriers is mandatory to enhance gene silencing efficacy in the airway epithelium. We recently demonstrated that lipid-polymer hybrid nanoparticles (hNPs), comprising a poly(lactic-co-glycolic) acid (PLGA) core and a lipid shell of dipalmitoyl phosphatidylcholine (DPPC), may assist the transport of the nucleic acid cargo through mucus-covered human airway epithelium. To study in depth the potential of hNPs for siRNA delivery to the lungs and to investigate the hypothesized benefit of PEGylation, here, an siRNA pool against the nuclear factor-κB (siNFκB) was encapsulated inside hNPs, endowed with a non-PEGylated (DPPC) or a PEGylated (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) or DSPE-PEG) lipid shell. Resulting hNPs were tested for their stability profiles and transport properties in artificial CF mucus, mucus collected from CF cells, and sputum samples from a heterogeneous and representative set of CF patients. Initial information on hNP properties governing their interaction with airway mucus was acquired by small-angle X-ray scattering (SAXS) studies in artificial and cellular CF mucus. The diffusion profiles of hNPs through CF sputa suggested a crucial role of lung colonization of the corresponding donor patient, affecting the mucin type and content of the sample. Noteworthy, PEGylation did not boost mucus penetration in complex and sticky samples, such as CF sputa from patients with polymicrobial colonization. In parallel, in vitro cell uptake studies performed on mucus-lined Calu-3 cells grown at the air-liquid interface (ALI) confirmed the improved ability of non-PEGylated hNPs to overcome mucus and cellular lung barriers. Furthermore, effective in vitro NFκB gene silencing was achieved in LPS-stimulated 16HBE14o- cells. Overall, the results highlight the potential of non-PEGylated hNPs as carriers for pulmonary delivery of siRNA for local treatment of CF lung disease. Furthermore, this study provides a detailed understanding of how distinct models may provide different information on nanoparticle interaction with the mucus barrier.

Vecuronium bromide and its advanced intermediates: A crystallographic and spectroscopic study.

Vecuronium bromide (Piperidinium, 1-[(2ß,3α,5α,16ß,17ß)-3,17-bis(acetyloxy)-2-(1-piperidinyl)androstan-16-yl]-1-methyl-, bromide; Norcuron®) has been extensively used in anesthesiology practice as neuromuscular blocking agent since its launch on the market in 1982. However, a detailed crystallographic and NMR analysis of its advanced synthetic intermediates is still lacking. Hence, with the aim of filling this literature gap, vecuronium bromide was prepared starting from the commercially available 3ß-hydroxy-5α-androstan-17-one (epiandrosterone), implementing some modifications to a traditional synthetic procedure. A careful NMR study allowed the complete assignment of the 1H, 13C, and 15N NMR signals of vecuronium bromide and its synthetic intermediates. The structural and stereochemical characterization of 2ß,16ß-bispiperidino-5α-androstane-3α,17ß-diol, the first advanced synthetic intermediate carrying all the stereocenters in the final configuration, was described by means of single-crystal X-ray diffraction and Hirshfeld surface analysis, allowing a detailed conformational investigation.

Synthesis of the sulfonate analogue of seminolipid via Horner-Wadsworth-Emmons olefination.

The first synthesis of the sulfonate analogue of seminolipid, the main sulfoglycolipid in mammalian sperm, is reported. Installation of the sulfonate unit was accomplished by a quite unexplored strategy based on Horner-Wadsworth-Emmons olefination on a 3 '-keto-galactoside, followed by stereoselective double bond reduction.

2-O-beta-d-glucopyranosyl-sn-glycerol based analogues of sulfoquinovosyldiacylglycerols (SQDG) and their role in inhibiting Epstein-Barr virus early antigen activation.

New sulfoquinovosyldiacylglycerols derived from 2-O-beta-d-glucopyranosyl-sn-glycerol, carrying acyl chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure in which a sulfonate is introduced at the C-6 position of glucose by oxidation of a thioacetate in the presence of the unprotected secondary hydroxyl groups, and tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. Our study has allowed to ascertain the role of the 6'-sulfonate group and the need of a free hydroxyl group on the glycerol moiety in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).

Complete 1H and 13C assignments of (21R) and (21S) diastereomers of argatroban.

The complete 1H and 13C NMR assignments are reported for the antithrombotic (21R)- and (21S)-argatroban by 1D and 2D NMR experiments (HSQC, HMBC, NOESY and 1H--1H COSY). Some well-resolved signals could be used for an accurate measurement of the diastereomeric composition of argatroban.

Nonenzymatic Polymerization into Long Linear RNA Templated by Liquid Crystal Self-Assembly.

Self-synthesizing materials, in which supramolecular structuring enhances the formation of new molecules that participate to the process, represent an intriguing notion to account for the first appearance of biomolecules in an abiotic Earth. We present here a study of the abiotic formation of interchain phosphodiester bonds in solutions of short RNA oligomers in various states of supramolecular arrangement and their reaction kinetics. We found a spectrum of conditions in which RNA oligomers self-assemble and phase separate into highly concentrated ordered fluid liquid crystal (LC) microdomains. We show that such supramolecular state provides a template guiding their ligation into hundred-bases long chains. The quantitative analysis presented here demonstrates that nucleic acid LC boosts the rate of end-to-end ligation and suppresses the formation of the otherwise dominant cyclic oligomers. These results strengthen the concept of supramolecular ordering as an efficient pathway toward the emergence of the RNA World in the primordial Earth.