Design, virtual screening and structural studies of new molecules with potential antitumor and antiinflammatory activity

2010 - 2011Computational methodologies in combination with experimental techniques as Nuclear Magnetic Resonance (NMR) have become a crucial component in drug discovery process, from hit identification to lead optimization. The study of ligand-macromolecule interactions, in fact, has a crucial role for the design and the development of new and more powerful drugs. In this project, different aspects of interaction and recognition processes between ligand and macromolecule, and streostructure assignment has been studied through this kind of combined approach with the aim to identify novel potential antitumor and/or antiinflammatory molecules. In particular, because the strong interconnection between the tumoral and inflammatory pathology has led to the identification of new target utilizable for the therapy, in this project will be described proteins (Histone deacetilase, HDAC; Nicotinamide Phosphoribosyltransferase, NMPRTase or Nampt; microsomal prostaglandin E2 synthase, mPGES-1; human synovial Phospholipases A2, hsPLA2; human Farnesoid-X-Receptor, FXR; human Pregnane-X-Receptor, PXR; Bile Acid Receptor GPBAR-1, TGR5) involved in essential cellular processes and acting at diverse levels and phases of the tumor and inflammation diseases. The results obtained can be summarized in three main areas of activity, whose relative weight was varied according to the development of the overall project: a) Support in the design of original scaffolds for the generation of libraries potentially utilizable in therapy. This work was exclusively conducted in silico by a molecular docking technique in order to direct the design of the new molecules basing on the analysis of ligand-target interactions and the synthetic possibilities. This kind of approach was successfully applied leading to the identification of new potential inhibitors for HDAC enzymes with ciclic (mono and bis amides, paragraph 2.2; conformationally locked calixarenes, paragraph 2.4), and linear (hydroxamic tertiary amines, paragraph 2.3) structures, and isoform selective (paragraph 2.6), and of ligands for microsomal prostaglandin E2 synthase (mPGES)-1 (two series of triazole-based compounds; paragraphs 4.2 and 4.3). For each of this described studied, the good qualitative accordance between the calculated and experimental data has made possible the identifications of new lead compounds, rationalizing in this way the key features to the target inhibition. b) Rationalization of the biological activity of compounds by the study of the drug-receptor interactions. Molecular docking was used for the detailed study of antiinflammatory and antitumoral compounds whose activities are known a priori. In fact, thanks to this procedure, in this thesis several rationalizations of binding modes were reported related to Ugi products derivatives of CHAP 1 (HDAC inhibitors, paragraph 2.5), new and potent inhibitor of NMPRTAse analogs of FK866 and CHS 828 (chapter 3), marine natural products as inhibitors of hsPLA2 (BLQ and CLDA, chapter 5), 4-methylen sterols extracted from Theonella swinhoei as ligands of FXR and PXR (chapter 6), and known compounds as taurolitholic acid and ciprofloxacin (chapter 7), agonists of TGR5. Through the in silico methodology the putative binding modes for the reported molecules was described offering a complete rationalization of docking results, evaluating the influence of the ligand target interactions (e.g. hydrophobic, hydrophilic, electrostatic contacts) on the biological activity. c) Determination of relative configuration of natural products. The complete comprehension of the three dimensional structure of synthetic or isolated molecules is fundamental to design and characterize new platform potentially utilizable in therapy. On this basis, the combined approach between the quantum mechanical (QM) calculation of NMR parameters and NMR spectroscopy was revealed a very useful mean to lead the total synthesis of natural product toward the right isomer avoiding waste of time and resources (paragraph 8.1). Moreover, the stereostructure assignment of marine natural products conicasterol F and its analog thonellasterol I was reported in the paragraph 8.2. by a novel combined approach between the quantitative interproton distance determinations by ROE and quantum mechanical calculations of chemical shifts. (edited by author)X n.s

The Bile Acid Receptor GPBAR-1 (TGR5) Modulates Integrity of Intestinal Barrier and Immune Response to Experimental Colitis

BACKGROUND: GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation. AIMS: To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis. METHODS: Colitis was induced in wild type and GP-BAR1(-/-) mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies. RESULTS: GP-BAR1(-/-) mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn's disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1. CONCLUSIONS: GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn's disease. Ciprofloxacin is a GP-BAR1 ligand

A Chemical-Biological Study Reveals C-9-type Iridoids as Novel Heat Shock Protein 90 (Hsp90) Inhibitors

The potential of heat shock protein 90 (Hsp90) as a therapeutic target for numerous diseases has made the identification and optimization of novel Hsp90 inhibitors an emerging therapeutic strategy. A surface plasmon resonance (SPR) approach was adopted to screen some iridoids for their Hsp90 alpha binding capability. Twenty-four iridoid derivatives, including 13 new natural compounds, were isolated from the leaves of Tabebuia argentea and petioles of Catalpa bignonioides. Their structures were elucidated by NMR, electrospray ionization mass spectrometry, and chemical methods. By means of a panel of chemical and biological approaches, four iridoids were demonstrated to bind Hsp90 alpha. In particular, the dimeric iridoid argenteoside A was shown to efficiently inhibit the chaperone in biochemical and cellular assays. Our results disclose C-9-type iridoids as a novel class of Hsp90 inhibitors

Exploring the Anticancer Potential of Premna resinosa (Hochst.) Leaf Surface Extract: Discovering New Diterpenes as Heat Shock Protein 70 (Hsp70) Binding Agents

Premna, a genus consisting of approximately 200 species, predominantly thrives in tropical and subtropical areas. Many of these species have been utilized in ethnopharmacology for diverse medicinal applications. In Saudi Arabia, Premna resinosa (Hochst.) Schauer (Lamiaceae) grows wildly, and its slightly viscid leaves are attributed to the production of leaf accession. In this study, we aimed to extract the surface accession from fresh leaves using dichloromethane to evaluate the anticancer potential. The plant exudate yielded two previously unknown labdane diterpenes, Premnaresone A and B, in addition to three already described congeners and four known flavonoids. The isolation process was accomplished using a combination of silica gel column chromatography and semi preparative HPLC, the structures of which were identified by NMR and HRESIMS analyses and a comparison with the literature data of associated compounds. Furthermore, we employed a density functional theory (DFT)/NMR approach to suggest the relative configuration of different compounds. Consequently, we investigated the possibility of developing new chaperone inhibitors by subjecting diterpenes 1–5 to a Surface Plasmon Resonance-screening, based on the knowledge that oridonin, a diterpene, interacts with Heat Shock Protein 70 (Hsp70) 1A in cancer cells. Additionally, we studied the anti-proliferative activity of compounds 1–5 on human Jurkat (human T-cell lymphoma) and HeLa (epithelial carcinoma) cell lines, where diterpene 3 exhibited activity in Jurkat cell lines after 48 h, with an IC50 of 15.21 ± 1.0 µM. Molecular docking and dynamic simulations revealed a robust interaction between compound 3 and Hsp70 key residues

Natural iminosugar (+)-lentiginosine inhibits ATPase and chaperone activity of Hsp90

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. The relevance of Hsp90 as a therapeutic target for numerous diseases states has prompted the identification and optimization of novel Hsp90 inhibitors as an emerging therapeutic strategy. We performed a screening aimed to identify novel Hsp90 inhibitors among several natural compounds and we focused on the iminosugar (+)-lentiginosine, a natural amyloglucosidases inhibitor, for its peculiar bioactivity profile. Characterization of Hsp90 inhibition was performed using a panel of chemical and biological approaches, including limited proteolysis, biochemical and cellular assays. Our result suggested that the middle domain of Hsp90, as opposed to its ATP-binding pocket, is a promising binding site for new classes of Hsp90 inhibitors with multitarget anti-cancer potentia

Carnosol Attenuates LPS-Induced Inflammation of Cardiomyoblasts by Inhibiting NF-κB: A Mechanistic in Vitro and in Silico Study

Carnosol possesses several beneficial pharmacological properties. However, its role in lipopolysaccharide (LPS) induced inflammation and cardiomyocyte cell line (H9C2) has never been investigated. Therefore, the effect of carnosol and an NF-kappa B inhibitor BAY 11-7082 was examined, and the underlying role of the NF-kappa B-dependent inflammatory pathway was analyzed as the target enzyme. Cell viability, inflammatory cytokines levels (tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, IL-6, and prostaglandin E-2 (PGE(2))), and related gene expression (TNF-alpha, IL-1 beta, IL-6, and cyclooxygenase-2 (COX-2)) were analyzed by ELISA and real-time PCR. In addition, docking studies analyzed carnosol's molecular interactions and binding modes to NF-kappa B and IKK. We report that LPS caused the reduction of cell viability while enhancing both cytokines protein and mRNA levels (P < 0.001, for all cases). However, the BAY 11-7082 pretreatment of the cells and carnosol increased cell viability and reduced cytokine protein and mRNA levels (P < 0.001 vs. LPS, for all cases). Furthermore, our in silico analyses also supported the modulation of NF-kappa B and IKK by carnosol. This evidence highlights the defensive effects of carnosol against sepsis-induced myocardial dysfunction and, contextually, paved the rationale for the next in vitro and in vivo studies aimed to precisely describe its mechanism(s) of action

Pharmacological and molecular docking assessment of cryptotanshinone as natural-derived analgesic compound

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Discovery That Theonellasterol a Marine Sponge Sterol Is a Highly Selective FXR Antagonist That Protects against Liver Injury in Cholestasis

Background: The farnesoid-x-receptor (FXR) is a bile acid sensor expressed in the liver and gastrointestinal tract. Despite FXR ligands are under investigation for treatment of cholestasis, a biochemical condition occurring in a number of liver diseases for which available therapies are poorly effective, mice harboring a disrupted FXR are protected against liver injury caused by bile acid overload in rodent models of cholestasis. Theonellasterol is a 4-methylene-24-ethylsteroid isolated from the marine sponge Theonella swinhoei. Here, we have characterized the activity of this theonellasterol on FXR-regulated genes and biological functions. Principal Findings: Interrogation of HepG2 cells, a human hepatocyte cell line, by microarray analysis and transactivation assay shows that theonellasterol is a selective FXR antagonist, devoid of any agonistic or antagonistic activity on a number of human nuclear receptors including the vitamin D receptor, PPARs, PXR, LXRs, progesterone, estrogen, glucorticoid and thyroid receptors, among others. Exposure of HepG2 cells to theonellasterol antagonizes the effect of natural and synthetic FXR agonists on FXR-regulated genes, including SHP, OSTa, BSEP and MRP4. A proof-of-concept study carried out to investigate whether FXR antagonism rescues mice from liver injury caused by the ligation of the common bile duct, a model of obstructive cholestasis, demonstrated that theonellasterol attenuates injury caused by bile duct ligation as measured by assessing serum alanine aminostrasferase levels and extent of liver necrosis at histopathology. Analysis of genes involved in bile acid uptake and excretion by hepatocytes revealed that theonellasterol increases the liver expression of MRP4, a basolateral transporter that is negatively regulated by FXR. Administering bile duct ligated mice with an FXR agonist failed to rescue from liver injury and downregulated the expression of MRP4. Conclusions: FXR antagonism in vivo results in a positive modulation of MRP4 expression in the liver and is a feasible strategy to target obstructive cholestasis

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients