Concettina La Motta

Sampling protein motion and solvent effect during ligand binding

An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one.

Adenosine Deaminase in the Modulation of Immune System and its Potential as a Novel Target for Treatment of Inflammatory Disorders

The adenosine pathway is a powerful evolutionarily selected mechanism aimed at a fine modulation of inflammatory responses and protection of tissues from injuries. Adenosine exerts its modulatory effects via interaction with G protein-coupled receptors, designated as A(1), A(2A), A(2B) and A(3). In this regard, extracellular adenosine concentrations are critical in determining its ability of regulating several biological functions. The levels achieved by adenosine in close proximity of its receptors are strictly regulated by a variety of dynamic mechanisms, including intracellular and extracellular biosynthesis, transport and metabolism, based on tissue energy status. In this context, the catabolic enzyme adenosine deaminase (ADA) represents a critical checkpoint in the regulation of extracellular adenosine levels and, consequently, in the control of receptor stimulation, thus playing a pivotal role in the modulation of purinergic responses to several pathophysiological events, such as chronic pulmonary diseases, rheumatoid arthritis, inflammatory bowel diseases and sepsis. This article reviews current data on the role played by ADA in the regulation of immune system activity through its modulation of adenosine pathways. Particular attention has been paid to the involvement of ADA in the pathophysiology of relevant inflammatory diseases. In addition, the interest in designing and developing novel ADA inhibitors, as new tools potentially useful for the therapeutic management of inflammatory disorders, has been discussed.

Acetic Acid Aldose Reductase Inhibitors Bearing a Five-Membered Heterocyclic Core with Potent Topical Activity in a Visual Impairment Rat Model

A number of 1,2,4-oxadiazol-5-yl-acetic acids and oxazol-4-yl-acetic acids were synthesized and tested for their ability to inhibit aldose reductase (ALR2). The oxadiazole derivatives, 7c, 7f, 7i, and 8h, 8i, proved to be the most active compounds, exhibiting inhibitory levels in the submicromolar range. In this series, the phenyl group turned out to be the preferred substitution pattern, as its lengthening to a benzyl moiety determined a general reduction of the inhibitory potency. The lead compound, 2-[3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl]acetic acid, 7c, showed an excellent in vivo activity, proving to prevent cataract development in severely galactosemic rats when administered as an eye-drop solution in the precorneal region of the animals. Computational studies on the ALR2 inhibitors were performed to rationalize the structure-activity relationships observed and to provide the basis for further structure-guided design of novel ALR2 inhibitors.

Computational studies of epidermal growth factor receptor: docking reliability, three-dimensional quantitative structure-activity relationship analysis, and virtual screening studies.

An aberrant activity of the epidermal growth factor receptor (EGFR) has been shown to be related to many human cancers, such as breast and liver cancers, thus making EGFR an attractive target for antitumor drug discovery. In this study we evaluated the reliability of various kinds of docking software and procedures to predict the binding disposition of EGFR inhibitors. By application of the best procedure and use of more than 200 compounds, a receptor-based 3D-QSAR model for EGFR inhibition was developed. On the basis of the results obtained, the possibility of developing virtual screening studies was also evaluated. The VS procedure that proved to be the most reliable from a computational point of view was then used to filter the Maybridge database in order to identify new EGFR inhibitors. Enzymatic assays revealed that among the eight top-scoring compounds, seven proved to inhibit EGFR activity at a concentration of 100 microM, two of them exhibiting IC(50) values in the low micromolar range and one in the nanomolar range. These results demonstrate the validity of the methodologies followed. Furthermore, the two low micromolar compounds may be considered as very interesting leads for the development of new EGFR inhibitors.

Indole amide derivatives: synthesis, structure–activity relationships and molecular modelling studies of a new series of histamine H1-receptor antagonists

A number of indole amide derivatives bearing a basic side chain, in which the indole ring replaces the isoster benzimidazole nucleus typical of some well-known antihistamines, were prepared and tested for their H1-antihistaminic activity. The 1-benzyl-3- indolecarboxamides 32-42 showed antihistaminic (H1) activity (pA2 6-8); the 3-indolylglyoxylylamides 7-16 and the 2-indolecarboxamides 48-56 showed little or no activity. Insertion of the basic side chain of the active 3-indolecarboxamide derivatives into a piperazine ring (compounds 57-59) led to a dramatic loss of activity. All the active compounds proved to be competitive antagonists, since the values of the regression slope were not statistically different from 1. The most active compounds, 32, 33, 38-41, were also tested both in vitro for their anticholinergic activity and in vivo for their ability to antagonize histamine-induced cutaneous vascular permeability in rats. The biological results and the structure-activity relationships of the novel compounds are discussed in the light of molecular modelling studies, taking the molecule of astemizole as a model, and referring to proposed H1-receptor pharmacophore models.

The Blockade of Adenosine Deaminase Ameliorates Chronic Experimental Colitis through the Recruitment of Adenosine A2A and A3 Receptors

Adenosine modulates immune/inflammatory reactions. This study investigates the expression of adenosine deaminase in the inflamed colon, the effects of adenosine deaminase inhibitors on established colitis, and the recruitment of adenosine receptors by endogenous adenosine after adenosine deaminase blockade. Adenosine deaminase expression was determined by Western blot. The effects of 4-amino-2-(2-hydroxy-1-decyl)pyrazole[3,4-d]pyrimidine (APP; a novel adenosine deaminase inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; a reference adenosine deaminase inhibitor), dexamethasone, and selective adenosine receptor antagonists were tested in rats with 2,4-dinitrobenzenesulfonic acid-induced colitis. Systemic (food intake, body and spleen weight) and colonic [macroscopic/microscopic damage, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and malondialdehyde (MDA)] inflammatory parameters were assessed. Test drugs were administered intraperitoneally for 6 days, starting at day 5 from colitis induction. Adenosine deaminase was detected in normal colon, and its expression was increased in inflamed tissues. Colitis was associated with decreased food intake and body weight, augmented spleen weight, and increased levels of colonic TNF-α, IL-6, and MDA. APP or EHNA, but not dexamethasone, improved food intake and body weight. APP, EHNA, and dexamethasone counteracted the increments of spleen weight, ameliorated macroscopic and microscopic indexes of inflammation, and reduced TNF-α, IL-6, and MDA levels. The beneficial effects of APP and EHNA on inflammatory parameters were prevented by the pharmacological blockade of A2A or A3 receptors, but not A1 or A2B. The present results show that: 1) bowel inflammation is associated with an enhanced adenosine deaminase expression; and 2) the anti-inflammatory actions of adenosine deaminase inhibitors against chronic established colitis depend on the sparing of endogenous adenosine, leading to enhanced A2A and A3 receptor activation.

Exploiting the Pyrazolo[3,4-d]pyrimidin-4-one Ring System as a Useful Template To Obtain Potent Adenosine Deaminase Inhibitors

A number of pyrazolo[3,4-d]pyrimidin-4-ones bearing either alkyl or arylalkyl substituents in position 2 of the nucleus were synthesized and tested for their ability to inhibit adenosine deaminase (ADA) from bovine spleen. The 2-arylalkyl derivatives exhibited excellent inhibitory activity, showing Ki values in the nanomolar/subnanomolar range. The most active compound, 1-(4-((4-oxo-4,5-dihydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea, 14d, was tested in rats with colitis induced by 2,4-dinitrobenzenesulfonic acid to assess its efficacy to attenuate bowel inflammation. The treatment with 14d induced a significant amelioration of both systemic and intestinal inflammatory alterations in animals with experimental colitis. Docking simulations of the synthesized compounds into the ADA catalytic site were also performed to rationalize the structure-activity relationships observed and to highlight the key pharmacophoric elements of these products, thus prospectively guiding the design of novel ADA inhibitors.

CLM3, a Multitarget Tyrosine Kinase Inhibitor With Antiangiogenic Properties, Is Active Against Primary Anaplastic Thyroid Cancer In Vitro and In Vivo

CONTEXT AND OBJECTIVE We have studied the antitumor activity of a pyrazolo[3,4-d]pyrimidine compound (CLM3) proposed for a multiple signal transduction inhibition [including the RET tyrosine kinase, epidermal growth factor receptor, and vascular endothelial growth factor (VEGF) receptor and with antiangiogenic activity] in primary anaplastic thyroid cancer (ATC) cells, in the human cell line 8305C (undifferentiated thyroid cancer), and in an ATC-cell line (AF). DESIGN AND MAIN OUTCOME MEASURES CLM3 was tested in primary ATC cells at the concentrations of 5, 10, 30, and 50 μM; in 8305C cells, in AF cells, at 1, 5, 10, 30, 50, or 100 μM; and in AF cells in CD nu/nu mice. RESULTS CLM3 significantly inhibited the proliferation of 8305C and AF cells, also inducing apoptosis. A significant reduction of proliferation with CLM3 in ATC cells (P < .01, ANOVA) was shown. CLM3 increased the percentage of apoptotic ATC cells dose dependently (P < .001, ANOVA) and inhibited migration (P < .01) and invasion (P < .001). The AF cell line was injected sc in CD nu/nu mice, and tumor masses became detectable 15 days later. CLM3 (50 mg/kg per die) significantly inhibited tumor growth (starting 16 d after the beginning of treatment). CLM3 significantly decreased the VEGF-A expression and microvessel density in AF tumor tissues. Furthermore, CLM3 inhibited epidermal growth factor receptor, AKT, and ERK1/2 phosphorylation and down-regulated cyclin D1 in 8305C and AF cells. CONCLUSIONS The antitumor and antiangiogenic activity of a pyrazolo[3,4-d]pyrimidine compound (CLM3) is very promising in anaplastic thyroid cancer, opening the way to a future clinical evaluation.

FOXD1–ALDH1A3 Signaling Is a Determinant for the Self-Renewal and Tumorigenicity of Mesenchymal Glioma Stem Cells

Glioma stem-like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1-ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1-ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219-30. ©2016 AACR.

Pursuing Aldose Reductase Inhibitors through in Situ Cross-Docking and Similarity-Based Virtual Screening

Aldose reductase (ALR2) is a critical enzyme in the development of the major complications of diabetes mellitus. Herein, new molecular entities active against ALR2 were discovered through an integrated receptor- and ligand-based virtual screening campaign. Twelve candidates were found to inhibit this enzyme in the micromolar range including two ligands having an IC(50) below 3 muM. Six new compounds, structurally unrelated to the known ARIs, have been identified, opening up opportunity for lead optimization.