L. Mugnaini

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.

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.

Antiproliferative and proapoptotic activity of CLM3, a novel multiple tyrosine kinase inhibitor, alone and in combination with SN-38 on endothelial and cancer cells

AIMS To demonstrate the antiproliferative and pro-apoptotic activity of the novel pyrazolopyrimidine derivative multiple tyrosine kinase inhibitor CLM3, alone and in combination with SN-38 (the active metabolite of irinotecan), on endothelial and tumor cells and to show its mechanism of action. METHODS Proliferation and apoptotic assays were performed on microvascular endothelial (HMVEC-d) and lung (A549) and thyroid cancer (8305C, TT) cell lines exposed to CLM3 and to the simultaneous combination with SN38 for 72h. Cell-based phospho-VEGFR-2, phospho-EGFR and phospho-RET inhibition assays were performed and ERK1/2 and Akt phosphorylation were quantified by ELISA kits. Cyclin D1 gene expression was performed with real-time PCR and cyclin D1 intracellular concentrations were measured by ELISA. RESULTS A strong effect on antiproliferative and pro-apoptotic activity was found with the CLM3 on endothelial and cancer cells, synergistically enhanced by SN38. Phospho-VEGFR-2, phospho-EGFR and phospho-RET levels significantly decreased after CLM3 treatments in activated endothelial and cancer cells; ERK1/2 and Akt phosphorylation were significantly inhibited by lower concentrations of the pyrazolopyrimidine drug in endothelial cells if compared to cancer cells. Moreover, CLM3 treatment greatly inhibited the expression of the cyclin D1 gene in endothelial and cancer cells, decreasing the cyclin D1 protein intracellular concentration. CONCLUSIONS The pyrazolopyrimidine derivative CLM3 demonstrated a highly significant and promising antiproliferative and proapoptotic activity, alone and in combination with SN-38, for activated endothelial and cancer cells. These effects are mainly due to its inhibition of phosphorylation of VEGFR-2, EGFR and RET tyrosine kinases and their related signaling pathways.

Synthesis and biological evaluation on human glioblastoma cells of novel pyrazolo3,4-Dpyrimidine adenosine A3 receptor antagonists

Third joint Italian-German Purine Club meeting: “Purinergic receptors: new frontiers for novel therapies” Invited Lectures Abstracts Purinergic signalling: some exciting new directions Geoffrey Burnstock Autonomic Neuroscience Centre, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK Presenting author: g.burnstock@ucl.ac.uk After a brief initial description of some of the important steps in the establishment of purinergic signalling, the talk will focus on some of the exciting new directions in the field, particularly those relating to purinergic pathophysiology and potential therapeutic applications. There will be discussion of the long-term trophic effects of purines and pyrimidines in blood vessel remodelling in restenosis; plasticity of purinergic cotransmission of diseased urinary bladder; hypertensive rats; spermmotility in IVF; purinergic mechanosensory transduction in visceral pain and the role of spinal microglial purinoceptors in neuropathic pain; the potential for P2X7 receptor antagonists in osteoporosis and kidney failure and the growing literature about the roles of purinergic signalling of disorders of the central nervous system; the role of ATP in the treatment of cancer; transient trophic purinergic signalling in embryological development and in stem cell activities; and studies of the evolutionary origins of purinergic receptors will also be discussed. Finally, a novel hypothesis will be presented for the involvement of purinergic signalling in acupuncture. New frontiers for ligands of adenosine and P2Y receptors Kenneth A. Jacobson National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, MD, 20902, USA Presenting author: kajacobs@helix.nih.gov Selective agonists and antagonists are now available for each of the four adenosine receptor (AR) subtypes, making possible research advances leading to the implementation of new therapeutic concepts. Development of selective P2Y receptor ligands is also underway and has been aided by computer modeling and in silico screening. Nevertheless, the field of purine and pyrimidine signaling has particular challenges for medicinal chemists and pharmacologists. The bioavailability and stability of polar, charged, or hydrolyzable compounds are often limited. Effective and selective inhibitors of nucleotidases are lacking. The interplay of chemically related signaling molecules, their metabolism and formation, and time-dependent signaling, in any given cell or tissue, is to be considered as a system rather than as isolated components. The relationship of disease treatment to Gprotein-dependent and independent pathways needs to be established, and new assays are needed. The definition of agonism vs partial agonism vs antagonism may depend on the model used. Large species differences, affecting both affinity and efficacy of new compounds, exist for these receptors. Novel drug delivery or prodrug approaches are needed to overcome side effects because receptors tend to be widespread. In addition, the effects of oligomerization of receptors on the pharmacology are mainly unknown. Thus, subtype selectivity and favorable pharmacokinetics alone are insufficient to propose a new clinical candidate. To address these needs, the predictive value and limitations of homology modeling were analyzed in light of the X-ray structure of the A2AAR in support of structure-based drug design. At the A3AR, we designed and synthesized novel Purinergic Signalling (2010) 6:49–115 DOI 10.1007/s11302-009-9171-1