Pharmacophore Hybridization To Discover Novel Topoisomerase II Poisons with Promising Antiproliferative Activity

We used a pharmacophore hybridization strategy to combine key structural elements of merbarone and etoposide and generated new type II topoisomerase (topoII) poisons. This first set of hybrid topoII poisons shows promising antiproliferative activity on human cancer cells, endorsing their further exploration for anticancer drug discovery

Design, Synthesis and in vitro Characterization of Novel Topoisomerase II Inhibitors and Poisons

Cancer is a term used for diseases characterized by out of control cell-growth and rapid creation of abnormal cells able to grow beyond their usual borders, invading adjoining parts of the body and spreading to other organs. Several biochemical targets have been recognized to play a fundamental role in its development. In particular, in cancer therapy, DNA topoisomerases have raised much interest as potential anticancer targets. Topoisomerases are ubiquitous enzymes essential for cell survival that regulate the topological state of DNA. They modulate DNA supercoiling and remove DNA knots and tangles. There are two major classes of topoisomerases, type I and type II, that are distinguished by the number of DNA strands that they cleave and the mechanism by which they alter the topological properties of the genetic material. Type II regulate the topological state of DNA in the cell by generating transient double-stranded breaks in the double helix. In order to maintain genomic integrity while the DNA is cleaved, topoisomerase II forms a covalent attachment, known as the “cleavage complex”, which is normally short-lived and is present at low steady-state levels in order to be tolerated by normal cells. Considering the key role of this enzyme for cell surviving, in this thesis project new small entities have been designed and synthesized in order to provide molecules able to block the activity of topoisomerase II by inhibiting the overall catalytic activity of the enzyme or by enhancing the concentration of the cleavage complex. To this aim, four different scaffolds known to act against topoisomerase II were selected: i) 4ꞌ-demethylepipodophyllotoxin, ii) xanthone, iii) thiobarbituric acid, and iv) 4-amino-2-pyrido-bicyclic pyrimidine moieties. All the new derivatives were investigated in biological assays and computational studies to evaluate their inhibitory activity and to identify their mechanism of action against topoisomerase IIα

Novel targets for \u201cold\u201d polyamine analogues: monoamine oxidases and benextramine derivatives

Benextramine is a well kown tetraamine disulfide acting as irreversible \u3b1-adrenergic antagonist and able to hit additional targets involved in neurodegeneration, such as nicotinic and muscarinic receptors and acetylcholinesterase enzyme. The disulfide group of benextramine can react with Cys residues of receptors and enzymes leading to irreversible alterations of their biological activity. On this basis, the potential inhibitory action of ten benextramine analogues was investigated on other well-known targets involved in neurodegenerative diseases, monoamine oxidases (MAOs), whose molecular structures contain various Cys residues. Most of the tested compounds were found able to inactivate both MAO isoforms, with inactivation rate constant values dependent on the polyamine structure as well as on MAO isoforms. Benextramine showed the highest kinact values (0.98 min-1) for MAO A and the \u201cshort\u201d CM367 was found the most potent MAO B inactivator (kinact/KIE =1x105 M-1min-1). CM331, with a cathecol moiety at the two terminal ends, showed the best selectivity vs MAO B. Docking experiments highlight the role of Cys323 (in MAO A) and Cys 172 (in MAO B) as targets of the reactive disulfide moiety of benextramine derivatives, suggesting thatthe sterical hindrace of the bound inhibitor may hinder the entrance of substrate towards the FAD cofactor of the active site. The effectiveness of benextramine in inactivating monoamine oxidases in cells was confirmed using human neuroblastoma cell line SH SY5Y. Optimization studies are now in progress with the aim to modify the structure of the described compounds in order to improve their affinity and selectivity for potential application in neurodegenerative diseases

Benextramine and derivatives as novel human monoamine oxidases inhibitors: an integrated approach

The two human monoamine oxidase isoforms (namely MAO A and MAO B) are enzymes involved in the catabolism of monoamines, including neurotransmitters, and for this reason are well-known and attractive pharmacological targets in neuropsychiatric and neurodegenerative diseases, for which novel pharmacological approaches are necessary. Benextramine is a tetraamine disulfide mainly known as irreversible alpha-adrenergic antagonist, but able to hit additional targets involved in neurodegeneration. As the molecular structures of monoamine oxidases contain nine cysteine residues, the aim of this study was to evaluate benextramine and eleven structurally related polyamine disulfides as potential MAO inhibitors. Most of the compounds were found to induce irreversible inactivation of MAOs with inactivation potency depending on both the polyamine structure and the enzyme isoform. The more effective compounds generally showed preference for MAO B. Structure-activity relationships studies revealed the key role played by the disulfide core of these molecules in the inactivation mechanism. Docking experiments pointed to Cys323, in MAO A, and Cys172, in MAO B, as target of this type of inhibitors thus suggesting that their covalent binding inside the MAO active site sterically impedes the entrance of substrate towards the FAD cofactor. The effectiveness of benextramine in inactivating MAOs was demonstrated in SH-SY5Y neuroblastoma cell line. These results demonstrated for the first time that benextramine and its derivatives can inactivate human MAOs exploiting a mechanism different from that of the classical MAO inhibitors and could be a starting point for the development of pharmacological tools in neurodegenerative diseases

Novel xanthone-polyamine conjugates as catalytic inhibitors of human topoisomerase II\uce\ub1

It has been proposed that xanthone derivatives with anticancer potential act as topoisomerase II inhibitors because they interfere with the ability of the enzyme to bind its ATP cofactor. In order to further characterize xanthone mechanism and generate compounds with potential as anticancer drugs, we synthesized a series of derivatives in which position 3 was substituted with different polyamine chains. As determined by DNA relaxation and decatenation assays, the resulting compounds are potent topoisomerase II alpha inhibitors. Although xanthone derivatives inhibit topoisomerase II alpha-catalyzed ATP hydrolysis, mechanistic studies indicate that they do not act at the ATPase site. Rather, they appear to function by blocking the ability of DNA to stimulate ATP hydrolysis. On the basis of activity, competition, and modeling studies, we propose that xanthones interact with the DNA cleavage/ligation active site of topoisomerase II alpha and inhibit the catalytic activity of the enzyme by interfering with the DNA strand passage step. (C) 2017 Elsevier Ltd. All rights reserved

An optimized polyamine moiety boosts the potency of human type II topoisomerase poisons as quantified by comparative analysis centered on the clinical candidate F14512

Combined computational-experimental analyses explain and quantify the spermine-vectorized F14512's boosted potency as a topoII poison. We found that an optimized polyamine moiety boosts drug binding to the topoII/DNA cleavage complex, rather than to the DNA alone. These results provide new structural bases and key reference data for designing new human topoII poisons