Stefania Terracciano

Effects of FR235222, a novel HDAC inhibitor, in proliferation and apoptosis of human leukaemia cell lines: Role of Annexin A1

FR235222, a novel histone deacetylase inhibitor (HDACi), at 50nM caused accumulation of acetylated histone H4, inhibition of cell proliferation and G1 cycle arrest accompanied by increase of p21 and down-regulation of cyclin E in human promyelocytic leukaemia U937 cells. The compound was also able to increase the protein and mRNA levels of annexin A1 (ANXA1) without effects on apoptosis. Similar effects were observed in human chronic myelogenous leukaemia K562 cells and human T cell leukaemia Jurkat cells. Cycle arrest and ANXA1 expression, without significant effects on apoptosis, were also induced by different HDACi like suberoylanilide hydroxamic acid (SAHA) and trichostatin-A (TSA). FR235222 at 0.5 microM stimulated apoptosis of all leukaemia cell lines associated to an increased expression of the full-length (37kDa) protein and the appearance of a 33kDa N-terminal cleavage product in both cytosol and membrane. These results suggest that ANXA1 expression may mediate cycle arrest induced by low doses FR235222, whereas apoptosis induced by high doses FR235222 is associated to ANXA1 processing.

9H-purine scaffold reveals induced-fit pocket plasticity of the BRD9 bromodomain

The 2-amine-9H-purine scaffold was identified as a weak bromodomain template and was developed via iterative structure based design into a potent nanomolar ligand for the bromodomain of human BRD9 with small residual micromolar affinity toward the bromodomain of BRD4. Binding of the lead compound 11 to the bromodomain of BRD9 results in an unprecedented rearrangement of residues forming the acetyllysine recognition site, affecting plasticity of the protein in an induced-fit pocket. The compound does not exhibit any cytotoxic effect in HEK293 cells and displaces the BRD9 bromodomain from chromatin in bioluminescence proximity assays without affecting the BRD4/histone complex. The 2-amine-9H-purine scaffold represents a novel template that can be further modified to yield highly potent and selective tool compounds to interrogate the biological role of BRD9 in diverse cellular systems.

Structural basis for the design and synthesis of selective HDAC inhibitors

Histone Deacetylases are considered promising targets for cancer epigenetic therapy, and small molecules able to modulate their biological function have recently gained an increasing interest as potential anticancer agents. In spite of their potential application in cancer therapy, most HDAC inhibitors unselectively bind the several HDAC isoforms, giving rise to different side-effects. In this context, we have traced out the structural elements responsible of selective binding for the therapeutically relevant different HDAC isoforms. The structural analysis has been carried out by molecular modeling, docking in the binding pockets of HDAC1-4 and HDAC6-8, 36 inhibitors presenting a well defined selectivity for the different isoforms. As quick proof of evidence, we have designed, synthesized and experimentally tested three selective ligands. The experimental data suggest that the obtained structural guidelines can be useful tools for the rational design of new potent inhibitors against selected HDAC isoforms.

Molecular modeling studies toward the structural optimization of new cyclopeptide-based HDAC inhibitors modeled on the natural product FR235222

The natural cyclopeptide FR235222 is a potent HDAC inhibitor displaying relevant multiple anticancer effects and is considered an attractive lead compound for the generation of new and more effective antitumor therapeutics. Recently, we have synthesized a small collection of FR235222 simplified analogues which showed interesting biological activities. These results encouraged us to further explore the structural determinants responsible for the activity of this class of HDAC inhibitors in order to gain guidelines for the rational design of new derivatives with putative higher affinity for this target. In the present paper, we report the results obtained, docking these ligands in the binding pocket of HDLP, an HDAC homologue.

Structural Insights for the Optimization of Dihydropyrimidin-2(1H)-one Based mPGES-1 Inhibitors

The recently crystallized structure of microsomal prostaglandin E2 synthase 1 (mPGES-1) in complex with the inhibitor LVJ (PDB code: 4BPM) offered new structural information for the optimization of the previously identified lead compound 1 (IC50 = 4.16 ± 0.47 μM), which contains the privileged dihydropyrimidin-2-one chemical core. Systematic optimization of 1, through accurate structure-based design, provided compound 4 with a 10-fold improved mPGES-1 inhibitory activity (IC50 = 0.41 ± 0.02 μM). Here we highlight the optimal scaffold decoration pattern of 4 and propose a three-dimensional model for the interaction with this complex trimeric membrane protein. The reported computational insights, together with the accessible one-pot synthetic procedure, stimulate for the generation of further potent dihydropyrimidine-based mPGES-1 inhibitors.

Exploration of the dihydropyrimidine scaffold for the development of new potential anti-inflammatory agents blocking prostaglandin E2 synthase-1 enzyme (mPGES-1)

Agents targeting microsomal prostaglandin E2 synthase-1 (mPGES-1) would inhibit only PGE2 production induced by inflammatory stimuli and thus could represent a valuable alternative to non-steroidal anti-inflammatory drugs (NSAIDs) as they should be free from the severe side effects of the classic anti-inflammatory drugs. Although several mPGES-1 inhibitors have been so far identified, none of them is currently in clinical trials, therefore the discovery of new molecular platforms, able to interfere with this interesting target, is urgently required. Here, we report the results of a focused collection of dyhidropyrimidin-2(1H)-one based molecules projected by Virtual Screening computational techniques. The key interactions with the receptor counterpart were introduced as a qualitative filter for the selection of the most promising compounds. The biological data obtained are consistent with the computer-aided suggestions and disclosed two interesting molecules showing in vitro mPGES-1 inhibitory activity in the low μM range.

Synthetic and pharmacological studies on new simplified analogues of the potent actin-targeting Jaspamide

In the recent years, we focused our attention on the cyclodepsipeptide Jaspamide 1, an interesting marine metabolite, possessing a potent inhibitory activity against breast and prostate cancer, as a consequence of its ability to disrupt actin cytoskeleton dynamics. Although its biological profile has been well determined, many mechanistic details are still missing in terms of molecular target identification. For this reason, we decided to synthetically modify the natural metabolite, obtaining small arrays of unnatural variants useful to illuminate the structural requirements essential for the activity. Here, we report the synthesis of seven new Jaspamide analogues 2-8, containing, as the parent compound, a beta-amino acid in the cyclopeptide backbone. Their biological profile is also described.

Design and synthesis of cyclopeptide analogues of the potent histone deacetylase inhibitor FR235222.

Various structurally modified analogues of FR235222 (1), a natural tetrapeptide inhibitor of mammalian histone deacetylases, were prepared in a convergent approach. The design of the compounds was aimed to investigate the effect of structural modifications of the tetrapeptide core involved in enzyme binding in order to overcome some synthetic difficulties connected with the natural product 1. The modifications introduced could also help identify key structural features involved in the mechanism of action of these compounds. The prepared molecules were subjected to in vitro pharmacological tests, and their potency was tested on cultured cells. Two of the components of the array were found to be more potent than the parent compound 1 and almost as efficient as trichostatin A (TSA). These results demonstrate that it is possible to synthesize highly active cyclic tetrapeptides using commercially available amino acids (with the exception of 2‐amino‐8‐oxodecanoic acid, Ahoda). The nature of the residue in the second position of the cyclic peptide and the stereochemistry of the Ahoda tail are important for the inhibitory activity of this class of cyclic tetrapeptide analogues.

Conformationally locked calixarene-based histone deacetylase inhibitors.

Alkyl- and arylamidocalix[4]arene derivatives 1-11 have been designed and theoretically evaluated by docking studies as potential histone deacetylase inhibitors (HDACi). On the basis of the trimodal distribution of the calculated inhibition constants (K(i)), five alkyl- or arylamido derivatives (3, 7, 8, 9, and 11) were synthesized and tested. A qualitative accordance between the experimental results and the theoretical predictions was obtained, confirming that appropriately substituted arylamidocalix[4]arenes are active HDACi.

Synthesis and biological activity of cyclotetrapeptide analogues of the natural HDAC inhibitor FR235222.

In the course of our ongoing efforts to discover new and more effective HDAC inhibitors useful for the development of promising anticancer candidates, we have recently undertaken a molecular modelling study on a small collection of FR235222 analogues, synthesized by us in the frame of a structure-activity relationship investigation, made in order to identify the key structural elements essential for the activity. Progress made in structure elucidation of HDAC active site, together with accurate docking calculations, provided new structural insights useful for a further refinement of the tetrapeptide scaffold which should assure an optimal interaction between the synthetic ligands and the biological target. Following the computer aided suggestions we synthesized six new cyclotetrapeptide analogues of the lead compound (3-8), bearing a carboxylic or an hydroxamic acid functionality as Zn binding moiety. Herein we describe their synthesis and their inhibition activity on different HDAC isoforms.