Diego Brancaccio

[2Fe-2S] cluster transfer in iron–sulfur protein biogenesis

Significance Biogenesis of iron–sulfur proteins is a complex process requiring a large number of accessory proteins. In eukaryotes, [2Fe-2S] clusters are synthesized in mitochondria on a scaffold protein. The cluster is then released to monothiol glutaredoxin 5 (GRX5), which was proposed to mediate the transfer of [2Fe-2S] clusters from the scaffold protein to several target proteins, but its precise molecular function remains to be clarified. By investigating the molecular recognition between human GRX5 and its partner proteins (human ISCA1 and ISCA2) and characterizing at the molecular level the cluster transfer process between them, we have shown that a switch between two conformational states of holo GRX5 drives the cluster transfer event, which occurs by a specific protein–protein recognition process. Monothiol glutaredoxins play a crucial role in iron–sulfur (Fe/S) protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of [2Fe-2S] clusters from scaffold proteins to target apo proteins, possibly by acting as cluster transfer proteins. The molecular basis of [2Fe-2S] cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe/S proteins and participates in cellular iron regulation. Here we show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein–protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in solution in the presence of physiological concentrations of glutathione and forming a transient, cluster-mediated protein–protein intermediate with two physiological protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins.

Novel Octreotide Dicarba-analogues with High Affinity and Different Selectivity for Somatostatin Receptors

A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7 and/or 10 were synthesized. Their affinity toward the ssts1-5 was determined. Derivative 4 exhibited a pan-somatostatin activity, except sst4, and derivative 8 exhibited high affinity and selectivity toward sst5. Actually, compound 8 has similar sst5 affinity (IC50 4.9 nM) to SRIF-28 and octreotide. Structure-activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the receptors. The NMR study on the conformations of these compounds in SDS(-d25) micelles solution shows that all these analogues have the pharmacophore beta-turn spanning Xaa7-D-Trp8-Lys9-Yaa10 residues. Notably, the correlation between conformation families and affinity data strongly indicates that the sst5 selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded conformational states.

[4Fe-4S] Cluster Assembly in Mitochondria and Its Impairment by Copper

The cellular toxicity of copper is usually associated with its ability to generate reactive oxygen species. However, recent studies in bacterial organisms showed that copper toxicity is also strictly connected to iron-sulfur cluster proteins and to their assembly processes. Mitochondria of eukaryotic cells contain a labile copper(I) pool localized in the matrix where also the mitochondrial iron-sulfur (Fe/S) cluster assembly machinery resides to mature mitochondrial Fe/S cluster-containing proteins. Misregulation of copper homeostasis might therefore damage mitochondrial Fe/S protein maturation. To describe, from a molecular perspective, the effects of copper(I) toxicity on such a maturation process, we have here investigated the still unknown mechanism of [4Fe-4S] cluster formation conducted by the mitochondrial ISCA1/ISCA2 and GLRX5 proteins, and defined how copper(I) can impair this process. The molecular model here proposed indicates that the copper(I) and Fe/S protein maturation cellular pathways need to be strictly regulated to avoid copper(I) ion from blocking mitochondrial [4Fe-4S] protein maturation.

Discovery of PTPRJ Agonist Peptides That Effectively Inhibit in Vitro Cancer Cell Proliferation and Tube Formation

PTPRJ is a receptor protein tyrosine phosphatase involved in both physiological and oncogenic pathways. We previously reported that its expression is strongly reduced in the majority of explored cancer cell lines and tumor samples; moreover, its restoration blocks in vitro cancer cell proliferation and in vivo tumor formation. By means of a phage display library screening, we recently identified two peptides able to bind and activate PTPRJ, resulting in cell growth inhibition and apoptosis of both cancer and endothelial cells. Here, on a previously discovered PTPRJ agonist peptide, PTPRJ-pep19, we synthesized and assayed a panel of nonapeptide analogues with the aim to identify specific amino acid residues responsible for peptide activity. These second-generation nonapeptides were tested on both cancer and primary endothelial cells (HeLa and HUVEC, respectively); interestingly, one of them (PTPRJ-19.4) was able to both dramatically reduce cell proliferation and effectively trigger apoptosis of both HeLa and HUVECs compared to its first-generation counterpart. Moreover, PTPRJ-pep19.4 significantly inhibited in vitro tube formation on Matrigel. Intriguingly, while ERK1/2 phosphorylation and cell proliferation were both inhibited by PTPRJ-pep19.4 in breast cancer cells (MCF-7 and SKBr3), no effects were observed on primary normal human mammary endothelial cells (HMEC). We further characterized these peptides by molecular modeling and NMR experiments reporting, for the most active peptide, the possibility of self-aggregation states and highlighting new hints of structure-activity relationship. Thus, our results indicate that this nonapeptide might represent a great potential lead for the development of novel targeted anticancer drugs.

New insight into the binding mode of peptide ligands at Urotensin-II receptor: structure-activity relationships study on P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of hU-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized several analogues of P5U and urantide in which the Asp(4) residue in N-terminus position was replaced with coded and noncoded amino acids. The replacement of the Asp(4) residue by Tic led to an analogue, compound 14, more potent as antagonist (pK(B) = 8.94) compared to urantide. Furthermore, a different SAR was observed for the P5U compared to the urantide analogues. NMR and docking studies revealed a different binding mode for the agonist and antagonist ligands which could explain the observed SAR.

Design, synthesis, and cytotoxic evaluation of acyl derivatives of 3-aminonaphtho[2,3-b]thiophene-4,9-dione, a quinone-based system.

A series of 3-acyl derivatives of the dihydronaphtho[2,3-b]thiophen-4,9-dione system were studied with respect to cytotoxicity and topoisomerase II inhibitory activity. These analogues were designed as electron-deficient anthraquinone analogues with potential intercalation ability. Derivatives 3-(diethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)propanamide (11m) and 3-(2-(dimethylamino)ethylamino)-N-(4,9-dioxo-4,9-dihydronaphtho[2,3-b]thiophen-3-yl)propanamide (11p) showed a high efficacy in cell lines that were highly resistant to treatment with doxorubicin, such as MDA-MB435 (melanoma), IGROV (ovarian), and SF-295 (glioblastoma) human cell lines. Both compounds inhibit topoisomerase II mediated relaxation of DNA, while only 11p incites arrest at the S phase in Caco-2 cells, inducing a delay of cell cycle progression and an increase of cell differentiation. The ability of these derivatives to modulate small heat shock proteins and cardiotoxicy effects was also explored. In addition, the DNA-binding properties of these compounds were investigated and discussed.

Synthesis and Pharmacological Evaluation of Some 4-Oxo- quinoline-2-carboxylic Acid Derivatives as Anti-inflammatory and Analgesic Agents

The synthesis and the pharmacological activity of a series of 1‐aroyl derivatives of kynurenic acid methyl ester (4‐oxo‐quinolin‐2‐carboxy methyl (KYNA) esters), structurally related to NSAID indomethacin are described. The derivatives were screened in vivo for anti‐inflammatory and analgesic activities. Most of the compounds exhibited good anti‐inflammatory and analgesic activities. An automatic docking of the synthesized compounds was performed using X‐ray structures of COX‐1 and COX‐2. Docking results are in good accordance with the experimental biological data.

Structure-based lead optimization and biological evaluation of BAX direct activators as novel potential anticancer agents

The first direct activator of BAX, a pro-apoptotic member of the BCL-2 family, has been recently identified. Herein, a structure-based lead optimization turned out into a small series of analogues, where 8 is the most potent compound published so far. 8 was used as pharmacological tool to ascertain, for the first time, the anticancer potential of BAX direct activators and the obtained results would suggest that BAX direct activators are potential future anticancer drugs rather than venoms.

De Novo Designed Library of Linear Helical Peptides: An Exploratory Tool in the Discovery of Protein–Protein Interaction Modulators

Protein-protein interactions (PPIs) have emerged as important targets for pharmaceutical intervention because of their essential role in numerous physiological and pathological processes, but screening efforts using small-molecules have led to very low hit rates. Linear peptides could represent a quick and effective approach to discover initial PPI hits, particularly if they have inherent ability to adopt specific peptide secondary structures. Here, we address this hypothesis through a linear helical peptide library, composed of four sublibraries, which was designed by theoretical predictions of helicity (Agadir software). The 13-mer peptides of this collection fixes either a combination of three aromatic or two aromatic and one aliphatic residues on one face of the helix (Ac-SSEEX(5)ARNX(9)AAX(12)N-NH2), since these are structural features quite common at PPIs interfaces. The 81 designed peptides were conveniently synthesized by parallel solid-phase methodologies, and the tendency of some representative library components to adopt the intended secondary structure was corroborated through CD and NMR experiments. As proof of concept in the search for PPI modulators, the usefulness of this library was verified on the widely studied p53-MDM2 interaction and on the communication between VEGF and its receptor Flt-1, two PPIs for which a hydrophobic α-helix is essential for the interaction. We have demonstrated here that, in both cases, selected peptides from the library, containing the right hydrophobic sequence of the hot-spot in one of the protein partners, are able to interact with the complementary protein. Moreover, we have discover some new, quite potent inhibitors of the VEGF-Flt-1 interaction, just by replacing one of the aromatic residues of the initial F(5)Y(9)Y(12) peptide by W, in agreement with previous results on related antiangiogenic peptides. Finally, the HTS evaluation of the full collection on thermoTRPs has led to a few antagonists of TRPV1 and TRPA1 channels, which open new avenues on the way to innovative modulators of these channels.

New insight into the binding mode of peptides at urotensin‐II receptor by Trp‐constrained analogues of P5U and urantide

Urotensin II (U‐II) is a disulfide bridged peptide hormone identified as the ligand of a G‐protein‐coupled receptor. Human U‐II (H‐Glu‐Thr‐Pro‐Asp‐c[Cys‐Phe‐Trp‐Lys‐Tyr‐Cys]‐Val‐OH) has been described as the most potent vasoconstrictor compound identified to date.