Marilisa Leone

Structure-activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid

Bcl-2 family proteins play a crucial role in tissue homeostasis and apoptosis (programmed cell death). Bid is a proapoptotic member of the Bcl-2 family, promoting cell death when activated by caspase-8. Following an NMR-based approach (structure–activity relationships by interligand NOE) we were able to identify two chemical fragments that bind on the surface of Bid. Covalent linkage of the two fragments led to high-affinity bidentate derivatives. In vitro and in-cell assays demonstrate that the compounds prevent tBid translocation to the mitochondrial membrane and the subsequent release of proapoptotic stimuli and inhibit neuronal apoptosis in the low micromolar range. Therefore, by using a rational chemical–biology approach, we derived antiapoptotic compounds that may have a therapeutic potential for disorders associated with Bid activation, e.g., neurodegenerative diseases, cerebral ischemia, or brain trauma.

Structural Analysis of Siah1-Siah-interacting Protein Interactions and Insights into the Assembly of an E3 Ligase Multiprotein Complex

Siah1 is the central component of a multiprotein E3 ubiquitin ligase complex that targets β-catenin for destruction in response to p53 activation. The E3 complex comprises, in addition to Siah1, Siah-interacting protein (SIP), the adaptor protein Skp1, and the F-box protein Ebi. Here we show that SIP engages Siah1 by means of two elements, both of which are required for mediating β-catenin destruction in cells. An N-terminal dimerization domain of SIP sits across the saddle-shaped upper surface of Siah1, with two extended legs packing against the sides of Siah1 by means of a consensus PXAXVXP motif that is common to a family of Siah-binding proteins. The C-terminal domain of SIP, which binds to Skp1, protrudes from the lower surface of Siah1, and we propose that this surface provides the scaffold for bringing substrate and the E2 enzyme into apposition in the functional complex.

Selective incorporation of 19F-labeled trp side chains for NMR-spectroscopy-based ligand: Protein interaction studies

In the last few years NMR spectroscopy has increasingly been used in pharmaceutical drug discovery and development as a result of its ability to provide structural and dynamic information at atomic resolution. However, the attainment of structural information on protein ± ligand complexes by NMR spectroscopy is limited to small to medium size proteins (MW 30 kDa) and by the lengthy nature of the protein resonance assignment process. Nonetheless it is known that protein binding sites often contain only a small subset of residues–mainly tryptophan (Trp), tyrosine (Tyr), and arginine (Arg). This fact has led to a growing interest in the search for new techniques for the selective isotope labeling of these residues. For example, a new labeling method to selectively incorporate C at position 2 or 4 of Trp side chains has very recently been reported and has been successfully applied to the study of protein ± ligand interactions by use of 2D [C,H] correlation spectra. Herein, we report a simple and effective method to selectively label Trp side chains with F at position 5 or 6. Until now, F-Trp has generally been introduced into proteins by using Escherichia coli strains auxotrophic for Trp and by including the amino acid analogue in the growth medium. This method requires the timeconsuming extra steps of cloning the protein of interest into these strains, which may result in a loss in yield and poor incorporation levels. Instead of employing auxotrophic strains, we find that 3-indoleacrilic acid (IAA) (Figure 1a) can be used as an inhibitor of tryptophan biosynthesis. Addition of IAA (50 mgL ) to minimal media results in the complete inhibition of E. coli cell growth and the inhibition is reversed by L-Trp (50 mgL ) (Figure 1b). This observation is in agreement with the accumulation of indole observed in bacterial culture media Figure 1. a) Chemical structures of 5F-Trp and IAA. b) Inhibitory effect of IAA on E. coli cell growth.

NMR studies of a heterotypic Sam-Sam domain association: the interaction between the lipid phosphatase Ship2 and the EphA2 receptor.

Sterile alpha motif (Sam) domains are protein interaction modules that are implicated in many biological processes mainly via homo- and heterodimerization. It has been recently reported that the lipid phosphatase Ship2 regulates endocytosis of the EphA2 receptor, a process that has been investigated as a possible route to reduce tumor malignancy. A heterotypic Sam-Sam domain interaction is mediating this process. Here, we report NMR and ITC (isothermal titration calorimetry) studies on the Sam domain of Ship2 revealing its three-dimensional structure and its possible mode of interaction with the Sam domain from the EphA2 receptor. These studies have also resulted in the identification of a minimal peptide region of Ship2 that retains binding affinity for the Sam domain of the EphA2 receptor. Hence, this peptide and the detection of key structural elements important for EphA2 receptor endocytosis provide possible ways for the development of novel small molecule antagonists with potential anticancer activity.

The FRB Domain of mTOR: NMR Solution Structure and Inhibitor Design.

The mammalian target of rapamycin (mTOR) is a protein that is intricately involved in signaling pathways controlling cell growth. Rapamycin is a natural product that binds and inhibits mTOR function by interacting with its FKBP-rapamycin-binding (FRB) domain. Here we report on the NMR solution structure of FRB and on further studies aimed at the identification and characterization of novel ligands that target the rapamycin binding pocket. The biological activity of the ligands, and that of rapamycin in the absence of FKBP12, was investigated by assaying the kinase activity of mTOR. While we found that rapamycin binds the FRB domain and inhibits the kinase activity of mTOR even in the absence of FKBP12 (in the low micromolar range), our most potent ligands bind to FRB with similar binding affinity but inhibit the kinase activity of mTOR at much higher concentrations. However, we have also identified one low-affinity compound that is also capable of inhibiting mTOR. Hence, we have identified compounds that can directly mimic rapamycin or can dissociate the FRB binding from the inhibition of the catalytic activity of mTOR. As such, these ligands could be useful in deciphering the complex regulation of mTOR in the cell and in validating the FRB domain as a possible target for the development of novel therapeutic compounds.

NMR Structure of the Single QALGGH Zinc Finger Domain from the Arabidopsis thaliana SUPERMAN Protein

Zinc finger domains of the classical type represent the most abundant DNA binding domains in eukaryotic transcription factors. Plant proteins contain from one to four zinc finger domains, which are characterized by high conservation of the sequence QALGGH, shown to be critical for DNA‐binding activity. The Arabidopsis thaliana SUPERMAN protein, which contains a single QALGGH zinc finger, is necessary for proper spatial development of reproductive floral tissues and has been shown to specifically bind to DNA. Here, we report the synthesis and UV and NMR spectroscopic structural characterization of a 37 amino acid SUPERMAN region complexed to a Zn2+ ion (Zn–SUP37) and present the first high‐resolution structure of a classical zinc finger domain from a plant protein. The NMR structure of the SUPERMAN zinc finger domain consists of a very well‐defined ββα motif, typical of all other Cys2‐His2 zinc fingers structurally characterized. As a consequence, the highly conserved QALGGH sequence is located at the N terminus of the α helix. This region of the domain of animal zinc finger proteins consists of hypervariable residues that are responsible for recognizing the DNA bases. Therefore, we propose a peculiar DNA recognition code for the QALGGH zinc finger domain that includes all or some of the amino acid residues at positions −1, 2, and 3 (numbered relative to the N terminus of the helix) and possibly others at the C‐terminal end of the recognition helix. This study further confirms that the zinc finger domain, though very simple, is an extremely versatile DNA binding motif.

Gold nanoparticles capped by a GC-containing peptide functionalized with an RGD motif for integrin targeting.

Gold nanoparticles were obtained by reduction of a tetrachloroaurate aqueous solution in the presence of a RGD-(GC)(2) peptide as stabilizer. As comparison, the behavior of the (GC)(2) peptide has been studied. The (GC)(2) and RGD-(GC)(2) peptides were prepared ad hoc by Fmoc synthesis. The colloidal systems have been characterized by UV-visible, TGA, ATR-FTIR, mono and bidimensional NMR techniques, confocal and transmission (TEM) microscopy, ζ-potential, and light scattering measurements. The efficient cellular uptake of Au-RGD-(GC)(2) and Au-(GC)(2) stabilized gold nanoparticles into U87 cells (human glioblastoma cells) were investigated by confocal microscopy and compared with the behavior of (GC)(2) capped gold nanoparticles. A quantitative determination of the nanoparticles taken up has been carried out by measuring the pixel brightness of the images, a measure that highlighted the importance of the RGD termination of the peptide. Insight in the cellular uptake mechanism was investigated by TEM microscopy. Various important evidences indicated the selective uptake of RGD-(GC)(2) gold nanoparticles into the nucleus.

G-quadruplex DNA recognition by nucleophosmin: New insights from protein dissection

BACKGROUNDnNucleophosmin (NPM1, B23) is a multifunctional protein that is involved in a variety of fundamental biological processes. NPM1/B23 deregulation is implicated in the pathogenesis of several human malignancies. This protein exerts its functions through the interaction with a multiplicity of biological partners. Very recently it is has been shown that NPM1/B23 specifically recognizes DNA G-quadruplexes through its C-terminal region.nnnMETHODSnThrough a rational dissection approach of protein here we show that the intrinsically unfolded regions of NPM1/B23 significantly contribute to the binding of c-MYC G-quadruplex motif. Interestingly, the analysis of the ability of distinct NPM1/B23 fragments to bind this quadruplex led to the identifications of distinct NPM1/B23-based peptides that individually present a high affinity for this motif.nnnRESULTSnThese results suggest that the tight binding of NPM1/B23 to the G-quadruplex is achieved through the cooperation of both folded and unfolded regions that are individually able to bind it. The dissection of NPM1/B23 also unveils that its H1 helix is intrinsically endowed with an unusual thermal stability.nnnCONCLUSIONSnThese findings have implications for the unfolding mechanism of NPM1/B23, for the G-quadruplex affinity of the different NPM1/B23 isoforms and for the design of peptide-based molecules able to interact with this DNA motif.nnnGENERAL OBSERVATIONnThis study sheds new light in the molecular mechanism of the complex NPM1/G-quadruplex involved in acute myeloid leukemia (AML) disease.

Zinc to cadmium replacement in the A. thaliana SUPERMAN Cys2His2 zinc finger induces structural rearrangements of typical DNA base determinant positions

Among heavy metals, whose toxicity cause a steadily increasing of environmental pollution, cadmium is of special concern due to its relatively high mobility in soils and potential toxicity at low concentrations. Given their ubiquitous role, zinc fingers domains have been proposed as mediators for the toxic and carcinogenic effects exerted by xenobiotic metals. To verify the structural effects of zinc replacement by cadmium in zinc fingers, we have determined the high resolution structure of the single Cys₂ His₂ zinc finger of the Arabidopsis thaliana SUPERMAN protein (SUP37) complexed to the cadmium ion by means of UV-vis and NMR techniques. SUP37 is able to bind Cd(II), though with a dissociation constant higher than that measured for Zn(II). Cd-SUP37 retains the ββα fold but experiences a global structural rearrangement affecting both the relative orientation of the secondary structure elements and the position of side chains involved in DNA recognition: among them Ser17 side chain, which we show to be essential for DNA binding, experiences the largest displacement.

The Sam domain of the lipid phosphatase Ship2 adopts a common model to interact with Arap3-Sam and EphA2-Sam

BackgroundSterile alpha motif (Sam) domains are small protein modules that can be involved in homotypic or heterotypic associations and exhibit different functions. Previous studies have demonstrated that the Sam domain of the lipid phosphatase Ship2 can hetero-dimerize with the Sam domain of the PI3K effector protein Arap3.ResultsHere, we determine the NMR solution structure of Arap3-Sam and implement a multidisciplinary approach consisting of NMR spectroscopy, ITC (Isothermal Titration Calorimetry), mutagenesis and molecular modeling studies to analyze the interaction between Ship2-Sam and Arap3-Sam. This work reveals that Arap3-Sam may associate with Ship2-Sam by adopting a binding mode common to other Sam domains. This binding mode is identical to what we have very recently observed for the association between Ship2-Sam and the Sam domain from the Ephrin A2 receptor.ConclusionOur studies further clarify the structural features that are relevant for Sam-Sam interactions involving Ship2 and give additional hints that could be used for the identification of new molecules able to selectively inhibit Sam-Sam associations.