A long pentraxin-3-derived pentapeptide for the therapy of FGF8b-driven steroid hormone-regulated cancers

Fibroblast growth factor-8b (FGF8b) affects the epithelial/stromal compartments of steroid hormone-regulated tumors by exerting an autocrine activity on cancer cells and a paracrine pro-angiogenic function, thus contributing to tumor progression. The FGF8b/FGF receptor (FGFR) system may therefore represent a target for the treatment of steroid hormone-regulated tumors. The soluble pattern recognition receptor long pentraxin-3 (PTX3) binds various FGFs, including FGF2 and FGF8b, thus inhibiting the angiogenic and tumorigenic activity of androgen-regulated tumor cells. Nevertheless, the complex/proteinaceous structure of PTX3 hampers its pharmacological exploitation. In this context, the acetylated pentapeptide Ac-ARPCA-NH2 (ARPCA), corresponding to the N-terminal amino acid sequence PTX3(100-104), was identified as a minimal FGF2-binding peptide able to antagonize the biological activity of FGF2. Here, we demonstrate that ARPCA binds FGF8b and inhibits its capacity to form FGFR1-mediated ternary complexes with heparan sulphate proteoglycans. As a FGF8b antagonist, ARPCA inhibits FGFR1 activation and signalling in endothelial cells, hampering the angiogenic activity exerted in vitro and in vivo by FGF8b. Also, ARPCA suppresses the angiogenic and tumorigenic potential of prototypic androgen/FGF8b-dependent Shionogi 115 mammary carcinoma cells and of androgen/FGF8b/FGF2-dependent TRAMP-C2 prostate cancer cells. In conclusion, ARPCA represents a novel FGF8b antagonist with translational implications for the therapy of steroid hormone-regulated tumor

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

Inhibition of FGFR Signaling by Targeting FGF/FGFR Extracellular Interactions: Towards the Comprehension of the Molecular Mechanism through NMR Approaches

NMR-based approaches play a pivotal role in providing insight into molecular recognition mechanisms, affording the required atomic-level description and enabling the identification of promising inhibitors of protein–protein interactions. The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signaling pathway drives several pathologies, including cancer development, metastasis formation, resistance to therapy, angiogenesis-driven pathologies, vascular diseases, and viral infections. Most FGFR inhibitors targeting the intracellular ATP binding pocket of FGFR have adverse effects, such as limited specificity and relevant toxicity. A viable alternative is represented by targeting the FGF/FGFR extracellular interactions. We previously identified a few small-molecule inhibitors acting extracellularly, targeting FGFR or FGF. We have now built a small library of natural and synthetic molecules that potentially act as inhibitors of FGF2/FGFR interactions to improve our understanding of the molecular mechanisms of inhibitory activity. Here, we provide a comparative analysis of the interaction mode of small molecules with the FGF2/FGFR complex and the single protein domains. DOSY and residue-level NMR analysis afforded insights into the capability of the potential inhibitors to destabilize complex formation, highlighting different mechanisms of inhibition of FGF2-induced cell proliferation

Investigating the Dynamic Aspects of Drug-Protein Recognition through a Combination of MD and NMR Analyses: Implications for the Development of Protein-Protein Interaction Inhibitors

<div><p>In this paper, we investigate the dynamic aspects of the molecular recognition between a small molecule ligand and a flat, exposed protein surface, representing a typical target in the development of protein-protein interaction inhibitors. Specifically, we analyze the complex between the protein Fibroblast Growth Factor 2 (FGF2) and a recently discovered small molecule inhibitor, labeled <b>sm27</b> for which the binding site and the residues mainly involved in small molecule recognition have been previously characterized. We have approached this problem using microsecond MD simulations and NMR-based characterizations of the dynamics of the apo and holo states of the system. Using direct combination and cross-validation of the results of the two techniques, we select the set of conformational states that best recapitulate the principal dynamic and structural properties of the complex. We then use this information to generate a multi-structure representation of the <b>sm27</b>-FGF2 interaction. We propose this kind of representation and approach as a useful tool in particular for the characterization of systems where the mutual dynamic influence between the interacting partners is expected to play an important role. The results presented can also be used to generate new rules for the rational expansion of the chemical diversity space of FGF2 inhibitors.</p></div

3D representation of the sm27/FGF2 complex.

<p>NMR-constraints derived structure of the complex between <b>sm27</b> and FGF2, used as a starting structure for all simulations and analyses. Red surface represents protein region involved in ligand recognition.</p

Comparison between the experimental and calculated local dynamics.

<p>a) Comparison between local dynamics for the apo system, calculated over the 100 ns trajectory interval yielding the best match (800–900 ns). b) Comparison between local dynamics for the holo system, calculated over the 100 ns trajectory interval yielding the best match (200–300 ns). The insets show a magnification of the graphs to improve clarity.</p

Distances between FGF2 binding site aminoacids and sm27.

<p>Histogram of the distance between the COM of the ligand calculated over the 200–300 ns interval.</p

Representative structures of the ensemble of the sm27/FGF2 complex that recapitulates calculated and experimental data.

<p>From structural representations of two different orientations of the <b>sm27</b>-FGF2 complex (a and c) it is immediately evident that the ligand can occupy a diverse range of conformations/configurations and the protein dynamically adapts to them. Sm27 molecule is shown in magenta sticks, while protein side-chains involved in interaction (Y33, K35, N36, R53, K128, R129, K134, Q143, K144, A145) are shown as blue lines. In b and d the same views of sm27-FGF2 complex are offered and the position of heparin (yellow sticks) and FGFR1 (dark olive and orange cartoons), deduced from X-ray structure (PDB ID: 1FQ9), are shown.</p