Inhibitors of ADAM10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect.

Shedding of ADAM10 substrates, like TNFa or CD30, can affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. We have published two new ADAM10 inhibitors, LT4 and MN8 able to prevent such shedding in Hodgkin lymphoma (HL). Since tumor tissue architecture deeply influences the outcome of anti-cancer treatments, we set up a new threedimensional (3D) culture systems to verify whether ADAM10 inhibitors can contribute to, or enhance, the anti-lymphoma effects of the ADC brentuximab-vedotin (BtxVed). In order to recapitulate some aspects of lymphoma structure and architecture, we assembled two 3D culture models: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells) or collagen scaffolds repopulated with LN-MSC and HL cells. In these 3D systems we found that: i) the ADAM10 inhibitors LT4 and MN8 reduce ATP content or glucose consumption, related to cell proliferation, increasing lactate dehydrogenase release as a cell damage hallmark; ii) these events are paralleled by mixed spheroids size reduction and inhibition of CD30 and TNFa shedding; iii) the effects observed can be reproduced in repopulated HL LN-derived matrix or collagen scaffolds; iv) ADAM10 inhibitors enhance the anti-lymphoma effect of the anti-CD30 ADC BtxVed both in conventional cultures and in repopulated scaffolds. Thus, we provide evidence for a direct and combined antilymphoma effect of ADAM10 inhibitors with BtxVed, leading to the improvement of ADC effects; this is documented in 3D models recapitulating features of the LN microenvironment, that can be proposed as a reliable tool for anti-lymphoma drug testing.

Biophysical and in Vivo Studies Identify a New Natural-Based Polyphenol, Counteracting Aß Oligomerization in Vitro and Aß Oligomer-Mediated Memory Impairment and Neuroinflammation in an Acute Mouse Model of Alzheimer's Disease.

In this study natural-based complex polyphenols, obtained through a smart synthetic approach, have been evaluated for their ability to inhibit the formation of Aß42 oligomers, the most toxic species causing synaptic dysfunction, neuroinflammation, and neuronal death leading to the onset and progression of Alzheimer's disease. In vitro neurotoxicity tests on primary hippocampal neurons have been employed to select nontoxic candidates. Solution NMR and molecular docking studies have been performed to clarify the interaction mechanism of Aß42 with the synthesized polyphenol derivatives, and highlight the sterical and chemical requirements important for their antiaggregating activity. NMR results indicated that the selected polyphenolic compounds target Aß42 oligomeric species. Combined NMR and docking studies indicated that the Aß42 central hydrophobic core, namely, the 17-31 region, is the main interaction site. The length of the peptidomimetic scaffold and the presence of a guaiacol moiety were identified as important requirements for the antiaggregating activity. In vivo experiments on an Aß42 oligomer-induced acute mouse model highlighted that the most promising polyphenolic derivative (PP04) inhibits detrimental effects of Aß42 oligomers on memory and glial cell activation. NMR kinetic studies showed that PP04 is endowed with the chemical features of true inhibitors, strongly affecting both the Aß42 nucleation and growth rates, thus representing a promising candidate to be further developed into an effective drug against neurodegenerative diseases of the amyloid type.

Multicomponent Synthesis of Polyphenols and their in vitro Evaluation as Potential ß-Amyloid Aggregation Inhibitors.

While plant polyphenols possess a variety of biological properties, exploration of chemical diversity around them is still problematic. Here, an example of application of the Ugi multicomponent reaction to the combinatorial assembly of artificial, yet "natural-like", polyphenols is presented. The synthesized compounds represent a second-generation library directed to the inhibition of ß-amyloid protein aggregation. Chiral enantiopure compounds, and polyphenol-ß-lactam hybrids have been prepared too. The biochemical assays have highlighted the importance of the key pharmacophores in these compounds. A lead for inhibition of aggregation of truncated protein AßpE3-42 was selected.

Specific ADAM10 inhibitors localize in exosome-like vesicles released by Hodgkin lymphoma and stromal cells and prevent sheddase activity carried to bystander cells.

Shedding of ADAM10 substrates, like TNFα, MICA or CD30, is reported to affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. Soluble forms of these molecules and ADAM10 can be carried and spread in the microenvironment by exosomes released by tumor cells. We reported new ADAM10 inhibitors able to prevent MICA shedding in Hodgkin lymphoma (HL), leading to recognition of HL cells by cytotoxic lymphocytes. In this paper, we show that the mature bioactive form of ADAM10 is released in exosome-like vesicles (ExoV) by HL cells and lymph node mesenchymal stromal cells (MSC). We demonstrate that ADAM10 inhibitors are released in ExoV by MSC or HL cells, endocytosed by bystander cells and localized in the endolysosomal compartment in HL MSC. ExoV released by HL cells can enhance MICA shedding by MSC, while ExoV from MSC induce TNFα or CD30 shedding by HL cells. Of note, ADAM10 sheddase activity carried by ExoV is prevented with the ADAM10 inhibitors LT4 and CAM29, pretreating either the ExoV-producing or the ExoV-receiving cells. In particular, both inhibitors reduce CD30 shedding maintaining the anti-tumor effects of the ADC Brentuximab-Vedotin or the anti-CD30 Iratumumab on HL cells. Thus, spreading of ADAM10 activity due to ExoV can result in the release of cytokines, like TNFα, a lymphoma growth factor, or soluble molecules, like sMICA or sCD30, that potentially interfere with host immune surveillance or immunotherapy. ADAM10 blockers can interfere with this process, allowing the development of anti-lymphoma immune response and/or efficient ADC-based or human antibody-based immunotherapy.

Multicomponent, fragment-based synthesis of polyphenol-containing peptidomimetics and their inhibiting activity on beta-amyloid oligomerization.

A new and short fragment-based approach towards artificial (but "natural-based") complex polyphenols has been developed, exploiting the Ugi multicomponent reaction of phenol-containing simple substrates. The resulting library of compounds has been tested for its capacity to inhibit ß-amyloid protein aggregation, as a possible strategy to develop new chemical entities to be used as prevention or therapy for Alzheimer's disease. Some of the members of this library have demonstrated, in thioflavin assays, a highly promising activity in inhibiting aggregation for two ß-amyloid peptides: Aß1-42 and the truncated AßpE3-42.

Differential toxicity, conformation and morphology of typical initial aggregation states of Aß1-42 and Aßpy3-42 beta-amyloids.

Among the different species of water-soluble ß-peptides (Aß1-42, Aß1-40 and N-terminal truncated Aß-peptides), Aßpy3-42 is thought to play a relevant role in Alzheimer's pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Aßpy3-42, we separated different aggregation states of Aß1-42 and Aßpy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates. Conformational analysis, by circular dichroism showed a prevailing random coil conformation for sa and ma, and typical ß-sheet conformation for la. AFM and TEM show differential structural features between the three aggregates of a given ß-peptide and among the aggregate of the two ß-peptides. The potential toxic effects of the different aggregates were evaluated using human neuroblastoma SH-SY5Y cells in the MTT reduction, in the xCELLigence System, and in the Annexin V binding experiments. In the case of Aß1-42 the most toxic aggregate is la, while in the case of Aßpy3-42 both sa and la are equally toxic. Aß aggregates were found to be internalized in the cells, as estimated by confocal immunofluorescence microscopy, with a higher effect observed for Aßpy3-42, showing a good correlation with the toxic effects. Together these experiments allowed the discrimination of the intermediate states more responsible of oligomer toxicity, providing new insights on the correlation between the aggregation process and the toxicity and confirming the peculiar role in the pathogenesis of Alzheimer disease of Aßpy3-42 peptide.