Filippo Genovese

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.

Optimization of Peptides That Target Human Thymidylate Synthase to Inhibit Ovarian Cancer Cell Growth

Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The d-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.

Molecular and proteomic insight into Notch1 characterization in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) ranks fifth in frequency worldwide amongst all human cancers causing one million deaths annually. Despite many promising treatment options, long-term prognosis remains dismal for the majority of patients who develop recurrence or present with advanced disease. Notch signaling is an evolutionarily conserved pathway crucial for the development and homeostasis of many organs including liver. Herein we showed that aberrant Notch1 is linked to HCC development, tumor recurrence and invasion, which might be mediated, at least in part, through the Notch1-E-Cadherin pathway. Collectively, these findings suggest that targeting Notch1 has important therapeutic value in hepatocellular carcinoma. In this regard, comparative analysis of the secretome of HepG2 and HepG2 Notch1 depleted cells identified novel secreted proteins related to Notch1 expression. Soluble E-Cadherin (sE-Cad) and Thrombospondin-1 (Thbs1) were further validated in human serum as potential biomarkers to predict response to Notch1 inhibitors for a tailored individualized therapy.

Conveying a newly designed hydrophilic anti-human thymidylate synthase peptide to cisplatin resistant cancer cells: are pH-sensitive liposomes more effective than conventional ones?

Abstract Context: LR-peptide, a novel hydrophilic peptide synthetized and characterized in previous work, is able to reduce the multi-drug resistance response in cisplatin (cDPP) resistant cancer cells by inhibiting human thymidylate synthase (hTS) overexpressed in several tumors, including ovarian and colon-rectal cancers, but it is unable to enter the cells spontaneously. Objective: The aim of this work was to design and characterize liposomal vesicles as drug delivery systems for the LR peptide, evaluating the possible benefits of the pH-responsive feature in improving intracellular delivery. Materials and methods: For this purpose, conventional and pH-sensitive liposomes were formulated, compared regarding their physical-chemical properties (size, PDI, morphology, in vitro stability and drug release) and studied for in vitro cytotoxicity against a cDDP-resistant cancer cells. Results and discussion: Results indicated that LR peptide was successfully encapsulated in both liposomal formulations but at short incubation time only LR loaded pH-sensitive liposomes showed cell inhibition activity while for long incubation time the two kinds of liposomes demonstrated the same efficacy. Conclusions: Data provide evidence that acidic pH-triggered liposomal delivery is able to significantly reduce the time required by the systems to deliver the drug to the cells without inducing an enhancement of the efficacy of the drug.

Qualitative and semiquantitative analysis of the protein coronas associated to different functionalized nanoparticles

AIM The investigation on protein coronas (PCs) adsorbed onto nanoparticle (NP) surface is representing an open issue due to difficulties in detection and clear isolation of the adsorbed proteins. In this study, we investigated protocols able to isolate the compositions of PCs of three polymeric NPs. MATERIALS & METHODS Unfunctionalized NPs and two functionalized NPs were considered as proof-of-concept for the qualitative and semiquantitative analysis of both the corona levels (stably or weakly adsorbed coronas [SC/WC]) of these different nanocarriers. RESULTS The protocols applied were able to discriminate between the SC and WC. In particular, experimental results indicated that stably adsorbed coronas are prevalently composed by ApoE, while WC by albumin in all the NPs. Otherwise, some differences in WC could be correlated with surface functionalization. CONCLUSION This experimental approach allows characterizing the whole PCs, proposing a protocol for isolation of different types of proteins composing PCs.

Proteomic and Bioinformatic Studies for the Characterization of Response to Pemetrexed in Platinum Drug Resistant Ovarian Cancer

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.