Matteo Santucci

The Hippo Pathway and YAP/TAZ–TEAD Protein–Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment

The Hippo pathway is an important organ size control signaling network and the major regulatory mechanism of cell-contact inhibition. Yes associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are its targets and terminal effectors: inhibition of the pathway promotes YAP/TAZ translocation to the nucleus, where they interact with transcriptional enhancer associate domain (TEAD) transcription factors and coactivate the expression of target genes, promoting cell proliferation. Defects in the pathway can result in overgrowth phenotypes due to deregulation of stem-cell proliferation and apoptosis; members of the pathway are directly involved in cancer development. The pharmacological regulation of the pathway might be useful in cancer prevention, treatment, and regenerative medicine applications; currently, a few compounds can selectively modulate the pathway. In this review, we present an overview of the Hippo pathway, the sequence and structural analysis of YAP/TAZ, the known pharmacological modulators of the pathway, especially those targeting YAP/TAZ-TEAD interaction.

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.

Label-free fiber optic optrode for the detection of class C β-lactamases expressed by drug resistant bacteria

This paper reports the experimental assessment of an automated optical assay based on label free optical fiber optrodes for the fast detection of class C β-lactamases (AmpC BLs), actually considered as one of the most important sources of resistance to β-lactams antibiotics expressed by resistant bacteria. Reflection-type long period fiber gratings (RT-LPG) have been used as highly sensitive label free optrodes, while a higher affine boronic acid-based ligand was here selected to enhance the overall assay performances compared to those obtained in our first demonstration. In order to prove the feasibility analysis towards a fully automated optical assay, an engineered system was developed to simultaneously manipulate and interrogate multiple fiber optic optrodes in the different phases of the assay. The automated system tested in AmpC solutions at increasing concentrations demonstrated a limit of detection (LOD) of 6 nM, three times better when compared with the results obtained in our previous work. Moreover, the real effectiveness of the proposed optical assay has been also confirmed in complex matrices as the case of lysates of Escherichia coli overexpressing AmpC.

Alanine mutants of the interface residues of human thymidylate synthase decode key features of the binding mode of allosteric anticancer peptides.

Allosteric peptide inhibitors of thymidylate synthase (hTS) bind to the dimer interface and stabilize the inactive form of the protein. Four interface residues were mutated to alanine, and interaction studies were employed to decode the key role of these residues in the peptide molecular recognition. This led to the identification of three crucial interface residues F59, L198, and Y202 that impart activity to the peptide inhibitors and suggest the binding area for further inhibitor design.

Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology

Demonstrating a candidate drug’s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding.

Repurposing of Drugs Targeting YAP-TEAD Functions

Drug repurposing is a fast and consolidated approach for the research of new active compounds bypassing the long streamline of the drug discovery process. Several drugs in clinical practice have been reported for modulating the major Hippo pathway’s terminal effectors, namely YAP (Yes1-associated protein), TAZ (transcriptional co-activator with PDZ-binding motif) and TEAD (transcriptional enhanced associate domains), which are directly involved in the regulation of cell growth and tissue homeostasis. Since this pathway is known to have many cross-talking phenomena with cell signaling pathways, many efforts have been made to understand its importance in oncology. Moreover, this could be relevant to obtain new molecular tools and potential therapeutic assets. In this review, we discuss the main mechanisms of action of the best-known compounds, clinically approved or investigational drugs, able to cross-talk and modulate the Hippo pathway, as an attractive strategy for the discovery of new potential lead compounds.

Structure-based virtual screening for the discovery of novel inhibitors of New Delhi Metallo-β-lactamase-1

Bacterial resistance has become a worldwide concern after the emergence of metallo-β-lactamases (MBLs). They represent one of the major mechanisms of bacterial resistance against beta-lactam antibiotics. Among MBLs, New Delhi metallo-β-lactamase-1 NDM-1, the most prevalent type, is extremely efficient in inactivating nearly all-available antibiotics including last resort carbapenems. No inhibitors for NDM-1 are currently available in therapy, making the spread of NDM-1 producing bacterial strains a serious menace. With this perspective, we performed a structure-based in silico screening of a commercially available library using FLAPdock and identified several, non-β-lactam derivatives as promising candidates active against NDM-1. The binding affinities of the highest scoring hits were measured in vitro revealing, for some of them, low micromolar affinity toward NDM-1. For the best inhibitors, efficacy against resistant bacterial strains overexpressing NDM-1 was validated, confirming their favorable synergistic effect in combination with the carbapenem Meropenem.

Computational and biological profile of boronic acids for the detection of bacterial serine- and metallo-β-lactamases

Abstractβ-Lactamases (BLs) able to hydrolyze β-lactam antibiotics and more importantly the last resort carbapenems, represent a major mechanism of resistance in Gram-negative bacteria showing multi-drug or extensively drug resistant phenotypes. The early detection of BLs responsible of resistant infections is challenging: approaches aiming at the identification of new BLs inhibitors (BLI) can thus serve as the basis for the development of highly needed diagnostic tools. Starting from benzo-[b]-thiophene-2-boronic acid (BZB), a nanomolar inhibitor of AmpC β-lactamase (Ki = 27 nM), we have identified and characterized a set of BZB analogues able to inhibit clinically-relevant β-lactamases, including AmpC, Extended-Spectrum BLs (ESBL), KPC- and OXA-type carbapenemases and metallo-β-lactamases (MBL). A multiligand set of boronic acid (BA) β-lactamase inhibitors was obtained using covalent molecular modeling, synthetic chemistry, enzyme kinetics and antibacterial susceptibility testing. Data confirmed the possibility to discriminate between clinically-relevant β-lactamases on the basis of their inhibition profile. Interestingly, this work also allowed the identification of potent KPC-2 and NDM-1 inhibitors able to potentiate the activity of cefotaxime (CTX) and ceftazidime (CAZ) against resistant clinical isolates (MIC reduction, 32-fold). Our results open the way to the potential use of our set of compounds as a diagnostic tool for the sensitive detection of clinically-relevant β-lactamases.

Chroman-4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1–3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1–TbPTR1 and Leishmania major–LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants.

Conformational Propensity and Biological Studies of Proline Mutated LR Peptides Inhibiting Human Thymidylate Synthase and Ovarian Cancer Cell Growth

LR and [d-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pro n]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation compared with LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.