Exploiting the Features of Short Peptides to Recognize Specific Cell Surface Markers.

Antibodies are the macromolecules of choice to ensure specific recognition of biomarkers in biological assays. However, they present a range of shortfalls including a relatively high production cost and limited tissue penetration. Peptides are relatively small molecules able to reproduce sequences of highly specific paratopes and, although they have less biospecificity than antibodies, they offer advantages like ease of synthesis, modifications of their amino acid sequences and tagging with fluorophores and other molecules required for detection. This work presents a strategy to design peptide sequences able to recognize the CD44 hyaluronic acid receptor present in the plasmalemma of a range of cells including human bone marrow stromal mesenchymal cells. The protocol of identification of the optimal amino acid sequence was based on the combination of rational design and in silico methodologies. This protocol led to the identification of two peptide sequences which were synthesized and tested on human bone marrow mesenchymal stromal cells (hBM-MSCs) for their ability to ensure specific binding to the CD44 receptor. Of the two peptides, one binds CD44 with sensitivity and selectivity, thus proving its potential to be used as a suitable alternative to this antibody in conventional immunostaining. In the context of regenerative medicine, the availability of this peptide could be harnessed to functionalize tissue engineering scaffolds to anchor stem cells as well as to be integrated into systems such as cell sorters to efficiently isolate MSCs from biological samples including various cell subpopulations. The data here reported can represent a model for developing peptide sequences able to recognize hBM-MSCs and other types of cells and for their integration in a range of biomedical applications.

Salicylic Acid Release from Syndiotactic Polystyrene Staple Fibers.

Films and fibers of syndiotactic polystyrene (sPS), being amorphous or exhibiting nanoporous crystalline (NC) or dense crystalline phases, were loaded with salicylic acid (SA), a relevant non-volatile antimicrobial molecule. In the first section of the paper, sPS/SA co-crystalline (CC) δ form is characterized, mainly by wide angle X-ray diffraction (WAXD) patterns and polarized Fourier transform infrared (FTIR) spectra. The formation of sPS/SA δ CC phases allows the preparation of sPS fibers even with a high content of the antibacterial guest, which is also retained after repeated washing procedures at 65 °C. A preparation procedure starting from amorphous fibers is particularly appropriate because involves a direct formation of the CC δ form and a simultaneous axial orientation. The possibility of tuning drug amount and release kinetics, by simply selecting suitable crystalline phases of a commercially available polymer, makes sPS fibers possibly useful for many applications. In particular, fibers with δ CC forms, which retain SA molecules in their crystalline phases, could be useful for antimicrobial textiles and fabrics. Fibers with the dense γ form which easily release SA molecules, because they are only included in their amorphous phases, could be used for promising SA-based preparations for antibacterial purposes in food processing and preservation and public health. Finally, using a cell-based assay system and antibacterial tests, we investigated the cellular activity, toxicity and antimicrobial properties of amorphous, δ CC forms and dense γ form of sPS fibers loaded with different contents of SA.

Structural analysis of a simplified model reproducing SARS-CoV-2 S RBD/ACE2 binding site.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an RNA virus identified as the cause of the coronavirus outbreak in December 2019 (COVID-19). Like all the RNA viruses, SARS-CoV-2 constantly evolves through mutations in its genome, accumulating 1-2 nucleotide changes every month, giving the virus a selective advantage through enhanced transmissibility, greater pathogenicity, and the possibility of circumventing immunity previously acquired by an individual either by natural infection or by vaccination. Several SARS-CoV-2 variants of concern (VoC) have been identified, among which we find Alpha (Lineage B.1.1.7), Beta (Lineage B.1.351), and Gamma (Lineage P.1) variants. Most of the mutations occur in the spike (S) protein, a surface glycoprotein that plays a crucial role in viral infection; the S protein binds the host cell receptor, the angiotensin-converting enzyme of type 2 (ACE2) via the receptor binding domain (RBD) and catalyzes the fusion of the viral membrane with the host cell. In this work, we present the development of a simplified system that would afford to study the change in the SARS-CoV-2 S RBD/ACE2 binding related to the frequent mutations. In particular, we synthesized and studied the structure of short amino acid sequences, mimicking the two proteins' critical portions. Variations in the residues were easily managed through the one-point alteration of the sequences. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies provide insights into ACE2 and SARS-CoV-2 S RBD structure with its related three variants (Alpha, Beta, and Gamma). Spectroscopy data supported by molecular dynamics lead to the description of an ACE2/RBD binding model in which the effect of a single amino acid mutation in changing the binding of S protein to the ACE2 receptor is predictable.

Towards an Improvement of Anticancer Activity of Benzyl Adenosine Analogs.

N6-Isopentenyladenosine (i6A) is a naturally occurring modified nucleoside displaying in vitro and in vivo antiproliferative and pro-apoptotic properties. In our previous studies, including an in silico inverse virtual screening, NMR experiments and in vitro enzymatic assays, we demonstrated that i6A targeted farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway and prenylation of downstream proteins, which are aberrant in several cancers. Following our interest in the anticancer effects of FPPS inhibition, we developed a panel of i6A derivatives bearing bulky aromatic moieties in the N6 position of adenosine. With the aim of clarifying molecular action of N6-benzyladenosine analogs on the FPPS enzyme inhibition and cellular toxicity and proliferation, herein we report the evaluation of the N6-benzyladenosine derivatives' (compounds 2a-m) effects on cell viability and proliferation on HCT116, DLD-1 (human) and MC38 (murine) colorectal cancer cells (CRC). We found that compounds 2, 2a and 2c showed a persistent antiproliferative effect on human CRC lines and compound 2f exerted a significant effect in impairing the prenylation of RAS and Rap-1A proteins, confirming that the antitumor activity of 2f was related to the ability to inhibit FPPS activity.

Ganoderma lucidum Ethanol Extracts Enhance Re-Epithelialization and Prevent Keratinocytes from Free-Radical Injury.

Ganoderma lucidum or Reishi is recognized as the most potent adaptogen present in nature, and its anti-inflammatory, antioxidant, immunomodulatory and anticancer activities are well known. Moreover, lately, there has been an increasing interest from pharmaceutical companies in antiaging G. lucidum-extract-based formulations. Nevertheless, the pharmacological mechanisms of such adaptogenic and regenerative actions remain unclear. The present investigation aimed to explore its molecular and cellular effects in vitro in epidermal keratinocyte cultures by applying liquid chromatography coupled to ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) for analysis of ethanol extracts using ganoderic acid-A as a reference compound. The G. lucidum extract showed a keratinocyte proliferation induction accompanied by an increase of cyclic kinase protein expressions, such as CDK2 and CDK6. Furthermore, a noteworthy migration rate increase and activation of tissue remodelling factors, such as matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), were observed. Finally, the extract showed an antioxidant effect, protecting from H2O2-induced cytotoxicity; preventing activation of AKT (protein kinase B), ERK (extracellular signal-regulated kinase), p53 and p21; and reducing the number of apoptotic cells. Our study paves the path for elucidating pharmacological properties of G. lucidum and its potential development as cosmeceutical skin products, providing the first evidence of its capability to accelerate the healing processes enhancing re-epithelialization and to protect cells from free-radical action.

NMR for screening and a biochemical assay: Identification of new FPPS inhibitors exerting anticancer activity.

Farnesyl pyrophosphate synthase (FPPS) is a crucial enzyme for the synthesis of isoprenoids and the key target of nitrogen-containing bisphosphonates (N-BPs). N-BPs are potent and selective FPPS inhibitors that are used in the treatment of bone-related diseases, but have poor pharmacokinetic properties. Given the key role played by FPPS in many cancer-related pathways and the pharmacokinetic limits of N-BPs, hundreds of molecules have been screened to identify new FPPS inhibitors characterized by improved drug-like properties that are useful for broader therapeutic applications in solid, non-skeletal tumours. We have previously shown that N6-isopentenyladenosine (i6A) and its related compound N6-benzyladenosine (2) exert anti-glioma activity by interfering with the mevalonate pathway and inhibiting FPPS. Here, we report the design and synthesis of a panel of N6-benzyladenosine derivatives (compounds 2a-m) incorporating different chemical moieties on the benzyl ring. Compounds 2a-m show in vitro antiproliferative activity in U87MG glioma cells and, analogous to the bisphosphonate FPPS inhibitors, exhibit immunogenic properties in ex vivo γδ T cells from stimulated peripheral blood mononuclear cells (PBMCs). Using saturation transfer difference (STD) and quantitative 1H nuclear magnetic resonance (NMR) experiments, we found that 2f, the N6-benzyladenosine analogue that includes a tertbutyl moiety in the para position of the benzyl ring, is endowed with increased FPPS binding and inhibition compared to the parent compounds i6A and 2. N6-benzyladenosine derivatives, characterized by structural features that are significantly different from those of N-BPs, have been confirmed to be promising chemical scaffolds for the development of non N-BP FPPS inhibitors, exerting combined cytotoxic and immunostimulatory activities.