May 17th, 2021 by Katja Herzog
Abstract:
Combinatorial discovery of synthetic biohybrid ligands for RNA-hairpins and for the SARS-CoV-2- spike protein
Topic:
The de novo discovery of ligands for challenging and novel drug targets often requires the cumbersome screening of individual compounds from large libraries. Here we present a fully chemistry based affinity selection – mass spectrometry (AS-MS) platform: within days synthetic polyamide compound libraries with > 100 million members can be produced, screened against targets of interest and originate hits with nanomolar affinity for their targets. We use AS-MS for the rapid discovery of synthetic high-affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The peptides display excellent selectivity for RBD over human serum proteins and can detect picomolar RBD concentrations in a biological matrix. We further expanded the AS-MS platform for the discovery of compounds targeting oncogenic pre-miRNA hairpins.
In nature nucleic acids are often controlled by large supramolecular protein/oligonucleotide complexes as in the case of ribosomal protein synthesis. Rather than forming large complexes to coordinate the role of different biopolymers, we dovetail protein amino acids and nucleobases into a single low molecular weight precision polyamide polymer. We established efficient chemical synthesis and de novo sequencing procedures and prepared combinatorial libraries with up to 100 million biohybrid molecules. This biohybrid material has a higher bulk affinity to oligonucleotides than peptides composed exclusively of canonical amino acids. Using affinity selection mass spectrometry, we discovered variants with a high-affinity for pre-microRNA hairpins. Our platform points toward the development of high throughput discovery of sequence defined polymers with designer properties, such as oligonucleotide binding.
May 17th, 2021 by Katja Herzog
Chiara Pizzolitto received master’s degree in nanobiotechnology in 2019. Currently she is a second year PhD student at University of Trieste.
Abstract:
Dual cross-link hydrogels with tunable viscoelasticity control stem cell differentiation
Topic:
Mechanotransduction recapitulates the conversion of external mechanical information into intracellular biochemical response. To this, it was recently recognized that stem cell fate can be markedly influenced by surrounding extracellular matrix (ECM) mechanics. Most of our knowledge in cell mechanobiology has been built using purely elastic materials as model of ECM. However, native tissues do not exhibit purely elastic response but manifest viscoelasticity, that is a time- and frequencydependence to loading. In fact, recent works have proved that stress-relaxation or plasticity, and more broadly, viscosity-driven processes can be considered as potent modulators of stem cell behavior. To recapitulate native tissue mechanics and provide a more realistic ECM model, here we present unprecedented viscoelastic substrates showing adaptable viscoelasticity. In particular, I will present an innovative dual cross-link gel system based on a chitosan derivative, shortly named CTL, assembled via both temporary and permanent cross-linkers.[1] Concepts related to macromolecular chemistry and physics will be disclosed. Of note, temporary junctions are exploited to finely tune material viscoelasticity. I will show how resulting hydrogels result optimal substrates for cell anchoring upon coating with ECM proteins. Though endowed with similar elasticity, a viscositycell function relationship will be unveiled, identifying high viscosity hydrogels as superior materials in fostering stem cell differentiation toward a bone-like phenotype with respect to more elastic counterparts. Taken together, these results lay the groundwork for additional investigations on the role played by substrate viscoelasticity in directing cell-fate decisions.
May 17th, 2021 by Katja Herzog
Francesca Cardano is currently completing her postdoc focused on synthesis and investigations of novel chiral sensors, dyes and fluorophores for chembio applications.
Abstract:
Squaraine NIR dyes: a structure to function study for novel bilayer membrane probes
Topic:
In the last decade, Near infra-red (NIR) fluorophores have been largely tested for bioimaging applications.[1] They typically show red-shifted absorption and emission, outstanding brightness and low photodegradation along with deep tissue penetration, small biological photodamage and negligible autofluorescence.[2] Several families of NIR dyes have been designed, synthesized
and proposed to the market for selective staining of a plethora of biological structures, but a proper modernisation in the study of cutting edge probes specific for the complex and dynamic assemblies of the bilayer membrane, is still necessary.[3] Squaraines have been already introduced to visualize and deeply study biological membranes highlighting their relevance due to singular lightness and specificity.[4] In this work, we have proposed symmetric and asymmetric squaraine dyes, decorated with carboxylic groups on the chromophoric core and different lengths aliphatic chains on the quaternary nitrogen positions of the scaffold itself. The formers facilitate the solubilization in biological media and lock the probes on the outer side of the amphiphilic bilayer, while the latter have been varied to investigate the respective interactions with the hydrophobic portion of
the membranes. The photophysical properties, the kinetic of the insertion into large unilamellar vesicles (LUVs) bilayer membranes beside with the emission signal fluctuations related to the membrane phases properties have been analysed in relation to the probe molecular structures to provide key data to optimize the design of new NIR probes for bioimaging purposes
May 17th, 2021 by Katja Herzog
Alejandra Matamoros-Recio received a postgraduate from the University of Alcalá, Madrid (M.Sc. in Drug Discovery 2017). She is a predoctoral research fellow in the “Computational Biological Chemistry” group led by Dr. Sonsoles Mart́ın-Santamaría, at CIB Margarita Salas, CSIC (Spain), pursuing a Ph.D. at the University Complutense of Madrid. Her research project lies at the interface between Chemistry and Biology, by means of molecular modeling and computational chemistry, applied to the understanding of ligand-receptor interactions and molecular recognition processes relevant for drug design, with a particular focus on Toll-like receptors and Antimicrobial Resistance-related receptors
Abstract:
Computational approaches to the dynamics and activation mechanism of Toll-like receptor 4
Topic:
Toll-like receptors (TLRs) are pattern recognition receptors involved in innate immunity. In particular, TLR4 binds to lipopolysaccharides (LPS), a membrane constituent of Gram-negative bacteria and, together with MD-2 protein, forms a heterodimeric complex which leads to the activation of the innate immune system response. TLR4 activation has been associated with certain autoimmune diseases, noninfectious inflammatory disorders, and neuropathic pain, suggesting a wide range of possible clinical settings for the application of TLR4 antagonists, while TLR4 agonists would be useful as adjuvants in vaccine development and in cancer immunotherapy. Specific molecular features of extracellular, transmembrane, and cytoplasmic domains of TLR4 are crucial for coordinating the complex innate immune signaling pathway. Although structural and biochemical data is currently available for the independent TLR4 domains, this only provides a partial fragmented view, because full-length proteins are flexible entities and dynamics play a key role in their functionality. Therefore, many structural and dynamical features of the TLR4 mode of action remain largely unknown. Computational studies of the different independent domains composing the TLR4 were undertaken, using ab-initio calculations, homology modeling, protein-protein docking, all-atom molecular dynamics simulations, and thermodynamics calculations, to understand the differential domain organization of TLR4. From the information gathered from our independent TLR4 domains studies, we have modeled, by allatom MD simulations, the structural assembly of plausible full-length TLR4 models embedded into realistic plasma membranes, with different chemical compositions, accounting for the active (agonist) state of the TLR4. We have also applied computational techniques to characterize, at the atomic level, the molecular recognition processes by reported TLR4 modulators, thus proposing a mechanism for their biological activity. These observations unveil relevant molecular aspects involved in the mechanism of receptor activation, and adaptor recruitment in the innate immune pathways, and will promote the discovery of new TLR4 modulators and probes.
May 17th, 2021 by Katja Herzog
Elva Morretta (EM) obtained a Master’s degree in Pharmaceutical Chemistry and Technology (110/110 cum laude) in July 2016 at Salerno University (bio-organic chemistry thesis, tutor Prof. Monti). In September, EM obtained a fellowship for the Drug Discovery and Development Ph.D. program (tutor Prof. Casapullo). In 2018, EM spent six months at Prof. Heck Biomolecular Mass Spectrometry and Proteomics Lab. On March 2020, EM defended her Ph.D., discussing a thesis entitled “Interactome Analysis of Bioactive Molecules: Optimization of a Functional Proteomics Platform”. From January 16th 2020, EM works as a Post-Doctoral Researcher at the Pharmacy Department of Salerno University.
Abstract:
Label-free functional proteomics links the anti-angiogenic properties of the pyrazolyl-urea GeGe-3 to Calreticulin binding
Topic:
n the last twenty years, 5-pyrazolyl-ureas have been largely investigated for their poly-pharmacological potential. In this scenario, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl)ureido)-1H-pyrazole-4-carboxylate (GeGe-3) emerged as a promising anti-angiogenic compound, inhibiting Human Umbilical Vein Endothelial Cells (HUVEC cells) proliferation and endothelial tube formation, impairing inter-segmental angiogenesis during zebrafish embryos development and blocking tumour growth in transplanted subcutaneous Lewis Lung Carcinomas. Regrettably, although different primary targets implicated in cell division and/or calcium homeostasis have been hypothesized for this compound, all the binding tests gave negative results. Thus, to link GeGe-3 anti-angiogenic potential to a suitable protein partner, the molecule interactome has been deeply investigated in HUVEC cells through label-free functional proteomics approaches, namely Drug Affinity Responsive Target Stability (DARTS) and targeted Limited Proteolysis coupled to Multiple Reaction Monitoring Mass Spectrometry (t-LiPMRM). These approaches share the principle that, interacting with a molecule, a protein undergoes conformational changes that result in its lower sensitivity to limited proteolysis, when performed in native conditions. Thus, in a first step, the coupling of DARTS with high resolution mass spectrometry allowed the identification of GeGe-3 most reliable interacting protein, Calreticulin, as later on validated by Western Blotting. Subsequently t-LiP-MRM, which allows to discover the target protein structural alterations due to complex formation with GeGe-3, served the purpose of pinpointing Calreticulin regions directly or distally involved in the interaction with the compound. T-LiP-MRM obtained results were corroborated by molecular docking analyses. Calreticulin is a major Ca2+ binding protein involved in intracellular Ca2+ homeostasis, cells adhesion, migration, proliferation, differentiation and apoptosis, as well as in cell-cell interactions. To shed light on the biological consequences of GeGe-3 interaction with such an interesting protein partner, in cell assays were performed. The obtained results disclosed GeGe-3 potential mechanism of action as anti-angiogenic factor: due to its binding to calreticulin, the molecule is able to alter Ca2+ intracellular shift in HUVEC cells, consequently modifying their cytoskeletal proteins organization.
May 17th, 2021 by Katja Herzog
Abstract:
Development of small molecule inhibitors of the endocytic cytoskeleton
Topic:
Clathrin-mediated endocytosis (CME) is the primary cellular route of cell surface receptors uptake, which in turn regulate the strength and specificity of downstream signalling. Changes in CME have been linked to increasing of cancer cell survival, proliferation and migration. FCHSD2 protein is a key activator of actin polymerization during CME. Importantly, it has been linked to chemotheraphy resistance and other diseases such as diabetes. FCHSD2 is recruited to clathrin-coated pits (CCPs) by intersectin via an SH3-SH3 interaction. Such interaction might be an interesting therapeutic target for inhibition of cancer cell metastasis and chemoresistance. Here, we are presenting two biochemical assays for a compound screening for a molecule that can break the interaction between FCHSD2 and Intersectin. The first assay is based on a split Nanoluc-luciferase where the second SH3 domain of FCHSD2 (F2S2) and the fourth SH3 domain of Intersectin (ITSd) were fused to Large and Small Nanoluc fragments respectively. After initial screens of Specs Consortium collection (30 000 compounds) and MicroSource Spectrum library (2 000 compounds), we found 148 hits which showed significant inhibition of luminescent signal. To exclude compounds that directly affect on Nanoluc luminescence a second screen was performed using the split-FAST tag fluorescent labelling system. This confirmatory screen yield 9 compounds that will be followed up for their specificity, mechanism of inhibition and cellular effects.
May 17th, 2021 by Katja Herzog
Callum Rosser is a young principal investigator within the Chemical Biology and Drug Discovery group from the University of Sydney
Abstract:
The synthesis and activity of analogues of the HDAC inhibitor panobinostat with added hydrogen bonding capacity.
Topic:
Histone deacetylase (HDAC) enzymes are crucial structural modulators of chromatin, which affect differentiation, cell proliferation and homeostasis of eukaryotic cells [1]. Overexpression of HDACs plays a role in cancer, neurological diseases, infection, and inflammation [1]. Currently, HDAC inhibitors have been approved for use in non-solid cancers, with benefit coupled with serious adverse effects. The low specificity of inhibitors to the HDAC isoforms and other Zn(II) containing metalloproteinases is proposed to cause these adverse effects [2]. This has prompted a search for optimised inhibitors to reduce these side effects. Molecular modelling studies have identified acidic amino acid residues at the surface of HDAC2 capable of forming hydrogen bonds to the cap group region of an HDAC inhibitor [3]. This project aimed to develop a library of cinnamylhydroxamate compounds that incorporate a hydrogen bonding group (carboxamide) to probe these acidic amino acid residues. The naturally occurring amino acid tryptophan, allowed easy addition of the carboxamide group into two regions of the inhibitor using two different synthetic routes. The carboxamide group was incorporated in alternative stereochemical configurations, via the use of L- or D-tryptophan, to explore enantioselective effects. Four cinnamyl-hydroxamate HDAC inhibitors were synthesised and screened for HDAC inhibitory activity in HeLa nuclear extract. Compounds with the carboxamide group in the linker region (5 (225 nM, S), 6 (240 nM, R)) were less potent than approved HDAC inhibitor panobinostat. Compounds with the carboxamide on the cap group (7 (264 nM, S), 8 (1564 nM, R)) possessed similar inhibitory activity to compounds 5 and 6 in the S configuration but reduced inhibitory activity in the R configuration. This project has provided a rationale for the design of new HDAC inhibitors that probe interactions with the acidic amino acid residues at the surface of the HDAC binding pocket.
May 17th, 2021 by Katja Herzog
Cosmin Stefan Butnarasu is a PhD candidate in Pharmaceutical and Biomolecular Sciences at the University of Turin. His research is focused on exploiting mucus models to assess the permeability of drugs to uncover the different phenomena that limit drug diffusion through human mucosa. Recently, he started to investigate and develop a protein-based nanocarrier to efficiently deliver drugs ensuring readiness for future outbreaks. Among other research projects, he investigated the application of squaraine dyes as fluorescent probes to detect disease-associated biomarkers. He is (co)author of 6 publications in international peer-reviewed journals.
Abstract:
Understanding the mechanisms governing the interaction of drugs with mucus using a novel biosimilar mucus model
Topic:
A constitutive mucus layer covers all the wet epithelial tissues ensuring lubrication and protection against external threats. Mucus can represent a strong barrier to tackle even for oral or pulmonary administered drugs (Figure 1). Despite the critical role played on drug absorption, very little is known about the molecular properties that mediate the interaction of drugs with mucus (Butnarasu, 2019). Moreover, due to its high biological complexity and heterogeneity, it is difficult to recreate a robust and reproducible in vitro model suitable for high throughput screening purposes. We have developed a biosimilar mucus model that mimics a pathological mucus (Pacheco, 2019). A natural polysaccharide was used to reproduce the viscoelastic behaviour while the composition was mimicked by adding mucin which is the main glycoprotein forming mucus. An in vitro mucosal surface was recreated by coupling the mucus model to 96-well permeable supports pre-coated with structured layers of phospholipids (PAMPA). Eventually, the permeability of a library of commercially available drugs was investigated in the absence and presence of the mucus model loaded on PAMPA plates. The mucus model not only represented a physical barrier, but it really behaved as an interactive filter. Different molecular structures were differently retained by mucus. The diffusion of the majority of the tested compounds was reduced; for some of them, the effect was less pronounced while for a few the diffusion was even enhanced. Multivariate statistical analysis was used to decipher the molecular descriptors that play a pivotal role in drug retention on mucus. Since drug development is characterized by a high rate of failure, the mucus platform could help to reduce at an early drug discovery stage the number of poor performers that reach preclinical trials. Moreover, the model is completely tunable as other mucus components (lipids, DNA, proteins) could be included during the production phase.
May 17th, 2021 by Katja Herzog
Abstract:
Bacterial mannan polysaccharide: chemical structure and conformational studies
Topic:
Many microorganisms are known to produce extracellular polysaccharide. Bacterial EPSs usually occur as capsule and/ormedium released polysaccharides. EPSs are involved in several biological functions, such as bacteria adhesion to surface andbiofilm formation, ion sequestering, and protection from desiccation. Furthermore, the enhanced production of a high-molecular-weight polyanionic EPS at sub-optimal incubation temperatures lends support to theories that EPS may serve as acryoprotectant for microorganisms as well as their enzymes. The cryoprotectant role of EPS was established in the psychrophilicbacterium Colwellia psychrerythraea 34H grown at low temperatures). The EPS from the psychrotolerant bacteriumPseudoalteromonas could enhance the stability of the cold-adapted protease secreted by the same strain by preventing itsautolysis, avoiding enzyme diffusion, and helping the strain in enriching the proteinaceous particles and trace metals in thedeep-sea environment. The chemical characterization of these polymers is the starting point for obtaining relationshipsbetween their structures and their various functions. Here, the chemical structure and conformational studies of a mannanexopolysaccharide from the bacterium Psychrobacter arcticus strain 273-4 isolated from permafrost is presented. The mannanfrom the cold-adapted bacterium was compared with its dephosphorylated derivative and the commercial product fromSaccharomyces cerevisiae. Starting from the chemical structure a new approach through various physicochemical techniques todeepen the study of the structure/activity relationship was explored. Finally, the ice recrystallization inhibition activity of thepolysaccharides is reported.
May 17th, 2021 by Katja Herzog
Miguel Santos received his PhD degree in Chemistry in 2013 by the University of Aveiro. His thesis focused on the supramolecular recognition of organic and inorganic anions by azacalix[2]arene[2]triazine macrocycles and isoftalamides. He is currently a Researcher at the Cultural Heritage and Responsive Materials Group from LAQV-REQUIMTE focusing on the development of Organic Salts and Ionic Liquids from Active Pharmaceutical Ingredients as highly effective ionic formulations of commercial drugs.
Abstract:
Enhanced formulations of Hydroxychloroquine as Organic Salts and Ionic Liquids to fight COVID-19
Topic:
Since the beginning of the COVID-19 pandemic, SARS-CoV-2 has infected more than 134 million people worldwide, from which 2.9 million have died. Multiple advances in the pharmacological treatment of severe cases have been made over the last year such as dexamethasone to control inflammatory response and repurposed drugs such as hydroxychloroquine (HCQ) and remdesivir as antivirals. However, HCQ, and despite showing efficacy against SARS-CoV-2 by putatively increasing lysosomal pH, it is no longer recommended for treatment of severe COVID-19 due to its toxicity at the required therapeutic doses. Nonetheless, it has shown to be an effective alternative in avoiding complications in milder cases, especially in a combinatorial regime. Still there is a need for the development of more effective antiviral drugs against SARS-CoV-2, but a very long journey is expected in the search of novel drugs. A promising alternative is the enhancement of current drugs by associating them with chemical adjuvants. For more than a decade, the combination of Active Pharmaceutical Ingredients (APIs) with such adjuvants as Organic Salts and Ionic Liquids (OSILs) has risen in the academia, and has recently reached Pharma, as an alternative to improve the properties of current drugs, in particular bioavailability, chemical and thermal stability, safety and therapeutic efficiency. In our lab, several antibiotics (β-lactam, fluoroquinolones) and bone anti-resorbing agents (bisphosphonates), among others, have been successfully combined as anions and/or cations with biocompatible organic counter-ions, with very interesting chemical and biological improvements being observed. Most recently, we set out to explore the benefits of the OSILs approach with hydroxychloroquine (HCQ-OSILs) as effective antivirals against SARS-CoV-2. Hence, in this communication we present the synthesis and characterization of fourteen novel mono- and dicationic HCQ-OSILs, and also a comparison of their water solubility and octanol-water partition coefficients with HCQ sulfate. Moreover, in vitro cytotoxicity data on Vero E6 cells and antiviral activity profile against the SARS- CoV-2 virus of the prepared HCQ-OSILs will be rationalized and discussed. Acknowledgments This work was supported by FCT-MCTES (Research 4 COVID-19 nº 582, PTDC/QUIQOR/32406/2017, PEst-C/LA0006/2013, RECI/BBBBQB/0230/2012) and by the Associate Laboratory for Green Chemistry – LAQV (FCT-MCTES UIDB/50006/2020).
