Author: 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.

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.

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.

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.

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).

Prof. dr hab. Wieslaw Nowak is currently a head of Chair of Biophysics at the Institute of Physics, N Copernicus Univ. Torun, Poland. He got MSc in 1979 in chemistry, PhD in 1989 in theoretical physics, habilitation in 2000 in Biophysics, after spending several years as a postdoc at UIC Chicago, Ecole Polytechnique (Paris) and Kyoto University. His current interest are computational studies of medically important proteins, optogenetics and single molecule AFM force spectroscopy

Abstract:

Nanomechanics of nanobodies-protein S SARS-CoV-2 virus complexes revealed by a Virtual Atomic Force Microscope

Topic:

World population is to a large extent saved by development of antibodies against pathogens such as SARS-CoV-2. Despite natural antibodies induced by a viral infection or vaccination, application of nanobodies is extremely promising technology in fight with the current pandemic. Nanobodies are single-domain antibodies that can be effectively produced in the lab. The main step in the research towards new tools against SARS-associated viruses is effective screening (or de novo design) of nanobodies. Here we propose a computational strategy called Virtual Atomic Force Microscopy to monitor the strength of a nanobody interaction with protein S from the SARS-CoV-2 virus. Starting from recently published cryo-EM structures of Nb6 nanobody bound to closed and open SpikeS2P protein, we use Steered Molecular Dynamics computational modeling method to assess unbinding forces and to determine molecular mechanisms of binding-unbinding processes. Our approach allows for efficient ranking of synthetic nanobodies affinities to protein S. The proposed computational pipeline may be particularly useful for initial checking of already developed nanobodies against new variants of SARS viruses. Acknowledgement: This project is funded by IDUB N. Copernicus ANTICO and #MEMOBIT grants. ICNT UMK computer facilities are acknowledged