Author: Katja Herzog

Gavin read Chemistry at UMIST and was awarded an MChem in Medicinal Chemistry.

Abstract:

Chemoenzymatic synthesis of sugar nucleotide chemical biology tools to explore the GDP-D-mannose dehydrogenase from Pseudomonas aeruginosa

Topic:

The opportunistic human pathogen Pseudomonas aeruginosa (PA) causes chronic bacterial infections in cystic fibrosis patients, contributing to a reduction in lung function and increased mortality rates. The lung environment induces a switch of P. aeruginosa to its mucoid phenotype, which is characterised by an overproduction of the exopolysaccharide alginate. Composed of β-D-mannuronic acid and its C5 epimer α-L-guluronic acid, alginate is a key component in the formation of a bacterial biofilm, which increases persistence of the bacteria in the airways and retards antimicrobial treatments. Inspection of the PA biosynthetic pathway reveals a key enzyme involved in alginate production, GDP-mannose dehydrogenase (GMD), which catalyses an NAD+-dependent oxidation of GDP-D-Man to GDP-D-ManA: the alginate feedstock monosaccharide. We have designed and synthesised a series of GDP-Man probes to interact with the GMD active site, providing mechanistic insight and identifying the first sugar nucleotide inhibitor of GMD.

Abstract:

A Modular, Dynamic, DNA-based Platform for Regulating Cargo Distribution and Transport between Lipid Domains

Topic:

Biological membranes feature highly evolved proteo-lipid machinery able to co-localise in lipid rafts, nano-scaled assemblies believed to underpin signal transduction [1], amongst other cellular processes. Bottom-up synthetic biology aims to replicate life-like behaviours in model artificial cells [2], often using synthetic lipid bilayers as passive enclosures that lack the functional complexity associated to their biological analogues. DNA nanotechnology has emerged as a popular choice for biomimicry, coupling bio-inspired nano-devices with model membranes using amphiphilic oligonucleotides [3]. In fact, amphiphilic DNA nanostructures also undergo partitioning in lipid domains [4], evoking the affinity of proteins for raft microenvironments.

Here, we regulate the lateral distribution of DNA nanostructures in phase-separated membranes by exploiting the tendency of cholesterol and tocopherol motifs to respectively enrich liquid-ordered (Lo) and liquid-disordered (Ld) domains. By prescribing combinations of multiple anchors, changes to nanostructure topology, and size, our DNA architectures are programmed to achieve partitioning states that span the energy landscape. In addition, the functionality of our approach is showcased with a responsive biomimetic DNA device that dynamically achieves ligand-induced reconfiguration and mediates cargo transport between lipid domains. Our synergistic platform [5] paves the way for the development of next-generation biomimetic DNA-based architectures, that can achieve sensing and communication in synthetic cellular systems

Abstract:

Immobilized Metal Affinity Chromatography as a Potential Drug Discovery Platform

Topic:

In 1990, approximately 80% of medicines approved in the U.S. were either natural products or their derivatives. In the early 2000s there was a significant drop in the number of natural products in clinical studies, coinciding with the expansion of high throughput screening (HTS) techniques. However, the limited structural diversity inherent to HTS and the emerging threat of antimicrobial resistance has reinvigorated the focus on exploiting natural products for the discovery and development of new medicines. Immobilized metal affinity chromatography (IMAC), a technique originally designed for the isolation of histidinetagged proteins, has shown promise in isolating and purifying bioactive compounds. IMAC relies on the fundamentals of coordination chemistry to reversibly retain compounds with known metal ion affinity. Although originally developed for recombinant protein purification, this simple method has been shown to readily purify hydroxamic acid siderophores, such as desferrioxamine B (DFOB) and other clinical agents, from bacterial cultures. Most IMAC work, in the context of siderophore isolation and purification, has utilized Ni(II) as the metal ion, but there is potential in substituting Ni(II) with other metal ions, such as Cu(II), Fe(III), Ga(III) and Zn(II). The modified IMAC resin beds may consequently act as metalloenzyme surrogates and select for different metabolites as directed by distinct coordination chemistries. This could open up a new platform to discover metalloenzyme inhibitor drug candidates as the isolated metabolites, by virtue of their metal binding affinity, may demonstrate activity against various metalloenzymes. As an initial proof of concept, we have exposed a mixture of in use metalloenzyme inhibitors to IMAC columns charged with various biologically relevant metals with promising results. The IMAC ligand-metal complex is a reasonable surrogate of the active site of a metalloenzyme and the method is capable of reversibly binding a variety of antihypertensive, anti-inflammatory and anticancer drugs.

Katarzyna Walczewska-Szewc is an assistant professor in the Theoretical Biophysics group, Nicolaus Copernicus University in Toruń, Poland. In her research she uses a number of computer-aided methods to model biomolecular interactions and to reveal the structural mechanisms of action of medically important proteins. She obtained her PhD degree in biology and physics jointly from the Australian National University and University of Gdansk, Poland. Katarzyna’s PhD project involved the development and use of numerical and simulation methods to design and interpret Förster resonance energy transfer (FRET) experiments in physics and biology

Abstract:

The changes in prolyl oligopeptidase structure upon inhibition modify its ability to decrease alpha-synuclein aggregation

Topic:

The formation of extended misfolded protein aggregates is one of the main reasons for neuronal malfunction and, eventually, brain damage in many neurodegenerative diseases. In Parkinson’s disease alpha-synucleins are implicated in the accumulation of the aggregates. The origin of such aggregation is not yet known, however, there is a compelling evidence that it can be reduced by inhibition of prolyl oligopeptidase (PREP). This effect cannot be simply related to the inhibition of the catalytic function of the enzyme, as not all PREP inhibitors stop the alpha-synuclein aggregation. Finding differences in the dynamics of the enzyme inhibited with diverse compounds would allow us to pinpoint the regions of the protein involved in the interaction between PREP and alpha-synuclein. Here, we study the action of three PREP inhibitors, each of which affects alphasynuclein aggregation to different extent. Using molecular dynamics modelling, we determine molecular mechanisms underlying the PREP inhibition and identify structural differences in each inhibitor-PREP system. We suggest that even subtle differences in the dynamics of the enzyme affect its interactions with alpha-synucleins. Thus, identification of these regions may be crucial in preventing formation of alpha-synuclein aggregates. Acknowledgement: The computational results were obtained using the facilities of the Interdisciplinary Centre for Modern Technologies, NCU, Poland

Topic:

Printing Biology: where printing meets synthetic biology

Abstract:

The assembly of life-like artificial systems is an emerging topic that contributes to the fundamental understanding of the molecular origins of life, and fuels the development of life-inspired platforms usable in different fields (e.g. molecular sensing, artificial biology, tissue engineering) [1]. The implementation of these platforms depends upon the ability to recapitulate the structural and functional features of biological systems, including multi-scale organization, adaptivity to environmental stimuli, collective behaviors [2]. The resulting research efforts have led to the recent definition of Printing Biology [3], a field resulting from the intersection between printing and the bottom up Synthetic Biology. Printing Biology aims at realizing reconfigurable multiscale systems (from nanometers to millimeters) with bespoke molecular composition, allowing for the determination of molecular interactions and features in conditions mimicking those of the living systems. As representative examples, the reproducible fabrication process of stable fL-scale compartments (the size selected by Nature for the formation of organelles and molecular condensates) by inkjet printing (IJP) and microcantilever spotting (μCS) will be shown.

The molecular composition of the compartments will be varied with the final aim to demonstrate the activity retention of different classes of the encapsulated biomolecules. Three different model applications will be shown, including DNA, proteins and phospholipids ink printable formulations. At first, the mechanism of DNA oligonucleotides ink imbibition by μCS into nylon porous supports is demonstrated (Figure 1). Subsequently, the immobilized DNA oligonucleotides (printed at different concentrations) are hybridized with a fluorolabeled complementary sequence permits to demonstrate the retaining of biological function [4], and the optical detection of oligonucleotides down to few tents of zeptomoles. As a second application, the retaining of CYP2E1 enzymatic activity from IJP compartments mimicking mitochondria is shown, highlighting the possibility to further induce spatial organization of the reaction products (Figure 2) [5]. Finally, preliminary experiments showing the realization of ordered phospholipids compartments containing fluorescein tagged phospholipids by μCS onto glass surfaces are reported. These systems allow for the realization of artificial platforms that could find applications in membrane-protein interaction studies.

Dr. Kamyar Hadian is the Head of the Research Group ‘Assay Development and Screening Platform’ at the Helmholtz Zentrum München in Munich/Germany. Dr. Hadian’s laboratory focuses on two main research aspects: (i) unraveling mechanisms that drive or block ferroptosis, a regulated cell-death pathway and (ii) identifying and characterizing protein interactions and regulators of ubiquitin signaling pathways. In addition, his research group has deep understanding in employing biochemical high-throughput and phenotypic high-content imaging approaches combined with machine learning methods to identify and validate small molecule modulators for treatment of distinct diseases related to the fields of Cancer, Virology and Immunology.

Dr. Hadian studied Biology at the Technical University of Munich (TUM) and received his PhD at the Helmholtz Zentrum München/Ludwig Maximilians University (LMU) in the field of Virology (HIV research). After a short PostDoc period in the field of NF-kB signaling, he was appointed the Head of ‘Assay Development and Screening Platform’ at the HelmholtzZentrum München in 2010. In 2015 he became a tenured Principal Investigator. From 2016-2017 he had a parallel appointment as an Adjunct Associate Research Scientist at the Columbia University in New York/USA, where he joined Dr. Stockwell’s lab to establish a long-lasting transatlantic collaboration. Dr. Hadian is the author of >50 publications, 5 patent applications and 2 issued patents.

Topic:

Acriflavine, a clinically aproved drug, inhibits SARS-CoV-2 and other betacoronaviruses

Abstract:

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and availability of vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, as well as in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.