Development of Dithiocarbamates as Anthelminthics and Inhibitors of RNA-helicase eIF4A as potential antiviral Agents

Project description:

Helminthic infections represent a major problem in large parts of the world. In comparison to the burden the arsenal of anthelminthic drugs is rather limited. Using successive cycles of design, synthesis and testing the novel class of dithiocarbamates shall be optimized regarding activity towards different helminths, host toxicity and drug-likeness. Derivatives are synthesized, tested against different helminths and human cell lines. For promising derivatives ADME-parameters are determined and selected compounds could be tested in vivo.

Different viruses pathogenic to humans, as for instance the Ebola- or the SARS-CoV-2 virus, are using host factors for intracellular replication. These host factors therefor represent a target for anti-viral drug design. One of these host factors is the RNA-helicase eIF4A. Based on the crystal structure of the eIF4A-RNA complex appropriate ligands are designed, evaluated by docking and in case of proper fit synthesized. Compounds are tested in regard to their effect on translation efficiency. Active derivatives are subsequently evaluated regarding their effect on virus replication in cell cultures.

Scientific goal:

Both projects should yield compounds which possess the quality of a lead structure or possibly a development candidate

 

DRUID Collaboration partners:

A2, A3, A4, A7, B2, B7P, C6 NWG, D4, E1, E4, E6

References B5: –

Schistosoma mansoni: Biological Targets and Inhibitor Development

Project description:

Background: In the human and animal parasite Schistosoma mansoni, the sexual maturation of the female depends on a continuous pairing contact with the male (Abb. 1). Pairing is a prerequisite for egg produc-tion and as such decisive for the pathogenesis of schistosomiasis, which is induced by the eggs. Our research showed that different classes of molecules are involved in organizing reproductive processes in schisto-somes.1 Among others, experiments with kinase inhibitors and by kinase RNA interference demonstrated that kinases regulate cell division, sper-matogenesis, oogenesis, egg production and vitality of schistosomes. For instance, the Abl-tyrosine kinase inhibitor imatinib (cancer drug Glivec) negatively affected reproduction and caused the degradation of the gastrodermis of adult S. mansoni in vitro with lethal consequences.2,3

Besides kinases, which we study in cooperation with the Falcone group4, we investigate further enzymes such as aldehyde dehydrogenases (ALDHs) and an aldehyde reductase (AR). In schistosomes, as in other organisms, these molecules are putatively involved in regulating responses to molecular stress. Inhibiting these molecules might devitalize the parasite. First in vitro experiments with inhibitors against these molecules, like the ALDH inhibitor disulfiram, caused morphologic alterations, a decrease of pairing stability, reduced vitality and finally, the death of adult schistosomes within days in vitro. Helicases might also represent interesting targets as suggested by studies using the RNA helicase-specific inhibitor Silvestrol.5 In adult S. mansoni, Silvestrol reduced the vitality of adult worms in vitro but also stem cell-proliferation in gonad cells (Abb. 2). Together with the working groups of Prof. Schlitzer and Prof. Grünweller (Marburg), we will investigate these potential target molecules and develop synthetic inhibitors6,7 to study their physiological und morphological effects, first in vitro. Further, top candidates will be used for in vivo tests in a rodent infection model.

Abb. 1. Bright-field microscopy of a S. mansoni couple. During the constant pairing contact, the female (arrow) resides within the ventral groove of the male. Pairing is the essential pre-requisite for the production of eggs (stars).

Abb. 2. EdU-staining of female S. mansoni before (left) and after Silvestrol treatment (right). The num-ber of proliferating stem cells (green) is significantly reduced (right).

Scientific goal:

We will clone and characterize two RNA helicases of S. mansoni at the molecular and biochemical levels. In different cooperations within DRUID (see below), we will test the recombinant proteins in enzyme assays against Silvestrol and synthetic derivatives of this substance. Furthermore, we continue our analyses of the recombinantly expressed enzymes SmALDH312 and SmAR, further potential target molecules, and perform enzyme tests with inhibitors. Among these are disulfiram derivatives, which exhibited anti-schistosomal effects in vitro as shown in previous experiments. In addition, we plan to crystallize these molecules for structure analyses. Aim is to develop substances with inhibitor activity against the selected target molecules with high specificity, bioavailability and reduced toxicity for the host.8 Finally, results of the in vitro and in vivo experiments provide a basis for using this knowledge also for other parasitic systems, in which effective compounds will be tested (platform projekt E1).

 

DRUID Collaboration partners:

A2 AG Grünweller, B3 AG Rahlfs/Kolb/van Zandbergen, B5 AG Schlitzer, E1 Platformprojekt/Häberlein, E3 AG Rahlfs/Przyborski, E5 AG Czermak/Salzig lab

References B4: 1Beckmann et al. (2010) PLoS Pathog 6:e1000769; 2Beckmann et al. (2010) Int J Parasitol 40:521-6; 3Beckmann et al. (2012) Curr Pharm Des 18:3579-94; 4Moreira et al. (2022) Molecules (in press); 5Taroncher-Oldenburg et al. (2021) Microorganisms 9(3):540; 6Peter Ventura et al. (2021) ChemMedChem 14(21):1856-62; 7Peter Ventura et al. (2012) Arch Pharm (Weinheim) 354(12): e2100259; 8Mäder et al. (2018) ChemMedChem 13(22):2374-89.

NAD(P)H-dependent metabolic pathways as targets for new anti-infective agents

Project description:

Cellular redox balance plays an essential role in pathogenic microorganisms. Enzymes of the NAD(P)H-dependent glutathione and thioredoxin system1,2, as well as the glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD), which significantly contribute to the NADPH and ribose 5-phosphate pool via the pentose phosphate pathway3, are centrally involved. G6PD of the malaria parasite Plasmodium falciparum and P. vivax (GluPho), present as a bifunctional enzyme, differs functionally and structurally from the human host enzyme4 and is essential for malaria parasites5. Together with the Sanford-Burnham Institute/UCSD, La Jolla, we have established a high-throughput compatible assay for PfGluPho and have screened about 400,000 compounds (i.a. NIH MLSMR Collection)6. Following structure-activity relationship studies and lead optimization, we identified the highly selective PfGluPho inhibitor SBI-750, which is active in the lower nanomolar range7. The concept could already be transferred to Leishmania; the 3D crystal structure of Leishmania donovani G6PDs and PGDs could be solved and based on this a first in silico screening of small molecules could be realized. In addition, a high-throughput compatible assay for the recombinant G6PDs and 6PGDs from Leishmania was established to screen for inhibitors at the Novartis FAST lab in Cambridge (USA).

Workflow-Diagramm zur Inhibitor Identifizierung gegen G6PDs/PGDs

3D-Kristallstruktur LdG6PD ©Isabell Berneburg

Scientific goal:

The aim of this project is to functionally and structurally characterize the enzymes G6PD and 6PGD from Leishmania and Plasmodium as targets for drug development, and to identify and further develop inhibitors against these enzymes (in silico and HTS). The concept will also be transferred to other pathogens within the DRUID consortium, such as Schistosoma.

 

DRUID Collaboration partners:

B1 Diederich/Kolb; B4 Grevelding; D3 van Zandbergen; E1 Grevelding/Häberlein; E3 Rahlfs/Przyborski; E4 Spengler; E6 Schiffmann/Laux

References B3: 1. Fritz-Wolf et al. (2011) Nature Comm. 2:383* 2. Koncarevic et al. (2009) PNAS 106: 13323-8* 3. Bozdech and Ginsburg (2005) Malaria J 3:23 4. Jortzik et al. (2011) Biochem J Energy 436:641-50* 5. Allen et al. (2015) FEBS J 282:3808-23*
6. Preuss et al. (2012) J Med Chem 55:7262-72* 7. Berneburg et al. (2022) Antimicrob Agents Chemother (accepted)*

*own puplications

Protein de-ADP-ribosylation und NMPylation activities as potential therapeutic targets against coronaviruses

Project description:

Coronaviruses are important human and animal pathogens. They are mainly associated with respiratory and intestinal infections and have significant zoonotic potential, resulting in several outbreaks of severe respiratory infections in humans over the past 2 decades including the SARS-CoV-2 pandemic starting in 2019. Therapeutic options to treat severe forms of COVID-19 and other coronavirus infections are very limited, indicating a high priority for the development of novel antiviral drugs. To address this need, project B2 focuses on two coronaviral proteins that are conserved among all coronaviruses: the coronavirus macrodomain (macD) in nonstructural protein 3 (nsp3) and the recently discovered nucleotidyltransferase (NiRAN) which is linked to the viral RNA-dependent RNA polymerase domain in nsp12 and was recently shown to be essential for coronavirus replication.

Coronavirus replicase polyprotein 1ab. Proteolytic processing by two or three viral proteases (PL, 3CL) results in the release of up to 16 nonstructural proteins (nsp) with numerous enzymatic and other functions.

Scientific goal:

The project aims to comprehensively characterize the biochemical properties of two coronavirus proteins/enzymes (macD, NiRAN) and their functions in the viral replication cycle using appropriate cell culture systems. Based on the conserved substrate specificity of the NiRAN domain, HTS assays will be developed and used to identify potential inhibitors.

 

DRUID Collaboration partners:

A2 Grünweller, A3 Weber, C5 Kraiczy, E3 Rahlfs/Przyborski

References B2: 1. *Slanina et al. (2021) Proc Natl Acad Sci USA 118: e2022310118 2. *Krichel et al. (2021) Sci. Adv. 7: eabf1004  3. *Müller et al. (2021) Antiviral Res 175: 1004706 4. *Snijder et al. (2016) Adv Virus Res 96: 59-126 5. *Putics et al. (2005) J Virol 79: 12721-31.

Dengue and Zika virus, design of inhibitors of NS3/NS2B serine protease.

Project description:

Infections with the dengue virus have reached a new high in recent years, with around 50-100 million new infections per year. In the vast majority of cases, the infection progresses with mild, flu-like symptoms, but a small percentage of those affected, often children, develop hemorrhagic fever, which is fatal if severe. Although a vaccine is now available, since this is only approved for a very limited group of people, the development of agents that efficiently suppress the multiplication of the virus is essential. Our work focuses on the virus’s own serine protease NS3/NS2B, which cleaves the viral precursor protein into functional proteins and is essential for the maturation of the virus.

Abb. B1. Kristallstruktur der NS3/NS2B Protease DENV 3.1

Scientific goal:

The aim of the project is to further develop allosteric inhibitors of the viral serine protease NS3/NS2B, not only with respect to their affinity, but also with respect to their pharmacokinetic properties and toxicity (hit-to-lead development) using a combined approach of computer-aided design, synthesis, biological assays and crystal structure analysis. In addition, inhibitors of the related Zika protease will be developed based on the knowledge gained.

 

DRUID Collaboration partners:

A1 Becker, A2 Grünweller, B3 Rahlfs/Kolb/van Zandbergen, XY Herker, E6 Schiffmann/Laux

References B1: [1] Noble et al. (2012) J Virol 86(1):438-446; *[2] Chevillard et al. (2015), J Chem Inf Model 55(9):1824-1835; *[3] Chevillard et al. (2018) J Med Chem 61(3):1118-1129

Targeting the highly divergent actin superfamily in malaria parasite transmission

Project description

Malaria remains one of the most devastating diseases and is caused by single celled parasites called Plasmodium. These parasites are transmitted between people by Anopheles mosquitoes. The parasite needs a set of diverse proteins that enable it to transmit to the mosquito vector, including members of the highly divergent actin cytoskeleton and its regulators. The parasite cytoskeleton has unique properties in order to transmit and, given its essential nature in various parasite processes at different life cycle stages, contains promising targets for novel malaria therapies. We make use of target validation approaches to identify and characterize novel transmission blocking targets that could be used to control disease spread.

Target validation approach has identified novel transmission blocking targets.

Scientific goal:

We aim to characterize identified proteins of interest using a variety of in vitro and in vivo methods with a view to identify novel compounds that selectively target these proteins and thus serve as transmission blocking drug candidates.

 

DRUID Collaboration partners:

A7 Przyborski, B1 Diederich/Kolb, B7 P Falcone, E3 Rahlfs/Przyborski

References A6: [1] Douglas et al. (2018) PLOS Bio e2005345; [2] Douglas et al. (2018) Malaria J 17:3191898-905; [3] Douglas et al. (2015) Trends Parasitol 31(8):357-362.

Search for lead structures to inhibit the chaperone IpgC from Shigella

Project description

Bacteria of the genus Shigella invade the epithelial cells of the colon, which results in the severe inflammation of the large intestine. Known as bacterial dysentery or Shigellosis, this causes a large number of deaths, foremost in developing countries. The Shigella specific chaperone IpgC interacts with numerous further pathogenicity factors and is prerequisite for the virulence of this organism. In the absence of a “substrate protein”, IpgC forms a homodimer, which is essential for its stability. We use IpgC as a target protein for the structure-based design of compounds against Shigellosis by preventing IpgC homodimer formation and/or binding to substrate proteins. By now, we have established a protocol which reproducibly yields excellently diffracting IpgC crystals. Using a fragment-based approach, we have identified a number of IpgC “binders”, some of which we were able to expand significantly. In addition to protein crystallography, we use “Microscale Thermophoresis”, Isothermal Titration Calorimetry” and a “Thermal Shift” assay to study the influence of such molecules on homodimer formation, on the ability to interact with “substrates” and on stability.

Crystal struc-ture of ho-modimeric IpgC. ©Klaus Reuter

“Follow up” compound bound to IpgC. ©Marina Gardonyi

Scientific goal:

In addition to further structural information on IpgC, our main goal is the optimization of the compounds identified so far using them as lead structures in the development of anti-Shigellosis compounds.

 

DRUID Collaboration partners:

A1 Stephan Becker, B1 Wibke Diederich / Peter Kolb, B7 Franco Falcone, D1 Eva Friebertshäuser / Torsten Steinmetzer

References A4: [1] Agerberth et al. (2005) World Health Organ [2] Williams & Berkley (2018) Paediatr Int Child Health 38:50-65. [3] Sansonetti (2001) Am J Physiol Liver Physiol 280:319-323. [4] Parsot et al. (2003) Curr Opin Microbiol 6:7-14. [5] Lunelli et al. (2009) Proc Natl Acad Sci USA 106:9661-9666.

Development of eIF4A inhibitors as drug candidates and characterization of eIF4A-variants in Pathogens

Project description

The cellular RNA helicase eIF4A is an excellent target for the development of broad-spectrum antivirals. During initiation of viral protein synthesis, many viruses, especially corona viruses, rely on this enzyme, which can efficiently and specifically be inhibited by rocaglates. In project A2, we would like to further develop rocaglates for potential clinical trials by e.g. nebulizing these compounds for local application into the respiratory tract and by creating a detailed side effect profile. In addition, new eIF4A inhibitors will be screened and characterized.  The systematic mutagenesis of known coronavirus sequences should allow a prediction of rocaglate sensitivity in newly emerging coronaviruses. Finally, the therapeutic relevance of rocaglates in different DRUID-relevant pathogens expressing eIF4A variants will be investigated.

RNA clamping onto the surface of eIF4A by the rocaglate Silvestrol and evidence of inhibition of (corona)viral protein synthesis by the rocaglate CR-31-B (-).

Scientific goal:

Rocaglates will be further developed for their testing in clinical trials and the possibility to predict rocaglate sensitivity in emerging corona viruses and other DRUID-relevant pathogens will be evaluated.

 

DRUID Collaboration partners:

B2 Ziebuhr lab, B4 Grevelding lab, B5 Schlitzer lab, C6 Häberlein lab, D1 Friebertshäuser/Steinmetzer lab, D3 van Zandbergen lab, D4 Hermosilla/Mazurek/Taubert lab, E4 Spengler lab, E6 Schiffmann lab

References A2: 1.*Biedenkopf et al., (2017), Antiviral Res. 137: 76-81; 2. *Müller et al., (2018), Antiviral Res. 150:123-129; 3. *Elgner et al., (2018), Viruses. 10(4): 149; 4. *Glitscher et al., (2018), Viruses.  10(6): 301; 5. *Henß et al., (2018), Viruses.  10(11): 592; 6. *Müller et al., (2020), Antiviral Res. 175:104706; *7. *Müller et al., (2021), Antiviral Res. 186: 105012; 8. *Blum et al., (2020), J Cell Mol Med. 24(12): 6988-6999; [*own publications].

Neglected infectious diseases with focus on imaging

Project description:

We apply imaging techniques to understand how pathogens interact with cells to cause disease, focusing on electron microscopy (EM) and correlated light- and electron microscopy (CLEM). To generate robust protocols that can easily be adapted, we use our model virus, the large DNA-virus vaccinia (VACV).

The SARS CoV2 pandemic illustrates how viruses have a world-wide socio-economical impact with dramatic consequences for the neglected tropical diseases (NTDs). There is an urgent need to contain the SARS CoV2 pandemic and refocus resources on NTDs. In a collaborative effort we showed that the spike-surface protein (S) of SARS COV2, that mediates infection, shows unexpected flexibility. The latter could facilitate receptor binding and cell entry of the virus, which remains to demonstrated.

Cryo-electron tomography and molecular dynamic simulation of SARS-CoV-2 spike shows unexpected flexibility of the stalk.

Upon budding at intracellular membranes coronaviruses leave the cell in a poorly explored way. Vesicular trafficking as well as regulated lysosomal exocytosis have been proposed.

 

Scientific goal:

The project aims to interfere with the flexibility of S and test for the outcome of infection using antibodies, drugs and genetics. Virus release from infected cells will be studied by a CLEM approach, combining live cell imaging with EM.  The expertise gained from our VACV- and SARS CoV2 host system is then applied to answer questions related to virus-host systems used in other teams of the DRUID-consortium.

 

DRUID Collaboration partners:

A3 Weber lab, B2 Ziebuhr lab, B6 P Herker lab, C1 Hildt lab, D3 van Zandbergen lab, E5 Czermak lab

References E7 P: Turoňová, B., et al. (2020). Science (80-. ). 370, 203–208. Blanco-Rodriguez, G., et al. (2020). J. Virol. 94 e00135-20. Quemin, E.R.,  et al. (2018). J. Mol. Biol.430, 1714-1724. Chlanda, P., and Krijnse Locker, J. (2017). Biochem. J. 474, 1041–1053. Sartori-Rupp, A., et al. (2019). Nat. Commun. 10, 342.

Protein production, assay development, crystallisation and interaction analysis

Project description:

Within the framework of this platform project, we offer methods for protein production, for the development of high-throughput assays, for crystallisation and structural analysis as well as for interaction analyses. A variety of optimisation options for cloning/expression and purification of native proteins (affinity chromatography, gel filtration, ion exchange) are available. Once recombinant protein has been obtained, provide support in the development of assay systems up to high throughput formats. Furthermore, the platform proves access to the crystallisation platform at the iFZ. A pipetting and a crystallisation robot as well as starter kits and various additive screens for optimisation are available for crystallisation screens on the nl scale. The system is currently being updated with funding from the latest DRUID funding round. Dr Fritz-Wolf acts as crystallographer and contact person and can carry out the first tests on the crystals. The X-ray sources of the Max Planck Institute for Medical Research (Heidelberg) and the AG Klebe/Heine [A5] (Marburg) are available for this purpose.

Crystal structure of sfroGFP (Heimsch et al. 2022)

DRUID Collaboration partners:

Protein-Expression: A1, A3, A6NWG, A7, B2, B3, B4, C3
Crystallisation: B2, B3, C3
Assay-Devlopment: A7, B3

References E3: 1. Heimsch et al. (2022) Antioxid & Redox Signal doi: 10.1089/ars.2021.0234 2. Harnischfeger et al. (2020) Electronic Journal of Biotechnology 3. Fritz-Wolf et al. (2011) Nat Commun 2:383 4. Koncarevic et al. (2009) Proc Natl Acad Sci USA 106:13323-8