Hit-to-lead development of selective HBV/HDV entry inhibitors

Project description:

Infections with the Hepatitis B (HBV) and D (HDV) viruses are the main cause of hepatocellular carcinoma and liver cirrhosis as a consequence of chronic hepatitis. Although an effective prophylactic vaccine is available, therapeutic options are highly limited, in particular for HDV. A promising novel drug target to block HBV/HDV virus entry into hepatocytes is represented by the hepatic bile acid carrier NTCP (Na+/taurocholate co-transporting polypeptide) that has been identified as the bona fide hepatic receptor for HBV/HDV. So far, more than 200 compounds from different compound classes have been tested and corresponding 3D structure-activity-relationship (QSAR) models have been generated. Furthermore, a pharmacophore model for HBV/HDV entry inhibitors was established. Virtual compound libraries have already been screened with these models and further effective hits have been identified.

NTCP is a physiological bile acid transporter in the plasma membrane of liver cells. NTCP is also the hepatic receptor for HBV and HDV. HBV/HDV entry inhibitors should selectively block NTCP viral receptor function. 3D QSAR and pharmacophore models help identify novel NTCP inhibitors.

 

Scientific goal:

Development of oral and selective HBV/HDV entry inhibitors that specifically block virus binding to NTCP, without tackling its physiological bile acid transport function. Hit compounds will be further developed into lead structures by means of molecular drug design.

 

DRUID Collaboration partners:

B1 Diederich/Kolb, B3 Rahlfs/Kolb/van Zandbergen, D2 Pfeiffer/Zeuzem/Hildt, E3 Rahlfs/Przyborski, E6 Schiffmann/Laux

References C5:1. *Kirstgen et al. (2020) Sci Rep 10:21772 2. *Grosser et al. (2021) Front Mol Biosci 8:689757 3. *Kirstgen et al. (2021) Viruses 13:666 4. *Kirstgen et al. (2021) Viruses 13:1489.

Improved diagnosis and therapy of visceral Leishmaniasis

Project description:

A reliable diagnosis and treatment of humans and dogs (reservoir host) suffering from visceral leishmaniasis (VL) is crucial for the control of this infection. Currently available diagnostic tests based on the antibody reaction with the Leishmania kinesin protein show poor sensitivity in some endemic areas. We have developed and patented a kinesin antigen (rKLi8.3) with an improved diagnostic performance in humans and animals. This was possible by developing a kinesin antigen with an optimized kinesin structure (repeats) and sequence. Currently various test formats with the rKLi 8.3 antigen are produced and tested with sera from infected humans and dogs.

The treatment of VL is problematic in terms of effectiveness and side effects. An inhibitor (GNF-6702) that selectively inhibits the proteasome of the Kinetoplastida (T. brucei, T. cruzei and L. donovani) has been developed. GNF-6702 binds to a proteasome subunit of Kinetoplastida (beta 4 subunit) that is structurally distinct from humans. The specific bind-ing to the kinetoplast proteasome seems to be the reason for the very low toxicity in mam-malian cells. We could demonstrate that the proteasomal beta 4 subunit is conserved in all Leishmania isolates, tested to date. Thus, the proteasome is a promising therapeutic target.

 

Scientific goal:

In the diagnostic part of the project, various formats of a sero-diagnostic VL rapid test will be manufactured and tested in cooperation with our industrial partner. In the treatment part of the project, we validate the specificity, effectiveness and toxicity of the new, kinetoplastid-specific proteasome inhibitors in cell culture experiments of Leishmania infected macrophages.

 

DRUID Collaboration partners:

B1 Diederich/Kolb lab, C2 Kempf lab, D3 van Zandbergen lab

References C4: [1] Abass et al. (2013) PLoS Negl Trop Dis. 18;7; [2] Abass et al. (2015) PLoS One. 3;10; [3] Martínez Abad et al. (2017) Acta Trop. 166:133-138; [4] Pereira et al. (2020) Eur J Microbiol Immunol 27;10:165-171. [5] Khare et al. (2016) Nature 537: 229-233

New ways towards the control of schistosomiasis and echinococcosis

Project description:

Echinococcosis and Schistosomiasis are two co-called Neglected Tropical Diseases, which have a severe impact on the health of affected individuals in endemic countries. Our group is developing new tools that can be used for a better control of these two diseases. For schistosomiasis, we are developing new drugs that target the schistosomal Mitogen Activated Protein Kinases [1],  in an effort to find new drugs, which are larvicidal as well as adulticidal. For Echinococcosis, we are developing a novel diagnostic platform based on detection of parasite-specific IgE using humanized IgE reporter cell lines [2-4], which can be used in a variety of formats. We are also working on extending this diagnostic platform to other parasitic infections (e.g. Fasciola, Cysticercosis, Clonorchis, Opisthorchis).

Larvicidal and adulticidal activity of compound 38_8, chosen from a compound library using our bioinformatic approach.

Structural prediction of S. mansoni JNK compared to the human orthologue.

Scientific goal:

The Schistosome project aims to identify lead kinase inhibitor molecules using a bioinformatics in silico analysis pipeline and further develop these into suitable drugs, while in the Echinococcus project we are pursuing a better understanding of the protective immune responses against echinococci and aim to incorporate this knowledge into the development of vaccines and greatly improved, innovative diagnostic technologies.

 

DRUID Collaboration partners:

A4 Heine lab, B4 Grevelding lab, C6 NWG Häberlein lab, E5 Czermak/Salzig lab

References B7P: [1] *Pereira-Moreira et al. (2020) ACS Omega 5:9064-9070 [2] Kalli M et al. (2020) Sci Rep. 10:18208 [3] Kalli M. et al. (2020) Methods Mol Biol.;2163:155-162. [4] Prakash PS et al. (2021)  Parasitol Res.

Pharmaceutical development, translational medicine and drug repurposing

Project description:

The SARS-CoV2 pandemic clearly demonstrates that there is an unmet medical need for anti-pathogenic drugs. In order to identify new treatment options, promising substances from other working groups will be characterized preclinical. This includes the generation of a safety profile and/or bioavailability testing of the drug candidates. Since supportive therapy with immune-modulating drugs is a proven therapeutic approach, an interaction of the drug candidates with the immune system will be tested.

Surface marker expression of treated M2 macrophages ©Marina Henke

Cytokine expression of treated M2 macrophages. ©Leonard Blum

Scientific goal:

As part of the project, a safety and/or immunomodulatory profile will be created for small molecules/peptides that have a potential antiviral, antiparasitic or antibacterial property. Furthermore, human proteins (e.g. TMPRSS2) which represent new targets for combating pathogens will be investigated.

 

DRUID Collaboration partners:

A1 Becker lab, A2 Grünweller lab, A3 Weber lab, B1 Diederich/Kolb lab, B3 Rahlfs/Kolb/van Zandbergen, B5 Schlitzer lab, D1 Friebertshäuser lab

References E6: 1. Blum et al. (2021) J Mol Med 99(2):261-72; 2. Blum et al. (2020) Sci Rep 10(1):7534; 3. Blum et al. (2020) J Cell Mol Med 24(12):6988-99.

Plasmodium chaperones, co-chaperones and their interactions as a target for drug development

Project description

Overview of assay design

Malaria parasites invade and live within mature human red blood cells RBC). To enable their survival, the parasite renovates it’s chosen host cell to its own advantage. Infected red blood cells become sticky and adhere to the lining of small blood vessels, and also coat themselves with proteins which enable them to become invisible the immune system. This unfortunately causes disease in the patient, and eventually leads to death. We have recently identified a number of important molecular players which are essential for this renovation process, including members of the so-called HSP70 and HSP40

families. It is the goal of this project to block the function of HSP40 and HSP70. If we can do this, parasites are likely to be cleared from the bloodstream, relieving the severity of disease. To do this, we will establish a number of assays to measure the activity of HSP40/HSP70, and use these to search for compounds which reduce this interaction. Promising compounds will then be tested directly on parasites for their ability to reduce host cell modification.

References A7: 1. Diehl et al. (2021) PLoS Pathogens 17:e1009969 2. Zhang et al. (2017) Sci Rep 7: 42188 3. Charnaud et al. (2017) PLoS One 12: e0181656 4. Külzer et al. (2012) Cell Micro 14: 1784-95 5. Külzer et al. (2010) Cell Micro 12: 1398-1420

Complement-interacting proteins of relapsing fever Borrelia

Project description:

Borrelia recurrentis is considered as a “neglected arthropod-borne pathogen” and the causative agent of louse-borne relapsing fever. If left untreated, the case-fatality rate of this epidemic disease can exceed >40%. The hematogenous dissemination suggests that borreliae developed efficient immune evasion strategies to overcome innate immunity, in particular complement. Proteins interacting with complement are known to play a crucial role in host-pathogen interaction. Bioinformatic analyses revealed a cluster of five proteins all of which inhibit complement at different activation levels. Of importance, molecules interacting with the immune system represent promising candidates for the development of in vitro diagnostics. Two test systems (Line immunoblot and ELISA) for the diagnosis of louse-borne relapsing fever have already been developed and evaluated.

Immunofluorescene microscopy of B. recurrentis.

Schematic representation of the complement inactivation on the outer surface of B. recurrentis.

Scientific goal:

The focus of this project deals with the functional and structural characterization of additional complement-inhibiting proteins of Borrelia recurrentis as well as the optimization of the evaluated test systems and the development of a point-of-care antigen test for the diagnosis of relapsing fever.

 

DRUID Collaboration partners:

B2 Ziebuhr lab, C2 Kempf lab, C4 Steinhoff lab, D1 Steinmetzer lab, E3 Rahlfs/Przyborski, Gold Standard Diagnostics (formerly NovaTec)

References C3: [1] Cordes et al. (2005) Nat Struct Mol Biol 12:276-277; [2] Röttgerding et al. (2017) Sci Rep 7:303; [3] Nguyen et al. (2018) Front Cell Infect Microbiol. 8:23; [4] Walter et al. (2019) Front Immunol 10:2722; [5] Röttgerding and Kraiczy (2020) Front Immunol 11:1560; [6] Schmidt et al. (2021) Sci Rep 11:4964.

Bartonella bacilliformis pathogenicity factors as diagnostic and therapeutic targets

Project description

Bartonella bacilliformis is the causative agent of Carrións disease, a vector borne illness restricted to the South American Andes. The bacteria cause severe hemolytic fever with high fatality rates. The inhibition of hemolysis represents a promising therapeutic approach. Two pathogenicity factors play a crucial role in hemolysis of which at least one represents a promising drug target. Humans are the only known reservoir host for B. bacilliformis and represent the source of new outbreaks. It is therefore of particular importance for disease control to identify asymptomatic carriers. For this, an alpha version of a B. bacilliformis IgG ELISA and line blot were developed.

Scanning electron microscopy of infected human erythrocytes with B. bacilliformis.

B. bacilliformis NovaLisa® KIT.

Scientific goal:

The project aims to analyze the role of the two pathogenicity factors in the process of hemolysis to develop a novel, anti-virulence-based therapeutic strategy. Furthermore, the alpha version of the B. bacilliformis IgG ELISA and line blot will be evaluated in field studies in cooperation with our partners from Lima/Peru.

 

DRUID Collaboration partners:

A7 Przyborski lab, C1 Bender / Hildt, E7 Locker lab, Gold Standard Diagnostics (formerly NovaTec)

References C2: [1] Garcia-Quintanilla et al. (2019) Parasites&Vectors 12(1):141, [2] Riess et al. (2004) J Exp Med 200:1267-78, [3] Dichter et al. (2019) Microbiology Res Announc, DOI: 10.1128/MRA.01377-19, [4] Dichter et al. (2021) Lancet Microbe 2:e685–94.

Posttranslational protein modifications as Achilles’ heel of pathogenic RNA viruses

Project description

Due to their small genome, viruses are highly dependent on functions of the host organism. Many of those functions are regulated by cell-encoded posttranslational protein modifications for which a substantial number of pharmaceutical inhibitors are available.

Rift Valley Fever Virus (RVFV) is a mosquito-borne zoonotic pathogen endemic in parts of Africa. In large and devastating outbreaks, it typically kills thousands of farm animals and hundreds of humans. In the preceding funding period, we used a high-throughput genetic screen and identified a pro-viral host cell factor for RVFV that binds to posttranslational protein modifications. Inhibition of this factor in a human organoid model reduced viral RNA synthesis and progeny particle production. In addition, proteomic analyses showed that a viral protein is modified is a manner that the host cell factor can bind, and mutation of the relevant site led to a reduction of viral RNA synthesis.

 

Scientific goal:

We aim to elucidate the molecular mechanism and exploit it to specifically inhibit RVFV infection. Moreover, we will test available pharmaceutical inhibitors, and also screen for other pathogenic RNA viruses that may depend on this mechanism.

 

DRUID Collaboration partners:

A1 Becker, A2 Grünweller, B2 Ziebuhr, C1 Bender/Hildt, D1 Friebertshäuser/Steinmetzer, E3 Rahlfs/ Przyborski, E4 Spengler, E6 Schiffmann, E7P Krijnse Locker

References A3: 1. Wuerth & Weber (2016) Viruses 8, 174*. 2. Barr, Weber, Schmaljohn (2020) Fields Virology, vol 1, p 706-749*

Protein/protein interactions of Ebola virus proteins as targets for new antiviral strategies

Electronmicroscopic picture of Ebola virus leaving an infected cell (red arrow). ©Schauflinger

Project description

The Ebola virus (EBOV) causes severe fever with extraordinarily high fatality rates. The matrix protein VP40 of EBOV plays key roles for the virus replication cycle and is regulated by homooligomerization. VP40 dimerization is crucial for the protein’s transport towards the plasma membrane where the dimers polymerize resulting in filament formation which enables virus budding. VP40 octamerization results in the down-regulation of viral RNA synthesis. Due to their central role as building blocks of the higher-order oligomers, dimers represent a promising target for antiviral intervention. A fragment-based approach was used to identify hits binding to VP40 crystals which will be developed into lead compounds in order to inhibit VP40 oligomerization.

Scientific goal:

High-resolution crystal Structure of Ebola virus VP40 Dimers. ©Anke Werner

The project aims to develop lead molecules into antiviral compounds using structure-based drug design – a combination of protein crystallography with in silico methods – as well as cell culture experiments under BSL4- conditions for validation.

DRUID collaboration partners:

B1 Diederich/Kolb lab, A4 Heine/Reuter lab, D1 Steinmetzer lab, E3 Rahlfs/Przyborski lab

References A1: 1. *Hartlieb et al. (2007) PNAS 104: 624-9 2. *Hartlieb et al., (2003) J. Biol. Chem. 278: 41830-6 3. *Hoenen et al. (2005) J Virol. 79: 1898-905 4. *Möller et al. 79, 14876-86 (2005) J Virol. 5. Hoenen et al. (2010) J Virol 84: 7053-63. 6. *Gomis-Ruth et al. (2003) Structure 11: 423-33.
* own project-specific preliminary work