User Story: Prof. Jan Rybniker, University Hospital Cologne

User:

Prof. Jan Rybniker, University Hospital Cologne, Germany

EU-OPENSCREEN screening partner site:

Prof. Ursula Bilitewski, Prof. Mark Brönstrup, Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany

Tuberculosis is one of the leading causes of death by infectious disease globally, responsible for over one million mortalities per annum. Several factors contribute to this high mortality rate, including the emergence of strains resistant to existing antibiotic treatments. In pursuit of new therapeutic avenues, Prof. Jan Rybniker collaborated with EU-OPENSCREEN to screen for compounds with novel activity against the tuberculosis-causing bacteria.

Tuberculosis (TB), a highly infectious airborne disease most commonly affecting the lungs, represents a widespread global health crisis. Despite its high mortality rate, TB is generally considered both preventable and curable. Several antibiotics have been developed to treat patients with TB, which can successfully manage and cure infections in many cases. However, complications arise when the disease-causing bacteria, Mycobacterium tuberculosis (Mtb), become resistant to these existing therapies. Such is the case in patients with multidrug-resistant (MDR) TB – defined as a resistance to some of the antibiotics in first-line TB therapies – and extensively drug-resistant (XDR) TB – resistance to both first- and second-line therapies.

Novel therapeutic approaches are needed to combat this crisis, but discovering such approaches is not trivial – it requires both innovation in screening techniques and access to diverse chemical compounds for said screening. These gaps fall within the research scope of Prof. Jan Rybniker, University Hospital Cologne, Germany, whose lab aims to identify novel therapeutic approaches for severe bacterial infections, such as MDR and XDR TB. Prof. Rybniker’s group have developed several high-throughput screening (HTS) methods to identify compounds with activity against Mtb and with host cell protective effects, as well as various secondary assays to validate novel lead structures. To make the most of these methods, Prof. Rybniker’s group sought a diverse compound library to screen against their assays. This led them to EU-OPENSCREEN's platform for screening collaboration projects, which allow users to test their assays against the 100,000+ compounds in EU-OPENSCREEN's European Chemical Biology Library (ECBL).

Under a typical EU-OPENSCREEN screening project, the collaborating researcher provides their assay to one of the consortium’s screening partner sites, where it can be screened against the ECBL. However, since Prof. Rybniker’s assay involved highly infectious bacteria, shipping it to a partner site would have posed undue risks and logistical challenges. To mitigate these obstacles, Prof. Rybniker’s EU-OPENSCREEN partner site collaborators, Prof. Ursula Bilitewski and Prof. Mark Brönstrup of the Helmholtz Centre for Infection Research (HZI), Braunschweig, Germany, instead opted to ship the ECBL compounds directly to Prof. Rybniker’s institute in Cologne so his group could conduct the screen locally using their dedicated biosafety level (BSL)-3 screening platform. The compound plates were prepared and shipped by Susanne Daenicke, a technician at HZI.

Prof. Rybniker’s group initially exploited resazurin microtiter assays (REMA) using a single concentration against whole-cell Mtb, resulting in a hit rate of 0.486%. They then subjected these hits to a secondary screening and cytotoxicity profiling against Mtb and the human HepG2 cell line, both in a dilution series. Overall, HTS of the ECBL identified 247 compounds with both Mtb growth-inhibiting and non-cytotoxic features. Preliminary evaluation of one selected hit suggested activity as a mycobacterial respiratory chain inhibitor, and two other hits exhibited cell wall biosynthesis inhibitory features.

Building on this successful and comprehensive screening campaign, Prof. Rybniker's group are now performing additional secondary assays to validate the identified hits. These assays include screening hits for efficacy against intracellular replication of Mtb and Mtb-induced toxicity in macrophages and lung fibroblasts, respectively. Attempts to identify the molecular targets of the most promising compounds will include, for example, resistant mutant generation and screening, whole genome sequencing, genetic and complementation experiments and target-specific assays.