Covalent ClpP Inhibitors

Identification of novel covalent ClpP inhibitors as inhibitors of Gram-negative bacteria (WP3)

Dr. Philip Gribbon & Dr. Björn Windshügel
Fraunhofer IME

The bacterial caseinolytic protease, ClpP, is a serine endoprotease (family S14) comprised of two subunits, A and P [Alexopoulos et al. J Struct Biol. 179, 202-10 (2012)]. The A subunit is regulatory and binds ATP; the P subunit is catalytic. ClpP plays a key role in controlling bacterial protein turnover, and is the main enzyme involved in protein homeostasis by removing damaged, denatured and aberrantly folded proteins that are harmful to the cell [Butler et al. Mol Microbiol. 60, 553-62 (2006)]. Interestingly, mutants in which ClpP is either absent or constitutively active both show fitness defects, suggesting that inappropriate activation of the enzyme can also wreak havoc on cell physiology. ClpP also plays a central role in allowing “persister” cells to survive exposure to conventional antibiotics, so inhibitors of ClpP should synergize the action of the latter. Our project aims to use both in silico and in vitro screening approaches to identify small molecules that modulate (inhibit or activate) ClpP activity, to investigate their MoA and characterize their potential as antimicrobial agents. To this end, we will work close collaboration with partners, which have complementary expertise in medicinal chemistry (WP2) and in silico structure based drug discovery (WP1). We will optimize ClpP expression and purification pipelines and develop HTP primary, secondary and counter assays for ClpP activity assays. “First pass” in silico screens will be used to prioritise our collection compounds for in vitro testing and confirmation, as well as HTS of diverse compound libraries to identify additional potential modulators. Hits will be confirmed and optimized with respect to efficacy, physico-chemical and lability properties through successive rounds of medicinal chemistry.

In this project, our aims are:

  1. Optimisation of protein over-expression and purification conditions, assay development and screening for activators of ClpP
  2. Development of primary, secondary and counter assays
  3. In silico screening of the Fraunhofer IME physical library of 200,000 compounds and commercially available compound libraries
  4. In vitro testing and confirmation of hits (in collaboration with University of Eastern Finland)
  5. HTS of 50,000 diverse compounds to identify potential covalent ClpP inhibitors
  6. Confirmation and profiling of hits in primary and secondary assays
  7. Optimisation of hits with respect to efficacy, physico-chemical and liability properties through successive rounds of medicinal and synthetic organic chemistry (in collaboration with University of Antwerp).

Objectives: Identification and optimization of covalent inhibitors of ClpP using in silico and in vitro screening approaches. Validation of hit compounds in target based functional assays and assessment of off-target compound activities.

Expected Results: Novel, potent and selective ClpP protease inhibitor with activity against Gram-negative bacteria.

This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska Curie grant agreement No  642620
European Union