RHOMBOSMALPS · Enabling malaria rhomboid proteases as drug targets: usage of molecular cookie cutters to shape novel activity assays and inhibitors.
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2017-10-01 → 2019-09-30
- EU contribution
- €160,800
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
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Results in brief
Enabling malaria rhomboid proteases as drug targets: usage of molecular cookie cutters to shape novel activity assays and inhibitors.
Intramembrane proteases (IMP) are proteolytic enzymes that are embedded in the lipid bilayer. Rhomboids a serine IMP subfamily which has been linked to important human diseases. For example, Plasmodium falciparum rhomboids 1 and 4, are thought to be key for the parasite invasion. In addition, human PARL and RHBDL-4 have a role in neurodegeneration. Nevertheless, despite their relevance in pathological processes, their specific role and druggability are unclear. The transmembrane packing interactions of IMPs are responsible for their thermodynamic stability, which translates in a high dependence on the environment. Hence, IMPs protease activity is substantially influenced by membrane composition. Unfortunately, the study of these IMPs in their native environment has rendered impractical to date. The bottleneck is that the current purification techniques use detergents that ravage the physiological membrane, yielding low enzyme stability and, in some cases, activity. In its turn, this rules out the use of activity assays and chemical probes to study their function. Encapsulating these proteins in their lipid environment will address these shortcomings. Our objective was set to allow the study of Rhomboids in order to enable them to function as drug targets. We have developed a detergent free purification method, based on maleic acid copolymers: SMA and DIBMA. Those function as a “molecular cookie cutter”, creating polymer-lipid-protein nanodiscs, which retain their biological properties upon purification. We have tested the suitability of these nanodiscs to maintain the activity and stability of rhomboids with promising results. Likewise, we have evaluated their suitability to undergo activity tests. Our results confirm that the nanodiscs are suitable to run inhibitor screening. Our work enables the study of rhomboids. This is, it has opened a door to finding inhibitors against rhomboids which will serve to enable them as new therapeutic targets for malaria or Alzheimer's disease.
Data: CORDIS, © European Union
Project objective
Rhomboid proteases from P. falciparum, the causative agent of malaria, play a role in invasion of human red blood cells. The exact role of the individual members is challenging to track, because of difficulties in genetic manipulation of the P.falciparum and the inviability of some loss-of-function mutants. Hence, a chemical strategy is an attractive alternative. Unfortunately, the study of these eukaryotic rhomboids (PfROMs) has rendered impractical to date.The bottleneck is that the current purification techniques use detergents that eliminate the physiological membrane, yielding low enzyme stability and activity. In its turn, this rules out the use of activity assays and chemical probes to study their function. Encapsulating these proteins in their lipid environment will address these shortcomings. I will develop a detergent free purification method, based on a styrene maleic acid (SMA) polymer that functions as a “molecular cookie cutter”, creating SMA-lipid-protein nanodiscs, which retain their biological properties upon purification.Using this “molecular cookie cutter” to create lipid nanodiscs, I will isolate PfROMs and develop activity assays in order to identify and optimize novel inhibitors. The most potent and selective candidates will be evaluated in a malaria invasion model to verify the druggability of malaria rhomboids. Furthermore, these novel compounds may serve as leads for a new generation of therapeutic agents.The straightforward expansion of our approach to other intramembrane proteases may be the game-changer for drug discovery and future therapeutics directed against rhomboids from other species.
Original text from CORDIS.
Participants
- KATHOLIEKE UNIVERSITEIT LEUVEN · LeuvenCoordinatorBelgium
Links
Data: CORDIS, © European Union