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Final Report Summary - STARPI4K (Structural targeting of PI4 kinases)

StarPI4K - Structural targeting of PI4 kinases
Phosphatidylinositol phosphates (PIPs) are universal markers of intracellular membranes. Phosphatidylinositol 4-phosphate (PI4P) is the single most abundant mono phosphoinositide that defines the membranes of the Golgi and TGN. It has been reported to play a key role in membrane biogenesis, vesicular transport, lipid dynamics, and protein and lipid sorting in the TGN. PI4P is biosynthesized by phosphatidylinositol kinases (PI4Ks). Humans have two type II PI4Ks – PI4K2A and PI4K2B (also known as α and β) – and two type III PI4Ks –PI4KA and PI4KB (also known as IIIα and IIIβ). Importantly, the type III PI4Ks are hijacked by positive strand RNA (+RNA) viruses such as the Hepatitis C virus, poliovirus or Aichi virus to create replication organelles, an extensively phosphorylated and modified membrane system dedicated to their replication. Highlighting medicinal importance of PI4P, it is produced by PI4K2A hijacked by intracellular bacteria (e.g. Legionella pneumophila, Chlamydia trachomatison). Thus, PI4Ks are potential pharmacological targets. The goal of this project was to get atomic insight in the PI4P biosynthesis that takes place at intracellular membranes of all eukaryotic organisms and to use the structural information to design specific inhibitors targeting PI4Ks.
Four years ago we have started to investigate the structure and function of PI4Ks. We have prepared constructs to express PI4Ks in E. coli and in insect cells. We had to screen for domain boundaries and use techniques that improve yields and solubility. For instance, the PI4K2A has a CysCysProCysCys motif that is palmitoylated; we replaced this motif with T4 lysozyme and removed the disordered N- and C- termini to obtain soluble protein suitable for structural studies. We also established collaboration with the group of Dr. Radim Nencka (also IOCB, Prague) to develop small molecule inhibitors of PI4KB that could be used as virostatics. Upon intensive screening, we obtained crystals both of PI4KB (that is an important target for antiviral therapy) and for PI4K2A. Our findings were published in prestigious journals (EMBO Reports and Journal of Medicinal Chemistry)1,2.
In the final 3rd and 4th year of the project we synthesized fluorescently labeled inhibitors that still preserved their excellent inhibitory and selectivity capacity. The most useful one proved to be coumarin labeled inhibitor that had an excellent fluorescence signal (lifetime to be exact) response to PI4KB binding. They proved to be useful biochemical tools (we have used them to measure dissociation constants even for unlabeled inhibitors via a replacement experiment) and fluorescence properties of the coumarin label make them ideal for FLIM microscopy. These findings were published very recently3.
We have also focused on improvement of our inhibitors into sub-nanomolar compounds suitable as drug candidates for animal tests in mice. These results were also published very recently in the Journal of Medicinal Chemistry4.
In a parallel effort, we focused on the structural aspects of PI4KB recruitment to the cellular membrane in normal cellular physiology and on PI4KB hijacking by +RNA viruses (Fig. 4). We discovered that ACBD3 recruits PI4KB to cellular membranes and we solved the NMR structure of the interacting domains5. We were also able to solve the crystal structure of viral 3A protein in complex with the ACBD3 GOLD domain to elucidate how viruses hijack PI4KB through the Golgi resident ACBD3 protein6.
Taken together, thanks to the StarPI4K project, I have established a successful structural biology laboratory that focuses on medically important enzymes. Importantly, students of the Charles University (currently I train three PhD students and several undergrads) in Prague have the opportunity to learn structural biology. The lab is now focused on i) testing the inhibitors against PI4KB in mice model (collaboration with the veterinary Institute in Brno) ii) structural biology of host factor hijacking by +RNA viruses iii) reconstituting the biogenesis of viral replication organelles in vitro – we use the biomimetic Giant Unilamellar Vesicle (GUV) system to learn how viruses use cellular membranes for their own benefits. If successful, we will prepare compounds effective against +RNA viruses (inhibitors of PI4KB) and, in addition, we will significantly expand our knowledge about replication of +RNA viruses in human cells.

Most important results
1 Baumlova, A. et al. The crystal structure of the phosphatidylinositol 4-kinase IIalpha. EMBO reports 15, 1085-1092, doi:10.15252/embr.201438841 (2014).
2 Mejdrova, I. et al. Highly Selective Phosphatidylinositol 4-Kinase IIIbeta Inhibitors and Structural Insight into Their Mode of Action. Journal of medicinal chemistry 58, 3767-3793, doi:10.1021/acs.jmedchem.5b00499 (2015).
3 Humpolickova, J., Mejdrova, I., Matousova, M., Nencka, R. & Boura, E. Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIbeta (PI4KB). Journal of medicinal chemistry 60, 119-127, doi:10.1021/acs.jmedchem.6b01466 (2017).
4 Mejdrova, I. et al. Rational Design of Novel Highly Potent and Selective Phosphatidylinositol 4-Kinase IIIbeta (PI4KB) Inhibitors as Broad-Spectrum Antiviral Agents and Tools for Chemical Biology. Journal of medicinal chemistry 60, 100-118, doi:10.1021/acs.jmedchem.6b01465 (2017).
5 Klima, M. et al. Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein. Scientific reports 6, 23641, doi:10.1038/srep23641 (2016).
6 Klima, M. et al. Kobuviral Non-structural 3A Proteins Act as Molecular Harnesses to Hijack the Host ACBD3 Protein. Structure 25, 219-230, doi:10.1016/j.str.2016.11.021 (2017).
More information is available at the laboratory web page: http://www.uochb.cz/infochem/boura

Related information

Documents and Publications

Reported by

USTAV ORGANICKE CHEMIE A BIOCHEMIE, AV CR, V.V.I.
Czech Republic

Subjects

Life Sciences
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