Allosteric activators of Ocrl1: a novel therapeutic strategy against Lowe syndrome

Lowe Syndrome (LS) is a lethal genetic disease caused by mutations in the OCRL1 gene that unfortunately leads to the early death of affected children and has no cure. However, this project aims to change such scenario. Although most OCRL1 mutations found in patients impair the enzymatic activity of the encoded lipid phosphatase Ocrl1, about half of these changes do not affect residues involved in binding or modification of the substrate (Fig. 1). In other words, there is a high number of patients expressing Ocrl1 mutated proteins with intact binding/catalytic sites but locked in a conformation unable to process lipid substrates. Therefore, we hypothesized that a substantial subset of Ocrl1 patient mutated proteins could re‐acquire functionality by action of drugs able to stabilize the enzymatically active conformer (allosteric activators) . Indeed, Using biochemical and cell biological approaches we identified the FDA‐approved drug and chemical chaperone, 4‐Phenyl Butyric Acid (4‐PBA) as able to restore catalytic activity of and to suppress LS cellular phenotypes caused by two different conformationally‐affected Ocrl1 patient mutants (Fig. 2 ) . In addition, we identified 2 additional FDA‐approved drugs ( not acting on Ocrl1 but on downstream pathways) able to ameliorate signaling deficiencies and associated LS phenotypes in cells from patients and in an animal model for LS. This project will produce a novel LS theoretical framework (introducing conformational/misfolding disease component valid for a subgroup of LS patients) and since it will address the lack of therapeutic approaches designed to suppress the causes of LS, has high significance. Importantly, drugs like 4‐PBA are currently used in children to ameliorate other conditions ( e.g. , urea cycle disorders, cystic fibrosis) and can be readily repurposed to LS ( i.e. , known safety and low risk of adverse toxicology). Therefore, the translational potential and impact of this project is very high. Further, since our investigations unveiled that LS shares some characteristics with other genetic diseases such as ciliopathies. Support or Funding Information Lowe Syndrome Association, Clinical and Translational Science InstitutePurified GST‐Ocrl1 1‐542 . Bacterial cells wire induced to produce GST‐Ocrl1 1‐542 WT, D451G and H524R using standard methods. A. Example of purified GST‐Ocrl1 fusion proteins (under different dilutions) as detected by Western blotting using a specific antibody. B–D: The enzymatic activity (measured as phosphate released from PIP 2 as function of time) of 3 different concentrations (closed circles>open circles>closed triangles) of purified GST‐Ocrl1 1‐542 WT (B). D451G (C) and H524R (D) was assayed using the malachite green method. Substrate concentration: 37μM. E. Plot of inital rate (V0) against substrate [PI(4,50P 2 ] concentration for WT Ocrl1 adjusted to a Michaelis ‐Menten equation.Phenotypes depending on Ocrl1 phosphates activity A. Cells expressing Ocrl1 [text missing] were treated with Vehicle (methanol) or 5mM 4‐PBA for 24h. Note the dispersed pattern of localization of the Ocrl1 [text missing] conformationally‐affected variant treated with vehicle as opposed to the tightly perinuclear normal localisation acquired in the presence of 4‐PBA. Scale bars: 10μm. B. Ocrl1 D451G characteristic dispersed pattern (Fig. 2) and Golgi apparatus fragmentation was also suppressed by 5mM 4‐PBA treatment. C. Catalytic activity of OCrl1 D451G was partially restored by 4‐PBA treatment in a dose‐dependent manner.