Aminocyclitol and iminosugar derivatives related to Gauche disease

Gaucher disease (GD) is one of the most prevalent lysosomal storage diseases.A mutation on gene gba1 generates a misfolded enzyme acid β-glucosidase. Due to this, this enzyme is not properly transported from the endoplasmic reticulum (ER) to the lysosome, with the consequent reduction of its lysosomal activity. This leads to the accumulation of the substrate glucosylceramide (GluCer) in the lysosomes of macrophages that originate the clinical symptoms. Besides the expensive Enzymatic Replacement Therapy (ERT), different approaches could be useful to minimize this accumulation of GluCer. One option could be the inhibition of Glucosylceramide Synthase (CGS) in order to reduce the formation of GluCer. Another approach is the use of Pharmacologic Chaperones (PC) that bind to the misfolded GCase in the ER, assisting the correct protein folding and allowing its transport to the lysosome, where the enzymatic degradation of GluCer takes place. In order to facilitate the correct folding of GCase without blocking its hydrolase activity, an ideal PC should show high affinity for GCase in the ER environment (neutral pH) and lower affinity in the lysosome (pH 5.2). For this purpose, we have designed and synthesized two small libraries of compounds in order to study the influence on their affinity to bind GCase of the pKas of the substituents in the side chain of several DNJ derivatives, as well as the pKas of the nitrogen in the sugar mimetic cores, was modulated by introduction of different β- substituents. The synthesized compounds were evaluated as imiglucerase (recombinant Gcase) inhibitors at pH 5.2 and pH 7.0 in presence of detergents that emulate the lipids naturally presents in the cell. In this assay conditions, most of the compounds showed better imiglucerase inhibition at neutral pH than at pH 5.2, but no correlation could be established between the affinity for GCase and their pKas. Even though, it has been discovered the DNJ derivative with higher potency as GCase inhibitor described so far. After analyzing the some representative compounds as GBA1 inhibitors in a detergent-free assay with cell homogenates it has been noticed the importance of the assay conditions when studying the inhibitory potency of different compounds. In this way, the IC50 values obtained in the assay with imiglucerase and detergents drive to totally different conclusions than when the assay was performed with cell homogenates without addition of exogenous detergents. Even so, neither direct correlation between the pKas of the inhibitors and their affinity for GCase could be established at this point. Moreover, the capacity of GCS inhibition of the synthesized compounds was also analyzed, revealing a family of compounds that act as GCS inhibitors more potent that NB-DNJ, a drug that is actually administered as GCS inhibitor for treatment of GD. Going one step further, a family of compounds with potential dual behavior GCS inhibitors and PC for GCase was discovered. On the other hand, GBA2 inhibition studies of the synthesized compounds revealed some nanomolar inhibitors of this enzyme. Finally, a new GBA inhibition assay in intact cell has been developed for the analysis of GBA1 or GBA2 inhibition without lysate cells, and allowing the study of the posterior recovery of GBA activity after inhibitor removal.La malaltia de Gaucher és una de les malalties d’emmagatzematge lisosomal més freqüent. Una mutació al gen gba1 provoca un incorrecte plegament de l’enzim Glucocerebrosidasa (GCase) que impedeix el seu transport del reticle endoplasmàtic (ER) fins al lisosoma, on té lloc la hidròlisi de la glucosilceramida (GluCer). Això provoca una acumulació d’aquest substrat, originant els símptomes clínics. En aquesta tesi s’ha sintetitzat una petita col·lecció de compostos per tal estudiar la influència del pKa de diferents inhibidors en la seva potencial activitat chaperona, és a dir, estudiar la seva capacitat de unir-se a la GCase mal plegada del reticle per facilitar-ne el correcte plegament i permetre el seu transport cap a lisosoma. Per aquest motiu, es buscaven compostos que presentessin una alta afinitat per la GCase en les condicions neutres del reticle endoplasmàtic, però que tinguessin baixa afinitat per aquest enzim a pHs lleugerament àcids com el del lisosoma (pH 5.2), per tal que no bloquegés l’activitat hidrolasa d’aquest enzim al lisosoma. Desprès d’analitzar els compostos amb diferents assajos, es va posar de manifest la importància de les condicions d’assaig a l’hora d’estudiar la inhibició d’un enzim, ja que diferents assajos poden conduir a conclusions diferents. Malgrat que no es va poder establir cap correlació directa entre el pKa dels inhibidors i la diferència d’activitat segons el pH de l’assaig, es va descobrir un dels derivats de DNJ amb millor potència inhibitòria de imiglucerasa (Gcase recombinant) descrit fins el moment. Per altra banda, es va analitzar la capacitat d’inhibició de GCS dels compostos sintetitzats, descobrint-se alguns inhibidors d’aquest enzim més potents que la NB-DNJ, compost que s’utilitza actualment com a inhibidor de GCS per al tractament de la malaltia de Gaucher, i possible comportament dual (inhibidors de GCS i chaperones per GCase)