Many genetic diseases are caused by mutations that result in improperly folded proteins, with key examples being the lysosomal storage diseases (LSDs), each one of them due to the deficiency of an enzyme involved in the stepwise degradation of glycolipids or glycosaminoglycans in the lysosome. In these cases, degradation stops and the residual macromolecule remains trapped within the cell . Gaucher disease, the most prevalent of these rare diseases (ca. 1/40000 births), is a consequence of missense mutations in the encoding gene GBA1, ultimately causing insufficient glucocerebrosidase (GCase) activity . This enzyme is a retaining beta-glucosidase responsible for catalyzing the last step in the degradation of glycosphingolipids . The work proposed here represents an alternative to the currently employed enzyme replacement therapy (ERT), and is based on the use of small molecules that specifically bind the functional form of the enzyme, compensating for the destabilizing effects of the mutation, thus stabilizing the folded form. This then allows more enzyme to avoid endoplasmic reticulum-associated degradation and travel to the lysosome, where it can degrade its substrate. This is the basis of the enzyme enhancement therapy (EET), which has the potential for being much less expensive than ERT and for treating neuronal forms of the disease .
Recently, the Withers lab reported on a library of iminoxylitol derivatives as potent inhibitors and chaperones of GCase. The synthetic strategy relied on a common iminosugar-alkene precursor (IMX), prepared from D-xylose in seven synthetic steps, from which 16 dideoxyiminoxylitols bearing various different lipophilic substituents were generated in the last step via a divergent thiol-ene coupling. Enzyme kinetic analyses revealed that a number of these products were potent, low-nanomolar inhibitors of human GCase that stabilize the enzyme to thermal denaturation by up to 20 ºC. Moreover, cell-based assays conducted on Gaucher disease patient-derived fibroblasts demonstrated that administration of the compounds can increase lysosomal GCase activity levels by therapeutically relevant amounts . Interestingly, NMR studies revealed that the conformation of these iminoxylitols in solution depends on the pH value, and that a proper 5C2 conformation, mimicking the 4C1 conformation of beta-glucopyranose, is only adopted at high pH values (free amine), while the opposite chair conformation (2C5), displaying most of its substituents in an axial configuration, is prevalent at low pH (protonated amine). This conformational change is related to the amine pKa, and therefore engineering of its basicity allowed the formation of second generation iminoxylitols with pKa values in the range of 7.0-4.5, thus optimizing inhibition towards GCase at the physiological conditions of the ER, but showing lower affinity at the more acidic enviroment of the lysosome. In this communication, I shall present progress in this direction, as well as supporting in vitro and in vivo results.
- a) Futerman, A.H.; van Meer, G. Nat. Rev. Mol. Cell Biol. 2004, 5, 554-565. b) Wennekes, T.; van der Berg, R.J.; Boot, R.G.; van der Marel, G.A.; Overkleeft, H.S.; Aerts, J.M. Angew. Chem. Int. Ed. 2009, 48, 8848-8869.
- Grabowski, G.A. Lancet 2008, 372, 1263-1271.
- Vocadlo, D.J.; Davies, G.J. Curr. Opin. Chem. Biol. 2008, 12, 539-555.
- Benito, J.M.; García-Fernández, J.M.; Mellet, C.O. Expert Opin. Ther. Pat. 2011, 21, 885-903.
- Goddard‐Borger, E.D.; Tropak, M.B.; Yonekawa, S.; Tysoe, C.; Mahuran, D.J.; Withers, S.G. J. Med. Chem. 2012, 55, 2737-2745.