Identifying Novel Therapies for Cystinuria Using Genetic Tools

Abstract

Cystinuria is a rare inherited renal stone disorder that effects 1 in every 2000 ‎people in the UK. Available drug treatment like Tiopronin, D-Penicillamine, and Captopril used to decrease the Cystine concentration in the urine. However, these drugs impose huge disadvantages to ‎patients through agonising side-effects such as: weight gain, excessive fatigue, loss of taste, and ‎breathing difficulties, all of which lead to poor quality of life. This necessitates the need for ‎other therapeutic approaches that can treat the disorder without causing major side-effects. ‎Cystinuria is caused by a mutation in one or both subunits of the Cystine transporter rBAT or b0,+AT which are ‎encoded by SLC3A1 or SLC7A9 genes, respectively. These mutations lead to mis-‎localization of functioning rBAT and b0,+AT proteins from the plasma membrane which ‎translate into a disruption of urinary Cystine re-absorption. This in turn leads to increased Cystine flow into ‎urine which results in Cystine accumulation that eventually forms Cystine stones in kidneys, ‎ureter, or bladder. This research hypothesizes that repurposing established drug compounds ‎‎(LOPAC1280) to re-direct b0,+AT protein back into the plasma membrane could be ‎a new and improved therapeutic approach. ‎

In this study, transduced human proximal tubule epithelial ‎cells (PTECs) were used to investigate the localisation of fluorescently tagged rBAT and b0,+AT proteins in wild-type and three ‎rBAT-based mutated cell lines. All cellular localization studies showed consistent results. Both rBAT and b0,+AT proteins were found to be trafficked together. In the fluorescently‎ tagged wildtype type expressing cell line, both proteins were located at the plasma memrbane, with the wild-type b0,+AT being the dominant trafficked subunit. All three mutants expessing cell lines suggested that both proteins were predominantly trapped within the ER. A unique screening assay model that can measure the exiting of b0,+AT from the ER using a high content fluorescent microscope system was developed, optimized and validated, and used to screen the LOPAC1280 library of compounds using the p. Met467Thr-rBAT expressing cell line. Nanchangmycin (NCM) was identified as a final target hit as it induced exiting of the b0,+AT from the ER and translocated it to the plasma membrane.