Mice were coadministered vehicle (water) or AT1001 (3 or 10?mg/kg, which result in plasma KO mouse tissues were 0.5 0.04, 0.1 0.04, and 0.3 0.02, nmol/mg protein/hour, respectively. GUID:?AC309688-11A5-40EE-B3AF-2AC1B5D7A3CD Abstract Fabry disease is an X-linked lysosomal storage disorder (LSD) caused by mutations in the gene (at neutral pH and 37?C. Coincubation of Fabry fibroblasts with rh-Gal A and AT1001 resulted in up to fourfold higher cellular -Gal A and ~30% greater GL-3 reduction compared to rh-Gal A alone. Furthermore, coadministration of AT1001 to rats increased the circulating half-life of rh-Gal A by 2.5-fold, and in knockout mice resulted in up to fivefold higher -Gal A levels and fourfold greater Ansamitocin P-3 GL-3 reduction than rh-Gal A alone. Collectively, these data spotlight the potentially beneficial effects of AT1001 on rh-Gal A, thus warranting clinical investigation. Introduction Fabry disease (OMIM 301500) is an X-linked lysosomal storage disorder (LSD) caused by inherited mutations in the gene (and cellular uptake and tissue uptake of the recombinant enzymes used to treat Gaucher and Pompe diseases, respectively.23,24 Similarly, AT1001 was shown to increase the cellular uptake of rh-Gal A in Fabry patient-derived cells knockout (KO) mice indicate that AT1001 coadministration increases the circulating half-life of rh-Gal A, resulting in significant and Ansamitocin P-3 dose-dependent increases in rh-Gal A levels in disease-relevant tissues. Most importantly, we NCR3 show that AT1001-mediated increases in cellular and tissue levels of rh-Gal A result in greater reduction of GL-3 compared to rh-Gal A alone in patient-derived cells and tissues of KO mice, indicating a boost in the net lysosomal activity from your exogenous recombinant enzyme. Taken together, these data show that AT1001 can increase the stability and improve the pharmacokinetic properties of ERT, thereby leading to greater tissue levels and substrate reduction. As such, a therapeutic approach for Fabry disease that combines a small molecule PC with ERT may warrant clinical investigation. Results AT1001 stabilizes rh-Gal A, preventing denaturation and loss of activity The effect of AT1001 binding around the stability of rh-Gal A was assessed using a fluorescence-based thermal denaturation assay.25 At neutral pH, rh-Gal A was significantly less stable [melting temperature (= 4) versus 48 14?nmol/l (= 4), respectively; 0.05 by = 4 each], but the magnitudes of increase did not reach those seen with either rh-Gal A incubation alone or with rh-Gal A and AT1001 coincubation (Table 1). Open in a separate window Physique 2 AT1001/recombinant human -Gal A (rh-Gal A) coincubation prospects to greater -Gal A levels and activity in Fabry fibroblasts. (a) Fabry fibroblasts expressing C52S -Gal A were incubated with rh-Gal A (0.5?nmol/l) alone or in the presence of increasing concentrations of AT1001 for 5 hours. -Gal A protein levels in cell lysates were measured by western blot 2 days later. The data shown are representative of four impartial experiments (Table 1). (b) C52S Fabry fibroblasts were incubated with rh-Gal A (0.5?nmol/l) alone or in the presence of increasing concentrations of AT1001 (0.1, 1, 10, 100, or 1,000?mol/l) for 5 hours. -Galactosidase A (-Gal A) activity (blue bars) in cell lysates was then measured 2 days later. The data points shown are the mean SEM of four wells tested in parallel from one representative of three independent experiments (Table 1). Globotriaosylceramide (GL-3) levels (red bars) were measured 10 days later. The data points represent mean SEM of three wells tested in parallel from one representative of three independent experiments (Table 1). The arrows indicate 10?mol/l AT1001. (c) C52S Fabry fibroblasts were incubated with increasing concentrations of rh-Gal A alone or in the presence of 1?mmol/l AT1001 for 5 hours. -Gal A activity (blue lines) and GL-3 levels (red lines) in cell lysates were measured 2 Ansamitocin P-3 and 10 days later, respectively. The data points shown are the mean SEM of four wells tested in parallel from one representative experiment. Similar effects of coincubation described in panels a and b were seen in R301Q and L300P fibroblasts (Table 1). * 0.05 compared to baseline; # 0.05 compared to rh-Gal A alone, as.