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  • During the course of the development of aminothiazole derive

    2022-06-27

    During the course of the development of aminothiazole-derived small molecule GSMs, SGSM-36 (Fig. 1A) was discovered and exhibited good activity for suppressing the production of Aβ42 (Aβ42 IC50=63nM) (Rynearson et al., 2016; Wagner et al., 2014). In addition, SGSM-36 exhibited improved physicochemical properties with respect to the parent compound including good aqueous solubility, as well as acceptable in vivo pharmacokinetic behavior in mice (Wagner et al., 2014). In order to build upon prior studies, a thorough in vivo efficacy analysis of SGSM-36 using Tg2576 mice showed that treatment at 25mg per kg for 3 consecutive days was effective at reducing Canrenone (Fig. 1B–C) and plasma (Fig. 1D) Aβ42 levels and Aβ (42:40) ratios in both brain and plasma (Fig. 1E). In comparison to the parent compound NGP-555 (or compound-4) (Kounnas et al., 2010), a nearly 2-fold superior decrease in brain Aβ42 levels was achieved following treatment with SGSM-36, although there is a greater than six-fold disparity between the in vitro Aβ42 IC50 values of the parent compound and SGSM-36 (Kounnas et al., 2010; Wagner et al., 2014). Furthermore, the parent compound NGP-555 was 2-fold superior to SGSM-36 for reducing plasma Aβ42 levels, suggesting that the highly lipophilic nature of this compound is resulting in sequestration of the drug in the periphery which likely associated with high plasma protein binding thereby limiting the effect in the brain. Additionally, the efficacy data belies the brain to plasma ratio of the parent compound and SGSM-36 (brain:plasma=0.93 and 0.54, respectively), demonstrating that the ratio can be a valuable tool to assess whether a compound can cross the blood-brain barrier but is unlikely to accurately capture the in vivo activity of a given compound. Based on the efficacy data, SGSM-36 exhibits good therapeutic potential, since the overall goal of treatment is to reduce Aβ42 in the brain. Furthermore, SGSM-36 clearly demonstrates that strategies which account for physicochemical properties can be applied to the identification analogs with improved potential. To address target specificity in vivo, samples from Tg2576 mouse brains and cell lysates transfected with Notch constructs were subjected to western blot analysis. Distinct from GSIs, SGSM-36 did not affect the levels of cleaved Notch1 protein in mouse brains (Fig. 3A) and NICD levels in cells (Fig. 3A) indicating a unique mechanism of action that does not interfere with γ-secretase, thereby precluding inhibition related side-effects. Moreover, detection of upstream cleavage products, sAPPα and sAPPβ, shows that SGSM-36 does not impact enzymatic processing of APP by BACE-1 or α-secretase, indicating that the observed shift in the Aβ profile results from modulation of the γ-secretase enzyme (Fig. 2A and B). A comparison of the mode of action of SGSM-36 and semagacestat, a GSI, was also conducted using a CHO cell line stably expressing human APP751 bearing the V717F Indiana mutation. Although the sequence of APP is distinct from the aforementioned in vivo study, SGSM-36 treatment resulted in dose-dependent decreases in Aβ42 and Aβ40, as well as a dose-dependent increase Aβ38, whereas reduction of all three Aβ analyzed was associated with semagacestat treatment (Fig. 2C and D). Consistent with discrete mechanisms of action, SGSM-36 modulates the constitution of the Aβ peptide profile, while semagacestat inhibits enzyme function. Analysis of APP-CTFs further supports the observed activities. SGSM-36 did not influence the levels APP-CTFs; however, the GSI, semagacestat showed dose-dependent accumulation of the γ-secretase substrate (Fig. 2E). The effects of SGSM-36 on Aβ levels in the CHO cell line were validated in our human 3D–differentiated neural cell culture expressing FAD mutations containing APP K670N/M671L (Swedish) and V717I (London) mutations and PSEN1 δE9 mutation (Fig. 2F). The presented characterization of the mechanisms of these pharmacological agents which target γ-secretase serves to distinguish GSMs from GSIs with respect to their impact upon the biological roles of γ-secretase. By avoiding inhibition of the γ-secretase enzyme, SGSM-36 will avoid instigating the side effects of GSIs while still efficaciously impacting the primary biomarker associated with AD, Aβ42. These studies encourage further evaluation of SGSM-36 toward the development of an amyloid directed therapy. The results of this study also warrant investigation of the roles of SGSM-36 on other γ-secretase related activity and proteins, e.g. AICD protein.