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. 2022 May 16;11:e72512. doi: 10.7554/eLife.72512

Figure 3. Stereospecific methyl additions onto the pyrrolidine of lasofoxifene (Laso) impact estrogen receptor alpha (ERα) levels in T47D breast cancer cells.

(A) Chemical structures of Laso and the synthesized stereospecific methyl derivatives. (B) Dose-response curves of hormone (E2) alongside LASOLaso and derivatives after 24 hr treatment for WT halo-ERα. (C) Dose-response curves of chirally purified LA3 and LA5 alongside E2 and 4-hydroxytamoxifen (4OHT) for WT halo-ERα. LA3/5-1 and LA3/5-2 represent the first and second major peaks separated by chiral affinity chromatography. (D/E) Dose-response curves of LA-Deg and LA-Stab for Y537S and D537G halo-ERα after 24 hr compared to E2, Laso, and 4OHT. (F–H) TMR fluorescence measured every 4 hr in T47D breast cancer cells with WT halo-ERα (F), Y537S (G), and D538G (H) treated over 100 hr at 1 μM LA-Stab or LA-Deg following induction of expression. Data are shown as the mean of two biological replicates ± SD.

Figure 3.

Figure 3—figure supplement 1. Chiral separation of 3 R (LA-Deg) and 2S-methylpyrrolidine (LA-Stab) lasofoxifene derivatives.

Figure 3—figure supplement 1.

(A) Chromatogram of LA-Deg chiral separation. (B) Purification of the LA-Deg peak 1. (C) Purification of LA-Deg peak 2. (D) Chromatogram of LA-Stab chiral separation. (E) Purification of LA-Stab peak 1. (F) Purification of LA-Stab peak 2.
Figure 3—figure supplement 2. In-cell western of T47D breast cancer cells treated with lasofoxifene (Laso).

Figure 3—figure supplement 2.

(A), LA-Stab (B), or LA-Deg (C) for 24 hours. Data shown are the mean of 3 replicates ± SD.