Wild-type MC4R, Rm1, and Rm2 were transiently transfected into HEK293 cells, which were stimulated with increasing concentrations of β-MSH <i>(A)</i> or of γ-MSH <i>(B)</i>, and cAMP was measured to generate dose-response curves.

<p>Data shown are mean±s.e.m. of quadruplicate determinations. The dose-response curves are representative of two independent experiments.</p

Wild-type (WT) and mutant MC4Rs were transiently transfected into HEK293 cells, which were stimulated with increasing concentrations of α-MSH <i>(A)</i> or of THIQ <i>(B)</i>, and cAMP was measured to generate dose-response curves.

<p>Data shown are mean±s.e.m. of quadruplicate determinations. The dose-response curves are representative of at least three independent experiments. The amino acid sequence of α-MSH and the chemical structure THIQ are shown below panels A and B, respectively.</p

HEK293 cells stably expressing wild-type MC4R <i>(A)</i>, Rm1 (L106P) <i>(B),</i> and Rm2 (D122A) <i>(C)</i> were exposed to increasing concentrations of α-MSH or THIQ for 2 h.

<p>Cell-surface FLAG-tagged receptors were measured by ELISA. Receptor sequestration was calculated as the loss of receptor expression from the cell surface. Wild-type MC4R was internalized significantly more (<i>p</i><0.05) in response to α-MSH than THIQ at doses of 10⁻⁷ M and higher. Data represent the mean±s.e.m. of two independent experiments, each performed in quadruplicate.</p

Cell-surface expression levels of wild-type and mutant MC4R were measured in transiently transfected HEK293 cells by ELISA.

<p>The prolactin signal sequence was added to the N-terminal domain of these receptors to ensure maximal membrane expression. MC4R mutation C271Y from TM6/EL3 significantly reduced membrane expression (<i>p</i><0.05) and was used as a control. Empty vector (pcDNA3.1) was transfected as a negative control to detect background.</p

Functional characterization of MC4R EL1 mutations.

<p>Values are mean±s.e.m. (<i>n</i>≥3).</p>a<p>EC50 could not be determined. WT, wild-type.</p

Discovery of (<i>R</i>)‑4-Cyclopropyl-7,8-difluoro-5-(4-(trifluoromethyl)phenylsulfonyl)-4,5-dihydro‑1<i>H</i>‑pyrazolo[4,3‑<i>c</i>]quinoline (ELND006) and (<i>R</i>)‑4-Cyclopropyl-8-fluoro-5-(6-(trifluoromethyl)pyridin-3-ylsulfonyl)-4,5-dihydro‑2<i>H</i>‑pyrazolo[4,3‑<i>c</i>]quinoline (ELND007): Metabolically Stable γ‑Secretase Inhibitors that Selectively Inhibit the Production of Amyloid‑β over Notch

Herein, we describe our strategy to design metabolically stable γ-secretase inhibitors which are selective for inhibition of Aβ generation over Notch. We highlight our synthetic strategy to incorporate diversity and chirality. Compounds <b>30</b> (ELND006) and <b>34</b> (ELND007) both entered human clinical trials. The in vitro and in vivo characteristics for these two compounds are described. A comparison of inhibition of Aβ generation in vivo between <b>30</b>, <b>34</b>, Semagacestat <b>41</b>, Begacestat <b>42</b>, and Avagacestat <b>43</b> in mice is made. <b>30</b> lowered Aβ in the CSF of healthy human volunteers