Discovery of Aromatic Carbamates that Confer Neuroprotective Activity by Enhancing Autophagy and Inducing the Anti-Apoptotic Protein B‑Cell Lymphoma 2 (Bcl-2)

Neurodegenerative diseases share certain pathophysiological hallmarks that represent common targets for drug discovery. In particular, dysfunction of proteostasis and the resultant apoptotic death of neurons represent common pathways for pharmacological intervention. A library of aromatic carbamate derivatives based on the clinically available drug flupirtine was synthesized to determine a structure–activity relationship for neuroprotective activity. Several derivatives were identified that possess greater protective effect in human induced pluripotent stem cell-derived neurons, protecting up to 80% of neurons against etoposide-induced apoptosis at concentrations as low as 100 nM. The developed aromatic carbamates possess physicochemical properties desirable for CNS therapeutics. The primary known mechanisms of action of the parent scaffold are not responsible for the observed neuroprotective activity. Herein, we demonstrate that neuroprotective aromatic carbamates function to increase the Bcl-2/Bax ratio to an antiapoptotic state and activate autophagy through induction of beclin 1

Metabolic abnormalities in <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) Graphs depicting female (left) and male (right) mean body weight data from wild-type (diamonds), heterozygous (squares), and homozygous (triangles) <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice at ages between 11 and 20-weeks are shown (n = 5–10 mice per genotype/sex/age). No significant genotypic differences were observed. Error bars represent SEM. (B) Mean ± SEM rectal body temperatures are shown for male (black bars) and female (gray bars) wild-type (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) littermate mice are shown. Rectal body temperatures, which were measured at rest, were slightly elevated in male and female, heterozygous and homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice, compared to wild-type mice. *, p<0.001 (heterozygous versus wild-type, homozygous versus wild-type). (C) Mean ± SEM values for minimum oxygen consumption (ml/hr) are shown for male (black bars) and female (gray bars) wild-type (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) littermate mice are shown. Minimum oxygen consumption was elevated in male and female heterozygous and homozygous <i>Cln3^Δex7/8</i> mice, compared to wild-type mice. 5–10 mice per group (genotype/sex) were analyzed. *, p<0.001 (heterozygous versus wild-type, homozygous versus wild-type).</p

Vacuolation of diverse cell types in homozygous <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) Representative images are shown of Wright-Giemsa stained peripheral blood smears from <i>Cln3^+/+</i> and <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} littermate mice (scale bar = 10 µm). Note the presence of vacuoles in the cytoplasm of the dark blue stained peripheral blood lymphocyte. (B) Representative images are shown of H&E-stained sections of epididymis from 19-week-old <i>Cln3^+/+</i> and <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} littermate male mice (scale bar = 50 µm). A representative image of a section of mutant (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) epididymis immunostained for vacuolar ATPase (V-ATPase, green) and aquaporin-9 (AQP9, red), which highlight the apical (luminal) membrane of clear/narrow cells or principal cells, respectively (scale bar = 25 µm). (C) Representative TEM images of <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} epididymis cross-sections are shown. Note both the giant vacuoles and the multiple smaller vacuoles filling the cytoplasm of the clear cells. Also note the relative absence of electron-dense material inside the vacuoles. Scale bars, left panel = 10 µm; right panel = 2 µm. (D) Representative images of subunit c immunostained <i>Cln3^+/+</i> and <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} epididymis sections are shown. Asterisks (*) mark some of the large vacuoles. Scale bars = 50 µm. Blood smears and epididymides from at least 10 mice per genotype were analyzed in total, and abnormal vacuolation was observed in all of the <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} mice and in none of the wild-type or <i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>} mice.</p

Summary of neurological and behavioral testing of <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice.

<p>A summary of genotypic differences observed in the neurological and behavioral screens is shown, with male and female results shown separately. Ages at which the indicated tests were performed are also shown.</p

Electroretinography of 16-month-old <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) Scotopic ERG traces are shown for 5-, 9-, and 16-month old wild-type (<i>Cln3^+/+</i>, black trace, n = 7) and homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}, red trace, n = 8) mice. The relative amplitudes of the a-wave do not dramatically differ between the wild-type and homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice. However, the b-wave is drastically reduced in aged homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice, compared to wild-type littermates. Thus, homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice exhibit an electronegative ERG at 16-months of age (b/a ratio = 1, versus b/a ratio = 2.4 in wild-type mice). (B) Photopic ERG traces, reflecting cone response, are shown for 5-, 9-, and 16-month-old wild-type (<i>Cln3^+/+</i>, black trace, n = 7) and homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}, red trace, n = 8) mice. There was a significant genotypic difference in the relative mean amplitudes already at 5 months of age.</p

Subtle genotypic differences in performance of young adult <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice in sensory and motor neurological assays.</b> Shown are results of behavioural analyses in a vertical pole-climbing test (A), prepulse inhibition to the acoustic startle response (PPI) (B), acoustic startle response (C), and thermal nociception (D) for female (left) and male (right) littermate control (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) mice (n = 9–10 mice per group). Data are presented as mean ± standard error of the mean (SEM). (A) Homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> female mice had an increased latency to descend the pole, compared to female wild-type or heterozygous littermates. In a Kruskal-Wallis test, the genotype effect was p<0.01 (*) for females, with or without heterozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice included in the analysis. (B) Mean %PPI to an acoustic startle, with four prepulse intensities (67, 69, 73, 81 decibels [db]), or with all prepulse intensities averaged (‘global’) are shown. *, ANOVA, p<0.05. (C) The mean ± SEM of the acoustic startle response to 70–120 db sounds is shown for littermate control (<i>Cln3^+/+</i>, circles), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}, squares) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}, triangles) <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice. NS = no startle sound. For females, ANOVA, genotype effect was F_(7,11) = 4.63, p<0.05, and post-hoc tests revealed that this was significant at 90 and 100 db (*p<0.05, ***<i>P</i><0.001). No statistically significant differences were detected in the acoustic startle response of males. (D) The mean ± SEM latency to the first sign of pain (seconds = s) in a hot plate assay is shown. *, ANOVA genotype effect p<0.05.</p

Abnormal hematology in peripheral blood from homozygous <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) Mean corpuscular volume (MCV, fL) of peripheral red blood cells from ∼12-week-old mice was measured on an automated analyzer. *, p<0.05, WT and heterozygous mutant mice vs. homozygous mutant mice, unpaired, two-tailed <i>t</i> test. Data shown as mean ± SEM. Percentage of reticulocytes (B) and absolute reticulocyte counts (C) on the specimens analyzed in (A) were determined manually by new methylene blue staining. *, p<0.05, WT and heterozygous mutant mice vs. homozygous mutant mice, unpaired, two-tailed <i>t</i> test. Data shown as mean ± SEM. (D) Linear regression analysis of data from (A) and (C). <i>r²</i> = 0.32, p = 0.02. Datapoints represent individual mice.</p

Heart analysis of <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> (A) The bar graph depicts normalized heart weights for wild-type (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}), and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) littermate 19–20 week old mice. Normalized heart weights represent a ratio of heart weight (mg = milligrams)/body weight (g = grams). Normalized heart weights were slightly increased in heterozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice, and more so in homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice, compared to wild-type littermates. ANOVA analysis suggested a significant genotype effect (p<0.05). (B) Representative micrographs of hematoxylin and eosin (H&E) stained heart sections from wild-type (<i>Cln3^+/+</i>, n = 8) and homozygous (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}, n = 10) littermate 19–20 week old mice are shown, which do not obviously differ from one another in their morphology. Scale bar = 100 µm. (C) Representative micrographs are shown of α-subunit c immunostained heart sections from 19-week old <i>Cln3^+/+</i> and <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} littermate mice. Note the abundance of subunit c-immunopositive deposits in the <i>Cln3^?ex7/8/</i>^{<b>Δ</b><i>ex7/8</i>} section. Only sparse punctate subunit c immunostaining is present in the <i>Cln3^+/+</i> section. Scale bar = 200 µm. Inset scale bar = 25 µm.</p

T cell frequencies in peripheral blood from <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> mice.

<p>The frequencies of T-cells [% T cells (CD45+)], the ratios of CD4+/CD8+ T cells, and the percentage of Ly6c+ cells among the CD8+ and CD4+ T cell populations, determined by flow cytometry, are shown for female and male wild-type (<i>Cln3^+/+</i>), heterozygous (<i>Cln3^+/</i>^{<b>Δ</b><i>ex7/8</i>}), and homozygous (<i>Cln3^Δex7/8/</i>^{<b>Δ</b><i>ex7/8</i>}) littermate mice. p values, determined in a two-tailed, unpaired Student’s t-test of the heterozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> values versus wild-type (<i>Cln3^+/+</i>) values, or homozygous <i>Cln3</i><b>^Δ</b>^<i>ex7/8</i> values versus wild-type (<i>Cln3^+/+</i>) values, are shown. Bold typeface highlights parameters that were significantly different versus wild-type controls. Samples from 9–10 mice per group (genotype/sex) were analyzed, as indicated.</p

Bone marrow analysis of <i>Cln3</i>^Δ

<p>^{<b><i>ex7/8</i></b>}<b> mice.</b> Representative images are shown of Wright-Giemsa-stained bone marrow brush cytology, H&E stained sections of formalin-fixed, paraffin embedded tibias, and iron stained brush cytology, from wild-type (<i>Cln3^+/+</i>) and homozygous mutant (<i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>}) mice (n = 3 mice per genotype). Stained iron appears blue. Note the reduced amount of stained iron in <i>Cln3</i><b>^Δ</b>^{<i>ex7/8/</i><b>Δ</b><i>ex7/8</i>} marrow, compared to wild-type marrow. Arrow, erythroid element; arrowhead, myeloid element; asterisk, megakaryocyte. Scale bars, top and bottom panels = 25 µm; middle panels = 100 µm.</p