An analog of glibenclamide selectively enhances autophagic degradation of misfolded α1-antitrypsin Z

The classical form of α1-antitrypsin deficiency (ATD) is characterized by intracellular accumulation of the misfolded variant α1-antitrypsin Z (ATZ) and severe liver disease in some of the affected individuals. In this study, we investigated the possibility of discovering novel therapeutic agents that would reduce ATZ accumulation by interrogating a C. elegans model of ATD with high-content genome-wide RNAi screening and computational systems pharmacology strategies. The RNAi screening was utilized to identify genes that modify the intracellular accumulation of ATZ and a novel computational pipeline was developed to make high confidence predictions on repurposable drugs. This approach identified glibenclamide (GLB), a sulfonylurea drug that has been used broadly in clinical medicine as an oral hypoglycemic agent. Here we show that GLB promotes autophagic degradation of misfolded ATZ in mammalian cell line models of ATD. Furthermore, an analog of GLB reduces hepatic ATZ accumulation and hepatic fibrosis in a mouse model in vivo without affecting blood glucose or insulin levels. These results provide support for a drug discovery strategy using simple organisms as human disease models combined with genetic and computational screening methods. They also show that GLB and/or at least one of its analogs can be immediately tested to arrest the progression of human ATD liver disease.</div

Rescue of the mild Egl phenotype shown by <i>kcnl-2</i>(<i>tm1885</i>) worms.

<p>A. <i>kcnl-2</i>(tm1885) animals retain a significantly greater number of eggs <i>in utero</i> relative to N2 animals (P<0.0001). The number of eggs <i>in utero</i> in heterozygotes (<i>kcnl-2</i>(-/+)) is significantly greater than that of the N2 animals (p=0.007), but significantly less than the average number of eggs found in <i>kcnl-2</i>(tm1885) animals (p=0.008). B. Transformation of <i>kcnl-2</i>(tm1885) with 1 ng/µl, 10 ng/µl or 90 ng/µl of <i>p</i>_{<i>kcnl-2</i>}<i>kcnl-2</i>(taa2)<i>::gfp</i> gives the transgenic lines KCNL-2(OE-3) (strain VK2220), KCNL-2(OE-4) (strain VK1004) and KCNL-2(OE-5) (strain VK1041), respectively. The average number of eggs retained <i>in utero</i> by KCNL-2(OE-3) was not significantly different from the N2 animals (P=0.8) but was significantly different from the <i>kcnl-2</i>(tm1885) organisms (p<0.0001). KCNL-2(OE-4) and KCNL-2(OE-5) retain fewer eggs <i>in utero</i> than N2 animals (p<0.005; p<0.0001, respectively) and <i>kcnl-2</i>(tm1885) animals (p<0.0001) (Student’s <i>t</i> test). C. Egg-staging assays show that the proportions of young eggs from KCNL-2(OE-2) (54.0±4.3%), KCNL-2(OE-4) (46.0±3.9%), and KCNL-2(OE-5) (46.6±6.7%) are significantly greater than those of N2 (7.2±1.3%) & <i>kcnl-2</i>(tm1885) worms (1.6±1.6%) (p<0.05; n=3, Kruskal–Wallis <i>H</i> test).</p

Phenotypic analysis of <i>kcnl-2</i>(<i>tm1885</i>) and KCNL-2(OE), a transgenic line that overexpresses <i>p</i>_{<i>kcnl-2</i>}<i>kcnl-2</i>(<i>taa2</i>)<i>::gfp</i> in the N2 background.

<p>A,C. Kaplan-Meier Survival Curves showing the longevities of N2 vs. <i>kcnl-2</i>(tm1885) (n=80; p=0.06, Logrank test) or N2 vs. KCNL-2(OE-2) (n=50; p=0.12, Logrank test). B,D. Brood size of N2 vs. <i>kcnl-2</i>(tm1885) (n≥17; p<0.001, Student’s <i>t</i> test) or N2 vs KCNL-2(OE-2) (n≥13; p=0.77, Student’s <i>t</i> test).</p

Confocal fluorescent images showing the expression pattern of the KCNL-2 promoter-GFP constructs.

<p>A–C. <i>p</i>_{<i>kcnl-2</i>(<i>atg1</i>)}<i>gfp</i> is expressed in neurons of the head and NR (A), the vulval muscles (B), and tail ganglia (C) (strain <i>vk1323</i>). D–F. <i>p</i>_{<i>kcnl-2</i>(<i>atg2</i>)}<i>gfp</i> is expressed in head neurons and NR (D); VNC, VC4 & VC5, and DC (E); and tail ganglia (F) (strain <i>vk1327</i>).</p

Structural analysis of KCNL-2 isoforms.

<p>A. The WRM063DE08 fosmid encodes six isoforms of KCNL-2, which vary in their amino and carboxy termini of the predicted protein structure. The exon structures show that there are two initiation sites that are 9.5 kb apart and two stop codons that are 68 bp apart. B) KCNL-2 isoforms have 37% identity with K _Ca2.2, while KCNL-2-aii and -b are 71% identical. The greatest degree of conservation occurs in the core of the channel where the S1-S6 transmembrane domains (underlined solid), the potassium-selective pore filter (underlined dotted), and the calmodulin binding domain (underlined dashed) are encoded. C) <i>kcnl-2</i>(tm1885), a 962 bp deletion, results in a premature stop codon before the first TMD of KCNL-2-aii and -b. D) Kyte-Doolittle hydropathy plot of the KCNL-2-a isoform showing seven domains that are highly hydrophobic, which represent the 6 TMDs and the pore motif (dashed line).</p

Overexpression of KCNL-2 in the N2 background causes a Hyperactive Egg-Laying Phenotype.

<p>A. An unlaid egg assay was carried out by dissolving the cuticle of adult worms in 1.8% sodium hypochlorite and shows that the number of eggs <i>in utero</i> in transgenic lines that overexpress KCNL-2 are significantly less than the number of eggs retained <i>in utero</i> in N2 and <i>kcnl-2</i>(tm1885) strains (p<0.001, Student’s <i>t</i> test), while <i>kcnl-2</i>(tm1885) has a significantly increased number of eggs retained <i>in utero</i> relative to N2 worms (p<0.001, Student’s <i>t</i> test). B) Representative images of worms from each strain. C) Egg-staging assays reveal that the proportion of young eggs from KCNL-2(OE-1) (strain VK1000) (54.82±7.65%) and KCNL-2(OE-2) (strain VK1065) (60.57±12.48%) was significantly greater than those of N2 worms (2.78±4.81%) (n=3, p<0.05; Kruskal–Wallis <i>H</i> test).</p

Expression pattern of KCNL-2.

<p>A. The <i>kcnl-2</i> gene was amplified from the WRM063DE08 fosmid and GFP was fused to the latter stop codon to produce a translational gene fusion that tagged the channel at the carboxy terminus. <i>p</i>_{<i>kcnl-2</i>}<i>kcnl-2</i>(taa2)<i>::gfp</i> localized to the neurons of the head, the NR, motor neurons of the VNC, the DC, the pharyngeal nervous system, and to tail ganglia (widefield fluorescent image of strain VK1567). B–G. Magnified confocal images of <i>p</i>_{<i>kcnl-2</i>}<i>kcnl-2</i>(taa2)<i>::gfp</i> (B–D) and <i>p</i>_{<i>kcnl-2</i>}<i>gfp</i> :<i>:</i>(atg2) <i>kcnl-2</i> (E-G; strain VK1401) when expressed in the N2 background (Scale bar=10 µm). B,E. Neurons of the head, the pharyngeal nervous system, and cell bodies of the nerve ring. C. Neuronal processes innervating the vulva. F. Neuronal processes at the vulva, including the VC4 & VC5 cell bodies (arrows). D,G. Tail ganglia.</p

SERPINB12 Is a Slow-Binding Inhibitor of Granzyme A and Hepsin

The clade B/intracellular serpins protect cells from peptidase-mediated injury by forming covalent complexes with their targets. SERPINB12 is expressed in most tissues, especially at cellular interfaces with the external environment. This wide tissue distribution pattern is similar to that of granzyme A (GZMA). Because SERPINB12 inhibits trypsin-like serine peptidases, we determined whether it might also neutralize GZMA. SERPINB12 formed a covalent complex with GZMA and inhibited the enzyme with typical serpin slow-binding kinetics. SERPINB12 also inhibited Hepsin. SERPINB12 may function as an endogenous inhibitor of these peptidases

Immunoblots of AT variant transgenic lines.

<p>Western blot analysis of lysates from animals expressing different AT variant transgenes. The steady-state levels of WT and mutant sGFP::AT fusion proteins under denaturing conditions (A, <i>upper panel</i>). Actin serves as a loading control (A, <i>lower panel</i>). Relative AT protein levels as determined by densitometry (B). Statistical significance was determined using two-tailed student's <i>t</i>-test, comparing each AT variant line to ATM. Relative mRNA levels as determined by qPCR (C). Statistical significance was determined using two-tailed student's <i>t</i>-test, comparing each AT variant line to ATM. Lysates from null mutants sGFP::Saar and sGFP::NHK, exposed to <i>vector(RNAi)</i> or <i>sel-1(RNAi)</i> probed with GFP (D, <i>upper panel</i>) or α-tubulin (D, <i>lower panel</i>).</p

Effect of AT variant protein expression on longevity.

<p>Representative Kaplan-Meier curves of deficient mutants (A). N2 (<i>black</i>), ATM (<i>green</i>), Siiyama (<i>red</i>), Mmalton (<i>blue</i>) and ATZ (<i>lavender</i>). Median survival times (in <i>parenthesis</i>). Representative Kaplan-Meier curves of ATS and null alleles (B). N2 (<i>black</i>), ATS (<i>green</i>), NHK (<i>red</i>) and Saar (<i>blue</i>). Statistical significance determined using the Log-Rank (Mantel-Cox) test, with probabilities of results reported as **<i>P</i><0.001, or ***<i>P</i><0.0001.</p