Brief Communication

Nature Chemical Biology 3, 99 - 100 (2006)
Published online: 31 December 2006 | doi:10.1038/nchembio852

Selective inhibitors of death in mutant huntingtin cells

Hemant Varma1, Cindy Voisine2, C Todd DeMarco3, Elena Cattaneo4, Donald C Lo3, Anne C Hart2 & Brent R Stockwell1,5

Huntington disease (HD) is an inherited neurodegenerative disorder with unclear pathophysiology. We developed a high-throughput assay in a neuronal cell culture model of HD, screened 43,685 compounds and identified 29 novel selective inhibitors of cell death in mutant huntingtin–expressing cells. Four compounds were active in diverse HD models, which suggests a role for cell death in HD; these compounds are mechanistic probes and potential drug leads for HD.

Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by polyglutamine expansion in a stretch of glutamine repeats in the huntingtin (htt) protein1. Mutant htt (>35 glutamine repeats) has been proposed to cause toxicity by multiple mechanisms2 (Fig. 1a). However, a clear understanding of how these mechanisms contribute to HD remains elusive. Striatal neuronal cell loss is ubiquitous in HD-afflicted individuals1. Though the role for cell death in HD pathophysiology is debated1, prevention of neuronal death can ameliorate disease phenotype in mouse models3, 4. However, the mechanisms by which mutant htt perturbs the cell death machinery are unclear. To address the role of mutant htt in cell death, we devised a screen to identify small molecules that selectively reverse cell death in a mutant htt–expressing neuronal cell line. This approach could potentially identify (i) chemical probes for elucidating the mechanisms underlying mutant htt toxicity and (ii) potential drug leads.

Figure 1: Novel compounds selectively suppress cell death and caspase-3 activation in N548 mutant cells.

Figure 1 : Novel compounds selectively suppress cell death and caspase-3 activation in N548 mutant cells.

(a) Diagram of multiple mechanisms implicated in HD. (b) Structures of compounds R1R1, R2R2, R3R3 and R4R4. R1R1, R2R2 and R3R3 (colored blue) are structurally related, whereas R4R4 (red) is structurally distinct. (c) Dose-response curves of nonselective rescue by the pan-caspase inhibitor Boc-D-FMK (R30R30) (Supplementary Methods). Viability is shown as fold increase above DMSO-treated cells. Error bars represent one s.d. (d) Selective rescue of N548 mutant cells by R1R1 (see Supplementary Fig. 3 for dose responses for R2R2, R3R3 and R4R4). (e) Effect of R1R1 and R4R4 on cleavage of caspase-3 and caspase-7 induced by serum-deficient medium for 24 h; DMSO vehicle (–) and 10% serum (ser) were controls (see Supplementary Fig. 4 for data on R2R2 and R3R3).

Full size image (91 KB)

We chose a cell culture HD model that uses immortalized striatal neurons (ST14A). ST14A cells were stably transfected with the N-terminal 548-amino-acid fragment of mutant (120 glutamine repeats) human htt to generate the N548 mutant cell line5 (Supplementary Methods online). In serum-deficient medium at 39 °C, N548 mutant cells undergo cell death at a rate greater than that of the parental ST14A5. We developed a high-throughput screening assay for detecting N548 mutant cell death in a 384-well plate format (Supplementary Methods). We screened 43,685 compounds and identified 29 novel compounds, designated R1R1 through R29R29 (Fig. 1b and Supplementary Table 1 online), that selectively rescue N548 mutant cell viability, without rescuing cell viability in ST14A cells. We confirmed the enhanced viability by two additional viability criteria (Supplementary Methods).

To prioritize hits for further study, we subjected these 29 compounds to additional filters (Supplementary Fig. 1 online). We excluded a decrease in mutant htt protein expression upon treatment with selective hits as a mechanism for selective suppression of cell death in N548 mutant cells (Supplementary Fig. 2 online). Most compounds rescued cell death in at least one additional striatal cell line expressing a distinct mutant htt construct (N63 or full-length htt)5, thereby assuring us of selectivity for mutant htt–expressing cells relative to parental ST14A cells. We tested all compounds for rescue of mutant htt–induced phenotype in rat pheochromocytoma (PC12)6, yeast7 and Caenorhabditis elegans8 HD models (results in Supplementary Table 2 online). Four new compounds (Fig. 1b) were active in multiple HD models (Table 1 and Supplementary Fig. 3 online); of these four, R1R1, R2R2 and R3R3 are structurally related. We next tested these four compounds in an in vivo–like brain slice HD model (Supplementary Methods). R1R1 and R2R2 rescued degeneration in this HD model, whereas R3R3 and R4R4 were inactive (Table 1 and Supplementary Fig. 3).


Because these compounds were selective for cells expressing mutant htt, we used them to gain insight into the mechanisms of mutant htt toxicity. Caspases have been implicated in htt toxicity, both as targets activated by mutant htt9 and as regulators of mutant toxicity that act by cleaving mutant htt to generate toxic htt fragments10. We found that caspase activation contributes to cell death in both ST14A and N548 mutant cells (Fig. 1c and Supplementary Fig. 4 online). We tested the possibility that mechanisms leading to caspase activation differ between N548 mutant and ST14A cells. Both R1R1 and R4R4 selectively inhibited cleavage of caspase-3, but not caspase-7, in a dose-dependent manner; inhibition of caspase-3 cleavage correlated with selective rescue of N548 mutant cells by these compounds (Fig. 1d,e). These results, together with reports of enhanced caspase-3 processing in N548 mutant cells5 and increased caspase-3 expression in the R6/2 HD mouse model9, suggest a role for aberrant caspase-3 activation in HD.

As these compounds were active in multiple HD models, we also assessed their potential as drug leads. R1R1, R2R2 and R4R4 passed in silico medicinal chemistry filtering, which suggests adequate oral bioavailability (Supplementary Table 3 online). We analyzed the structure-activity relationship by testing 57 analogs of R1R1 and 23 analogs of R4R4; we identified several active analogs for both scaffolds (Supplementary Table 3). This information will be useful for synthesizing more potent analogs. These compounds are potential drug leads and should be tested in other HD models and in models of other polyglutamine diseases11.

Given that these compounds were active in different assays, they may target mechanisms of mutant htt toxicity that are conserved across the models tested. Considering the undefined mechanisms involved in neuronal loss in HD, these compounds should be valuable tools for investigating the mechanisms by which mutant htt affects caspases and induces neuronal loss. Furthermore, the assays and hit prioritization strategies described should be useful for identifying other HD drug leads.

Note: Supplementary information and chemical compound information is available on the Nature Chemical Biology website.

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Author contributions

H.V. and B.R.S. designed the screen and wrote the manuscript. E.C. provided essential reagents, cell lines and advice. H.V. performed the high-throughput screen and tested compounds in PC12 and yeast HD models. H.V., C.V. and A.C.H. designed the C. elegans assay; H.V. and C.V. performed the C. elegans assay. C.T.D. and D.C.L. designed the brain slice assay, C.T.D. tested compounds in the assay and D.C.L. performed analysis of the data.



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Acknowledgments

We gratefully acknowledge the support of the High Q Foundation (H.V.), the Hereditary Disease Foundation (C.V. and B.R.S.), Cure Huntington Disease Initiative (B.R.S. and D.C.L.), Fondazione Telethon Onlus (E.C.) and the US National Institutes of Health (A.C.H.). B.R.S. is supported in part by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.

Competing interests statement:

The authors declare no competing financial interests.

Received 15 September 2006; Accepted 6 December 2006; Published online 31 December 2006.

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  1. Department of Biological Sciences, Columbia University, Fairchild Center, MC 2406, 1212 Amsterdam Avenue, New York, New York 10027, USA.
  2. Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Department of Pathology, Charlestown, Massachusetts 02129, USA.
  3. Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, 4321 Medical Park Drive, Suite 200, Durham, North Carolina 27704, USA.
  4. Department of Pharmacological Sciences, University of Milano, Via Balzaretti 9, Milano 20133, Italy.
  5. Department of Chemistry, Columbia University, New York, New York 10027, USA.

Correspondence to: Brent R Stockwell1,5 e-mail: stockwell@biology.columbia.edu

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Selective inhibitors of death in mutant huntingtin cells

Nature Chemical Biology Brief Communication