Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2022 Feb 16;13(3):396–402. doi: 10.1021/acsmedchemlett.1c00500

Conversion of a PROTAC Mutant Huntingtin Degrader into Small-Molecule Hydrophobic Tags Focusing on Drug-like Properties

Keigo Hirai †, Hiroko Yamashita ‡, Shusuke Tomoshige †,*, Yugo Mishima †, Tatsuya Niwa §, Kenji Ohgane ‡, Mayumi Ishii ∥, Kayoko Kanamitsu ∥, Yui Ikemi ⊥, Shinsaku Nakagawa ⊥, Hideki Taguchi §, Shinichi Sato †,#, Yuichi Hashimoto ‡, Minoru Ishikawa †,*
PMCID: PMC8919385  PMID: 35300080

Abstract

graphic file with name ml1c00500_0008.jpg

The onset of neurodegenerative disorders (NDs), such as Alzheimer’s disease, is associated with the accumulation of aggregates of misfolded proteins. We previously showed that chemical knockdown of ND-related aggregation-prone proteins can be achieved by proteolysis targeting chimeras (PROTACs). However, hetero-bifunctional PROTACs generally show poor permeability into the central nervous system, where NDs are located. Here, we document the conversion of one of our PROTACs into hydrophobic tags (HyTs), another class of degraders bearing hydrophobic degrons. This conversion decreases the molecular weight and the number of hydrogen bond donors/acceptors. All the developed HyTs lowered the level of mutant huntingtin, an aggregation-prone protein, with potency comparable to that of the parent PROTAC. Through IAM chromatography analysis and in vivo brain penetration assay of the HyTs, we discovered a brain-permeable HyT. Our results and mechanistic analysis indicate that conversion of protein degraders into HyTs could be a useful approach to improve their drug-like properties.

Keywords: PROTACs, hydrophobic tagging, protein degradation, neurodegenerative disorders, drug-like properties

Neurodegenerative disorders (NDs), represented by Alzheimer’s disease, Parkinson’s disease, and various polyglutamine (polyQ) diseases, are an array of progressive diseases causing impairments of motility and/or cognitive functions. Several NDs occur in association with the accumulation of aggregation-prone misfolded proteins, e.g., amyloid β in Alzheimer’s disease, α-synuclein in Parkinson’s disease, and mutant proteins with abnormally extended polyQ repeats in polyQ diseases.1 These proteins form β-sheet-rich structures and aggregate into insoluble fibrils via cytotoxic soluble oligomeric intermediates. Removal of such aggregation-prone proteins is considered to be a promising approach for the treatment of NDs.2 However, because the misfolded proteins form aggregates independently of their intrinsic functions, conventional drug discovery strategies targeting chemical modulators of the functions of disease-related proteins is not effective. For this reason, novel approaches are needed to develop definitive small-molecule treatments for NDs.3

In recent years, various chemical protein knockdown technologies to lower the levels of target proteins have emerged. In almost all cases, the concept of hybrid molecules with a dual mode of action4 is employed, and the most developed modality so far is hetero-bifunctional proteolysis targeting chimeras (PROTACs), which induce degradation of target proteins mediated by the ubiquitin-proteasome system (UPS).5 PROTACs are hetero-bifunctional molecules comprised of a ligand of a protein of interest (POI) linked to a ligand of ubiquitin ligase (E3); thus, PROTACs serve to bring the POI and E3 into close proximity, thereby inducing ubiquitination and subsequent proteasomal degradation of the POI. In the early phase of PROTAC development, our group developed a subclass of small-molecule PROTACs harnessing inhibitor of apoptosis (IAP) E3 ligase, and we termed these compounds specific and nongenetic IAP-dependent protein erasers (SNIPERs).6−8 While PROTACs/SNIPERs have already attracted great attention in cancer therapy, our group and other groups have also proposed a strategy for the treatment of NDs by applying PROTACs/SNIPERs to aggregation-prone proteins such as mutant huntingtin (mHTT), mutant ataxin-3, mutant ataxin-7, mutant atrophin-1, and tau (Figure 1).9−13

Figure 1.

Figure 1

Open in a new tab

Structures of small-molecule protein degraders of aggregation-prone proteins. The degraders 1, 2, and QC-01-175 are hetero-bifunctional molecules, each composed of a protein aggregate binder and a ligand for E3. These compounds serve to bring protein aggregates and E3 into close proximity, leading to ubiquitination and proteasomal degradation of the aggregates.

Because of the lack of specific small-molecule ligands for ND-related aggregation-prone proteins, the PROTACs/SNIPERs used in this strategy exploit aggregate binders as POI ligands. Aggregate binders are small molecules first used as positron emission tomography probes to detect protein aggregates in patients with NDs, via interaction with the β-sheet-rich structure of the aggregates.14 We have shown that SNIPERs 1 and 2 interact with aggregates of 62-repeat glutamine polypeptide and treatment with 1 and 2 at 10 μM lowered the level of endogenous mHtt in fibroblasts from a Huntington’s disease (HD) patient.9 Furthermore, fluorescent-protein-fused mHtt expressed in living cells is known to form aggregates that appear as bright foci in fluorescence-microscopic images, and the number of the foci was decreased following treatment with 1 at 10 μM. However, this modality is not ideal from the viewpoint of central nervous system (CNS) drug discovery, because the hetero-bifunctional structure results in violations of Lipinski’s rule of 5, such as the fairly high molecular weight and large number of hydrogen bond donors/acceptors (HBDs/HBAs).15 In addition, this low drug-likeness is suggested to influence the degradation-inducing activity of PROTACs.16 Hydrophobic tagging technology is another class of chemical knockdown technologies, which harnesses the heat shock protein 70 kDa (HSP70)/C-terminus of HSC70-interacting protein (CHIP) protein quality control machinery to decrease the level of a POI (Figure 2).17,18

Figure 2.

Figure 2

Open in a new tab

Protein quality control machinery and mode of action of HyTs. HyTs are hetero-bifunctional molecules consisting of POI ligands linked to small, hydrophobic degrons. These degron moieties function as mimics of the exposed hydrophobic regions of misfolded proteins, which are recognized by the HSP70/CHIP machinery, leading to proteasomal degradation.

This technology uses molecules termed hydrophobic tags (HyTs), consisting of a POI ligand linked to a hydrophobic degron that mimics the exposed hydrophobic region of misfolded proteins and is recognized by HSP70. Since the hydrophobic degron structures in HyTs generally have lower molecular weight than the E3 ligands employed in PROTACs/SNIPERs and have no HBDs/HBAs, we hypothesized that the application of HyTs would improve the CNS drug-like properties of our SNIPERs (Figure 3).

Figure 3.

Figure 3

Open in a new tab

Conversion of our SNIPER into HyTs. Since the degron moieties of HyTs are small and contain no HBAs/HBDs, improved drug-like properties are expected. Numbers of HBDs/HBAs were calculated by ChemOffice Excel add-in.

Herein, we report the discovery of small-molecule HyTs that induce the degradation of mHtt. The brain permeability properties of these HyTs predicted in vitro are superior to those of the parent SNIPER.

To achieve both degradation of protein aggregates and high brain permeability, we designed and synthesized HyT 3 (Figure 4A, Scheme S1). In this design, the E3 ligand moiety of 2 was replaced with an adamantyl group, a well-established hydrophobic degron, while the aggregate binder moiety was retained. We then evaluated the aggregate-degradation-inducing activity, using mHtt as a target aggregation-prone protein. HeLa cells transiently expressing C-terminally EGFP-fused exon 1 products of HTT containing a much extended 145-CAG repeat (mHtt exon 1-Q145-EGFP) were treated with HyT 3, and the level of intracellular mHtt exon 1-Q145 was evaluated by Western blotting and measuring the GFP fluorescence intensity of lysates (Figures 4B and S1A). We found that treatment with 10 μM HyT 3 for 24 h or longer decreased the level of intracellular mHtt exon 1-Q145-EGFP. HyT 3 also decreased the endogenous mHtt level in fibroblasts derived from a patient with HD in a dose-dependent manner (Figure S1B).

Figure 4.

Figure 4

Open in a new tab

Hydrophobic tagging strategy targeting protein aggregates. (A) Chemical structure of HyT 3. (B) HeLa cells transiently expressing mHtt exon 1-Q145-EGFP were treated with DMSO or HyT 3 at the indicated concentrations for 48 h.

In our previous study, the reduction of number of aggregates by SNIPER 1 was confirmed by fluorescence microscopy, based on the fact that mHtt exon 1-Q145 forms intracellular bright foci in fluorescence-microscopic images. Therefore, we examined the effect of HyT 3 on the number of aggregates by counting the number of bright foci in cells (Figure 5). The number of foci was decreased by approximately 60% after 24 h treatment with 10 μM HyT 3.

Figure 5.

Figure 5

Open in a new tab

HyT 3 decreases mHtt aggregates in cells. HeLa cells transiently expressing mHtt exon 1-Q145-EGFP were treated with DMSO or 10 μM HyT 3 and incubated for 24 h. Fluorescence microscopy images were captured, and aggregates of mHtt exon 1-Q145-EGFP (the number of fluorescent foci with fluorescence intensity above the threshold) in the cells were counted and normalized by the area of Hoechst33342 staining. Bars in the left panel indicate the mean ± SD of three individual experiments. Asterisks indicate a significant difference (**p < 0.01) from DMSO treatment according to the unpaired, two-sided Student’s t test. The two images on the right are representative microscopic images. Fluorescence signals in these images were colored and adjusted to improve visibility. Original images are included in the Supporting Information (Figure S2).

Next, we evaluated the influence of the HyT structure on the degradation-inducing activity against aggregation-prone protein by designing and synthesizing a series of derivatives of HyT 3 having various hydrophobic degrons (Figure 6A, Schemes S2–S5). To our knowledge, this is the first time that 6-undecanyl, 1-naphthyl, and 1-diamantyl, incorporated in 4, 5, and 7, respectively, have been employed as hydrophobic degrons for HyTs. We also designed and synthesized HyT 3′, an HyT 3 derivative with shorter linker, and HyTs 8 and 9 bearing an aliphatic linker instead of an ethylene glycol linker (Figure 6A, Scheme S6). We evaluated the degradation-inducing activity of these HyTs against mHtt exon 1-Q145-EGFP by measuring the GFP fluorescence intensity of lysates. We confirmed that HyTs 4–6 reduced mHtt exon 1-Q145-EGFP levels but these compounds required higher concentrations to show activity (Figure 6B). On the other hand, HyT 7 decreased the mHtt exon 1-Q145-EGFP level to 60% at 5 μM, whereas no activity was observed when HyT 3 was used at the same concentration, suggesting that the diamantyl group is a potent hydrophobic degron for HyTs; however, we could not evaluate its activity at higher concentrations due to its cytotoxicity. In addition, HyT 3′ also showed the activity to the same extent as that of HyT3. We could not evaluate the activity of HyTs 8 and 9 because of their poor aqueous solubility. These results indicate that the efficacy of HyTs depends on the rigidity and bulkiness of the hydrophobic degron structure. Linker modification revealed that shortening the linker length of HyT 3 tolerates its activity and the aliphatic linker confers too much hydrophobicity on HyTs.

Figure 6.

Figure 6

Open in a new tab

Hydrophobic tagging strategy targeting protein aggregates. (A) Chemical structures of HyT 4–9 and 3′. (B) HeLa cells transiently expressing mHtt exon 1-Q145-EGFP were treated with DMSO or HyTs 3–7 and 3′ at the indicated concentrations for 24 h. The amount of mHtt exon 1-Q145-EGFP was evaluated by measuring the GFP intensity of the lysate and normalized with the protein concentration of each lysate. Data are the mean ± SD of more than three individual experiments.

Next, the degradation mechanism of HyT 3 was examined. As the first step, we examined the importance of conjugation between the aggregate binder and hydrophobic degron. The cells were treated with aggregate binder 10, adamantane derivative 11, a combination of compounds 10 and 11, or HyT 3 (Figure 7A). As shown in Figure 7B, no significant degradation was observed when the cells were treated with 10, 11, or their combination, while HyT 3 clearly reduced the amount of mHtt exon 1-Q145-EGFP. This result shows that conjugation between the aggregate binder and the hydrophobic degron is essential for the activity, supporting our mechanistic hypothesis. Next, we confirmed the involvement of proteasome in the activity of HyT 3 by demonstrating that the proteasome inhibitor bortezomib blocked the activity of HyT 3 (Figure 7C). As described above, HSP70 is also involved in the proteolysis induced by HyTs. Therefore, we also confirmed the involvement of HSP70 in the aggregate-degradation activity of our HyTs (Figure 7D). Geldanamycin (GA), a specific inhibitor of HSP90, was used as an HSP70 activator, because GA is known to stabilize the transcriptional factor heat shock factor 1, an HSP90 client protein, leading to enhancement of HSP70 transcription.19−21 HeLa cells were preincubated with GA for 6 h and then treated with HyT 3, and the influence of GA on HyT 3 activity was evaluated by measuring the GFP fluorescence intensity of cell lysates (Figure 7D). Indeed, GA increased the expression level of HSP70 (Figure S3), and we found that the activity of HyT 3 was enhanced in the presence of GA. These results indicate that HSP70 is involved in the degradation-inducing activity of HyT 3. We also examined whether the degradation-inducing activity of HyTs is mHtt-specific. HeLa cells transiently expressing Htt exon 1-Q23-EGFP were treated with HyT 3 (note that this length of polyQ repeats is less than the minimum pathogenic threshold of 36–40 repeats). We measured the Htt exon 1-Q23-EGFP level by Western blotting, and found no significant decrease (Figure S4). In addition, a quantitative proteomic analysis of 3′-treated cells vs control cells showed no significant changes in the abundances of most of the detected other proteins (Figure S5). These results indicate that degradation-inducing activity of HyT 3 and 3′ would be specific for mHtt. For more details on the proteome-wide analysis, see the list of significantly decreased/increased proteins in Figure S5. All the decreased proteins in the list are likely contaminants. As for significantly increased proteins in 3′-treated cell, we noticed a trend that proteins involved in transcription and translation accounted for more than one-third. However, precise mechanisms by which the abundances of these proteins are increased by HyT 3′ are unknown. Although a few proteins associated with apoptosis, HyT 3′ is not cytotoxic. Increase of HSP90 might be attributed to HSP70-mediated activity of HyT 3′.

Figure 7.

Figure 7

Open in a new tab

Functional validation of mHtt degradation by HyT 3. (A) Chemical structure of aggregate binder 10 and linker-linked hydrophobic degron 11. (B) HeLa cells transiently expressing mHtt exon 1-Q145-EGFP were treated with DMSO or the indicated compounds at 10 μM for 24 h. +, transfection reagents or compounds added; −, not added. (C, D) HeLa cells transiently expressing mHtt exon 1-Q145-EGFP were treated with DMSO, HyT 3, bortezomib, GA, or their combination for 24 h. The amount of mHtt exon 1-Q145-EGFP was evaluated by measuring the EGFP intensity of the lysate and normalized with the protein concentration of each lysate. Concentrations were: bortezomib: 300 nM, HyT 3: 10 μM, GA: 200 nM. Data are the mean ± SD of three individual experiments.

Since HyTs synthesized in this study have a lower molecular weight and a smaller number of HBDs/HBAs than those of the corresponding SNIPER, as shown in Figure 3, the HyTs might be expected to cross the blood-brain barrier (BBB) more effectively. To test this idea, we carried out a preliminary investigation employing immobilized artificial membrane (IAM) column analysis, which has been used to predict the brain permeability of compounds.23,24 Yoon et al. have shown that an index of membrane permeability (defined as PI here) derived from the capacity factor of the drug (kIAM, see the Supporting Information for more details) correlates with CNS penetration.24 Progesterone, a known brain-permeable steroid,25 was used as a standard, and its PI value was compared with those of SNIPERs and HyTs (Tables 1 and S1). All the HyTs developed in this study have better PI values than the parent SNIPER. In particular, HyT 4 and 7 showed higher values than progesterone. This suggests that HyT 4 and 7 can cross the BBB. Then, we evaluated the Caco-2 permeability and in vivo BBB penetration property of selected HyTs 3, 4, 7, 3′, and SNIPER 2 (Table 2). Unexpectedly, Caco-2 assay revealed that SNIPER 2 shows the best efflux ratio among compounds tested. HyTs 4 and 7 showed moderate–high apical to basolateral permeation (A to B influx), whereas their efflux ratios were comparable to that of SNIPER 2. On the other hand, A to B influx of 3 was much lower and B to A efflux of 3′ was much higher in comparison with those of others. In contrast, in vivo mouse brain penetration analysis showed that HyT 3′ crosses the BBB and is taken up in the brain actively and HyT 7 slightly crosses the BBB, whereas brain penetration of SNIPER 2 and the other HyTs was hardly observed. These results suggest that, although HyT 3′ is unlikely to be orally available, 3′ is promising as a CNS-penetrating mHtt degrader.

Table 1. Physicochemical Parameters and PI Values of Parent SNIPERs and Newly Synthesized HyTs.

compd MW no. of HBDsa no. of HBAsa TPSAb PI
progesterone         2.3
1 661.86 5 8 147.3 0.5
2 719.90 4 11 172.5 0.4
3 608.80 0 9 86.47 1.6
4 628.87 0 9 86.47 5.8
5 600.73 0 9 86.47 1.9
6 640.80 0 9 86.47 1.4
7 660.87 0 9 86.47 3.6
3′ 520.69 0 7 68.01  
Open in a new tab
a

Number of HBDs/HBAs was calculated by ChemOffice Excel add-in.

b

Total polar surface area (TPSA) values were calculated by ChemDraw Professional 20. Blank, not tested.

Table 2. Summary of Caco-2 Permeability Data and In Vivo BBB Penetration Data of Parent SNIPERs and Newly Synthesized HyTs.

  Caco-2
  
compd A to Ba B to Aa efflux ratio Kp,brainb
2 1.03 0.196 0.190 ND
3 0.098 0.118 1.20 ND
4 2.46 0.592 0.241 ND
7 5.32 1.25 0.235 0.13
3′ ND 4.71 NC 4.89
Open in a new tab
a

10–6 cm/s.

b

Mice, 4 h post i.v.; ND, not determined due to below measurement limit; NC, not calculated.

In summary, we designed and synthesized small-molecule HyTs that lower mHtt levels by modifying our SNIPER-based mHtt degrader 2. The conversion to HyTs decreased both the molecular weight and the number of HBDs/HBAs. Preliminary prediction of brain permeability using an IAM column suggested that these HyTs may have better permeability than the parent SNIPER 2. Indeed, HyT 3′ successfully crossed BBB in in vivo analysis. Wager and co-workers have reported desirable physicochemical criteria for CNS drugs: MW ≤ 360, 40 ≤ TPSA ≤ 90, HBD ≤ 0.5, CLogD ≤ 2, CLogP ≤ 3, pKa ≤ 8.26 TPSA and number of HBDs of HyT 3′ meet the criteria, and its MW got closer to the criteria compared with the parent PROTAC while some of its physicochemical properties still lie outside this criteria. In particular, its ChromLogD7.4 value is much greater than the criteria (predicted value from extrapolation is 10.3, see Table S2). However, surprisingly, ChromLogD7.4 experiments indicated SNIPER 2 is more lipophilic than HyTs at pH 7.4. Kihlberg et al. have reported that conformation of PROTACs is flexible and can be folded by interaction between each warhead to mask polar groups in nonpolar circumstances, e.g. cell membrane interior and ODS silica in HPLC column.27 We speculate that the conformation of HyTs is also flexible but HyTs could not mask the polar groups of another warhead because hydrophobic degron structures do not have groups to interact with them, resulting in lower lipophilicity compared with SNIPER 2. Taken together, our findings suggest that conversion of hetero-bifunctional protein degraders into HyTs could be a general approach to improve brain permeability. For example, it should be feasible to convert the PROTAC-based tau degrader QC-01-175 shown in Figure 1 into HyTs with a lower molecular weight and a decreased number of HBDs/HBAs.

Li and colleagues have reported peptide-based HyTs that decrease the levels of aggregation-prone tau and TDP43 associated with Alzheimer’s disease and amyotrophic lateral sclerosis, respectively.28,29 However, to our knowledge, the compounds developed in the present study are the first small-molecule HyTs targeting an aggregation-prone protein. Furthermore, our small-molecule HyTs were more potent than Li’s peptide-based HyTs, which show activity at 100 μM. The difference in potency seems likely to be attributable to the difference of cell permeability between small molecules and peptides. Here, we also found that the diamantane moiety is a potent hydrophobic degron, providing insight into the relationship between bulkiness and degradation activity. So far, our HyT mHtt degraders have the lowest molecular weight and the fewest HBDs/HBAs among reported hetero-bifunctional mHtt degraders. The best reported CNS drug-like mHtt degraders are the autophagosome-tethering compound (ATTEC)-based mHtt degraders reported by Lu’s group.30 However, these are molecular glues that are difficult to design rationally and generally require well-crafted screening systems to discover. In contrast, HyTs can be designed rationally and the same design concept is expected to be applicable for HyTs targeting other aggregation-prone proteins.

Our SNIPER-based mHtt degrader also lowers the levels of other polyQ proteins.11 Therefore, the HyTs described in this study are also expected to show activity against other polyQ proteins. Indeed, HyTs might work as pan-degraders of aggregation-prone proteins, since a protein aggregates binder is employed as the POI ligand. Further studies of the HyTs are underway in our laboratory.

Acknowledgments

The work described in this Letter was partially supported by the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number JP19K16326, S.T.; 18H02551 and 18H05502, M.I.), Japan Science and Technology Agency (JST ACT-X Grant number JPMJAX2018, S.T.), The Uehara Memorial Foundation (201920310, M.I.), and AMED-CREST, AMED (JP21gm1410007, M.I.). The cells GM04281 and DNA sample CH00019 were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research. Caco-2 permeability assay was carried out by Eurofins Discovery. In vivo brain permeability analysis was supported by AMED-BINDS program (JP21am0101087, JP21am0101123, 3198).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00500.

  • Western blot results of HyT 3 mechanistic analysis; original fluorescence microscopic images; proteome-wide analysis data and method; complete IAM chromatographic data and method; ChromLogD data and method; methods for cell culture, transfection, quantitative fluorescence analysis, Western blotting, fluorescence microscopic analysis, Caco-2 A-B permeability analysis, and in vivo BBB-penetration analysis; synthetic procedures, characterization, NMR charts, schemes, and purity of compounds (PDF)

  • Data set of the detected proteins in the proteome-wide analysis (XLSX)

Author Contributions

K.H., S.T., Y.M., K.O., S.S., Y.H., and M.I. designed the project and wrote the manuscript. K.H. and H.Y. performed organic syntheses and biological evaluations. S.T and K.O. constructed mHtt exon 1-Q145-EGFP plasmid. Y.M. performed IAM column analysis. T.N. and H.T. designed, carried out, and wrote the methods for proteomic analysis. M.I., K.K., Y.I, and S.N. designed, conducted, and wrote the methods for in vivo experiments.

The authors declare no competing financial interest.

Special Issue

Published as part of the ACS Medicinal Chemistry Letters virtual special issue “New Drug Modalities in Medicinal Chemistry, Pharmacology, and Translational Science”.

Supplementary Material

ml1c00500_si_001.pdf (1.4MB, pdf)
ml1c00500_si_002.xlsx (631.4KB, xlsx)

References

  1. Soto C.; Pritzkow S. Protein Misfolding, Aggregation, and Conformational Strains in Neurodegenerative Diseases. Nat. Neurosci. 2018, 21, 1332–1340. 10.1038/s41593-018-0235-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boland B.; Yu W. H.; Corti O.; Mollereau B.; Henriques A.; Bezard E.; Pastores G. M.; Rubinsztein D. C.; Nixon R. A.; Duchen M. R.; Mallucci G. R.; Kroemer G.; Levine B.; Eskelinen E.-L.; Mochel F.; Spedding M.; Louis C.; Martin O. R.; Millan M. J. Promoting the Clearance of Neurotoxic Proteins in Neurodegenerative Disorders of Ageing. Nat. Rev. Drug Discovery 2018, 17, 660–688. 10.1038/nrd.2018.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Eisele Y. S.; Monteiro C.; Fearns C.; Encalada S. E.; Wiseman R. L.; Powers E. T.; Kelly J. W. Targeting Protein Aggregation for the Treatment of Degenerative Diseases. Nat. Rev. Drug Discovery 2015, 14, 759–780. 10.1038/nrd4593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Meunier B. Hybrid Molecules with a Dual Mode of Action: Dream or Reality?. Acc. Chem. Res. 2008, 41, 69–77. 10.1021/ar7000843. [DOI] [PubMed] [Google Scholar]
  5. Burslem G. M.; Crews C. M. Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery. Cell 2020, 181, 102–114. 10.1016/j.cell.2019.11.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Itoh Y.; Ishikawa M.; Naito M.; Hashimoto Y. Protein Knockdown Using Methyl Bestatin-Ligand Hybrid Molecules: Design and Synthesis of Inducers of Ubiquitination-Mediated Degradation of Cellular Retinoic Acid-Binding Proteins. J. Am. Chem. Soc. 2010, 132, 5820–5826. 10.1021/ja100691p. [DOI] [PubMed] [Google Scholar]
  7. Naito M.; Ohoka N.; Shibata N. SNIPERs—Hijacking IAP Activity to Induce Protein Degradation. Drug Discovery Today Technol. 2019, 31, 35–42. 10.1016/j.ddtec.2018.12.002. [DOI] [PubMed] [Google Scholar]
  8. Ishikawa M.; Tomoshige S.; Demizu Y.; Naito M. Selective Degradation of Target Proteins by Chimeric Small-Molecular Drugs, PROTACs and SNIPERs. Pharmaceuticals 2020, 13, 74. 10.3390/ph13040074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Tomoshige S.; Nomura S.; Ohgane K.; Hashimoto Y.; Ishikawa M. Discovery of Small Molecules That Induce the Degradation of Huntingtin. Angew. Chem., Int. Ed. 2017, 56, 11530–11533. 10.1002/anie.201706529. [DOI] [PubMed] [Google Scholar]
  10. Tomoshige S.; Nomura S.; Ohgane K.; Hashimoto Y.; Ishikawa M. Degradation of Huntingtin Mediated by a Hybrid Molecule Composed of IAP Antagonist Linked to Phenyldiazenyl Benzothiazole Derivative. Bioorg. Med. Chem. Lett. 2018, 28, 707–710. 10.1016/j.bmcl.2018.01.012. [DOI] [PubMed] [Google Scholar]
  11. Yamashita H.; Tomoshige S.; Nomura S.; Ohgane K.; Hashimoto Y.; Ishikawa M. Application of Protein Knockdown Strategy Targeting β-Sheet Structure to Multiple Disease-Associated Polyglutamine Proteins. Bioorg. Med. Chem. 2020, 28, 115175. 10.1016/j.bmc.2019.115175. [DOI] [PubMed] [Google Scholar]
  12. Tomoshige S.; Ishikawa M. PROTACs and Other Chemical Protein Degradation Technologies for the Treatment of Neurodegenerative Disorders. Angew. Chem., Int. Ed. 2021, 60, 3346–3354. 10.1002/anie.202004746. [DOI] [PubMed] [Google Scholar]
  13. Silva M. C.; Ferguson F. M.; Cai Q.; Donovan K. A.; Nandi G.; Patnaik D.; Zhang T.; Huang H. T.; Lucente D. E.; Dickerson B. C.; Mitchison T. J.; Fischer E. S.; Gray N. S.; Haggarty S. J. Targeted Degradation of Aberrant Tau in Frontotemporal Dementia Patient-Derived Neuronal Cell Models. Elife 2019, 8, e45457 10.7554/eLife.45457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Harada R.; Okamura N.; Furumoto S.; Yanai K. Imaging Protein Misfolding in the Brain Using β-Sheet Ligands. Front. Neurosci. 2018, 12, 585. 10.3389/fnins.2018.00585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Edmondson S. D.; Yang B.; Fallan C. Proteolysis Targeting Chimeras (PROTACs) in ‘beyond Rule-of-Five’ Chemical Space: Recent Progress and Future Challenges. Bioorg. Med. Chem. Lett. 2019, 29, 1555–1564. 10.1016/j.bmcl.2019.04.030. [DOI] [PubMed] [Google Scholar]
  16. Maple H. J.; Clayden N.; Baron A.; Stacey C.; Felix R. Developing Degraders: Principles and Perspectives on Design and Chemical Space. Medchemcomm 2019, 10, 1755–1764. 10.1039/C9MD00272C. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Neklesa T. K.; Tae H. S.; Schneekloth A. R.; Stulberg M. J.; Corson T. W.; Sundberg T. B.; Raina K.; Holley S. A.; Crews C. M. Small-Molecule Hydrophobic Tagging-Induced Degradation of HaloTag Fusion Proteins. Nat. Chem. Biol. 2011, 7, 538–543. 10.1038/nchembio.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gustafson J. L.; Neklesa T. K.; Cox C. S.; Roth A. G.; Buckley D. L.; Tae H. S.; Sundberg T. B.; Stagg D. B.; Hines J.; McDonnell D. P.; Norris J. D.; Crews C. M. Small-Molecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging. Angew. Chem., Int. Ed. 2015, 54, 9659–9662. 10.1002/anie.201503720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Prodromou C.; Roe S. M.; O’Brien R.; Ladbury J. E.; Piper P. W.; Pearl L. H. Identification and Structural Characterization of the ATP/ADP-Binding Site in the Hsp90 Molecular Chaperone. Cell 1997, 90, 65–75. 10.1016/S0092-8674(00)80314-1. [DOI] [PubMed] [Google Scholar]
  20. Zou J.; Guo Y.; Guettouche T.; Smith D. F.; Voellmy R. Repression of Heat Shock Transcription Factor HSF1 Activation by HSP90 (HSP90 Complex) That Forms a Stress-Sensitive Complex with HSF1. Cell 1998, 94, 471–480. 10.1016/S0092-8674(00)81588-3. [DOI] [PubMed] [Google Scholar]
  21. Griffin T. M.; Valdez T. V.; Mestril R. Radicicol Activates Heat Shock Protein Expression and Cardioprotection in Neonatal Rat Cardiomyocytes. Am. J. Physiol. - Hear. Circ. Physiol. 2004, 287, 1081–1088. 10.1152/ajpheart.00921.2003. [DOI] [PubMed] [Google Scholar]
  22. Reichel A.; Begley D. J. Potential of Immobilized Artificial Membranes for Predicting Drug Penetration across the Blood-Brain Barrier. Pharm. Res. 1998, 15, 1270–1274. 10.1023/A:1011904311149. [DOI] [PubMed] [Google Scholar]
  23. Yoon C. H.; Kim S. J.; Shin B. S.; Lee K. C.; Yoo S. D. Rapid Screening of Blood-Brain Barrier Penetration of Drugs Using the Immobilized Artificial Membrane Phosphatidylcholine Column Chromatography. J. Biomol. Screen. 2006, 11, 13–20. 10.1177/1087057105281656. [DOI] [PubMed] [Google Scholar]
  24. Pardridge W. M.; Mietus L. J. Transport of Thyroid and Steroid Hormones through the Blood-Brain Barrier of the Newborn Rabbit: Primary Role of Protein-Bound Hormone. J. Clin. Invest. 1979, 64, 145–154. 10.1172/JCI109433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wager T. T.; Hou X.; Verhoest P. R.; Villalobos A. Moving beyond Rules: The Development of a Central Nervous System Multiparameter Optimization (CNS MPO) Approach to Enable Alignment of Druglike Properties. ACS Chem. Neurosci. 2010, 1, 435–449. 10.1021/cn100008c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Atilaw Y.; Poongavanam V.; Svensson Nilsson C.; Nguyen D.; Giese A.; Meibom D.; Erdelyi M.; Kihlberg J. Solution Conformations Shed Light on PROTAC Cell Permeability. ACS Med. Chem. Lett. 2021, 12, 107–114. 10.1021/acsmedchemlett.0c00556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gao N.; Chu T. T.; Li Q. Q.; Lim Y. J.; Qiu T.; Ma M. R.; Hu Z. W.; Yang X. F.; Chen Y. X.; Zhao Y. F.; Li Y. M. Hydrophobic Tagging-Mediated Degradation of Alzheimer’s Disease Related Tau. RSC Adv. 2017, 7, 40362–40366. 10.1039/C7RA05347A. [DOI] [Google Scholar]
  28. Gao N.; Huang Y. P.; Chu T. T.; Li Q. Q.; Zhou B.; Chen Y. X.; Zhao Y. F.; Li Y. M. TDP-43 Specific Reduction Induced by Di-Hydrophobic Tags Conjugated Peptides. Bioorg. Chem. 2019, 84, 254–259. 10.1016/j.bioorg.2018.11.042. [DOI] [PubMed] [Google Scholar]
  29. Li Z.; Wang C.; Wang Z.; Zhu C.; Li J.; Sha T.; Ma L.; Gao C.; Yang Y.; Sun Y.; Wang J.; Sun X.; Lu C.; Difiglia M.; Mei Y.; Ding C.; Luo S.; Dang Y.; Ding Y.; Fei Y.; Lu B. Allele-Selective Lowering of Mutant HTT Protein by HTT–LC3 Linker Compounds. Nature 2019, 575, 203–209. 10.1038/s41586-019-1722-1. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml1c00500_si_001.pdf (1.4MB, pdf)
ml1c00500_si_002.xlsx (631.4KB, xlsx)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES