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Targeted Protein Degradation by Chimeric Compounds Using Hydrophobic E3 Ligands and Adamantane Moiety

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Targeted Protein Degradation by Chimeric Compounds Using Hydrophobic E3 Ligands and Adamantane Moiety pharmaceuticals Article Targeted Protein Degradation by Chimeric Compounds using Hydrophobic E3 Ligands and Adamantane Moiety 1, , 2, 1 1,3 3 Takuji Shoda y *, Nobumichi Ohoka y, Genichiro Tsuji , Takuma Fujisato , Hideshi Inoue , 1 2 1, Yosuke Demizu , Mikihiko Naito and Masaaki Kurihara z 1 Division of Organic Chemistry, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki, Kanagawa 210-9501, Japan; [email protected] (G.T.); [email protected] (T.F.); [email protected] (Y.D.); [email protected] (M.K.) 2 Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki, Kanagawa 210-9501, Japan; [email protected] (N.O.); [email protected] (M.N.) 3 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-44-270-6579; Fax: +81-44-270-6579 These authors contributed equally to this study. y Current address: School of Pharmacy, Department of Pharmaceutical Sciences, International University of z Health and Welfare, 2600-1 Kitakanemaru, Otawara, Tochigi, Japan. Received: 20 January 2020; Accepted: 19 February 2020; Published: 25 February 2020 Abstract: Targeted protein degradation using small chimeric molecules, such as proteolysis-targeting chimeras (PROTACs) and specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs), is a promising technology in drug discovery. We recently developed a novel class of chimeric compounds that recruit the aryl hydrocarbon receptor (AhR) E3 ligase complex and induce the AhR-dependent degradation of target proteins. However, these chimeras contain a hydrophobic AhR E3 ligand, and thus, degrade target proteins even in cells that do not express AhR. In this study, we synthesized new compounds in which the AhR ligands were replaced with a hydrophobic adamantane moiety to investigate the mechanisms of AhR-independent degradation. Our results showed that the compounds, 2, 3, and 16 induced significant degradation of some target proteins in cells that do not express AhR, similar to the chimeras containing AhR ligands. However, in cells expressing AhR, 2, 3, and 16 did not induce the degradation of other target proteins, in contrast with their response to chimeras containing AhR ligands. Overall, it was suggested that target proteins susceptible to the hydrophobic tagging system are degraded by chimeras containing hydrophobic AhR ligands even without AhR. Keywords: protein degradation; chimeric compound; hydrophobic tagging; adamantane 1. Introduction A technology for selectively degrading a target protein is expected not only to elucidate the physiological function of proteins but also to develop a therapeutic drug for a disease caused by the aberrant protein. Previous studies reported a series of chimeric compounds that recruit E3 ubiquitin ligases to specifically degrade target proteins via the ubiquitin-proteasome system [1,2]. These chimeras, which are called specific and nongenetic inhibitors of apoptosis protein [IAP]-dependent protein erasers (SNIPERs) and proteolysis-targeting chimeras (PROTACs), contain two ligands connected with a linker: one ligand specific to an E3 ubiquitin ligase and another ligand specific to the target protein. These chimeric compounds are designed to cross-link the E3 ligase with the target protein within Pharmaceuticals 2020, 13, 34; doi:10.3390/ph13030034 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 34 2 of 12 the target protein. These chimeric compounds are designed to cross-link the E3 ligase with the target protein within the cells, and thereby, induce ubiquitination and subsequent degradation by Pharmaceuticalsproteasomes.2020 , Several13, 34 chimeric degraders have successfully been employed to degrade several2 of 13 proteins such as nuclear receptors [3–7], oncogenic kinases [8], epigenetic regulators [9,10], transcription factors [11], and others [12,13]. Some compounds can also degrade target proteins in the cells, and thereby, induce ubiquitination and subsequent degradation by proteasomes. Several vivo, suggesting the possibility of clinical applications [5,9,10,14,15]. In contrast to traditional chimericinhibitor-based degraders pharmaceuticals, have successfully such been as employedreversible/irreversible to degrade inhibitor several proteins and antagonists, such as nuclear the receptorschimeras [3 –require7], oncogenic only the kinases transient [8 ],binding epigenetic to any regulators surface of [the9,10 target], transcription to catalytically factors induce [11], its and othersubiquitination. [12,13]. Some Thus, compounds this technology can also has degrade emer targetged as proteins a novelin therapeutic vivo, suggesting approach the possibilityknown as of clinical“undruggable” applications proteins. [5,9,10 ,14,15]. In contrast to traditional inhibitor-based pharmaceuticals, such as reversibleRecently,/irreversible we developed inhibitor a and new antagonists, class of small the molecu chimerasle chimeras require that only recruit the transient the aryl hydrocarbon binding to any surfacereceptor of the (AhR) target E3 toligase catalytically and induce induce the AhR-dependent its ubiquitination. degradation Thus, this of target technology proteins has [16]. emerged β-NF- as a novelATRA, therapeutic 1 (Figure approach1), a chimeric known compound as “undruggable” directed against proteins. cellular retinoic acid-binding proteins (CRABPs),Recently, induced we developed the AhR-depe a new classndent of degradation small molecule of CRABP-1 chimeras and that CRABP-2 recruit in the MCF-7 aryl hydrocarbon and IMR- receptor32 cells (AhR) expressing E3 ligaseAhR [16]. and Interestingly, induce the the AhR-dependent 1-induced significant degradation reduction of of target CRABP-2, proteins but not [16 ]. β-NF-ATRA,of CRABP-1,1 was(Figure also1 ),observed a chimeric in SH-SY5Y compound cells, directedwhich do against not express cellular AhR retinoic (Figure acid-bindingS1 and S2). proteinsSimilar (CRABPs), results were induced also obtained the AhR-dependent by the treatmen degradationt of the ITE-ATRA of CRABP-1 [16], and in CRABP-2which an inalternative MCF-7 and IMR-32AhR cellsligand expressing was used AhR(Figure [16 S3).]. Interestingly, The ligand-bound the 1-induced AhR forms significant a CUL4B-based reduction E3 ligase of CRABP-2, complex but [17]. The NEDD8-activating E1 enzyme inhibitor MLN4924 [18,19] completely inhibited the 1- not of CRABP-1, was also observed in SH-SY5Y cells, which do not express AhR (Figures S1 and S2). induced reduction of CRABP-1 but partly that of CRABP-2 (Figure S4). These results suggested that Similar results were also obtained by the treatment of the ITE-ATRA [16], in which an alternative AhR these chimeric compounds degrade CRABP-1 and CRABP-2 via an AhR-dependent mechanism, ligand was used (Figure S3). The ligand-bound AhR forms a CUL4B-based E3 ligase complex [17]. whereas CRABP-2 is also degraded via an AhR-independent mechanism. The NEDD8-activatingIn this study, we E1 synthesized enzyme inhibitor several MLN4924compounds [ 18to, 19investigate] completely the inhibitedmechanisms the of1-induced AhR- reductionindependent of CRABP-1 degradation but partlyand evaluate that of their CRABP-2 activities. (Figure These S4).results These will provide results suggesteduseful information that these chimericfor the compounds development degrade of chimeric CRABP-1 compounds and CRABP-2 using a via highly an AhR-dependent hydrophobic E3 mechanism, ligand and whereastheir CRABP-2pharmaceutical is also degraded applications. via an AhR-independent mechanism. FigureFigure 1. 1.Chemical Chemical structurestructure of ββ-NF-ATRA-NF-ATRA (1 (1).). 2. InResults this and study, Discussion we synthesized several compounds to investigate the mechanisms of AhR-independent degradation and evaluate their activities. These results will provide useful information2.1. Design forand theSynthesis development of Compounds of chimeric compounds using a highly hydrophobic E3 ligand and theirSince pharmaceutical CRABP-2 was applications. degraded by 1 and ITE-ATRA in SH-SY5Y cells that do not express AhR (Figure S1-S3), we assumed that the high hydrophobicity of the β-NF ligand module might play a 2. Resultsrole in the and AhR-independent Discussion degradation of CRABP-2. Appending a hydrophobic moiety to a protein surface induces protein degradation; the hydrophobic moiety mimics a state of partial unfolding, 2.1. Design and Synthesis of Compounds inducing the degradation of the target protein via an intracellular quality control system [20–22]. BocSince3-Arg- CRABP-2 and adamantane-based was degraded hydrophobic by 1 and ITE-ATRA tagging (H inyT) SH-SY5Y strategies cells have thatbeen do applied not express to a range AhR (Figuresof exogenous S1–S3), we[23,24] assumed and endogenous that the high proteins hydrophobicity [25,26]. To of investigate the β-NF ligandwhether module CRABP might proteins play are a role in thedegraded AhR-independent by the HyT system, degradation we designed of CRABP-2. and synthesized Appending 2 (Ad-ATRA-1) a hydrophobic and 3 moiety (Ad-ATRA-2) to a protein by surface induces protein degradation; the hydrophobic moiety mimics a state of partial unfolding, inducing the degradation of the target protein via an intracellular quality control system [20–22]. Boc3-Arg- and adamantane-based hydrophobic tagging (HyT) strategies have been applied to a
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