Simple and efficient knockdown of His-tagged proteins by ternary molecules consisting of a His-tag ligand, a ubiquitin ligase ligand, and a cell-penetrating peptide

Article abstract of DOI:10.1016/j.bmcl.2017.08.001

We designed and synthesized hybrid molecules for a protein knockdown method based on the recognition of a His-tag fused to a protein of interest (POI). The synthesized target protein degradation inducers contained three functional moieties: a His-tag liga

Full text of DOI:10.1016/j.bmcl.2017.08.001

Accepted Manuscript
Simple and efficient knockdown of His-tagged proteins by ternary molecules
consisting of a His-tag ligand, a ubiquitin ligase ligand, and a cell-penetrating
peptide
Takayuki Hattori, Koyo Okitsu, Norikazu Yamazaki, Nobumichi Ohoka,
Norihito Shibata, Takashi Misawa, Masaaki Kurihara, Yosuke Demizu,
Mikihiko Naito
PII:
S0960-894X(17)30789-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.001
BMCL 25195
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 July 2017
31 July 2017
1 August 2017
Please cite this article as: Hattori, T., Okitsu, K., Yamazaki, N., Ohoka, N., Shibata, N., Misawa, T., Kurihara, M.,
Demizu, Y., Naito, M., Simple and efficient knockdown of His-tagged proteins by ternary molecules consisting of
a His-tag ligand, a ubiquitin ligase ligand, and a cell-penetrating peptide, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.001
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Simple and efficient knockdown of His-tagged proteins by ternary
molecules consisting of a His-tag ligand, a ubiquitin ligase ligand, and a
cell-penetrating peptide
Takayuki Hattori,a Koyo Okitsu,b Norikazu Yamazaki,b Nobumichi Ohoka,a Norihito Shibata,a
Takashi Misawa,b Masaaki Kurihara,b Yosuke Demizu,b,* and Mikihiko Naitoa,*
aDivision of Molecular Target and Gene Therapy Products, National Institute of Health Sciences,
1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, Japan
bDivision of Organic Chemistry, National Institute of Health Sciences, 1-18-1, Kamiyoga,
Setagaya, Tokyo 158-8501, Japan
KEYWORDS: protein knockdown; His-tag-fused protein; ubiquitin-proteasome system; carrier
peptide
*Corresponding authors: Tel: +81-3-3700-1141, Fax: +81-3-3707-6950, E-mail:
demizu@nihs.go.jp (Y. Demizu), miki-naito@nihs.go.jp (M. Naito)
1

Abstract
We designed and synthesized hybrid molecules for a protein knockdown method based on the
recognition of a His-tag fused to a protein of interest (POI). The synthesized target protein
degradation inducers contained three functional moieties: a His-tag ligand (nickel nitrilotriacetic
acid [Ni-NTA]), an E3 ligand (bestatin [BS] or MV1), and a carrier peptide (Tat or nonaarginine
[R9]). The designed hybrid molecules, BS-Tat-Ni-NTA, MV1-Tat-Ni-NTA, BS-R9-Ni-NTA,
and MV1-R9-Ni-NTA, efficiently degraded His-tagged cellular retinoic acid binding protein 2
via the ubiquitin-proteasome system (UPS). This system will become a useful tool for research
into selective protein degradation inducers that act via the UPS.
2

Elucidating the functions of intracellular proteins is quite important for the development of
innovative drugs, and the artificial control of protein expression is a useful technique for
clarifying protein functions. A protein knockdown strategy in which the degradation of proteins
of interest (POI) is induced via the ubiquitin-proteasome system (UPS) has recently attracted
attention. Based on this approach, novel small molecules composed of two different ligands
connected by a linker (one is a ligand for the POI, and the other is a ligand for ubiquitin ligases
[E3]) have been developed. These molecules, which are called proteolysis-targeting chimeras
(PROTAC)1-9 or specific and non-genetic inhibitors of apoptosis protein [IAP]-dependent protein
erasers (SNIPER)10-21 are able to induce the ubiquitylation and subsequent proteasomal
degradation of POI via the UPS, and a variety of PROTAC and SNIPER that target pathogenic
proteins have been reported.
We have recently described the development of a small hybrid molecule, SNIPER(CH6),
which mediates the degradation of CH6 tag-fused proteins.22-26 The CH6 tag is a modified
version of the conventional His-tag and consists of hexa-His and a Cys residue. It attaches to the
N- or C-terminus of the POI. SNIPER(CH6) is composed of three functional moieties, nickel
nitrilotriacetic acid (Ni-NTA; a His-tag ligand), a maleimide moiety covalently conjugated to the
Cys residue in the CH6 tag, and the IAP antagonist (MV1) as an E3 ligand. However,
SNIPER(CH6) itself cannot penetrate into cells, and therefore, CPP-His, a cell-penetrating
peptide (CPP) conjugated with a His-tag was used to deliver SNIPER(CH6) into cells. As
expected, the combination of SNIPER(CH6) and CPP-His efficiently degraded CH6-tagged
POI within cells.26 This system could be an effective screening tool for finding proteins that are
susceptible to degradation via the UPS and for identifying target proteins for drug discovery.
However, there are two main disadvantages of this method. The first disadvantage is that it
3

involves the use of two types of molecules; i.e., SNIPER(CH6) and CPP-His, and the other
concerns the fact that SNIPER(CH6) is also decomposed by the UPS together with the CH6-
tagged POI because SNIPER(CH6) covalently binds to the protein (Figure 1).
Figure 1. Mechanism responsible for the degradation of CH6 tag-fused POI using a
combination of SNIPER(CH6) and CPP-His24
4

The SNIPER(CH6)/CPP-His complex penetrates the plasma membrane efficiently. In the
cytosol, SNIPER(CH6) is released from CPP-His and forms another complex with the CH6-
tagged POI through the covalent binding of its maleimide moiety with the Cys residue of the
CH6 tag. Finally, the POI is ubiquitylated and degraded via the UPS.
In this study, we designed a new type of His-tagged POI degradation inducer. The
synthesized hybrid molecules were composed of three functional moieties, a His-tag ligand (Ni-
NTA), an IAP antagonist (bestatin [BS]10,12 or MV119,26) as an E3 ligand, and a representative
carrier peptide (Tat or nonaarginine [R9]). The mechanism proposed for the degradation of His-
tagged POI is shown in Figure 2. 1) The degradation inducer MV1-CPP-Ni-NTA is taken up
into the target cells. 2) In the cells, the degradation inducer non-covalently binds to the His-tag
moiety of the POI. 3) Finally, E3 is recruited to the E3 ligand, and the POI is ubiquitylated and
degraded by the UPS. The designed hybrid molecules, BS-Tat-Ni-NTA, MV1-Tat-Ni-NTA,
BS-R9-Ni-NTA, MV1-R9-Ni-NTA, were synthesized, and their His-tagged POI degradation
activities were evaluated.
5

Figure 2. Proposed mechanism for the degradation of His-tag-fused POI using a hybrid molecule
(see the main text for details)
The synthetic route for the designed molecules is shown in Scheme 1. The Ni-NTA moiety 3
was synthesized starting from the lysine derivative 1. Carrier peptides containing BS or MV1,
BS-Tat, MV1-Tat, BS-R9, MV1-R9 were prepared via the conventional solid-phase peptide
synthesis method. The conjugation of the Ni-NTA moiety 3 with the abovementioned peptides
was performed under basic conditions, producing BS-Tat-NTA, MV1-Tat-NTA, BS-R9-NTA,
and MV1-R9-NTA, and then a nickel ion (Ni2+) was added to each molecule to give the desired
6

molecules; i.e., BS-Tat-Ni-NTA, MV1-Tat-Ni-NTA, BS-R9-Ni-NTA, and MV1-R9-Ni-
NTA.27
Scheme 1. Synthesis of the His-tagged POI degradation inducers BS-Tat-Ni-NTA, MV1-Tat-
Ni-NTA, BS-R9-Ni-NTA, and MV1-R9-Ni-NTA
Then, we evaluated the effects of the synthesized compounds on 10×His-cellular retinoic acid
binding protein 2 (CRABP2)-T7 protein degradation in HT1080 cells by comparing with the
protein level of mock treatment (lane 1) (Figure 3).28,29 As demonstrated in previous studies,
treatment with the IAP antagonist (BS10,12 or MV119,24) moiety-containing molecules resulted in
a reduction in the level of the c-IAP1 protein. The control compound, MV1-all-trans retinoic
acid (ATRA),19 which targets the CRABP2 protein, induced the degradation of the 10×His-
CRABP2-T7 protein (compare lane 3 with lane 1), which was blocked by the addition of the
7

proteasome inhibitor MG132 (compare lane 4 with lane 3). Treatment with the carrier peptide
Tat alone did not affect the expression levels of c-IAP1 or the 10×His-CRABP2-T7 protein
(compare lane 21 with lane 1). A slight reduction in the protein level was observed in the cells
treated with 10 µM BS-Tat-Ni-NTA (compare lane 5 with lane 1), whereas treatment with 3 µM
MV1-Tat-Ni-NTA successfully induced protein degradation (compare lane 9 with lane 1). A
similar tendency was observed for 10 µM BS-R9-Ni-NTA (compare lane 13 with lane 1) and 3
µM MV1-R9-Ni-NTA (compare lane 17 with lane 1). Thus, the MV1-conjugated molecules
(MV1-Tat-Ni-NTA, MV1-R9-Ni-NTA) showed higher activities than BS-conjugated ones (BS-
Tat-Ni-NTA, BS-R9-Ni-NTA) in lower concentration (3-5 µM for MV1-conjugated molecules
and 10 µM for BS-conjugated molecules, respectively). These results were consistent with those
of a previous study, in which it was demonstrated that the E3 ligand activity of MV1 was greater
than that of BS.19 Furthermore, BS-R9-Ni-NTA and MV1-R9-Ni-NTA, which contained R9
(lanes 13 and 17), exhibited greater activity than BS-Tat-Ni-NTA and MV1-Tat-Ni-NTA,
which contained the Tat peptide (lanes 5 and 9), indicating that R9 is a superior carrier for
transporting cargo molecules into HT1080 cells. The reductions in the levels of CRABP2 and c-
IAP1 induced by the synthesized compounds were suppressed by MG132 (compare the protein
levels in the presence of MG132 with in the absence of MG132), indicating that these
compounds also induced proteasomal degradation of the target proteins.
8

Figure 3. The degradation of 10×His-CRABP2-T7 by the synthesized compounds
HT1080 cells that constitutively expressed 10×His-CRABP2-T7 were treated with the indicated
dose of the relevant compound with or without MG132 for 6 h. The lysates were analyzed with
the indicated antibodies.
In summary, we developed a protein knockdown method for His-tagged POI involving the use
of a hybrid degradation inducer containing three components, a His-tag ligand (Ni-NTA), an E3
ligand (BS or MV1), and a carrier peptide (Tat or R9). The compound MV1-R9-Ni-NTA
efficiently induced the degradation of His-tagged CRABP2 proteins. This method will become a
useful technique for research into selective UPS-based protein degradation inducers. The
development of innovative protein degradation inducers that target various types of proteins is
currently underway.
9

Acknowledgements
This study was supported, in part, by the Japan Society for the Promotion of Science KAKENHI
grant numbers 26460169 and 17k08385 (Y.D.), 16H05090, and 16K15121 (M.N.); by a Grant-
in-Aid from the Research Foundation for Pharmaceutical Sciences (Y.D.); by the Shorai
Foundation for Science and Technology (Y.D.); and by grants for the Project for Cancer Research
And Therapeutic Evolution (P-CREATE) (Y.D.) and Research on the Development of New
Drugs (M.N.) from the Japan Agency for Medical Research and Development (AMED).
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27. See the supplementary materials.
28. HT1080 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% heat-
inactivated fetal bovine serum, and 100 µg/ml of kanamycin at 37 °C in a humidified
atmosphere of 5% CO2 in air. The cells were transfected with the expression vectors for
10×His-CRABP2-T7 using Lipofectamine 2000 (Invitrogen), according to the
12

manufacturer’s instructions. After 48 h, the cells were selected with 500 µg/ml G418
(Roche) for 2 weeks to isolate single-cell clones.
29. The cells were treated as described in the figure legends, washed with phosphate-buffered
saline (-), lysed in sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 0.1 M Tris-HCl, and
10% glycerol; pH 7.4), and boiled for 10 min. Protein concentrations were determined using
the bicinchoninic acid protein assay kit (Thermo Fisher Scientific) and normalized to the
total protein concentration of each lysate. After being boiled for 5 min with Laemmli buffer,
each lysate was resolved via SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 5-
20% gradient gel, before being transferred onto a polyvinylidene difluoride membrane. After
being blocked with Tris-buffered saline-Tween 20 (25 mM Tris-HCl, 137 mM NaCl, and
0.1% Tween 20; pH 7.4) containing 5% skimmed milk, the membranes were probed with the
antibodies as described in the figure legends. The immunoblots were visualized via
enhanced chemiluminescence with the Clarity western ECL substrate (BIO-RAD).
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Graphical Abstract
Simple and efficient knockdown of His-tagged proteins by ternary molecules consisting
of a His-tag ligand, a ubiquitin ligase ligand, and a cell-penetrating peptide
Takayuki Hattori, Koyo Okitsu, Norikazu Yamazaki, Nobumichi Ohoka, Norihito Shibata,
Takashi Misawa, Masaaki Kurihara, Yosuke Demizu,* and Mikihiko Naito*
His tag
Ni-NTA
CPPs
BS/MV1
IAP
POI
Ub
Ub
Ub
Ub