Characterization of novel non-peptide thrombopoietin mimetics, their species specificity and the activation mechanism of the thrombopoietin receptor

Article abstract of DOI:10.1016/j.ejphar.2008.02.060

A series of non-peptide small compounds discovered to be thrombopoietin receptor agonists showed species specificity to humans. Compound I could induce megakaryocyte lineage from human bone marrow cells, but not from mouse, guinea pig or cynomolgus monkey bone marrow cells. To elucidate the mechanism, we identified the pivotal amino acid residue for the receptor activation by compound I by taking advantage of its species specificity. The response of compound I to three human/mouse chimeric receptors indicated the importance of the transmembrane domain. Comparison of amino acid sequences of the transmembrane domain of the thrombopoietin receptor between human and three animal species led us to hypothesize that histidine 499 is necessary for the reactivity to the thrombopoietin mimetics. We verified the hypothesis using two mutant receptors: the human thrombopoietin receptor mutant His499Leu and the mouse thrombopoietin receptor mutant Leu490His. These results should be helpful for structure-activity relationship studies and conducting in vivo studies of thrombopoietin mimetics.

Full text of DOI:10.1016/j.ejphar.2008.02.060

Available online at www.sciencedirect.com
European Journal of Pharmacology 586 (2008) 44–51
www.elsevier.com/locate/ejphar
Characterization of novel non-peptide thrombopoietin mimetics, their species
specificity and the activation mechanism of the thrombopoietin receptor
a,
⁎
b
b
b
b
Noriko Yamane , Yoshikazu Tanaka , Naoki Ohyabu , Shoji Yamane , Kazuhiko Maekawa ,
b
c
d
b
Jun Ishizaki , Ryuji Suzuki , Tsunetoshi Itoh , Hiroshi Takemoto
a
Discovery Research Laboratories, Shionogi & Co., Ltd., 1-1, Futaba-cho 3-chome, Toyonaka-shi, Osaka 561-0825, Japan
Discovery Research Laboratories, Shionogi & Co., Ltd., 12-4, Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan
b
c
Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital, Sakuradai 18-1, Sagamihara,
Kanagawa 228-8522, Japan
d
Department of Immunology and Embryology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku Sendai 980-8575, Japan
Received 22 July 2007; received in revised form 3 February 2008; accepted 13 February 2008
Available online 29 February 2008
Abstract
A series of non-peptide small compounds discovered to be thrombopoietin receptor agonists showed species specificity to humans. Compound
I could induce megakaryocyte lineage from human bone marrow cells, but not from mouse, guinea pig or cynomolgus monkey bone marrow cells.
To elucidate the mechanism, we identified the pivotal amino acid residue for the receptor activation by compound I by taking advantage of its
species specificity. The response of compound I to three human/mouse chimeric receptors indicated the importance of the transmembrane domain.
Comparison of amino acid sequences of the transmembrane domain of the thrombopoietin receptor between human and three animal species led
us to hypothesize that histidine 499 is necessary for the reactivity to the thrombopoietin mimetics. We verified the hypothesis using two mutant
receptors: the human thrombopoietin receptor mutant His499Leu and the mouse thrombopoietin receptor mutant Leu490His. These results should
be helpful for structure–activity relationship studies and conducting in vivo studies of thrombopoietin mimetics.
©
2008 Elsevier B.V. All rights reserved.
Keywords: Thrombopoietin; Human thrombopoietin mimetics; Species specificity; Chimera/mutant receptor; Loss/gain of function; Megakaryocytosis
1
. Introduction
the Ras-mitogen-activated protein kinase (MAPK) pathway, and
phospholipase Cγ (PLCγ), as well as recombinant human throm-
bopoietin. However, as these compounds are specific for the
human receptor, we could not evaluate their in vivo activity in
common experimental animals. We tried to elucidate the mecha-
nism of their thrombopoietin-like activities and species specificity.
The thrombopoietin receptor is a type I cytokine receptor
consisting of an extracellular domain (485 amino acids), a trans-
membrane domain (22 amino acids), and a cytoplasmic domain
(122 amino acids). The extracellular domain contains two cytokine
receptor homology regions (CRH1, CRH2). The cytoplasmic
domain (lacking kinase activity) is defined by BOX1 and BOX2
motifs, through which various signaling molecules are regulated
In trying to develop a drug to treat thrombocytopenic patients
in place of platelet transfusion (Kuter, 2007), we found a novel
series of non-peptide thrombopoietin receptor agonists. They
directly stimulated the thrombopoietin-dependent cell line Ba/
F3-huMPL to proliferate in a dose-dependent manner and pro-
moted in vitro megakaryocytopoiesis from human bone marrow
cells (Yamane, N. et al. in submission). They could activate the
thrombopoietin signal transduction pathway in Ba/F3-huMPL,
such as the thrombopoietin receptor, Janus kinase/signal
transducers and activators of transcription (JAK/STAT) pathway,
(Bazan, 1990; Kaushansky, 2005; Vigon et al., 1992).
⁎
In this study, we engineered three human/mouse chimeras
Corresponding author. Tel.: +81 6 6331 8081; fax: +81 6 6331 8612.
E-mail address: noriko.yamane@shionogi.co.jp (N. Yamane).
and two mutants of the thrombopoietin receptor to identify the
0014-2999/$ - see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2008.02.060

N. Yamane et al. / European Journal of Pharmacology 586 (2008) 44–51
45
body was purchased from StemCell Technologies (Vancouver,
Canada). Compound I, with the structure shown in Fig. 1A, was
synthesized by Shionogi (Osaka, Japan). Briefly, it was syn-
thesized from the amidation reaction with intermediate III and
intermediate V. Intermediate III was synthesized from the com-
mercially available compound II (2-chloro-1-(3,4-difluoro-
phenyl)-ethanone, Enamine Ltd., Kiev, Ukraine), which was
allowed to react with thiourea. Similarly intermediate V was
synthesized from commercially available compound IV (tereph-
thalaldehydic acid, Tokyo Chemical Industry Co., Ltd., Tokyo,
Japan), which was allowed to react with 2, 4-thiazolidinedione.
The structure and the synthesis method are shown in Fig. 1B.
2.2. Cells and cultures
An interleukin (IL)-3-dependent mouse pre-B cell line Ba/
F3 (Riken Cell Bank, Tsukuba, Japan) was maintained in
RPMI1640 medium containing L-glutamine, 10% fetal bovine
serum (HyClone), and 10% WEHI-3 conditioned medium as a
source of mouse interleukin-3 (mIL-3). A packaging cell line
Fig. 1. (A) Chemical structure of compound I (N-[4-(3,4-Difluoro-phenyl)-
thiazol-2-yl]-4-[2,4-dioxo-thiazolidin-(5Z)-ylidenemethyl]-benzamide), a tool
compound of non-peptide thrombopoietin mimetics used in this study. Mole-
cular weight is 443.45 Da. (B) Synthesis of Compound I.
2
93GP was purchased from Clontech Laboratories Inc. (CA,
+
USA). Human CD34 bone marrow hematopoietic progenitor
cells were purchased from Cambrex Bio Science Walkersville,
Inc. (MD, U.S.A). This study was approved by the institutional
ethics committee, and all specimens were obtained with in-
formed consent.
pivotal site for the receptor activation by these compounds,
which should help clarify their in vivo activity.
2
. Materials and methods
2.3. Animals
2
.1. Reagents
Balb/c mice and Hartley guinea pigs were purchased from
Recombinant human thrombopoietin and recombinant mouse
thrombopoietin were purchased from Genzyme (MA, U.S.A).
Anti-human Glycoprotein IIb/IIIa (CD41a) monoclonal anti-
Charles River Japan, and cynomolgus monkeys from Japan
E.D.M., Inc. (Tokyo, Japan). The experimental protocol for
this study was approved by the local animal ethics research
Fig. 2. Schematic presentation of the constructions of cDNA encoding chimeric and mutant thrombopoietin receptors. This figure shows primers used in their
construction. The construction procedures are described in detail in Materials and methods. The nucleotide sequences of the primers are shown in Table 1.

46
N. Yamane et al. / European Journal of Pharmacology 586 (2008) 44–51
Table 1
committee. All efforts were made to minimize animal suffer-
ing and to reduce the number of animals used.
Nucleotide sequences of primers used for construction of chimeras and mutants
of the thrombopoietin receptor
2
.4. Construction of expression plasmids of wild-type
thrombopoietin receptors
Human thrombopoietin receptor gene, c-mpl cDNA was am-
plified from human leukemia cell cDNA and cloned into a mam-
malian expression vector pGEM3-SRα-neo (pGEM3-huMPL)
in our recent study (Yamane, N. in submission). Mouse c-mpl
cDNA was amplified from total cDNA of Balb/c mouse bone
marrow cells using PCR primers: 5′-ATG CCC TCT TGG GCC
CTC TTC A-3′ and 5′-CTG CCT TCA GGG CTG CTG CCA A-
3′, and cloned into the expression vector pcDNA3.1(−) containing
a Cytomegalovirus (CMV) promoter (Invitrogen Corp., Carlsbad,
CA, U.S.A.). The mouse c-mpl cDNA constructed into the
expression plasmids (pcDNA3.1-muMPL) was verified by DNA
sequencing.
Underlining; sequences corresponding to mouse c-mpl, Bold lettering; tri-
nucleotides corresponding to the amino acid replaced at residue 499 of human
thrombopoietin receptor or at residue 490 of mouse thrombopoietin receptor,
Boxed sequences; TCTAGA, GAGCTC, and GCTCAGC are recognition sites
of restriction enzymes Xba I, Sac I and Bpu1102 I, respectively.
2
.5. Construction of the human/mouse thrombopoietin receptor
chimeras A, B, C, H499L-mutated human thrombopoietin
receptor, and L490H-mutated mouse thrombopoietin receptor
using primers of H499LS and C-Xba I. The purified 1st PCR
products were subjected to the 2nd PCR using primers H-Bgl
II and C-Xba I. After the digestion by Bgl II and Xba I, the Bgl
II-Xba I fragment of pGEM3-huMPL was replaced by the PCR
product. To construct L490H-mutated mouse c-mpl (L490H-
muMPL), PCR using primers M-Sac I and L490HA was per-
formed on pcDNA3.1-muMPL. The PCR product was
digested using Sac I and Bpu1102 I, and the Sac I-Bpu1102
I fragment of pcDNA3.1-muMPL was replaced by the PCR
fragment. The L490H-muMPL was recloned into a retroviral
vector pQCXIP (Clontech). These newly constructed expres-
sion plasmids were all verified by DNA sequencing.
All chimeras and mutants were prepared according to the
PCR-based overlap extension method (Horton et al., 1990). The
positions and nucleotide sequences of each primer used in these
constructions are indicated in Fig. 2 and Table 1. To make
chimera A, part of human c-mpl was amplified using primers H-
Bgl II and HTMR, and part of mouse c-mpl was amplified using
primers MCPF and C-Xba I (1st PCR). HTMR and MCPF
contain partial sequences of the human transmembrane domain
and the mouse cytoplasmic domain (shown in Table 1). These
1
st PCR fragments were purified and mixed for use as a
template for the next PCR using primers H-Bgl II and C-Xba I
2nd PCR). The 2nd PCR fragment was digested by Bgl II and
(
Xba I, and the Bgl II-Xba I fragment of pGEM3-huMPL was
replaced by the PCR product. For chimera B, the 1st PCR using
primers H-Bgl II and HECR was done for human c-mpl cDNA
and that using primers MTMF and C-Xba I for mouse c-mpl
cDNA. HECR and MTMF contain partial sequences of the human
extracellular domain and the mouse transmembrane domain.
The purified 1st PCR fragments were combined and subjected
to the 2nd PCR using primer set of H-Bgl II and C-Xba I. After
digestion by Bgl II and Xba I, the Bgl II-Xba I fragment of
pGEM3-huMPL was replaced by the 2nd PCR product. For
chimera C, the 1st PCR was performed on mouse c-mpl with
a primer set of M-Sac I and MTHR or another set of MTHS
and C-Xba I. The primers MTHR and MTHS have sequences
of the transmembrane domain of human c-mpl and a small
portion of an extracellular domain or cytoplasmic domain of
mouse c-mpl on the 3′ end, respectively. The purified 1st PCR
products were mixed and subjected to the 2nd PCR using a
primer set of M-Sac I, C-Xba I. After the digestion by Sac I and
Xba I, the Sac I-Xba I fragment of pcDNA3.1-muMPL was
replaced by the 2nd PCR product. To construct H499L-mutant
human c-mpl (H499L-huMPL), the 1st PCR was performed on
human c-mpl cDNA using primers of H-Bgl II and H499LA or
2.6. Transfection of the thrombopoietin receptor expression
plasmids
7
Parental Ba/F3 cells were washed twice and 1×10 cells
were suspended in 0.6 ml PBS. Cells were mixed with 20 µg of
each expression plasmid carrying the human or mouse thrombo-
poietin receptor gene, chimeric thrombopoietin receptor genes
A, B, or C, or the H499L mutant thrombopoietin receptor gene,
and transfected by electroporation at 220 Vand 960 µFD. After
24-h incubation at 37 °C in RPMI medium supplemented
with 10% fetal bovine serum (HyClone) and mouse IL-3, the
cells were collected by centrifugation and resuspended in the
same medium containing 2 mg/ml Geneticin (G418, Invitrogen-
Gibco) as a selection marker. Ba/F3-huMPL, Ba/F3-muMPL,
Ba/F3-chimeras A, B, C, and Ba/F3-huMPL(H499L) were
established and maintained in RPMI1640 medium containing
10% fetal bovine serum, mouse IL-3 and 2 mg/ml Geneticin.
A retroviral packaging cell line 293GP2 was cotransfected
with pQCXIP-L490H-muMPL and pVSV-G plasmids using
Fugene 6 (Roche). The culture supernatant was collected as a
viral suspension and added to Ba/F3 cells in the presence of
polybrene. After 24 h of infection, Ba/F3-muMPL(L490H)

N. Yamane et al. / European Journal of Pharmacology 586 (2008) 44–51
47
+
Fig. 3. Effects of compound I on human and mouse megakaryocyte colony formation. (A) Human CD34 cells were cultured with 1 nM recombinant human
thrombopoietin or 0.2, 1, 5 μM compound I, respectively. After 12 days, megakaryocyte colonies were labeled with anti-CD41 antibody and the number of them was
counted. (B) Mouse bone marrow cells were cultured with 0.135 nM (10 ng/ml) recombinant mouse thrombopoietin, 1 nM recombinant human thrombopoietin, or 1,
2
μM compound I, respectively. After 7 days, megakaryocyte colonies were stained for acetylcholinesterase activity and the number of them was counted. All results
are shown as the mean±S.D. of four determinations. Lower panels of (A) and (B) show their typical images. Bar=250 µm.
cells were selected by 3-day incubation with 1.25 µg/mL
puromycin.
slides (StemCell Technologies) in the presence of different
concentrations of compound I, recombinant human throm-
bopoietin, or vehicle (0.1% DMSO). The slides were incubated
2
.7. Cell proliferation assay
at 37 °C in humidified air containing 5% CO for 12 days.
2
After being dehydrated, fixed, and blocked with 5% human
serum in Tris-buffered saline, the cells on the slides were
incubated with 10 µg/ml anti-human CD41a antibody, then
biotin-conjugated goat anti-mouse IgG and avidin alkaline
phosphatase conjugate and alkaline phosphatase substrate were
applied in sequence. Evans blue counter-staining was done for
Cells growing in log-phase were collected and washed once in
RPMI1640 medium (without any supplements) to remove resi-
5
dual mouse IL-3 and resuspended at a density of 5×10 cells/ml
in RPMI1640 medium containing 10% fetal bovine serum
but without IL-3). Fifty thousand cells were dispensed into
(
+
each well of 96-well tissue culture plates containing recombinant
human thrombopoietin or compound I at a variety of concen-
trations. At 20 h after incubation, WST-1 reagent (Takara Bio
Inc.) was added to each well (10 µl/well), and the plates were
incubated for a further 4 h at 37 °C. Finally the amount of viable
cells was measured as the absorbance at 450 nm. Data was
represented as a percentage of the maximum viability of each cell
stimulated by recombinant human thrombopoietin.
each slide. CD41a colonies were enumerated as megakaryocyte
2
.8. Human megakaryocyte colony formation assay
Human megakaryocyte colony formation assay was per-
formed according to the method of MegaCult™-C (StemCell
+
Technologies). Briefly, CD34 cells from human bone marrow
were washed and resuspended at a concentration of 3.3×
3
1
0 cells/ml in Iscoves modified Dulbecco medium (IMDM)
supplemented with 10 µg/ml recombinant human insulin,
00 µg/ml human transferrin (iron-saturated), 2 mM L-glutamine,
.1 mM 2-mercaptoethanol and 1.1 mg/ml collagen as serum-
free medium (Mega-IMDM). An aliquot of 0.75 ml of cell
suspension was dispensed into each well of double chamber
Fig. 4. Proliferative activity of compound I for each thrombopoietin-dependent
cell line expressing human or mouse receptor. Proliferative responses of Ba/F3-
huMPL stimulated by recombinant human thrombopoietin (open circles) or
compound I (closed circles), and Ba/F3-muMPL stimulated by recombinant
human thrombopoietin (open squares), or compound I (closed squares) are shown.
The values are shown as the means of duplicate determinations.
2
0

48
N. Yamane et al. / European Journal of Pharmacology 586 (2008) 44–51
5
concentration of 3.3×10 cells/ml and subjected to the same
assay as that for human megakaryocyte colony formation. After
being dehydrated and fixed in ice-cold acetone, megakaryocyte
colonies were stained for acetylcholinesterase activity with
0.5 mg/ml acetylthiocholiniodide in 75 mM sodium phosphate
buffer (pH 6.0) including 5 mM sodium citrate, 3 mM copper
sulfate, and 0.5 mM potassium ferricyanide at room temperature
for 3.5 h. Slides were refixed and counterstained with Harris'
hematoxylin solution. Megakaryocyte colonies were defined as
consisting of at least 3 megakaryocytes per colony and scored
with a stereoscopic microscope. The values are shown as the
means±S.D. of 4 determinations.
Fig. 5. Responses of three chimera receptors to compound I in proliferation
assay. Three thrombopoietin-dependent cell lines Ba/F3-chimera A (circles),
B (squares) or C (triangles) were stimulated by recombinant human throm-
bopoietin (open) or compound I (closed). The values are shown as the means of
duplicate determinations.
3
. Results
3.1. Species specificity of the thrombopoietin mimetics
We had discovered novel thrombopoietin mimetics with a
colony-forming units (CFU-Meg) using a stereoscopic micro-
scope (OPTIHOT-2, Nikon, Japan). The values are shown as the
means±S.D. of 4 determinations. Simultaneously, photographs
of the colonies were obtained with a stereoscopic microscope-
mounted camera.
cell-based assay using the thrombopoietin-dependent Ba/F3-
huMPL cells expressing the human thrombopoietin receptor on
their surface (Yamane et al. in submission). Fig. 1A shows the
chemical structure of compound I, a tool compound as a repre-
sentative of the thrombopoietin mimetics examined in this
study.
2
.9. Mouse megakaryocyte colony formation assay
Compound I stimulated not only proliferation of Ba/F3-
+
huMPL cells, but also the generation of CD41 megakaryocytic
+
Mouse megakaryocyte colony formation assay was per-
colonies from CD34 human bone marrow cells in a dose-
formed according to the method of MegaCult™-C (StemCell
Technologies Inc.). Female Balb/c mice 7 to 8 weeks old were
sacrificed and bone marrow cells were collected from their
femurs. Next, the cells were cleared of erythrocytes by lysing
buffer (Beckman Coulter, Tokyo, Japan) and washed with Mega-
IMDM twice. They were then resuspended in Mega-IMDM at a
dependent manner, as did recombinant human thrombopoietin
(Fig. 3A). Prior to evaluation of the in vivo activity of com-
pound I, megakaryocyte colony formation assay using mouse
bone marrow cells was performed. In this assay, acetylcholi-
nesterase activity (AchE) (Jackson, 1973) was measured to
detect mouse megakaryocytes. Although 1 nM of recombinant
Fig. 6. The amino acid residue unique to human in the transmembrane domain. (A) Comparison of mouse, guinea pig, cynomolgus monkey and human thrombopoietin
receptor transmembrane domains. Shaded boxes show the residues identical to human receptor. The results of megakaryocyte colony formation assay are shown by +/−
on the right. (B) Construction of loss-of-function mutant and gain-of-function mutant. Open rectangles show mismatched amino acid residues between human and
499
mouse receptors. Asterisk shows His at residue 499 is unique to the human receptor. Leu at residue 490 of mouse receptor corresponds to human His
.

N. Yamane et al. / European Journal of Pharmacology 586 (2008) 44–51
49
human thrombopoietin increased the equivalent number of
mouse megakaryocyte colonies that were induced by 0.135 nM
of recombinant mouse thrombopoietin, even 2 µM of compound
I had no effect on the mouse bone marrow cells (Fig. 3B).
To confirm the species-specificity of compound I, the pro-
liferation assay was conducted using thrombopoietin-dependent
cells expressing the human thrombopoietin receptor (Ba/F3-
huMPL) or the mouse thrombopoietin receptor (Ba/F3-muMPL).
While recombinant human thrombopoietin stimulated prolifera-
tion of both Ba/F3-huMPL and Ba/F3-muMPL cells, compound
I stimulated proliferation of only Ba/F3-huMPL and not Ba/F3-
muMPL cells (Fig. 4).
Fig. 7. Responses of the human H499L mutant receptor and the mouse L490H
mutant receptor to compound I in proliferation assay. Two thrombopoietin-
dependent cell lines expressing mutants, Ba/F3-huMPL(H499L) (circles) and
Ba/F3-muMPL(L490H) (squares), were stimulated by recombinant human
thrombopoietin (open) or compound I (closed). The values are shown as the
means of duplicate determinations.
3
.2. Responses of chimera thrombopoietin receptors to the
thrombopoietin mimetics
We tried to identify the pivotal site for the activation of the
thrombopoietin receptor by exploiting the species-specificity of
compound I. We established three thrombopoietin-dependent
Ba/F3 cell lines expressing different types of chimera receptors
two cell lines expressing mutant receptors: the human throm-
4
99
(
Ba/F3-chimeras A, B and C). As shown in Fig. 2, chimera A
bopoietin receptor in which His is replaced with Leu (Ba/F3-
huMPL(H499L)) for a loss of the function and the mouse
has the human extracellular and human transmembrane do-
mains. Chimera B has the human extracellular and mouse
transmembrane domains. Chimera C has the mouse extracellular
and human transmembrane domains. All chimera receptors have
the mouse cytoplasmic domain. A cell proliferation assay was
done using the cell lines expressing these chimera receptors
4
90
thrombopoietin receptor in which Leu
is replaced with His
(Ba/F3-muMPL(L490H)) for a gain of the function (Fig. 6B).
Although the two mutant receptors were functionally active as
both reacted to recombinant human thrombopoietin, the replace-
4
99
490
ment of the residue of H or L led Ba/F3-muMPL(L490H)
to gain the reactivity to compound I and Ba/F3-huMPL(H499L)
to lose it (Fig. 7).
(Fig. 5). All three chimera receptors were confirmed as being
functional because recombinant human thrombopoietin could
react with them. Compound I led to the proliferation of Ba/F3-
chimeras A and C, but not Ba/F3-chimera B. The peculiar do-
main to chimera B was the mouse transmembrane domain. These
results suggested that the human-type transmembrane domain
was pivotal for the receptor activation by compound I. Interest-
ingly, Ba/F3-chimera C proliferated at a lower concentration of
the compound than Ba/F3-chimera A or Ba/F3-huMPL shown in
Fig. 4.
4. Discussion
We discovered a series of novel non-peptide small molecules
with proliferation activities on the human thrombopoietin-
dependent cells (Ba/F3-huMPL) (Yamane et al., in submission).
These compounds activated signal molecules in the thrombo-
poietin signal transduction pathway that have been revealed by
previous studies (Drachman et al., 1995, 1997; Kaushansky,
2005; Miyakawa et al., 1995, 1997; Rojnuckarin et al., 1999;
Sattler et al., 1995). Our thrombopoietin mimetics induced
megakaryocytopoiesis from human hematopoietic progenitor
cells, but did not act on mouse bone marrow cells. Further
investigation using bone marrow cells of guinea pig and cyno-
molgus monkey revealed that the compound could stimulate
only the human thrombopoietin receptor. These results sug-
gested that it would be difficult to estimate the thrombopoietin-
like activity of our compounds in an in vivo system using
laboratory animals. Analysis of human/mouse chimera recep-
tors demonstrated that the transmembrane domain was essential
for the exhibition of the activity of the compound. Comparison
of the transmembrane domains among mouse, guinea pig,
3
.3. Comparison of amino acid sequences from humans and
other animals
Fig. 6A shows the comparison of the amino acid sequences
of the transmembrane domains from human and three other
animals. The human sequence differs from mouse, guinea pig,
and cynomolgus monkey sequences by four, two and one amino
acid, respectively. We examined the reactivity of bone marrow
cells from these animals to compound I by the megakaryocyte
colony formation assay, but found no response to it. These
results strongly suggested that the unique histidine at residue
4
99 of the human thrombopoietin receptor was the pivotal site
of the thrombopoietin receptor activation by compound I.
4
99
cynomolgus monkey and human revealed that His is a resi-
due unique to the human thrombopoietin receptor. To confirm
3
.4. Responses of point-mutated thrombopoietin receptors to
4
99
thrombopoietin mimetics
the hypothesis that His in the transmembrane domain of the
human thrombopoietin receptor is pivotal for the receptor acti-
vation by our compound, we conducted a cell proliferation
assay using two thrombopoietin-dependent cell lines expressing
To confirm that His at residue 499 in the transmembrane
domain is required for activity of compound I, we established

50
N. Yamane et al. / European Journal of Pharmacology 586 (2008) 44–51
the mutant receptor: Ba/F3-huMPL(H499L) or Ba/F3-muMPL
L490H). Ba/F3-huMPL(H499L) cells showed that the hu-
man thrombopoietin receptor lost the reactivity to compound
Acknowledgements
(
We thank Mr. Koji Takahashi, Ms. Kazue Kato, Mr. Kenzo
Koizumi, for their work in evaluating compounds, Mr. Etsuo
Nakamura, Dr. Satoshi Orita, Mr. Hiroyuki Nakai, for their
work in cloning and engineering receptors. We are very thankful
to Dr. Masami Takayama, Dr. Takeshi Shiota, Dr. Hideyuki
Takenaka, Dr. Yutaka Yoshida, Mr. Shinichiro Hara, Mr.
Takami Murashi for their work in developing compounds.
And we would like to thank Dr. Kiyoshi Nagata for his advice
on the planning of the experiments.
4
99
I by the replacement of His
with Leu. Conversely, Ba/F3-
muMPL(L490H) cells showed that the mouse thrombopoietin
receptor gained reactivity to compound I by the replacement
4
90
499
of Leu
with His. These results confirmed that His
in
the transmembrane domain of the human thrombopoietin re-
ceptor is essential for the thrombopoietin-like activity of the
compound.
Among the non-peptidyl thrombopoietin receptor agonists
reported to date, SB394725, NIP-004 and YM77 showed high
specificity to the human receptor, and SKF-57626, SB-497115
and NIP-004 were reported to need the transmembrane region
References
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99
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5
05
mutation of Ser to Asn in the transmembrane domain made
the thrombopoietin receptor constitutively active (Onishi et al.,
Drachman, J.G., Sabath, D.F., Fox, N.E., Kaushansky, K., 1997. Thrombo-
poietin signal transduction in purified murine megakaryocytes. Blood 89,
1
996). This mutation was found in patients with familial
4
83–492.
essential thrombocythemia (Ding et al., 2004). It is likely that
the transmembrane domain plays an important role in control-
ling the activation of the thrombopoietin receptor. An amphi-
pathic motif which keeps the unliganded receptor inactive has
been found at the transmembrane-cytoplasmic junction of the
thrombopoietin receptor (Staerk et al., 2006). Considering
hydrophobicity as a structural peculiarity of our thrombopoietin
mimetics, the compound might be inclined to interact with a
region abundant in hydrophobic amino acids like the trans-
membrane domain. A plausible explanation is that compound I
binds at some other position on the external part of the receptor
and needs the transmembrane His 499 to affect the conforma-
tional change in the receptor.
Erickson-Miller, C.L., Delorme, E., Iskander, M., Giampa, L., Hopson, C.B.,
Luengo, J., Duffy, K., Dillon, S.B., Rosen, J., Miller, S., Lamb, P., Tian, S.-S.,
2004. Species specificity and receptor domain interaction of a small molecule
TPO receptor agonist. Blood (ASH Annual Meeting Abstracts) 104, 2909
Abstract.
Erickson-Miller, C.L., DeLorme, E., Tian, S.-S., Hopson, C.B., Stark, K.,
Giampa, L., Valoret, E.I., Duffy, K.J., Luengo, J.L., Rosen, J., Miller, S.G.,
Dillon, S.B., Lamb, P., 2005. Discovery and characterization of a selective,
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8–64.
The in vivo activity of our thrombopoietin mimetics cannot
be evaluated using common experimental animals. In the case
of SB-497115, the chimpanzee was used for in vivo study
because the chimpanzee thrombopoietin receptor has a human-
Jackson, C.W., 1973. Cholinesterase as a possible maker for early cells of the
megakaryocytic series. Blood 42, 413–421.
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phosphorylation of Janus kinase 2 and Shc in human blood platelets. Blood
4
99
type transmembrane domain containing His (Erickson-Miller
et al., 2004; Sellers et al., 2004). For other in vivo assay sys-
tems, the transplantation of human bone marrow cells into
experimental animals (Nakamura et al., 2006) or the transgenic
4
99
mouse expressing human c-mpl should be useful. While His
in its transmembrane domain should be essential for the acti-
vation by compound I, there might be a region which regulates
the sensitivity to the compound in the extracellular domain of
the receptor. Figs. 4, 5 and 7 show that Ba/F3-chimera C and
Ba/F3-muMPL(L490H) containing mouse type extracellular
domains were more sensitive to compound I than Ba/F3-
chimera A and Ba/F3-huMPL. Further investigation using
chimeras or mutants of the thrombopoietin receptor should
help us understand the mechanism of the receptor activation by
these compounds and lead to the development of more active
thrombopoietin mimetics.
8
6, 23–27.
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Y., 1997. Thrombopoietin and thrombin induce tyrosine phosphorylation of
Vav in human blood platelets. Blood 89, 2789–2798.
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Ohnishi, Y., Ishiwata, N., Ikeda, Y., Tsuruzoe, N., 2006. A novel nonpeptidyl
human c-Mpl activator stimulates human megakaryopoiesis and thrombo-
poiesis. Blood 107, 4300–4307.
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Gorman, D.M., Miyajima, A., Kitamura, T., 1996. Identification of an
oncogenic form of the thrombopoietin receptor MPL using retrovirus-
mediated gene transfer. Blood 88, 1399–1406.

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cillins as thrombopoietin mimetic small molecules. Bioorg. Med. Chem. 13,
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TYK2 tyrosine kinases. Exp. Hematol. 23, 1040–1048.
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