Articles
https://doi.org/10.1038/s41589-019-0285-7

Macrocyclic peptide-based inhibition and imaging
of hepatocyte growth factor
Katsuya Sakai1,2, Toby Passioura3,12,13, Hiroki Sato1,2,13, Kenichiro Ito3, Hiroki Furuhashi4,
Masataka Umitsu5, Junichi Takagi 5, Yukinari Kato 6, Hidefumi Mukai7, Shota Warashina7,
Maki Zouda7, Yasuyoshi Watanabe8, Seiji Yano2,9, Mikihiro Shibata2,10, Hiroaki Suga 3* andKunio Matsumoto 1,2,11*
Activation of hepatocyte growth factor (HGF) by proteolytic processing is triggered in cancer microenvironments, and subsequent signaling through the MET receptor is involved in cancer progression. However, the structure of HGF remains elusive, andfew small/medium-sized molecules can modulate HGF. Here, we identified HiP-8, a macrocyclic peptide consisting of 12 aminoacids, which selectively recognizes active HGF. Biochemical analysis and real-time single-molecule imaging by high-speed
atomic force microscopy demonstrated that HiP-8 restricted the dynamic domains of HGF into static closed conformations,
resulting in allosteric inhibition. Positron emission tomography using HiP-8 as a radiotracer enabled noninvasive visualizationand simultaneous inhibition of HGF–MET activation status in tumors in a mouse model. Our results illustrate the conformational change in proteolytic activation of HGF and its detection and inhibition by a macrocyclic peptide, which may be useful for
diagnosis and treatment of cancers.

P

roteolytic processing is a fundamental mechanism of physiological regulation of protein function and is often coupled todisease conditions. Hepatocyte growth factor (HGF) is secreted
as a biologically inactive single-chain polypeptide (scHGF) and it
remains in this precursor form in plasma and tissues1–3. HGF consists of six individual domains––the amino terminus (N terminus; N), first to fourth kringle (K1–K4) and carboxy terminus (Cterminus) serine protease-like (SP) domains. On tissue damage
and in cancer microenvironment, serine proteases cleave scHGF
between the K4 domain and the SP domain, generating functionally active two-chain HGF (tcHGF)1–3. The tcHGF activates MET
receptor signaling, resulting in the growth, survival and promotionof the migratory behaviors of diverse cell types4–6. HGF and MET
are essential for the development and repair of tissues, and aberrant regulation of HGF/MET participates in oncogenesis and cancer
pathology4–6. Targeting active HGF is therefore attractive for both
diagnosis and treatment of cancers.
Various MET targeted tracers for imaging of tumors have beendeveloped and evaluated in preclinical studies or clinical settings7,8,
however, most of them monitor MET expression rather than activation. Since MET in cancer is activated through both HGF-dependentand HGF-independent (MET overexpression) mechanisms4–6,9–12,
the assessment of HGF-dependent MET activation, which can beassessed by molecules targeting active HGF, would be valuable forpatient selection for HGF–MET inhibitors5,8,13,14 or for detection ofearly metastatic cancers14–18.

Activation of HGF by proteolytic processing causes local structural change in the SP domain of HGF19–22. This confers MET receptor binding ability on the SP domain19–22. In addition, a change inmolecular shape, that is, closed structures of scHGF to elongatedstructures of tcHGF, has been suggested by electron microscopy23.
However, the limited structural information currently available for
HGF/MET and the nature of the interacting surface between HGF
and MET made the development of small/medium-sized molecules
targeting HGF extremely challenging24. For example, a known peptide inhibitor targeting the SP domain of HGF generated by phagedisplay has shown a modest inhibitory activity (a half-maximal
inhibitory concentration, IC50 = 20 µM) in cellular assays25.
Macrocyclic peptides have emerged as a new and exciting class ofdrug candidates for targets such as protein–protein interactions thathave proved challenging for small molecules26,27. The constrained
structures of macrocyclic peptides with molecular masses in therange of 500–2,500 Da can produce antibody-like binding affinity and specificity28, can target unique chemical space and exhibit
different pharmacokinetic profiles from either small molecules or
biologics26–28, thus bridging the gap between small molecules andbiologics. To synthesize and screen large libraries of macrocyclic
peptides against protein targets, several powerful combinatorial
library technologies have been developed29–34. One such technique
is random non-standard peptides integrated discovery (RaPID)35,
which integrates messenger RNA display36 with flexible in vitrotranslation (FIT) genetic code reprogramming37 and allows theDivision of Tumor Dynamics and Regulation, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. 2WPI-Nano Life Science Institute(WPI-NanoLSI), Kanazawa University, Kanazawa, Japan. 3Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
4
Mathematical and Physical Sciences, Graduate School of Natural Science & Technology, Kanazawa University, Kanazawa, Japan. 5Laboratory of Protein
Synthesis and Expression, Institute for Protein Research, Osaka University, Osaka, Japan. 6Medicine/New Industry Creation Hatchery Center, Tohoku
University, Sendai, Japan. 7Laboratory for Molecular Delivery and Imaging Technology, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
8
Laboratory for Pathophysiological and Health Science, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan. 9Division of Medical Oncology,
Cancer Research Institute, Kanazawa University, Kanazawa, Japan. 10High-speed AFM for Biological Application Unit, Institute for Frontier Science
Initiative, Kanazawa University, Kanazawa, Japan. 11Tumor Microenvironment Research Unit, Institute for Frontier Science Initiative, Kanazawa University,
Kanazawa, Japan. 12Present address: Sydney Analytical, The University of Sydney, Sydney New, South Wales, Australia. 13These authors contributed
equally: Toby Passioura, Hiroki Sato. *e-mail: hsuga@chem.s.u-tokyo.ac.jp; kmatsu@staff.kanazawa-u.ac.jp
1598

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NATurE CHEMicAl BiOlOGy
synthesis of very large (>1012 compound) libraries of natural product-like macrocycles that can be readily screened for binding to protein targets of interest.
Here, using RaPID selection, we identified HiP-8 (1), a fairly
small (1,678 Da) macrocyclic peptide, which selectively binds to
tcHGF and potently inhibited the HGF–MET interaction with subnanomolar potency. Biochemical analysis and high-speed atomic
force microscopy (HS-AFM) observations revealed conformational differences between scHGF and tcHGF, the latter of which
was selectively recognized and allosterically inhibited by HiP-8.
Intravenous administration of HiP-8 radiotracer and imaging by
positron-emission tomography (PET) demonstrated noninvasive
visualization and simultaneous inhibition of HGF–MET activationin tumors in a mouse model.

Results

Identification of HGF-inhibitory macrocyclic peptides. To identify macrocyclic peptide inhibitors of HGF, we employed a RaPID
approach using a peptide library initiated with N-chloroacetylD-Trp, which spontaneously reacts with a cysteine residue downstream of the translated 4–15 random amino acids, thereby yieldinga >1012 compound library of thioether-closed macrocyclic peptides(Fig. 1a, left). Biotinylated HGF protein (Supplementary Fig. 1)
was used as bait for the in vitro translated thioether-macrocycles
displayed on their cognate mRNAs and HGF-binding species wereisolated and their mRNA/complementary DNA were enriched by
PCR. Recovery of HGF-binding macrocyclic peptides increased
after 3–5 rounds of selection (Supplementary Fig. 2), and enriched
cDNA pools after the fifth round of selection were cloned andsequenced. Sequence alignment of the 43 clones recovered identified two independent sequence families (Supplementary Fig. 3).
On the basis of sequence similarity, 16 candidates were chosen
for chemical synthesis to evaluate anti-HGF activity on the HGFinduced MET receptor phosphorylation in human mesothelioma
cells in culture38 (Fig. 1a, right). Most of these peptides showedsome degree of HGF-inhibitory activities and were named ‘HGFinhibitory peptides’ (HiPs).
Characterization of HiP-8. Remarkably, HiP-8, a macrocyclic peptide comprising 12 amino acids (Fig. 1b), bound tightly to HGF
with a dissociation constant (KD) of 0.4 nM and a remarkably slow
dissociation rate (koff = 0.4 × 10−3 s−1) (Supplementary Fig. 4a). HiP-8
demonstrated potent HGF-inhibitory activity in cellular assays withan IC50 of 8 nM (Supplementary Fig. 4b). HiP-8 effectively inhibited the interaction between fluorescein-tagged HGF and an immobilized MET-ectodomain-Fc, with an IC50 of 0.9 nM (Fig. 1c andSupplementary Figs. 4c and 5). We further explored HiP-8 analogs
by repeating RaPID selection using a focused library based on theconserved LSKW sequence motif found in HiP-8, and this identified three analogs, HiP-8-D-01 (2), HiP-8-D-08 (3) and PLSKW01 (4), which inhibited the HGF–MET interaction with IC50 valuesof 0.6, 1.3 and 1.2 nM, respectively (Supplementary Fig. 6). On thebasis of smaller size and greater potency, HiP-8 was chosen for further characterization.
To improve water-solubility and pharmacokinetics of HiP-8, we
modified its C-terminal region with a polyethylene glycol (PEG)
chain39 (Supplementary Fig. 7a). Since the C terminus of HiP-8 wasconnected to its mRNA during the selection, C-terminally modified
HiP-8 would be likely to retain wildtype HiP-8 activity. Indeed, on
modification with PEG5 or PEG11 at the C terminus, full inhibitory
activity was retained (Supplementary Fig. 7b,c). HiP-8 and HiP-8PEG11 were used for most of the subsequent experiments.
HiP-8 inhibits HGF-induced tumor cell responses. We evaluatedHiP-8 for its inhibitory activity on tumor cell responses induced by
HGF (Fig. 1d–f). HGF-induced activation of the MET receptor andsubsequent cell signaling through Gab1, Akt and Erk1/2 were all
potently inhibited by HiP-8 in human mesothelioma cells (Fig. 1d).
HGF-induced B16-F10 melanoma cell migration was significantly
inhibited by HiP-8-PEG11 at 4–20 nM and completely inhibited at
100 nM in a trans-well migration assay (Fig. 1e). This potency of
HiP-8-PEG11 was comparable to that of an anti-HGF neutralizing
antibody (Fig. 1e).
Activation of the MET receptor by HGF10–12 or overexpression of
MET9,12 is involved in acquired drug-resistance of various types ofcancer. We tested HiP-8-PEG11 on HGF-induced human lung cancer cell growth under treatment with the anti-cancer drug, gefitinib(Fig. 1f). HGF rescued cell growth in gefitinib-treated cells. HiP-8PEG11 significantly inhibited the HGF-induced gefitinib resistanceof the cells at 10–100 nM, and almost completely abolished resistance at 1,000 nM (Fig. 1f). These results indicate that HiP-8 potently
inhibited HGF-induced cellular responses in tumor cells in vitro.
Selectivity of HiP-8. We evaluated the selectivity of HiP-8 for
HGF compared with other growth factors; specifically, epidermal growth factor, basic fibroblast growth factor, platelet-derived
growth factor, transforming growth factor-β and vascular endothelial cell growth factor. For these experiments, a phospho-receptor
tyrosine kinase array (Supplementary Fig. 8) and assessment of
Akt phosphorylation following growth factor stimulation (Fig. 2a)
were used. HiP-8 selectively inhibited HGF–MET signaling inall cases. We further evaluated HiP-8’s selectivity against macrophage-stimulating protein (MSP) or murine HGF, which are
structurally homologous to human HGF, by surface plasmon resonance (SPR) (Fig. 2b–d). We observed almost no affinity of HiP-8PEG11 for MSP (Fig. 2c), whereas HiP-8-PEG11 exhibited binding
to murine HGF with 50-fold less affinity (Fig. 2d, KD = 53.7 nM)
than human HGF (Fig. 2b, KD = 0.9 nM). These results demonstrated that HiP-8 was both a potent and highly selective inhibitorof human HGF.
Allosteric inhibition of HGF by HiP-8. To study mechanism of
HiP-8 inhibition, we used SPR to assess the effect of HiP-8 on two
proposed interfaces of HGF with MET: the NK4 (N terminus)40–42
and SP (C terminus)19-22,42 domains of HGF (Supplementary Fig. 9).
Concentrations of 2.5 to 10 nM of HiP-8 inhibited the interaction between HGF (5 nM) and MET-Fc (Supplementary Fig. 9d),which was consistent with results from a competition binding assay(Fig. 1c). Notably, HiP-8 inhibited interactions neither between NK4
and MET-Fc nor between SP and MET-Fc (Supplementary Fig. 9d).
In agreement with these results, when HGF was divided into two
fragments, neither NK4 nor SP could individually bind to HiP-8PEG11 (Fig. 3a). A mixture of equimolar amounts of NK4 and SP
reconstituted binding to HiP-8-PEG11 (Fig. 3a). Varying concentrations of NK4 in combination with 600 nM SP bound to HiP-8PEG11 in a concentration-dependent manner (Supplementary
Fig. 10a). Conversely, varying concentrations of SP in combination
with 600 nM of NK4 also bound to HiP-8-PEG11 in a concentration-dependent manner (Supplementary Fig. 10b). Thus, both NK4
and SP contribute to the binding with HiP-8-PEG11. Because themixture of NK4 and SP reconstituted functional HGF that activates MET42 (Supplementary Fig. 11), these results suggest thatthe structure created by the interaction between these domains isessential for binding to HiP-8. Further evaluation of N-terminally
deleted HGF demonstrated the essential contribution of the K2–K4
domains to HiP-8 binding and the lesser contribution of the NK1
domains of HGF (Fig. 3a). These results demonstrated that HiP-8
allosterically inhibited the HGF–MET interaction through interactions with multiple domains of HGF.
Selectivity for active conformations of HGF. Proteolytic cleavage between the K4 and SP domains converts inactive HGF to the

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Articles
s

HN

MET
pGab1 (Y627)
Gab1
pAkt (S473)
Akt
pErk1/2 (T202/Y204)
Erk1/2
GAPDH

NH2

+

OO

HN

O
S

H2N

NH

O
NH2

NH
O

N
H

O
O

HN

HN

NH

N
H

NH

O

H
N

HN

O HO
O

HN

NH2

50
IC50 = 0.9nM
IC50 = 0.9 nM
0

NH

O

0.01 0.1

NH1
10 100 1,000
HiP-8 (nM)O
OH

HiP-8 (1)

e

10
10
0
1,
00
0
+

NH

HO

+

P = 0.00005
P = 0.0003
P = 0.0026

150
150

100
100
50
50
50
50

250

150
100
50
0
HGF

–

+

+

+

P = 0.00003
P = 0.0008
P = 0.0064

120

200

4
33
kDa

f

P = 0.00006
P = 0.0003
P = 0.0166

Cell viability (%)

pMET (Y1234/1235)

– + +

O

Migrated cell number

HGF

1

HiP-8 (nM)

100

N

HiP-12
HiP-13
HiP-1
HiP-9
HiP-10
HiP-6
HiP-7
HiP-16
HiP-14
HiP-8
HiP-4
HiP-5
HiP-11
HiP-3
HiP-2
HiP-15dcbPercent inhibitionof MET
0 20 40 60 80 100

Ac DW (random sequence) C-NH2

Percent inhibition of
HGF-MET bindinga

NATurE CHEMicAl BiOlOGy

+

+

+

+

20 10020 100 4
HGF-Ab
HiP-8-PEG11
(nM)
(nM)

100
80
60
40
20

0
Gefitinib

–

+

+

+

+

+

+

HGF

–

–

+

+

+

+

+

1 10 100 1,000
HiP-8-PEG11 (nM)

Fig. 1 | A potent macrocyclic peptide inhibitor of HGF. a, HGF-binding macrocyclic peptides identified by RaPID selection (left) and their inhibitory activity
at 1,000 nM on HGF-induced MET activation in EHMES-1 cells (right). Data represent mean (n = 2, distinct replicates for cell cultures). b, Structure of
HiP-8. c, Concentration–response curve of HiP-8 on binding between fluorescein-HGF and MET-beads. Data represent the mean (n = 2, distinct
replicates). d, HiP-8 inhibits phosphorylation of MET, Gab1, Akt and Erk1/2 induced by HGF in EHMES-1 meosthelioma cells. e, HiP-8-PEG11 inhibits
HGF-induced cell migration of B16-F10 melanoma cells in a manner comparable to an anti-HGF antibody. Data represent mean ± s.d. (n = 3, distinct
replicates for cell cultures, unpaired two-tailed t-test). f, HiP-8-PEG11 inhibits gefitinib resistance induced by HGF. PC-9 human lung cancer cells were
cultured ±1 μM gefitinib, 220 pM HGF and HiP-8-PEG11 for 3 d. Data represent mean ± s.d. (n = 3, distinct replicates for cell cultures, unpaired two-tailed
t-test). a–f, Experiments were repeated twice independently with similar results. Uncropped blots can be found in Supplementary Fig. 17.

active conformation1–3,20–22. Given the essential contribution of theK4–SP interface to HiP-8 binding (Fig. 3a), we sought to determine
whether HiP-8 could distinguish between the active and inactive
conformations of HGF. To this end, we used inactive scHGF andactive tcHGF prepared by introduction of a Factor Xa cleavage
sequence43. tcHGF bound tightly to immobilized HiP-8-PEG11,
with a KD of 2.5 nM (Fig. 3b), which was similar to the affinityof HGF for HiP-8-PEG11 (Fig. 2b). In contrast, scHGF did notinteract with HiP-8-PEG11 (Fig. 3b).
Active and inactive HGF observed by HS-AFM. The results above
highlight the structural difference of the K4–SP connection between
inactive and active HGF that is selectively recognized by HiP-8(Fig. 3b,c). We used HS-AFM to visualize inactive scHGF and active
tcHGF to reveal any differences in molecular shape and dynamics.
HS-AFM enables real-time observation of macromolecules withnanometer resolution under near physiological conditions44–47. To
verify the size, shape and dynamics of each domain of HGF, we first600

observed NK1, NK4 and SP on a mica surface treated with 3-aminopropyl-triethoxysilane (AP-mica) (Fig. 4a, Supplementary Fig. 12
and Supplementary Videos 1–3). The size analysis of these molecules
showed that the sum of the mean area of NK4 and SP was almost
equivalent to the mean area of tcHGF (Fig. 4a, 100 molecules of NK1,
NK4, SP and tcHGF were analyzed). The HS-AFM video of NK1
showed that NK1 molecules consist of two globular domains, consistent with the crystal structure48, and they attached onto the AP-mica
surface (Supplementary Fig. 12a and Supplementary Video 1). The
HS-AFM video of NK4 indicated that NK4 molecules also attachedto the AP-mica surface predominantly through NK1 domains, but
additional domains corresponding to K2–K4 appeared flexible(Supplementary Fig. 12b and Supplementary Video 2). In addition,
the HS-AFM video of SP revealed a weak interaction between SP
molecules and the AP-mica surface (Supplementary Fig. 12c andSupplementary Video 3). Collectively, these data have shown thatNK1 domains of HGF attach onto the AP-mica surface, while K2–
K4 and SP domains of HGF can move freely on the AP-mica surface.

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Stimulation:

–

HGF

EGF

bFGF

PDGF

TGF-β

VEGF

HiP-8:

–

–

–

–

–

–

–

+

+

+

+

+

+
kDa
50

pAkt
Akt

1

3

3.

1

3.

2.

.5
2.
9

.5

33

36

8
3.
6
4.
4
6.
8

1

3.

8

0.

7.

1.

0

50

pAkt/Akt level :120

Human HGF

100

(nM)

80

30
10
3.3
1.1

60
40

0.37

20

0.12
0

0
Time

200 s

120
100
80
60

KD = 0.93 nM

40
20
0

0

800

Human HGF
(10 nM)

600
400
200

MSP
(0-200 nM)

0
Time

200 s

1,200
1,000
800
600
400
200
0
0

50

100

150

200

1,200

Murine HGF
(nM)

1,000

200

800

100

600

50

400

25
12.5
6.3
3.1
0

200
0
Time

MSP (nM)

200 s

Binding to HiP-8-PEG11 (RU)

1,000

Binding to HiP-8-PEG11 (RU)

1,200

10

20

30

Human HGF (nM)dBinding to HiP-8-PEG11 (RU)

Binding to HiP-8-PEG11 (RU)cBinding to HiP-8-PEG11 (RU)b
Binding to HiP-8-PEG11 (RU)

a

1,200
1,000
800
600

KD = 53.7 nM

400
200
0

0

50

100

150

200

Murine HGF (nM)

Fig. 2 | HiP-8’s selectivity for other growth factors. a, Effect of HiP-8 on growth factor-induced Akt phosphorylation. EHMES-1 cells were stimulated for
10 min with 20 ng ml−1 of each growth factor with or without HiP-8 (1,000 nM). Akt and phosphorylation of Akt (pAkt) were analyzed and quantified bywestern blot. Membranes from representative experiments are shown. The quantified pAkt/Akt values are shown below. Uncropped blots can be foundin Supplementary Fig. 17. b–d, Binding kinetics of human HGF (b), human MSP (c) and murine HGF (d) to immobilized HiP-8-PEG11 were analyzed using
SPR. b–d, Data represent mean (n = 2, distinct replicates) for representative sensorgrams performed with multiple concentrations of human HGF, MSP or
murine HGF. The similarities of the amino acid sequences of human MSP and murine HGF to human HGF are 40.1 and 90.5%, respectively (CLUSTALW,
GenomeNet). RU, resonance unit. For a–d, experiments were repeated twice independently with similar results.

We next observed scHGF and tcHGF on the AP-mica surface.
HS-AFM videos of both scHGF and tcHGF showed that they
attached onto the AP-mica surface predominantly through NK1
domains (Fig. 4b, arrows) and have highly flexible domains corresponding to K2–SP (Fig. 4b (arrowheads) and Supplementary
Videos 4 and 5). To analyze conformational flexibilities of scHGF
and tcHGF, we calculated the correlation coefficients, which indicate differences of molecular shapes in sequential AFM images
against a reference first frame image of each molecule (Fig. 4c).
Changes in correlation coefficients indicated the conformational
flexibilities of both scHGF and tcHGF. Despite flexible molecularshapes of both scHGF and tcHGF, scHGF showed more bent molecular shapes compared to the elongated shapes of tcHGF (Fig. 4b andSupplementary Videos 4 and 5), consistent with previous observations by electron microscopy23. This difference was semi-quantified
by measuring the distances from the tips of the head and tail of HGF
molecules (Fig. 4d). The mean distances were 9.3 nm for tcHGF and
7.5 nm for scHGF, reflecting the elongated shape of tcHGF and bent
shape of scHGF, respectively. Thus, tcHGF and scHGF have flexible
inter-domain connections, but their shapes are different.
Allosteric action of HiP-8 observed by HS-AFM. We next visualized the allosteric action of HiP-8 to tcHGF. tcHGF and HiP-8 werepremixed in a 1:10 molar ratio in buffer solution, applied to the
AP-mica surface and observed by HS-AFM. tcHGF complexed with
HiP-8 showed two typical molecular shapes: one was an elongatedconformation but slightly more compact than free tcHGF (shape 1of tcHGF/HiP-8 in Fig. 4b,d) (Supplementary Video 6), while theother was a closed circular conformation (shape 2 of tcHGF/HiP-8
in Fig. 4b,d) (Supplementary Video 7). Remarkably, compared withthe flexible inter-domain connections of free tcHGF, both types oftcHGF complexed with HiP-8 clearly showed static conformations
as shown in Supplementary Videos 6 and 7, and as quantitatively
analyzed by the correlation coefficients (Fig. 4c; Mol 1 of tcHGF/
HiP-8 corresponds to the molecule of shape 1 in Fig. 4b. Mol 2 oftcHGF/HiP-8 corresponds to the molecule of shape 2 in Fig. 4b). As
a control, scHGF was treated with ten-times molar excess HiP-8, butshowed an indistinguishable molecular shape and flexibility compared to free scHGF (Fig. 4c and Supplementary Video 8), further
supporting the selectivity of HiP-8 to tcHGF.
To further validate the mode of action of HiP-8, we observed
the interaction between t5A11 anti-HGF monoclonal antibody andtcHGF using HS-AFM (Supplementary Fig. 13 and Supplementary
Video 9). t5A11 recognizes both tcHGF and scHGF, but does notinhibit HGF43. HS-AFM images indicated that t5A11 bound to
tcHGF between the NK1 domains and the SP domain, which agreewith its epitope mapped to K2–K3 (ref. 43). tcHGF molecules complexed with t5A11 maintained their flexible inter-domain connections and adapted elongated shapes, in contrast to the tcHGF/HiP-8
complex. This result further supports the unique action of HiP-8
and suggests use of HS-AFM for analysis of drug action on conformational dynamics.
To confirm a conformational change of tcHGF induced by HiP8, we performed limited proteolysis of free tcHGF and tcHGF/
HiP-8 complex (Fig. 4e). Both the NK4 and SP domains were sensitive to trypsin digestion in free tcHGF, whereas the SP domain wasprotected from trypsin digestion in tcHGF complexed with HiP-8.
This result supports a conformational change in tcHGF on HiP-8
binding and highlights the effect of HiP-8 on the surface exposureof the SP domain essential for MET activation19–22,42.
Immunohistochemical detection of active HGF. To validate
if HiP-8 selectively detects biologically active HGF (tcHGF) intumor microenvironments1–3, we performed immunohistochemical
detection of tcHGF and active MET in human lung cancer tissue
sections using biotinylated HiP-8-PEG11 and an anti-phosphoMET antibody (Fig. 5a, upper panels). MET activation (pMET)
was localized in some tumor cells at the tumor–stroma boundaries. Notably, tcHGF detected by HiP-8-PEG11 co-localized withthe staining of pMET. When a serial tissue section from the same

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K1 K2 K3 K4
SS
SP

SP
SS

SP

K2 K3 K4

K2–4–SP

100
NK4 + SP

80
60
40

NK4

20
0
0

K4

SS
tcHGF (Xa)

N K1 K2 K3 K4SPSS

scHGF (Xa)c

N K1 K2 K3 K4SP

Binding to HiP-8-PEG11 (RU)

SP
300

K2–4–SPKD = 250 nM

200

tcHGF

N
K1

N
K1

K2

K2

K4

SP

(nM)
30
10
3.3
1.1
0.37
0.12
0

40
30
20
10
0

200 s

Time
60
50
40
30

scHGF (Xa)

20
10
0
200s

Time
60

K4–SP
0
100 200 300 400 500 600
HGF fragment (nM)

HiP-8

K3

K3

tcHGF (Xa)

50

100

0
scHGF

60

SP

HGF or fragment (nM)

SS
K4–SP

100 200 300 400 500 600

Binding to HiP-8-PEG11
(RU)

NK4

SP

HGF

Binding to HiP-8-PEG11(RU)

N K1 K2 K3 K4

HGF

b

120

K4

Binding to HiP-8-PEG11
(RU)

SS

Binding to HiP-8-PEG11 (RU)a50

tcHGF (Xa)40

KD = 2.5 nM

30
20
10

scHGF (Xa)

0
0

SP

10

20

30

HGF (nM)

Fig. 3 | HiP-8 binds at interfaces of multiple domains only present in tcHGF. a, HGF, HGF fragments, active tcHGF and inactive scHGF. Xa, the cleavage
site of HGF (KQLR/V), was mutated to the recognition sequence of Factor Xa (IEGR/V)43. Binding kinetics of HGF, NK4, SP, mixture of NK4 and SP, K2–4–
SP and K4–SP to immobilized HiP-8-PEG11 were analyzed using SPR. b, Binding kinetics of tcHGF and scHGF to immobilized HiP-8-PEG11 were analyzed
using SPR. c, Schematic illustration of the interaction between HiP-8 and tcHGF. For a,b, data represent mean (n = 2, distinct replicates) for representative
sensorgrams performed with multiple concentrations of HGF and HGF fragments. These experiments were repeated twice independently with similar
results. Gels for purified proteins can be found in Supplementary Fig. 18. Sensorgrams can be found in Supplementary Fig. 19.

patient was stained using the t5A11 antibody reactive to both tcHGF
and scHGF43, HGF was abundantly expressed in most tumor cells inwhich MET was not activated (Fig. 5a, lower panels). Co-localizationof pMET and tcHGF probed by HiP-8-PEG11 or sc/tcHGF probed by
t5A11 was analyzed for lung cancer tissue sections from 37 patients(Fig. 5b). The percentage overlap of the pMET-positive area with
the HiP-8-PEG11-positive area (57.6 ± 25.1%) indicated statisticallyhigher co-localization compared with the percentage overlap of thepMET-positive area with the t5A11-positive area (14.3 ± 10.9%)(P = 4.2 × 10−12). When the stained area of pMET, HiP-8-PEG11 or
t5A11 for each tissue section were quantified and classified as − (nosignal), ± (very weak signal), + (weak signal), ++ (intermediatesignal) and +++ (strong signals), sc/tcHGF probed by t5A11 wasabundant in most cancer tissues and 26 cases were classified into+++ (Fig. 5c). In contrast, tcHGF probed by HiP-8-PEG11 andpMET distributed from − to +++, showing similar distributions(Fig. 5c). These results suggest that, compared to detection of sc/
tcHGF, selective detection of tcHGF by HiP-8 provides more precise
evaluation of MET activation status in human cancer tissues.
PET imaging and in vivo efficacy. We next validated the use ofa tagged HiP-8-PEG11 variant as an imaging probe for noninvasive direct imaging of HGF–MET activation in tumors using PET.
Since HiP-8-PEG11 binds to murine HGF with 50-fold less affinity
than human HGF (Fig. 2), we used human HGF knock-in, severe
combined immunodeficiency (hHGFki;scid) mice as an appropriate
602

model reflecting human subjects. Because both alleles of exons 3–6of the endogenous murine HGF gene were replaced by exons 2–18
of the human HGF gene in hHGFki mice, these mice express human
HGF under a native promoter but do not express murine HGF, asconfirmed by plasma HGF (Supplementary Fig. 14). We preparedhHGFki;scid mice bearing both PC-9 tumors (HGF−) and PC-9
tumors stably expressing human HGF (HGF+)10 (Supplementary
Fig. 15; 100–800 mm3). Immunohistochemistry of these tumors
confirmed the difference of HGF production and MET activation
status (Fig. 6a). A PET tracer 64Cu-labeled HiP-8-PEG11 variant wasadministered intravenously to the hHGFki;scid mice bearing thesetumors. 64Cu-labeled HiP-8-PEG11 distribution was characterizedby liver uptake and rapid renal clearance, with accompanying tumor
accumulation in high contrast images depending on the HGF–MET
activation levels (Fig. 6b and Supplementary Videos 10 and 11).
Rapid tumor uptake of 64Cu-labeled HiP-8-PEG11 reached a peakat 10–17 min, followed by a slow washout of radioactivity over theremaining 75 min (Fig. 6c). Tumor/muscle ratio of radioactivity, acontrast index, indicated excellent accumulation of 64Cu-labeled
HiP-8-PEG11 in HGF+ tumors with high selectivity (5.74 ± 0.62 for
HGF−, 20.16 ± 2.22 for HGF+) (Fig. 6d).
We further evaluated the in vivo efficacy of HiP-8-PEG11 byintravenously administering HiP-8-PEG11 to the hHGFki;scid micebearing PC-9 stably expressing human HGF (Supplementary Fig. 16,
arrow, 50–150 mm3). HiP-8-PEG11 prevented MET activation intumor tissues in a dose-dependent manner (0.5–4 mg kg−1, Fig. 6e) at

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a

b
NK1

0s

0.2 s

2.0 s

3.2 s

6.4 s

N K1

NK4

8.8 s

4.2

scHGF

N K1 K2 K3 K4

0 nm

SP

SP

tcHGF

N K1 K2 K3 K4

0s

SP

0.2 s

1.4 s

2.0 s

4.6 s

6.2 s 4.5

tcHGF
0 nm

30

NK4

20

0s

SP

1.6 s

4.2 s

30

60

90

120

150

d

Mol 1

Mol 2

Mol 2

Mol 3

Mol 3

15

Mol 4

Mol 4

10

HiP-8

tcHGF/HiP-8

20

0

5

10

15

20

scHGF treated with HiP-8

Mol 2

Mol 2

Mol 3

Mol 3

Mol 4

Mol 4

10

Mol 5

Mol 5

5

15

0
Times (s)

5

–

+

0.5

0

15

20

NK4

SP

5

10

15

7.5 nm
scHGF

5
0

0

15

10

42
34
28
17
kDa

10

Mol 1

10

tcHGF

15

Mol 1

5

52
9.3 nm500

5.6 s 4.2

72

0
15

3.2 s

Trypsin

Mol 5

10

1.0 s

e

Mol 1

5

0.8 s

0 nm

scHGF

0

0.2 s

tcHGF/HiP-8(shape 2)

tcHGF

Mol 5

5.8 s 6.0
0 nm

0s
0

Area (nm2)

Correlation coefficients

0.6 s

10
0

c

0.2 s

tcHGF/HiP-8(shape 1)

tcHGF

No. of frames (%)

No. of molecules

NK1

5

10

15

4.2 nm: shape 2
8.5 nm: shape 1
tcHGF/HiP–8

0

5

10

15

Distance (nm)Fig. 4 | HS-AFM observations of tcHGF, scHGF and tcHGF/HiP-8 complex. a, Size distributions of NK1, NK4, SP and tcHGF in HS-AFM images (totalof 100 individual molecules were analyzed for each sample). Histogram indicates the area of NK1, NK4, SP and tcHGF. b, Sequential HS-AFM imagesof scHGF, tcHGF and tcHGF/HiP-8 complexes. Two shapes were observed for tcHGF/HiP-8 complexes (shape 1 and shape 2). Arrows indicate theNK1 domains attached to the AP-mica surface. Arrowheads indicate the SP domain. The color (from black to white) corresponds to the heights of themolecules. These experiments were repeated three times independently with similar results. Scale bars, 10 nm. c, Time courses of correlation coefficientsfrom the sequential HS-AFM images. Five molecules were analyzed for each sample (Mol 1–Mol 5). Note the lack of conformational flexibility of the
tcHGF/HiP-8 complexes. d, The distribution of the distance between the tips of the head and tail in each molecule. Total of 500 frames from five individual
molecules (100 frames of the sequential HS-AFM images/molecule) were analyzed for tcHGF, scHGF and tcHGF/HiP-8 complexes. e, Limited proteolysis
of tcHGF (2.5 μM) ± HiP-8 (10 μM) with trypsin. Samples were analyzed by 5–20% SDS–PAGE under reducing conditions. HiP-8 protects the SP domain oftcHGF from trypsin digestion. This experiment was repeated twice independently with similar results.

1 h post-injection. A dose of HiP-8-PEG11 at 4 mg kg−1, inhibited MET
activation in tumor tissues within 0.5–2 h (Fig. 6f), which is consistent
with the rapid distribution and gradual clearance of 64Cu-labeled HiP8-PEG11 in tumor tissues (Fig. 6c). Collectively, this ability of HiP-8 tosensitively detect HGF activation and simultaneously inhibit tcHGF
strongly suggests that it is applicable to both diagnosis and therapy.

Discussion

Using RaPID selection, we successfully identified high-affinity
binders to HGF with KD values in the subnanomolar to nanomolar

range. Of these, HiP-8, harboring an LSKW sequence motif, exhibited the most potent HGF-inhibitory activity. The remarkably
slower dissociation rate of HiP-8 from HGF compared to other lesspotent analogs (for example, HiP-11–13, which exhibited 3–20-foldfaster dissociation rates, Supplementary Fig. 4) may be essential for
its potent inhibitory action. Future X-ray crystallographic analysis
of a HiP/HGF complex may give us further structural insight.
HS-AFM clearly visualized allosteric action of HiP-8 on HGF.
HiP-8 restricted the dynamic domains of HGF into static closed
conformations, under which conditions the SP domain is packed

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HiP-8-PEG11 (tcHGF)

pMET

Overlayc
pMET staining

t5A11 (scHGF & tcHGF)

bpMET+ / HiP-8-PEG11+
pMET+ / t5A11+
Co-localization (%)

100

pMET

Overlay

57.6 ± 25.1%
14.3 ± 10.9%

Number of tissue sample

HiP-8-PEG11 staining
t5A11 staining
30
20
10
0

–

+
+
++
+++
–
Immunostaining score

P = 4.2 × 10–12

80
60
40
20
0
A1 A2 A3 A4 A5 A6 A7 A8 B1 B2 B3 B4 B5 B6 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 E5Squamous cell carcinoma

Adenosquamous Small cell
carcinoma
carcinoma

Fig. 5 | HiP-8-PEG11 detects tcHGF co-localized with MET activation in clinical tissue sections. a, Co-localization of tcHGF probed by HiP-8-PEG11 and
pMET. Serial lung cancer tissue sections from the same patient with lung cancer were double stained for biotinylated HiP-8-PEG11 and anti-phosphoMET antibody (upper panels) or anti-HGF antibody and anti-phospho-MET antibody (lower panels). Nuclei were stained by DAPI (blue). The anti-HGF
antibody, t5A11, recognizes both scHGF and tcHGF43. Scale bars, 100 μm. The tissue sample presented in a is number D1. b, The percentages of pMETpositive area in HiP-8-PEG11-positive area (tcHGF) or in t5A11-positive area (sc/tcHGF). Tissue sections from 37 patients with lung cancer were analyzed.
The numerical values represent mean ± s.d. (n = 35 for pMET+/HiP-8-PEG11+; n = 37 for pMET+/t5A11+; P = 4.2 × 10−12, unpaired two-tailed t-test).
c, Distribution of immunostaining scores of tissue sections from 37 patients with lung cancer. The stained areas of pMET, HiP-8-PEG11 or t5A11 were
quantified and classified into five scores.

and protected from proteolysis. These findings support the functional importance of the SP domain of HGF for activation of MET
receptor in line with previous studies19–22,42. Mechanistically, theconformational stabilization of HGF by HiP-8 could be explainedby the multi-faceted interaction of HiP-8 with separate domainsof HGF, as demonstrated by SPR. Similarly, macrocyclic peptide
inhibitors bound at the bi-domain interface of phosphoglyceratemutases in co-crystal structures has suggested the inhibition ofphosphoglycerate mutases through the stabilization in an uncatalytic structure49. Because of their general size and circular structures, macrocyclic peptides can span greater distances between
flexible protein domains compared to more conventional small
molecules and thereby capture these separate domains in the staticstate as demonstrated here. This possibly unique binding propertyof macrocyclic peptides, which may differ from smaller molecules
and antibodies, may expand the range of druggable binding pockets
and target proteins.
It has been proposed that processing of scHGF to tcHGF alters
the shape of the HGF molecule. Electron microscopy observations
suggested that the SP domain of scHGF bends toward the N terminus domain, while tcHGF shows an elongated open structure24.
Small-angle X-ray scattering analysis of scHGF and tcHGF in solution suggested that the structural difference between the two proteins is attributable to the movement of one end of the molecule,presumed to be the SP domain23. Consistent with these observations, our real-time direct analysis of scHGF and tcHGF by HS-AFM
visualized flexible inter-domain connection of both proteins with
604

differences in their molecular shapes, that is, the SP domain of
scHGF bent toward the N terminus, while the SP domain of tcHGF
showed a more open conformation. Although we cannot specify
the details of the structure because of the limitations in resolution
and analysis of AFM images, the previous study23 and our HS-AFM
observations suggest that the inter-domain connection at the K4–SP
domain may be organized differently between scHGF and tcHGF.
The specificity of HiP-8 to tcHGF further supports the existenceof a conformational difference in the K4–SP connection between
scHGF and tcHGF.
HiP-8 detected active tcHGF that co-localized with MET
activation in clinical cancer tissue sections. We further demonstrated that 64Cu-labeled HiP-8-PEG11 is an excellent PET
probe for noninvasive imaging of HGF–MET activation status intumors on a mouse model, due to ideal properties of HiP-8 for thisapplication: feasibility of modification, high affinity and selectivity for active HGF and rapid clearance from non-targeted tissues
as well as rapid distribution and accumulation into tumor tissues. These are potential advantages of macrocyclic peptides forimaging compared to imaging with the use of antibodies, which
include problems of a long clearance half-life and concerns regarding immunogenicity50. HiP-8 also prevented HGF-induced MET
activation in tumor tissues in a mouse model. The relatively
short effective duration of HiP-8 (≤5 h) compared to antibodies(generally days to a week) may be explained by its rapid clearance asshown in PET studies. Sustained release of HiP-8 may improve in vivo
therapeutic efficacy, and the relatively small molecular mass of HiP-8

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NATurE CHEMicAl BiOlOGyab0.25 min15 min90 min1.0

HGF (–)HGF (+)

SUV

Liver

Kidney0.0

HGF pMET DAPI

c

d

0.7

P = 0.0033
25

SUV

0.5

Tumor/muscle ratio

0.6
HGF+

0.4
0.3

HGF–

0.2
0.1
0

20
15
10
5
0

0

20

40

60

80

100

HGF–

HGF+

Time (min)efHiP-8-PEG11 (mg kg–1)
0.25 0.5

1

2

4

pMET

141

MET

141

GAPDH

33
kDa

pMET (%)

0

HiP-8-PEG11 (4 mg kg–1)

120
100
80
60
40
20
0

0

0
1
2
3
4
HiP-8-PEG11 (mg kg–1)

0.5

1

2

5

10

20 (h)

pMET

141

MET

141

GAPDH
33
kDa

Fig. 6 | Imaging and targeting of HGF–MET activation in tumors using HiP-8-PEG11 in a mouse model. a–d, PET studies. a, Representative
immunohistochemistry for HGF (t5A11), MET activation (anti-phospho-MET antibody) and nuclei (DAPI) in PC-9 tumor tissues grown in human HGF
knock-in, severe combined immunodeficiency (hHGFki;scid) mice. Scale bars, 100 μm. b, Representative PET slice images in the frontal planes of micebearing PC-9 tumors intravenously administered with 64Cu-labeled HiP-8-PEG11 over a 90 min time course. Arrow indicates HGF− tumor. Arrowhead
indicates HGF+ tumor. Scale bars, 10 mm. c, Time–activity curves for HGF− and HGF+ tumors was expressed as the standardized uptake value (SUV). Data
represent mean ± s.d. (n = 3, distinct animals). d, Tumor/muscle ratios of radioactivity at approximately 2 h. Data represent mean ± s.d. (n = 3, distinct
animals, unpaired two-tailed t-test). For a–d, these experiments were repeated three times independently with similar results. e,f, HiP-8-PEG11 inhibits
MET activation in tumors. HiP-8-PEG11 or vehicle (PBS) was intravenously administered to tumor-bearing mice and MET activation was analyzed bywestern blot. Graph shows quantification of western blot. e, Dose responses. f, Time courses. These experiments were repeated twice independently withsimilar results. Uncropped blots can be found in Supplementary Fig. 17.

may allow flexible delivery options such as inhaled, transdermal or
sublingual delivery. Further in vivo pharmacokinetic studies to optimize chemical modifications of HiP-8, such as PEGylation, and preclinical efficacy studies relevant to clinical settings are required toestablish diagnostic and therapeutic applications of HiP-8.
In summary, using macrocyclic peptides, biochemical analysis and HS-AFM observations, the present study illustrates theconformational change at the SP domain allocation in proteolytic
activation of HGF and functional importance of the SP domain
for MET receptor activation. Our results also highlight the uniquebinding properties possible with macrocyclic peptides: recognizing
inter-domain structures of a protein through interaction with multiple domains, which can provide specificity and unique inhibitory
mechanisms. This binding property of macrocyclic peptides andtheir advantages in imaging applications present considerable diagnostic and therapeutic development potential.

Online content

Any methods, additional references, Nature Research reporting
summaries, source data, statements of code and data availability andassociated accession codes are available at https://doi.org/10.1038/
s41589-019-0285-7.
Received: 28 September 2018; Accepted: 3 April 2019;
Published online: 17 May 2019

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Acknowledgements

This work was supported by World Premier International Research Center Initiative (WPI),
MEXT, Japan. This work was supported in part by the A-STEP (Adaptable and Seamless
Technology Transfer Program through Target-driven R&D) (grant no. AS262Z) from theJapan Science and Technology Agency (JST), the Medical Research Fund of Takeda Science
Foundation, the Mitani Foundation for Research and Development, the Grant-in-Aid for
JSPS Scientific Research (C) (no. 16K08544) to K.S., a Grant-in-Aid for JSPS Scientific
Research (B) (no. 15K14473) to K.M., Project for Cancer Research and Therapeutic
Evolution (P-CREATE) from the Japan Agency for Medical Research and development(AMED) to Y.W., H.M., K.M. and T.P., Basic Science and Platform Technology Programfor Innovative Biological Medicine from AMED to H. Suga, a Grant-in-Aid for JSPS
Research Activity Start-up (no. 16H06830) to H. Sato, a Grant-in-Aid for JSPS Fellows(no. 23-7727) to K.I., a Grant-in-Aid for JSPS Scientific Research (B) (no. 18K01836)
to M.S. This work was performed under the Cooperative Research Program of the Institutefor Protein Research, Osaka University (no. CR15-05) and an Extramural Collaborative
Research Grant from the Cancer Research Institute (Kanazawa University). We thank
T. Ando (Kanazawa University) for providing HS-AFM apparatus, T. Uchihashi (Nagoya
University) for providing the analytical software of HS-AFM, Y. Kanayama and R. Zochi
for their assistance in 64Cu production, Y. Wada and E. Hayashinaka for their assistance inreconstructing the PET images and Enago (www.enago.jp) for the English language review.

Author contributions

K.S., H. Suga and K.M. conceived and designed the study. K.S. expressed and purified
HGF and HGF fragment proteins. M.U. and J.T. expressed and purified Xa-modified
scHGF, tcHGF, K2–4–SP and K4–SP proteins. K.I., K.S. and T.P. performed RaPID
and peptide synthesis. K.S., H. Sato and K.I performed cell-based assays. K.S. and K.I.
performed biochemical analysis. H. Sato performed immunohistochemistry and thein vivo efficacy studies. M.S. and H.F. performed HS-AFM observations. H.M., H. Sato,
S.W., M.Z. and Y.W. designed and performed PET studies. Y.K. developed t5A11. S.Y.
developed HGF-expressing PC-9. All authors analyzed the experimental data, discussed
the results and were involved in preparation of the manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https://doi.org/10.1038/
s41589-019-0285-7.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to H.S. or K.M.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.
© The Author(s), under exclusive licence to Springer Nature America, Inc. 2019

Nature Chemical Biology | VOL 15 | JUNE 2019 | 598–606 | www.nature.com/naturechemicalbiology

NATurE CHEMicAl BiOlOGy
Methods

Recombinant proteins. Full-length human HGF cDNA (NM_001010932.2) wasused in all plasmid constructions throughout this study. The residue numbering
was based on the sequence of variant 1, which contained five additional aminoacids in the K1 domain. HGF cDNA with or without point mutations to eliminate
N-linked glycosylation sites51 (N294Q, N402Q, T476G, N566Q and N653Q),
NK1 cDNA (residues Met1 to Glu210) and NK4 cDNA (residues Met1 to Val478)
were cloned into pEHX1.1 plasmid (TOYOBO). Recombinant HGF protein,NK1 protein and NfK4 protein were expressed in Chinese hamster ovary cells,
and secreted proteins were purified on an AKTApurifier system (GE Healthcare)using a HiTrap Heparin HP Column (GE Healthcare) followed by size-exclusion
chromatography on a Superdex 200 10/300 GL column (GE Healthcare)equilibrated in 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. For preparation ofrecombinant SP protein, HGF protein was cleaved with elastase (Sigma, HGF:
elastase = 100:1 mole ratio) at 37 °C for 90 min. The reaction was terminated byaddition of 1 mM phenylmethylsulfonyl fluoride (PMSF) and purified on a HiTrap
Heparin HP Column. The flow-through fraction containing SP protein was further
purified by size-exclusion chromatography on a Superdex 200 10/300 GL column
equilibrated in 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl.
Recombinant full-length HGF (Xa) protein and N-terminally truncated HGF
proteins (Glu208-Ser728 for K2–4–SP, Gly388-Ser728 for K4–SP), in which thecleavage site of wildtype HGF (KQLR/V) was mutated to the recognition sequenceof Factor Xa (IEGR/V) were prepared as previously described43. Briefly, these
proteins were appended with a hexahistidine tag at the C terminus and expressedin Expi293F cells (ThermoFisher scientific). Secreted proteins were purified ona Ni-nitrilotriacetic acid agarose column (Qiagen). The C-terminal His-tag waseliminated by overnight incubation with TEV protease. To prepare tcHGF (Xa),the TEV-treated samples from above were further cleaved with 6 μg ml−1 Factor
Xa (Novagen). Recombinant scHGF (Xa) and tcHGF (Xa) proteins were further
purified on a HiTrap Heparin HP Column (GE Healthcare) followed by sizeexclusion chromatography on a Superdex 75 10/300 GL column (GE Healthcare)equilibrated in 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. N-terminallytruncated recombinant HGF proteins contain mutations to eliminate N-linked
glycosylation sites (N294Q, N402Q, T476G, N566Q and N653Q) or an unpaired
cysteine (C561S), as previously described43. None of these mutations affect theactivity of HGF43,51. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS–PAGE) and Coomassie blue staining of recombinant proteins used for thisstudy are shown in Supplementary Fig. 18.
Macrocyclic library design. A thioether-macrocyclic peptide library wasconstructed by using N-(2chloroacetyl)-d-tryptophan (ClAcDW) as an initiator ina FIT system reaction37. The underlying mRNA library was designed to have an
AUG (ClAcDW) initiator codon followed by 4–15 NNK codons (N = G, C, A or
U; K = G or U), which code random proteinogenic amino acid residues, followed
by a fixed UGC codon that assigns Cys. The theoretical diversity of macrocyclic
peptides based on the quantitative assessment of the efficiencies of the individualtransformation steps is ≥1012. After in vitro translation, a thioether bond formed
spontaneously between the N-terminal ClAc group of the initiator D-Trp residue
and the sulfhydyl group of a downstream Cys residue to generate the macrocyclic
peptide backbone.
Selection of macrocyclic peptides binding to HGF. Affinity selection was
performed using the DW initiated library against full-length human HGF by
employing a RaPID approach35. The mRNA library and ClAc-d-Trp-tRNAfMetCAU
were prepared as previously reported38,49. Briefly, 1 μM mRNA library was ligatedto a puromycin-linked oligonucleotide (1.5 μM) using T4 RNA ligase at 25 °C
for 30 min. After purification by phenol–chloroform extraction and ethanol
precipitation, 1.2 μM mRNA-puromycin conjugate and was translated at 37 °C
for 30 min in a methionine-deficient FIT reaction containing 25 μM ClAc-d-TrptRNAfMetCAU to generate the peptide library. Following translation, incubation at
25 °C for 12 min was performed to facilitate mRNA-peptide complexes, EDTA
was added to a final concentration of 20 mM and the reaction was incubated at
37 °C for 30 min to remove the mRNA-peptide complexes from the ribosomes.
The product was subsequently reverse-transcribed using RNase H minus
reverse transcriptase (Promega) at 42 °C for 1 h. The final library was counterselected against streptavidin-coated beads (Dynabeads M-280 streptavidin, Life
Technologies) to remove undesired bead binders. Counter selection was repeated
twice for the first round of selection and six times for all later rounds. For affinity
selection to HGF, the peptide–mRNA/cDNA solution was incubated with
200 nM biotinylated full-length human HGF-immobilized on Dynabeads M-280
streptavidin for 30 min at 4 °C to isolate HGF-binders. Biotinylation of HGF was
performed with a Succinimidyl Biotin Labeling kit (Dojindo) according to the
manufacturer’s instructions. The bioactivity of biotinylated HGF was equivalent
to unlabeled HGF in the MET activation assay of EHMES-1 cells (Supplementary
Fig. 1). The fused peptide–mRNA/cDNA was isolated from the beads by
incubating in 1× PCR buffer heated for 5 min at 95 °C, and amount of eluted
cDNAs was measured by quantitative PCR. The remaining cDNAs were amplified
by PCR and then purified and transcribed to produce an enriched mRNA libraryfor the next round of selection. For sequencing of the recovered cDNAs fromNature Chemical Biology | www.nature.com/naturechemicalbiology

Articlesthe initial selection against HGF, ligation was performed into the pGEM-T-Easy
vector (Promega) by TA-cloning. The vectors were transformed into DH5αcompetent cells; individual clones were picked and sequenced (Supplementary
Fig. 3). Focused library screening was performed as above, with the NNK libraryreplaced with a focused library as shown in Supplementary Fig. 6, and sequencingof the final enriched cDNA carried out using a MiSeq next generation sequencer(Illumina).
Chemical synthesis of macrocyclic peptides. Macrocyclic peptides werechemically synthesized using a Syro Wave automated peptide synthesizer (Biotage)by standard Fmoc solid-phase peptide synthesis, as previously described38,49.
Following synthesis, the chloroacetyl group was coupled onto the N-terminal aminegroup for formation of peptide macrocycles. For Pegylated peptides, different
Fmoc-protected PEG linkers (Merck Millipore) were coupled as for any otheramino acid, such that the PEG linker was C-terminal to the cyclizing Cys residue
and separated from it by a beta-alanine residue. For biotinylated HiP-8-PEG11,an additional C-terminal Lys(Mmt) residue (separated from the PEG linker by anadditional beta-alanine residue) was selectively deprotected with 1% trifluoroacetic
acid (TFA) in dichloromethane following N-terminal chloroacetylation, andsubsequently coupled with three equivalents of d-biotin N-hydroxysuccinimide
ester for 3 hours at room temperature. Peptides were cleaved from the resin anddeprotected with 92.5% TFA, 2.5% water, 2.5% tri-isopropylsilane and 2.5%
ethanedithiol and then precipitated by diethyl ether addition. To conduct thecyclization reaction, the peptide pellet was dissolved in 10 ml DMSO/0.1% TFA inwater (1:1), adjusted to pH > 8 by addition of triethylamine and incubated for 1 h
at 42 °C. The cyclization reaction was quenched by acidification with TFA. Then,the peptides were purified by reverse-phase HPLC using a Shimadzu Prominence
LC-20AP system with a Merck Chromolith Prep column (200 × 25 mm i.d.), andmolecular masses were verified by MALDI–TOF mass spectrometry (AutoFlex
II instrument; Bruker Daltonics) (Supplementary Fig. 20). Alternatively, incases where high purity was not required, macrocyclic peptides were purified
on HyperSep SPE C18 columns (ThermoFisher Scientific). Analytical ultra
high-pressure liquid chromatography was performed using a Nexera X2 system(Shimadzu) fitted with a C18 reversed phase column. Approximately 1 nmolof peptide was separated using a 10–70 vol% aqueous acetonitrile gradientsupplemented with 0.1 vol% trifluoroacetic acid and monitored by absorbance at
280 nm. Chromatograms for all peptides are shown in Supplementary Fig. 21.
Cell-based assays. EHMES-1 cells were provided by Y. Hamada (Ehime University,
Japan). B16-F10 cells were obtained from the ATCC. PC-9 cells were obtained
from Immuno-Biological Laboratories Co. (Gunma). All cell lines were cultured in
RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM
l-glutamine at 37 °C and 5% CO2, unless otherwise stated.
The MET activation assay was performed as previously described38. Briefly,
EHMES-1 cells were stimulated with 20 ng ml−1 (220 pM) HGF protein with orwithout macrocyclic peptides in culture medium for 10 min, washed with PBS,
fixed with 4% paraformaldehyde in PBS for 30 min and washed three times
with PBS. The cells were blocked with 5% goat serum, 0.02% Triton X-100 in
PBS for 30 min, and then incubated for 12 h in phospho-MET (Tyr1234/1235)
XP rabbit mAb (D26; Cell Signaling Technology (CST)) diluted 1:1,000 in 1%
goat serum in PBS at 4 °C. The cells were washed three times with PBS andincubated for 1 h in horseradish peroxidase (HRP)-conjugated anti-rabbit goat
antibody diluted 1:1,000 in 1% goat serum in PBS. Following incubation, thecells were washed four times with PBS. Chemiluminescence was developed with
ImmunoStar LD reagent (Wako) and measured by ARVO MX (PerkinElmer).
Relative MET phosphorylation was calculated as (Chemiluminescence unit ofsample − Chemiluminescence unit of mock control)/(Chemiluminescence unit of
220 pM HGF − Chemiluminescence unit of mock control).
For western blot analysis, EHMES-1 cells were cultured in a six-well plate until
they were 80–90% confluent. After starvation for 6 h, the cells were stimulatedwith 2 nM HGF and 0, 10, 100, 1,000 or 10,000 nM macrocyclic peptides in culture
medium for 10 min. Cells were washed with PBS and lysed with 200 µl of lysis
buffer 17 (R&D Systems) containing 1× complete protease inhibitor cocktail(Roche). Proteins were measured by bicinchoninic acid assay (ThermoFisher
Scientific) and loaded into 10% polyacrylamide gel SDS–PAGE. Protein wastransferred onto a polyvinylidene difluoride membrane and probed with theprimary antibody, MET (CST, 25H2), phospho-MET (Tyr1234/1235; CST,
D26), Akt (CST, 11E7), phospho-Akt (Ser473; CST, D9E), Erk1/2 (CST, 137F5),
phospho-Erk1/2 (Thr202/Tyr204; CST, D13.14.4E), Gab1 (CST), phospho-Gab1(Tyr 627; CST, C32H2; 1:2,000) or glyceraldehyde-3-phosphate dehydrogenase(GAPDH; CST, 14C10; 1:2,000) in Can Get Signal Solution 1 and HRP-conjugatedsecondary antibodies (Dako; 1:5,000) in Can Get Signal Solution 2 (TOYOBO).
Chemiluminescent signals were developed with Luminata Forte HRP substrate(Merck Millipore) and observed using ImageQuant LAS 350 (GE Healthcare) or
Image Reader LAS-3000 mini v.2.2 (FUJIFILM). Alternatively, cell lysates wereanalyzed for phosphorylation of their receptors using the human phospho-receptor
tyrosine kinase array (R&D Systems).
For cell migration assays, B16–F10 cells were suspended at a density of
2.5 × 104 cells per ml in 10% FBS-containing RPMI medium and 0.2 ml of theArticles
cell suspension (5 × 103 cells) was placed into each upper chamber (6.5-mm
diameter trans-well with 8-µm pores; Corning). Lower chambers were filledwith the 0.8 ml of the same culture medium with each test sample. The cells wereincubated with HGF (220 pM) in the presence or absence of function-blocking
anti-HGF antibody52,53 or HiP-8-PEG11 at indicated concentrations. After the 20-h
incubation, non-migratory cells on the upper surface of the filters were removed
using a cotton swab. Migrated cells on the lower surface of the filters were fixedwith 4% (w/v) paraformaldehyde, stained with crystal violet and counted under amicroscope.
For the gefitinib resistance assay, PC-9 lung cancer cells were seeded into
each well of a 24-well suspension culture plate (EZ-BindShut II, IWAKI) in 10%
FBS-containing RPMI medium. After 24-h culture, the cells were treated withgefitinib (1 μM) with or without HGF (220 pM) and HiP-8-PEG11 (1–1,000 nM)
and cultured for a further 72 h, after which the surviving cells were counted on ahemocytometer.
Binding assay. Fluorescein labeling of HGF was performed using a Succinimidyl
Fluorescein Labeling kit (Dojindo). The bioactivity of fluorescein-HGF was
equivalent to unlabeled HGF in the MET activation assay of EHMES-1 cells(Supplementary Fig. 1). A 5 μg amount of MET-ectodomain-Fc fusion protein(R&D systems) was immobilized on 200 μl (50% v/v) of protein G beads(Spherotech) in Tris-buffered saline (TBS; pH 7.5), 0.1% (w/v) BSA and 0.05%
Tween-20. Titrated concentrations of fluorescein-HGF were incubated with 5 μl
(50% v/v) of protein G beads or MET-Fc-immobilized protein G beads in 200 μl of
Tris-buffered saline (pH 7.5), 0.1% BSA and 0.05% Tween-20 for 1 h at 25 °C. The
fluorescent intensity of beads was detected using a flow cytometry (FACSCanto
II; BD Biosciences). The binding affinity of fluorescein-HGF was 70 pM(Supplementary Fig. 5). For competition of macrocyclic peptides for HGF–MET
binding, titrated concentrations of macrocyclic peptides were mixed with 0.44 nMof fluorescein-HGF and MET-Fc-immobilized beads for 1 h at 25 °C, then the
fluorescent intensity of the beads was analyzed by flow cytometry.
SPR analysis. Binding of macrocyclic peptides to immobilized HGF was
measured using a Biacore T200 (GE Healthcare), as previously described38.
Briefly, biotinylated HGF was immobilized at a density of approximately
2,000 resonance units on a CAP sensor chip using a Biotin CAPture Kit (GEHealthcare). Macrocycle binding was tested by injecting varying concentrationsof the macrocycles at a flow rate of 30 μl min−1 in HBS EP+ buffer (10 mM Hepes
pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% (v/v) SurfactantP20) containing
0.1% DMSO. Kinetic parameters were determined using a 1/1 binding single-cycle
model of Biacore T200 Software v.3.0 (GE Healthcare).
Binding of HGF and HGF fragments to immobilized HiP-8-PEG11 wasmeasured using a Biacore 3000 (GE Healthcare). Biotinylated HiP-8-PEG11
was captured on the surface of streptavidin-coated chips (GE Healthcare) at~10 resonance units (Figs. 2b and 3a,b), ~50 resonance units (Fig. 2c,d) or~30 resonance units (Supplementary Fig. 10). A streptavidin sensor chip without
HiP-8-PEG11 was used as the reference. HGF or HGF fragments were tested byinjecting varying concentrations at a flow rate of 30 μl min−1 in 10 mM TBS pH 7.4,
300 mM NaCl, 0.05% (v/v) Tween 20. Binding affinity was analyzed using a multicycle method fitting a model of steady-state affinity of BIAevaluation Software v.4.1(GE Healthcare).
HiP-8 inhibition of HGF, NK4 and SP binding to MET-Fc was measured usinga Biacore 3000 (Supplementary Fig. 9d). His-tagged MET-Fc was captured on
the surface of Ni-nitrilotriacetic acid-coated tips (GE Healthcare) at a density of
approximately 1,000 resonance units. A sensor chip without MET-Fc was used as
the reference. The titrated concentrations of HiP-8 were premixed with 5 nM HGF,
30 nM NK4 or 30 nM SP protein and tested at a flow rate of 30 μl min−1 in 10 mM
TBS pH 7.4, 300 mM NaCl and 0.05% (v/v) Tween 20.
HS-AFM observations. HS-AFM observation was performed as described
previously44,47. Briefly, HS-AFM measurements were operated in tapping mode.
Cantilever deflection was detected with an optical beam deflection detector using
an infrared laser (0.7 mW, 780 nm). The infrared laser beam was focused onto
the back side of a cantilever covered with gold film (Olympus: BL-AC7DS-KU4)
through a ×60 objective lens (CFI S Plan Fluor ELWD ×60; Nikon). Reflection of
the infrared laser from the cantilever was detected using a two-segmented PIN
photodiode (MPR-1; Graviton). The free-oscillation amplitude was approximately
1 nm, and the set-point amplitude was approximately 90% of the free amplitude
for feedback control of HS-AFM. For the AFM probe, an amorphous carbon tip
with a length of approximately 500 nm grown by electron beam deposition wasused. For the AFM substrate, a mica surface treated with 0.01% 3-aminopropyltriethoxysilane (Shin-Etsu Silicone) was used. All HS-AFM observations were
performed in buffer solution containing 50 mM Tris-HCl (pH 7.4) and 100 mM
NaCl at room temperature. The complex of HGF (tcHGF or scHGF) and HiP-8
macrocyclic peptide was pre-assembled (HGF:HiP-8 = 1:10 molar ratio) in AFMimaging buffer before HS-AFM observations.
Correlation coefficient analysis of HS-AFM images. Two-dimensionalcorrelation coefficients were calculated between the HS-AFM images of the first

NATurE CHEMicAl BiOlOGy
frame and each of the frames in the region of interest (ROI) (that is, the first frame
is the reference), as described previously45. The size of the ROIs for tcHGF, scHGF,
tcHGF/HiP-8 complexes and scHGF treated with HiP-8 that included the whole
HGF molecule was 40 × 30 nm2. The two-dimensional correlation coefficient
was calculated frame-by-frame for each ROI. The two-dimensional correlation
coefficient is defined as:r=∑m ∑n (Hmn−H ) (R mn−R )(∑ ∑ (H −H ) )(∑ ∑ (R −R ) )
m

n

mn

2

m

n

mn

2

where, Hmn and Rmn are the heights at the pixel point (m,n) in the ROI to beanalyzed and the reference ROI of the reference frame, respectively. H and R are themean values of the height matrices H and R, respectively.
Limited proteolysis. HGF (2.5 μM) was incubated with or without HiP-8 (10 μM)
for 5 min at room temperature before adding varying amounts of trypsin (Wako
Pure Chemical Industries) for 30 min at room temperature. The trypsin:HGF(mol:mol) ratios were 0:1, 0.004:1, 0.012:1, 0.037:1, 0.11:1, 0.33:1 and 1:1. Reactions
were terminated by adding 2 mM PMSF for 5 min and heated at 95 °C in SDS sample
buffer. Digested products were analyzed by SDS–PAGE using 5–20% polyacrylamide
separating gels under reducing conditions and stained with Coomassie blue.
Immunohistochemistry. Frozen human lung cancer tissue arrays wereobtained from US Biomax (FLU401B). Tissue sections were fixed with 4%(w/v) paraformaldehyde in PBS for 30 min at room temperature, treated with3% (w/v) BSA in PBS for 1 h to block non-specific adsorption of probes andincubated overnight at 4 °C with HiP-8-PEG11-biotin or primary antibodies
against human HGF (clone: t5A11, final 5 μg ml−1)43 or phosphorylated MET(phospho Y1230/1234/Y1235) (Abcam, 1:200 dilution) diluted with Can Get Signal
immunostain (TOYOBO). After washing three times with PBS, these probes weredetected by Alexa Fluor 488-labeled streptavidin or goat anti-mouse-IgG and
Alexa Fluor 594-labeled goat anti-rabbit-IgG (ThermoFisher Scientific). Cell nuclei
were counterstained with a 4′,6-diamidino-2-phenylindole (DAPI; ThermoFisher
Scientific). Sections were analyzed by using Biozero BZ-9000 (KEYENCE).
To semi-quantitatively score the immunostaining, the stained area and merge
area in each tissue were quantified using ImageJ software (NIH). Each sample wasclassified as −, ±, +, ++ and +++ according to the same criteria (Fig. 5c). The
ratio of phosphorylated MET-positive area to total HGF (scHGF and tcHGF)positive or tcHGF-positive area was calculated on the basis of the above values(Fig. 5b).
PET studies. Production and purification of 64CuCl2 was performed as previously
described54. For preparation of chelator-conjugated HiP-8-PEG11, HiP-8-PEG11
was modified to include an azide group by addition of Fmoc-Lys(N3)-OH(Watanabe Chemical) C-terminal to the PEG linker and then conjugated to
DBCO-PEG4-CB-TE1K1P (ref. 55). 64CuCl2 was reacted with the chelatorconjugated HiP-8-PEG11 in 0.05 M sodium acetate (pH 7.0), followed by incubatedat 40 °C for 15 min. The radiochemical purity of 64Cu-labeled HiP-8-PEG11 wasdetermined to be ≥95% by radio-HPLC. Tumor-bearing mice were anesthetizedwith a mixture of 1.5% isoflurane and nitrous oxide:oxygen (7:3) and intravenously
injected with 64Cu-labeled HiP-8-PEG11 (6.5 MBq in 200 μl saline). Emission
data were acquired for 90 min with a microPET scanner (microPET Focus 220,
Siemens Co.) and PET images were reconstructed using microPET manager
v.2.4.1.1 (Siemens Co.) as previously described54. Time–activity curves of tumors
were generated using PMOD v.3.612 (PMOD Technologies LLC) as previously
described56. After the PET studies, tumors and muscle were collected from themice. Radioactivity in each tissue was determined using the 2480 WIZARD2
automatic gamma counter (PerkinElmer Life and Analytical Sciences) as previously
described54. This study was approved by the Institutional Review Boards ofKanazawa University and RIKEN Kobe Institute, and performed in accordancewith the guidelines and regulations.
In vivo evaluation of HiP-8. hHGFki;scid mice were obtained from the JacksonLaboratory (stock number 014543). Male hHGFki;scid mice (8–10 weeks old) weremaintained at the Advanced Science Research Center Institute for Experimental
Animals, Kanazawa University. Human lung adenocarcinoma cell line PC-9 orPC-9 overexpressing transfected human HGF10 were inoculated subcutaneously(5 × 106 cells per 0.1 ml per head for PET studies, 3 × 106 cells per 0.1 ml per headfor efficacy studies) into the flanks of hHGFki;scid mice. After 3 weeks, tumorbearing mice were subjected to PET studies or efficacy studies. For efficacy studies,tumor-bearing mice were intravenously administered with HiP-8-PEG11 or vehicle(PBS). For dose response, mice were euthanized and tumors were resected 1 hafter administration of HiP-8-PEG11. For time courses, HiP-8-PEG11 (4 mg kg−1)
was administered and tumor tissues were harvested at different times. The tumor
samples were immediately homogenized in buffer composed of 50 mM Tris-HCl(pH 7.4), 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 150 mM NaCl, 1 mM EDTA,
1 mM PMSF, 10 mM NaF, 1 mM Na3VO4 and 1× proteinase inhibitor mix (Nacalai
Tesque). Phosphorylated and total MET levels were determined by western
Nature Chemical Biology | www.nature.com/naturechemicalbiology

Articles

NATurE CHEMicAl BiOlOGy
blot. This study was approved by the Institutional Review Boards of Kanazawa
University and performed in accordance with the guidelines and regulations.
Measurement of human and murine HGF in plasma. C57BL/6 mice werepurchased from SLC. Blood samples were taken from the tail vein using heparin as
an anti-coagulant and were centrifuged at 1,000g for 30 min at 4 °C and plasma wascollected. HGF levels were measured by enzyme-linked immunosorbent assay kits
for human HGF and murine HGF, respectively (Institute of Immunology Co., Ltd).
Statistical analysis. Statistical parameters, including the definitions and exact
values of n, are reported in the figures and corresponding figure legends. Twogroup comparisons were analyzed using unpaired two-tailed t-test. A valueof P ≤ 0.05 was accepted as indicative of statistical significance. Prism 6.0d
(GraphPad) was used to generate and fit dose–response curves. The IC50 values of
the cell-based MET activation assay or competitive binding assay were determinedby plotting percentage inhibition versus log compound concentration using thedose–response (variable slope, four parameters) curve-fitting function of Prism
6.0d. No samples or animals were excluded from the analysis. No statistical method
was used to predetermine the sample size. The investigators were not blinded toallocation during the experiments and outcome assessment.
Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that all data supporting the findings of this study are available
within the article and its Supplementary information or from the authors upon
reasonable request.

Nature Chemical Biology | www.nature.com/naturechemicalbiology

Code availability

The authors declare that no custom code was used in this study. Software code ormathematical algorithm used in this study is available within the article and its
Supplementary Information or from the authors upon reasonable request.

References

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leads to suppression of tumor growth and migration in malignant pleural
mesothelioma. Int. J. Cancer 127, 1948–1957 (2010).
53. Isozaki, H. et al. Non-small cell lung cancer cells acquire resistance to the
ALK inhibitor alectinib by activating alternative receptor tyrosine kinases.
Cancer Res. 76, 1506–1516 (2016).
54. Mukai, H., Wada, Y. & Watanabe, Y. The synthesis of 64Cu-chelated porphyrin
photosensitizers and their tumor-targeting peptide conjugates for theevaluation of target cell uptake and PET image-based pharmacokineticsof targeted photodynamic therapy agents. Ann. Nucl. Med. 27,
625–639 (2013).
55. Zeng, D. et al. New cross-bridged cyclam derivative CB-TE1K1P, an improved
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Sequencing of the final enriched cDNA following focused library screening (supplementary Fig. 6) was collected using a MiSeq next
generation sequencer (Illumina).
HS-AFM images were collected using Igor Pro Ver. 6.3.6.0. (WaveMetrics).
Immunohistochemical staining images were captured using a Biozero BZ-9000 microscope (KEYENCE).
Western blotting images were captured using an ImageQuant LAS 350 (GE Healthcare) or Image Reader LAS-3000 mini Ver. 2.2(FUJIFILM) machine.
PET imaging was performed using a microPET scanner (microPET Focus 220, Siemens Co.) and PET images were reconstructed in
microPET manager 2.4.1.1 (Siemens Co.).
Radioactivity in each tissue was determined using the 2480 WIZARD2 automatic gamma counter (PerkinElmer Life and Analytical
Sciences).
SPR analysis was performed using a Biacore T200 (GE Healthcare) or Biacore 3000 (GE Healthcare) machine.
Chemiluminescence in MET activation assays was measured using an ARVO MX microplate reader (PerkinElmer).
Binding of fluorescein-HGF to MET-Fc immobilized beads in the presence or absence of HiPs were analyzed by flow cytometry, BD
FACSCanto (BD Biosciences).

Data analysis

Prism 6.0d (GraphPad) was used to generate and fit dose–response curves. The IC50 values of the cell-based MET activation assay orcompetitive binding assay were determined by plotting percentage inhibition versus log compound concentration using the dose–
response (variable slope, 4 parameters) curve-fitting function of Prism 6.0d.
Binding kinetics were analyzed using a 1:1 binding single-cycle model multi-cycle method fitting as part of Biacore T200 Software v3.0

April 2018

Data collection1For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers
upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:- Accession codes, unique identifiers, or web links for publicly available datasets- A list of figures that have associated raw data
- A description of any restrictions on data availability

nature research | reporting summary(GE Healthcare).
Binding affinity was analyzed using a multi-cycle method fitting a model of steady-state affinity as part of BIAevaluation Software v4.1 (GEHealthcare).
HS-AFM images were analyzed using Igor Pro Ver. 6.3.6.0. (WaveMetrics).
Immunohistochemical staining images were analyzed by ImageJ 1.51k software (NIH).
Peptide sequence was aligned by CLC Sequence Viewer 8 (Qiagen).
The similarities of the amino acid sequences of human MSP and murine HGF to human HGF were analyzed by CLUSTALW (GenomeNet).
Time-activity curves of tumors were generated using PMOD v. 3.612 (PMOD Technologies LLC).
Flow cytometry data were analyzed by BD FACSDiva Software v6.1.3. (BD Biosciences).

All data supporting the findings of this study are available within the article and its supplementary information files or from the authors upon reasonable request.

Field-specific reporting
Please select the best fit for your research. If you are not sure, read the appropriate sections before making your selection.
Life sciences

Behavioural & social sciences

Ecological, evolutionary & environmental sciences

For a reference copy of the document with all sections, see nature.com/authors/policies/ReportingSummary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size

The sample size for each experiment is indicated in the figures or corresponding figure legends. The data include error bars (standard
deviation). Based on the distribution of the means in our pilot experiments, we chose a sample size that would adequately power eachexperiment to detect a difference in outcomes between groups.
The library synthesis and the selections were performed only once. However, model reactions have been well optimized and routinely work
well [Hipolito, C.J. & Suga, H. Ribosomal production and in vitro selection of natural product-like peptidomimetics: the FIT and RaPID systems.
Curr Opin Chem Biol. 16, 196-203 (2012). Passioura, T. & Suga, H. Flexizyme-mediated genetic reprogramming as a tool for noncanonicalpeptide synthesis and drug discovery. Chemistry. 19, 6530-6536 (2013)].
The hits (HiPs) were synthesized only once or repeatedly, but purity and identity were experimentally confirmed.
Recombinant proteins were produced and purified only once or repeatedly, but purity and identity were experimentally confirmed.

Data exclusions

No data was excluded.

Replication

All experiments were repeated at least twice independently with similar results. Replication of each experiment was described in figurelegends.

Randomization

Mice in experimental groups were paired at the beginning of each study in order to age- and gender-match subjects. Otherwise, mice andother samples were randomly allocated into experimental groups.

Blinding

No blinding was applied. For PET study, animals were treated with single experimental condition, therefore not allocated to experimentalgroups.

Reporting for specific materials, systems and methods
April 20182

Methods
n/a Involved in the study

Unique biological materials

ChIP-seq

Antibodies

Flow cytometry

Eukaryotic cell lines

MRI-based neuroimaging

Palaeontology
Animals and other organisms
Human research participants

Unique biological materials
Policy information about availability of materials
Obtaining unique materials

All materials are available from the authors upon reasonable request.

nature research | reporting summary

Materials & experimental systems
n/a Involved in the study

Antibodies
Antibodies used

The anti-human HGF antibody used in figure 1e is a rabbit polyclonal antibody prepared by Suzuki, Y. et al.
The anti-human HGF antibody, t5A11 is a mouse monoclonal antibody prepared by Kato, Y. et al. For immunohistochemistry(IHC) (figure 5, figure 6a), used at 5 μg/ml. For AFM (Fig. 4c, supplementary figure 13, supplementary movie 9) used at 200 nM.
The anti-MET antibody used in figure 1d, 6e, 6f is a mouse monoclonal antibody from Cell Signaling Technology (CST) (#3127,
clone 25H2, Lot 8, 1:2000 dilution).
The anti-phospho-MET (Tyr1234/1235) antibody used in figure 1a, 1d, 6e, 6f, supplementary figure 1, 4b, 6c, and 7c is a rabbit
monoclonal antibody from CST (#3077, clone D26, Lot 9, 1:2000 dilution in WB, 1:1000 dilution in cell-based pMET assay).
The anti-phospho-MET (Tyr1230/1234/1235) antibody used for IHC in figure 5 and 6a is a rabbit polyclonal antibody from Abcam(#ab5662, Lot GR3191923-5, 1:200 dilution).
The anti-Akt antibody used in figure 1d and 2a is a rabbit monoclonal antibody from CST (#4685, clone 11E7, Lot 27, 1:2000
dilution).
The anti-phospho-Akt (Ser473) antibody used in figure 1d and 2a is a rabbit monoclonal antibody from CST (#4060, clone D9E,
Lot 23, 1:2000 dilution).
The anti-Erk1/2 antibody used in figure 1d is a rabbit monoclonal antibody from CST (#4695, clone 137F5, Lot 14, 1:2000
dilution)
The anti-phospho-Erk1/2 (Thr202/Tyr204) antibody used in figure 1d is a rabbit monoclonal antibody from CST (#4370, clone
D13.14.4E, Lot 12, 1:2000 dilution).
The anti-Gab1 antibody used in figure 1d is a rabbit polyclonal antibody from CST (#3232, Lot 5, 1:2000 dilution).
The anti-phospho-Gab1 (Tyr 627) antibody used in figure 1d is a rabbit monoclonal antibody from CST (#3233, clone C32H2, Lot
2, 1:2000 dilution).
The anti-GAPDH antibody used in figure 1d, 6e, and 6f is a rabbit monoclonal antibody from CST (#2118, clone 14C10, Lot 10,
1:2000 dilution).
The anti-Rabbit Immunoglobulins/HRP used for western blot or cell-based pMET assay was from Dako (#P0448, Lot 20053537,
1:5000 dilution in WB, 1:1000 dilution in cell-based pMET assay).
The anti-Mouse Immunoglobulins/HRP used for western blot was from Dako (#P0447, Lot 20030309, 1:5000 dilution).
The Alexa Fluor 488-labeled anti-mouse-IgG used for IHC was from ThermoFisher Scientific (#A11029, Lot 1829920, 1:200
dilution).

Validation

The anti-human HGF rabbit polyclonal antibody used in figure 1e has been prepared as described in Suzuki, Y. et al. Inhibition of
Met/HGF receptor and angiogenesis by NK4 leads to suppression of tumor growth and migration in malignant pleural
mesothelioma. Int J Cancer. 127, 1948-1957 (2010), and has been characterized and used for neutralizing human HGF [Isozaki, H.
et al. Non-Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor
Tyrosine Kinases. Cancer Res. 76, 1506-1516 (2016)].

April 2018

The Alexa Fluor 594-labeled goat anti-rabbit-IgG used for IHC was from ThermoFisher Scientific (#A11037, Lot 1915919, 1:200
dilution).

3

The anti-phospho-MET (Tyr1234/1235) antibody (CST, #3077, clone D26) has been characterized for cell-based pMET assay as
described in Ito, K. et al. Artificial human Met agonists based on macrocycle scaffolds. Nat Commun. 6, 6372 (2015).
All other antibodies below were commercially available and validated by manufacturers and such information is provided in the
manufacturer's website. Applications and species reactivity of these antibodies for the experiments used in this study (WB, IHC,
and immunocytochemistry) were already validated by manufactures and validated in our pilot experiments.
The anti-MET antibody (CST, #3127, clone 25H2); Application: WB, IP; Species reactivity to human, mouse, and rat.
The anti-phospho-MET (Tyr1234/1235) antibody (CST, #3077); Application: WB, IP, IHC, etc.; Species reactivity to human, mouse,
and rat.
The anti-phospho-MET (Tyr1230/1234/1235) antibody (Abcam, #ab5662); Application: WB, IHC; Species reactivity to human,
mouse, rat, etc.

nature research | reporting summary

The anti-human HGF mouse monoclonal antibody t5A11 has been prepared and characterized as described in Umitsu, M. et al.
Probing conformational and functional states of human hepatocyte growth factor by a panel of monoclonal antibodies. Sci Rep.
6, 33149 (2016). t5A11 recognizes both scHGF and tcHGF.

The anti-Akt antibody (CST, #4685, clone 11E7); Application: WB, IP, IHC, etc.; Species reactivity to human and mouse.
The anti-phospho-Akt (Ser473) antibody (CST, #4060, clone D9E); Application: WB, IP, IHC, etc.; Species reactivity to human,
mouse, rat, etc.
The anti-Erk1/2 antibody (CST, #4695, clone 137F5); Application: WB, IP, IHC, etc.; Species reactivity to human, mouse, rat, etc.
The anti-phospho-Erk1/2 (Thr202/Tyr204) antibody (CST, #4370, clone D13.14.4E); Application: WB, IP, IHC, etc.; Species
reactivity to human, mouse, rat, etc.
The anti-Gab1 antibody (CST, #3232); Application: WB; Species reactivity to human, mouse, rat, etc.
The anti-phospho-Gab1 (Tyr 627) antibody (CST, #3233, clone C32H2); Application: WB; Species reactivity to human.
The anti-GAPDH antibody (CST, #2118); Application: WB, IP, IHC, etc.; Species reactivity to human, mouse, rat, etc.

Eukaryotic cell lines
Policy information about cell lines
Cell line source(s)The human methothelioma cell line EHMES-1 was provided by Dr. Hamada (Ehime University, Ehime, Japan). EHMES-1 wasestablished as described in Yokoyama, A. et al. Origin of heterogeneity of interleukin-6 (IL-6) levels in malignant pleural
effusions. Oncol Rep. 1,507-511 (1994).
The murine melanoma cell line B16-F10 cells were obtained from the ATCC (CRL-6475, Manassas, VA).
The human lung adenocarcinoma cell line PC-9 was obtained from Immuno-Biological Laboratories Co. (Gunma, Japan).
The human lung adenocarcinoma cell line PC-9 exogenously expressing human HGF has been established as described in
Yano, S. et al. Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor
receptor-activating mutations. Cancer Res. 68, 9479-9487 (2008).

Authentication

All cell lines were expanded upon initial receipt to create large stocks of frozen vials. To limit the risks of cross-contaminationand over-subculturing, all cell lines used in our laboratory are passaged for no more than one month. Cell morphology ismonitored several times per week during culture of all cell lines. If there are any changes in cell appearance, growth kinetics,
or performance in routine assays in our laboratory, cells are discarded and a new culture established from frozen stocks.
PC-9 cells has been partially authenticated by the fact that the cells were sensitive for gefitinib and rescued by HGF (figure1h). PC-9 cells and PC-9 cells expressing HGF has been partially authenticated by the fact that they showed different HGF
production level (figure 6a).

Mycoplasma contamination

All cell lines tested negative.

Commonly misidentified lines

No commonly misidentified cell lines were used.

(See ICLAC register)Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research
C57BL/6 mice used for supplementary figure 14 were obtained from SLC. hHGFki;scid mice were used for figure 6,
supplementary figure 14, 15, 16, 21, supplementary video 10 and 11. The hHGFki;scid mice were obtained from JacksonLaboratory (stock number: 014543, Sacramento, CA) and were maintained at the Advanced Science Research Center Institute forExperimental Animals, Kanazawa University. Human lung adenocarcinoma cell line PC-9 or PC-9 overexpressing transfected
human HGF were inoculated subcutaneously into the flanks of male hHGFki;scid mice (8–10 weeks old). After 3 weeks, tumorbearing mice were subjected to PET or efficacy studies. All animal experiments were approved and performed in accordancewith the institutional experiment guidelines.

April 2018

Laboratory animals

4This study did not involve wild animals.

Field-collected samples

This study did not involve samples collected from the field.

Flow Cytometry
Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation

Binding of fluorescein-HGF to MET-Fc immobilized beads in the presence or absence of HiPs were analyzed by flow cytometry infigure 1e, supplementary figure 4b, 5, 6e, and 7b. Sample preparation is described in in the manuscript (methods). Briefly, a 5 μg
amount of MET-Fc fusion protein (R&D systems) was immobilized on 200 μl (50% v/v) of protein G beads (Spherotech) in Trisbuffered saline (TBS; pH 7.5), 0.1% (w/v) bovine serum albumin (BSA), and 0.05% Tween-20. Titrated concentrations offluorescein-HGF were incubated with 5 μl (50% v/v) of protein G beads or MET-Fc-immobilized protein G beads in 200 μl of TBS(pH 7.5), 0.1% BSA, and 0.05% Tween-20 for 1 h at 25°C. The fluorescent intensity of beads was detected using a flow cytometry(FACSCanto II; BD Biosciences). For competition of HiPs for HGF–MET binding, titrated concentrations of HiPs were mixed with
0.44 nM of fluorescein-HGF and MET-Fc-immobilized beads for 1 h at 25°C, and then the fluorescent intensity of the beads wasanalyzed by flow cytometry.

Instrument

BD FACSCanto (BD Biosciences).

Software

BD FACSDiva Software v6.1.3. (BD Biosciences).

Cell population abundance

Not applicable to this study.

Gating strategy

The gated single bead population in FSC/SSC expansion was analyzed for mean fluorescent intensity of fluorescein-HGF. Details
are shown in supplementary figure 5a.

nature research | reporting summary

Wild animals

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

April 20185