Hot spots in prion protein for pathogenic conversion

Kazuo Kuwata†‡§, Noriyuki Nishida¶, Tomoharu Matsumoto†, Yuji O. Kamatari†, Junji Hosokawa-Muto†, Kota Kodama†,
Hironori K. Nakamura†, Kiminori Kimura†, Makoto Kawasaki¶, Yuka Takakura¶, Susumu Shirabe储, Jiro Takata††,
Yasufumi Kataoka††, and Shigeru Katamine¶
†Center for Emerging Infectious Diseases, ‡Department of Gene and Development, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu
501-1194, Japan; ¶Department of Molecular Microbiology and Immunology, 储Internal Medicine I, Nagasaki University Graduate School of Biomedical
Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; and ††Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical
Sciences, Fukuoka University, 8-19-1 Nanakuma, Jyonann-ku, Fukuoka 814-0180, Japan

Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved June 6, 2007 (received for review March 22, 2007)

Prion proteins are key molecules in transmissible spongiform mutations related to familial forms of the prion diseases are
encephalopathies (TSEs), but the precise mechanism of the con- rather concentrated in helices B and C (SI Fig. 4b), and their
version from the cellular form (PrPC) to the scrapie form (PrPSc) is distribution is somewhat similar to that of slowly fluctuating
still unknown. Here we discovered a chemical chaperone to stabi- regions. Moreover, those residues form a major cavity (Fig. 1a,
lize the PrPC conformation and identified the hot spots to stop the green). Thus, a small substance capable of specifically binding to
pathogenic conversion. We conducted in silico screening to find those residues could stabilize the PrPC conformation because of
compounds that fitted into a ‘‘pocket’’ created by residues under- the decrease in the Gibbs free energy of PrPC upon binding (6),
going the conformational rearrangements between the native and as well as the suppression of the conformational rearrangements
the sparsely populated high-energy states (PrP*) and that directly by cross-linking of distant regions. We termed this strategy
bind to those residues. Forty-four selected compounds were tested in dynamics-based drug discovery. Because PrPSc is gradually de-
a TSE-infected cell culture model, among which one, 2-pyrrolidin-1- graded in ex vivo experiments (17, 18), such a population shift

BIOCHEMISTRY
yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, toward PrPC will result in a decrease in PrPSc population.
termed GN8, efficiently reduced PrPSc. Subsequently, administra- Based on dynamics-based drug discovery, we conducted a
tion of GN8 was found to prolong the survival of TSE-infected mice. search for chemical compounds that could specifically bind to
Heteronuclear NMR and computer simulation showed that the the unstable residues. We focused on 14 amino acid residues
specific binding sites are the A-S2 loop (N159) and the region from (M129, G131, N159, V161, Y162, D178, C179, T183, I184, L185,
helix B (V189, T192, and K194) to B-C loop (E196), indicating that the H187, T190, G195, and E196, shown in red with side chain in Fig.
intercalation of these distant regions (hot spots) hampers the 1a), located in the loop between helix A and S2 (A-S2 loop) and
pathogenic conversion process. Dynamics-based drug discovery the loop between helices B and C (B-C loop). A virtual ligand
strategy, demonstrated here focusing on the hot spots of PrPC, will screening program initially picked up 624 chemicals potentially
open the way to the development of novel anti-prion drugs. capable of binding to the pocket (Fig. 1a, green) with a binding
score better (i.e., less) than ⫺32 (SI Table 1), of 320,000
anti-prion compound 兩 binding sites 兩 chemical chaperone 兩 dynamics- candidates in a database. We further selected the compounds
based drug discovery 兩 transmissible spongiform encephalopathy that formed hydrogen bonds with at least one of the 14 amino
acids. With careful examination of binding modes, taking into
account Lipinski’s rules (19), we then selected the 59 compounds
T he accumulation of abnormal protease-resistant prion pro-
tein (PrPSc), a conformational isoform of cellular prion
protein (PrPC), is a key event in the pathogenesis of transmissible
showing the lowest predicted binding free energy.

Results
spongiform encephalopathies (TSEs) (1–3), and this host-
encoded PrPC has a crucial role in the development of the To evaluate the effect of the selected compounds on the
diseases (4, 5). Because details of the mechanism of conversion conversion of PrP, we next conducted ex vivo screening. We used
from PrPC to PrPSc still remain obscure at this stage, PrPC could a mouse neuronal cell culture uninfected (GT1-7) and persis-
be an appropriate molecular target for the drug treatment of tently infected with human TSE agent (Fukuoka-1 strain),
TSEs (6) for avoiding the problems associated with the strain designated GT⫹FK (20). Of the 59 compounds, we tested the 44
differences in PrPSc (7). PrPC is a membrane-anchored glycosy- that were commercially available (see SI Table 1). Among these,
lated protein and is well conserved in mammals, and its physi- 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-
ological function is currently argued (8). The three-dimensional benzyl]-phenyl]-acetamide (compound number 8, molecular
structure of recombinant PrPC has been elucidated by NMR weight 420) (Fig. 1b) was found to significantly inhibit the PrPSc
(9–13). Briefly, it contains a globular fold with three ␣-helices
(A, B, and C) and a small, imperfectly formed ␤-sheet (S1
Author contributions: K. Kuwata designed research; K. Kuwata, N.N., T.M., Y.O.K., J.H.-M.,
and S2). K. Kodama, H.K.N., K. Kimura, M.K., Y.T., S.S., J.T., Y.K., and S.K. performed research; H.K.N.
The pathogenic conversion process could be related to the analyzed data; K. Kuwata wrote the paper; K. Kuwata performed the NMR measurements
thermal stability or the global conformational fluctuation of and in silico screening; N.N. and J.H.-M. performed the ex vivo and in vivo screening; T.M.
prepared the labeled and nonlabeled recombinant PrP; Y.O.K. measured NMR spectra; K.
PrPC. Recently, a metastable state of the PrPC was characterized Kodama synthesized GN8; K. Kimura, M.K., Y.T., S.S., and S.K. were mainly engaged in the
by using a high-pressure NMR (14), where hydrostatic pressure in vivo experiment; and J.T. and Y.K. prepared GN8 aqueous solution for injection and the
was elevated up to 2,500 bar in an on-line high-pressure NMR in vivo test.
cell. The thermodynamical stability profile shows that diverse The authors declare no conflict of interest.
residues in helices B and C are less stable, indicating the This article is a PNAS Direct Submission.
formation of the intermediate conformation (PrP*) (14). Sub- Abbreviations: TSE, transmissible spongiform encephalopathy; PrP, prion protein; PrPC,
sequently, a Carr–Purcell–Meiboom–Gill relaxation–dispersion cellular isoform of PrP; PrPSc, scrapie isoform of PrP; PrP*, sparsely populated high-energy
study revealed that slow fluctuation on a time scale of micro- state of PrP; BSE, bovine spongiform encephalopathy; d.p.i., days postinoculation.
seconds to milliseconds occurs at the corresponding regions §To whom correspondence should be addressed. E-mail: [email protected].
[supporting information (SI) Fig. 4a], indicating the conforma- This article contains supporting information online at www.pnas.org/cgi/content/full/
tional rearrangements occurring between the native and the 0702671104/DC1.
sparsely populated high-energy states (15, 16). Interestingly, © 2007 by The National Academy of Sciences of the USA

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0702671104 PNAS 兩 July 17, 2007 兩 vol. 104 兩 no. 29 兩 11921–11926

 production in the GT⫹FK at 10 ␮M (Fig. 1c). The other
a compounds either had little effect even at a higher dose (IC50 ⬎
100 ␮M) or were highly toxic to the cells at 1 ␮M. The effect of
the compound (now designated GN8) was dose-dependent (Fig.
1d), and by repeating the experiment we established that the
effective concentration for 50% reduction of PrPSc (IC50) over
72 h was ⬇1.35 ␮M. The normal PrPC expression in uninfected
cells was unaffected. A similar effect was confirmed by using
other scrapie-infected GT cells (GT⫹22L and GT⫹Ch) (20),
and also by using GT⫹BSE cells stably infected with mouse-
adapted bovine spongiform encephalopathy (BSE) (Fig. 1e).
This confirms that the action of GN8 is not strain-specific. To see
the effect of GN8 in vivo, mice inoculated with 20 ␮l of 10% FK-1
mouse brain homogenate were given the compound at a dose of
250 ␮g/kg per day by intraventricular infusion using osmotic
pumps (Alzet Durect, Cupertino, CA) during 42–70 days post-
inoculation (d.p.i.) or 70–98 d.p.i. Although the vehicle-only
control (5% glucose/saline) showed that the average survival
time was 123.8 ⫾ 7.4 (n ⫽ 6), as expected, the GN8-treated mice
b showed slightly but significantly prolonged survival even after
the appearance of clinical signs [132.3 ⫾ 9.2 days (n ⫽ 7) in the
42–70 d.p.i. group and 141.5 ⫾ 18.8 days (n ⫽ 6) in the 70–98
d.p.i. group; P ⬍ 0.05], as shown in Fig. 1f. The effect on the
survival of infected mice is limited here because of the transient
c administration of GN8.
DM 4
compound #
5 6 7 8 PS
d GN8 (µM)
The mice administrated subcutaneously with GN8 at a dose of
DM 1 2 4 6 8 10 PS
8.9 mg/kg per day using infusion pumps survived longer than
control mice. Whereas the control [5% glucose/saline (63–120
d.p.i. group)] showed that the average survival time was 133.0 ⫾
4.9 days (n ⫽ 9), the GN8-treated mice showed slightly but
significantly prolonged survival [148.6 ⫾ 10.3 days (n ⫽ 8) in the
67–95 d.p.i. group and 151.4 ⫾ 15.3 days (n ⫽ 10) in the 67–123
d.p.i. group; P ⬍ 0.01], as shown in Fig. 1g. Pharmacological
e GN8 (µM) GN8 (µM) GN8 (µM)
analysis using labeled GN8 is currently going on. On the other
CR 20 10 1 0 CR 20 10 1 0 CR 20 10 1 0
hand, pentosan polysulfate was not effective at all on the survival
time when given peripherally (data not shown). Thus, GN8 could
PrPres
(PK+) be a potential lead compound for prion diseases.
Binding of GN8 to PrPC was confirmed by the surface plasmon
resonance (21), and its dissociation constant was estimated to be
PrPC 3.9 ⫾ 0.2 ⫻ 10⫺6 M from the Scatchard plot (Fig. 2a; see also SI
(PK–) Fig. 5 and SI Methods). To identify the putative sites for
interaction of GN8 with PrP, we analyzed the chemical shift
22L Chandler BSE perturbation of 1H-15N heteronuclear single quantum coherence
NMR spectra (22) of a uniformly 15N-labeled PrP. A comparison
f 100 g 100 of the spectra revealed that three cross peaks (corresponding to
Survival (%)

Survival (%)

80 80 V189, K194, and E196) shifted significantly upon the addition of

60 60
GN8 (Fig. 2b), apparently in a fast-exchange mode. The GN8
40 40
20 20
concentration did not appear to significantly affect line broad-
0 0 ening. Most of the perturbed residues were located in the S2-A
100 120 140 160 180 200 100 120 140 160 180 200 loop, the B-helix, or the B-C loop regions, indicating the specific
Survival time (days) Survival time (days) binding between GN8 and PrPC (Fig. 2c). Fig. 2d shows the
Fig. 1. In silico and ex vivo screening. (a) Residues undergoing global
markedly perturbed residues mapped onto a three-dimensional
fluctuation displayed as a wire frame in red mapped on the mouse PrPC PrP model.
structure (residues 124 –226) (12), and a binding pocket defined by those To investigate whether GN8 indeed stabilizes the PrPC con-
residues, colored green. S1, A, S2, B, and C indicate S1 strand, helix A, S2 strand, formation, we measured the thermal stability using CD. The
helix B, and helix C, respectively. The image was created by using thermal unfolding curves of recombinant mouse PrP, monitored
PyMol (www.pymol.org). (b) 2-Pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl- by the molar ellipticity at 222 nm, representing the helical
acetylamino)-benzyl]-phenyl]-acetamide, termed GN8. (c) Western blotting of content of PrP and the overall unfolding behavior with (red) or
PrPSc in GT⫹FK cells after treatment with different compounds picked up by without (blue) GN8, were compared quantitatively (Fig. 2e and
in silico screening. The cells treated with no. 8 compound showed significant
reduction of PrPSc, which was better than that with 10 ␮g/ml pentosan
polysulfate. DM, DMSO at 0.1%; PS, 10 ␮g/ml pentosan polysulfate. Molecular
masses (37, 25, and 15 kDa) are indicated by bars on the right side of the panels. at 123.8 ⫾ 7.4 days (black line). Average survival time of GN8-treated mice was
(d) PrPSc signals in serially diluted mock-treated samples and tested samples 132.3 ⫾ 9.2 days (42–70 d.p.i. group, n ⫽ 7, red line) and 141.5 ⫾ 18.8 days
were scanned and quantified. The IC50 of no. 8 (72-h treatment), as deter- (70 –98 d.p.i. group, n ⫽ 6, green line). (g) Kaplan–Meier’s survival curves of
mined by four repeated experiments, was 1.35 ␮M. (e) GN8 reduced PrPSc also FK-infected mice administered GN8 subcutaneously. The control group (n ⫽ 9)
in 22L-, Ch-, and BSE-infected cells. CR, Congo red at 10 ␮g/ml; PK, proteinase was killed at 133.0 ⫾ 4.9 days (black line). Average survival time of GN8-
K digestion. ( f) Kaplan–Meier’s survival curves of FK-infected mice adminis- treated mice was 148.6 ⫾ 10.3 days (67–95 d.p.i. group, n ⫽ 8, red line) and
tered GN8 by intraventricular infusion. The control group (n ⫽ 6) was killed 151.4 ⫾ 15.3 days (67–123 d.p.i. group, n ⫽ 10, green line).

11922 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0702671104 Kuwata et al.

 a b a
0.6 120
196
Req /C (µM)

N (ppm)
0.4
121
194
0.2

15
0.0 122
189
0.0 1.0 2.0 3.0
Req 7.9 7.8 7.7 7.6
1 H (ppm)

c 0.20
194
0.15 192
189
b
159
∆δ

0.10

Free energy
0.05 PrP*
PrP
0.00
50 100 150 200

d e
PrP -GN8 complex
[θ]MRW(x10-3 degree cm2 dmol-1 )

0.6
∆[θ]MRW

-4
0.4 PrP
0.2 PrPSc

BIOCHEMISTRY
-6 0.0
20 40 60 80 Conformational space
Temperature (ºC)
-8
Fig. 3. Inhibitory mechanism of GN8 for pathogenic conversion. (a) Stereo-
view of the optimized complex structure of the GN8 and mouse PrPC, with
-10
putative hydrogen bonds, calculated by using ICM version 3.0. Presumed
30 40 50 60 70 80 hydrogen bonds between GN8 (green) and E196 (red) and between GN8
Temperature (ºC) (green) and N159 (blue) are shown in orange (dotted lines). The images were
created with VMD (52). (b) Illustration of the Gibbs free energy as a function
Fig. 2. Interaction of an anti-prion compound, GN8, and a recombinant of the conformational space to explain the inhibitory mechanism of a chemical
mouse PrPC. (a) Scatchard plot (Req vs. Req/C, where Req and C are the chaperone, GN8. GN8 stabilizes the PrPC conformation and reduces the pop-
equilibrium response of SPR and the concentration of GN8, respectively) of the ulation of PrP*, PrPSc, and PrPU. PrP* may not interact with GN8, because the
specific binding of GN8 with the PrP obtained by a surface plasmon resonance specific conformation around the binding sites would be lost (14). Thus, the
sensorgram. From the slope of the line, Kd was estimated to be 3.9 ␮M. (Details free energy level of PrP*, PrPU, and PrPSc would not change much in the pres-
are shown in SI Fig. 5 and SI Methods.) (b) An overlay of the 1H-15N hetero- ence of GN8.
nuclear single quantum coherence NMR spectra of PrP in the absence and
presence of GN8. Blue contours show the spectrum of PrPC without GN8, and
red contours show the spectrum in the presence of 1.0 mM GN8 at pH 4.5. (c) Discussion
Plot of the weighted averages of the 1H and 15N chemical shift changes, The structure of the PrPC–GN8 complex was further analyzed by
calculated by using the function ⌬␦ ⫽ [(⌬␦1H)2 ⫹ 0.17(⌬␦15N)2]1/2 against the computer simulation using the refined energy minimization
residue number. The absence of bars in the plot indicates unassigned residues,
procedure with flexible receptor side chain. GN8 (Fig. 3a)
proline residues, or unmeasured shifts due to resonance overlaps. Perturbed
residues with ⌬␦ values of ⬎0.9 ppm are shown in red, and those with 0.9 ⬎
connected distant residues, N159 (A-S2 loop) and E196 (B-C
⌬␦ ⬎ 0.5 ppm are in orange. (d) Mapping of the perturbed residues on the loop), by hydrogen bonds. The regions with the significant
structure of mPrP(121–231) (PDB entry 1AG2). The perturbed residues with ⌬␦ chemical shift changes described above (Fig. 2d) were in good
values of ⬎0.9 ppm are shown in red, and those with 0.9 ⬎ ⌬␦ ⬎ 0.5 ppm are agreement with the binding regions of GN8 in the simulated
in orange. Binding pocket is overlaid in green. S1, A, S2, B, and C indicate S1 structure of the prion–GN8 complex (Fig. 3a). Intriguingly, K194
strand, helix A, S2 strand, helix B, and helix C, respectively. The image was at the C terminus of the helix B and E196 in the B-C loop
created by using PyMol. (e) Thermal unfolding profiles of recombinant mouse
undergo slow exchange dynamics (16) (SI Fig. 4b). Indeed, the
PrP (amino acids 23–231, 5 ␮M) without (blue) or with (red) GN8 (10 ␮M). CD
intensities of PrP in the presence of GN8 were normalized to those of PrP
mutation E196K causes a rapidly progressive dementia and
without GN8, and fitted curves (see SI Methods) are also shown. Binding with ataxia (23) and could be expected to greatly reduce the protein
GN8 stabilizes the conformation of PrPC. (Inset) The difference in extinction stability because of the elimination of salt bridges between E196,
coefficients at 222 nm of PrP without GN8 and those in the presence of GN8, R156, and K194 (23).
as a function of temperature. Intercalation of these two binding regions (A-S2 loop and B-C
loop) may be essential to stabilize the PrPC conformation. For
example, two representative binding sites, N159 and E196, are
SI Methods). Parameter sets obtained by a nonlinear fit, i.e., close together (distance between C␣ atoms ⬇ 15.4 Å) in PrPC
melting temperature (Tm) and enthalpy change (⌬H), without structure (12), but in the hypothetical PrPSc structure (24) they
GN8 were 65.3 ⫾ 0.4°C and 35.0 ⫾ 1.9 kcal/mol, respectively,
are considerably more distant (distance between C␣ atoms ⬇
whereas those with GN8 were 67.7 ⫾ 0.6°C and 41.8 ⫾ 2.2
kcal/mol, respectively. This indicates that the binding of GN8 45.2 Å). Because GN8 connects distant regions (N159 and E196)
stabilizes the PrPC conformation significantly. Intriguingly, the in the PrP sequence (36 aa) by hydrogen bonds, large confor-
accumulation of the intermediate was demonstrated by an early mational shift may be significantly prohibited, and thus inter-
increase in ellipticity at ⬇40°C before the global unfolding as mediate (PrP*) and further PrPSc or PrPU formation may be also
shown in Fig. 2e Inset. However, this is strongly suppressed in the blocked.
presence of GN8. Thus, GN8 suppressed the production of both Matsuda et al. (25) reported about the chemical chaperone
PrPSc (Fig. 1 c–e) and PrPU (thermally unfolded state) (Fig. 2e) therapy for GM1-gangliosidosis, but there has been no direct
by reducing the intermediate population (14). evidence for such a mechanism working on the anti-prion com-

Kuwata et al. PNAS 兩 July 17, 2007 兩 vol. 104 兩 no. 29 兩 11923
pounds. Experimental evidences presented here fully support the proach, based on the experimentally identified hot spot (43), will
concept that GN8 acts as a chemical chaperone to stabilize the make the mass screening of chemical compounds more efficient,
normal prion protein (PrP) conformation. As illustrated in Fig. 3b, especially for diseases related to protein misfolding (44).
free energy of PrPC–GN8 complex is significantly less than that of
PrPC, so the populations of the transition state, PrP*, PrPU, and Materials and Methods
PrPSc may be reduced accordingly. Virtual Ligand Screening. We performed in silico screening of
Over the past 10 years there have been various efforts to find ligands on 320,000 compounds in the Available Chemicals
out small compounds to reduce PrPSc population. These include Directory (MDL Information Systems, San Leandro, CA) for
porphyrins (26, 27), Congo red and its derivatives (28–30), specific binding to mouse PrPC (12). Residues with the exchange
acridine and phenothiazine derivatives (17, 31, 32), heparan time constant, ␶ex, between two sites of ⬎10 ms are displayed in
sulfate (33), aminoglycan, and polyamines (34, 35). Simulta- SI Fig. 4a (16). The software used was ICM version 3.0 (Molsoft,
neously, various technological developments have been reported La Jolla, CA). The program, by global optimization of the entire
including structure-based drug design (36) followed by the flexible ligand in the receptor (mouse PrPC) field (45), came up
structure–activity relationship study (37), small interfering RNA with 624 candidates potentially capable of binding to the pocket
(38), library screening (18), high-throughput screening (39), with a binding score better (i.e., less) than ⫺32, which roughly
chimeric ligand approach (40), and so on. Although strategies for corresponds to binding energy (kcal/mol). Then, a more refined
drug discovery used in these studies have a broad spectrum from energy-minimization procedure using a flexible receptor side
empirical to rational preponderance, there has been no report on chain was conducted and further selected the compounds that
the residue-specific evidences for the binding regions of anti- formed hydrogen bonds with at least one of the 14 amino acids.
prion compounds. For instance, the effect of BF-168 depends on
the strains (41), suggesting that BF-168 may not interact with Chemical Compounds. Chemical compounds selected from our
PrPC but with PrPSc in a strain-dependent manner, but this is still virtual ligand screening simulation were purchased from Aldrich
indirect evidence. Chemical Company (Milwaukee, WI), G & J Research Chem-
The structure of GN8 somewhat resembles a number of other icals (Devon, U.K.), Wako Pure Chemical (Osaka, Japan),
small PrPSc inhibitors, such as the Congo red, which is able to Interchim (Montlucon, France), Labotest (Niederschoena, Ger-
interact with the N-terminal domain of PrPC (42). However, we many), Florida Center for Heterocyclic Compounds (Gaines-
could not find out any evidence for the interaction between GN8 ville, FL), ChemBridge (San Diego, CA), Maybridge Chemical
and the N-terminal domain. The inhibitory mechanism of GN8 Company (Cornwall, U.K.), TimTec (Newark, NJ), Ambinter
and that of Congo red seem to be quite different because of the (Paris, France), Oak Samples (Kier, U.K.), Scientific Exchange
following reasons: (i) Congo red has two sulfonates on the edge (Center Ossipee, NH), ChemStar (Moscow, Russia), ChemDiv
of the molecule, but GN8 does not have negative charge. Thus, (San Diego, CA), and AsInEx (Moscow, Russia), or kindly
GN8 may not strongly interact with His⫹ at the octapeptide provided by the Drug Synthesis and Chemistry Branch, Devel-
repeats of the N-terminal domain. (ii) Although Congo red can opmental Therapeutic Program, Division of Cancer Treatments
cause aggregation of recombinant PrPC (42), GN8 never causes and Diagnosis, National Cancer Institute. Detailed information
aggregation even at a relatively high concentration (⬇0.03 mM) on the sources for all of the compounds is shown in SI Table 1.
in NMR tube. (iii) Chemical shift changes caused by binding with The compounds were dissolved with DMSO for the in vitro
GN8 are not significant at the N-terminal half region as shown screening and with distilled water for the spectral measurements.
in Fig. 2c. (iv) SPR affinity profile between GN8 and the GN8 hydrochloride salt was prepared for in vivo test as follows:
C-terminal half of PrPC(120–230) and that between GN8 and the GN8 was first dissolved with dioxane and added to 3 N HCl in
full-length PrPC(23–231) were quite similar, and the calculated dioxane. The solvent was evaporated in vacuo, and the residue
dissociation constants were also close (⬇5 ␮M), supporting our was recrystallized from acetone to give the hydrochloride salt of
conclusion that the major binding regions of GN8 locate at the GN8. GN8 hydrochloride salt (2-pyrrolidin-1-yl-N-[4-[4-(2-
C-terminal domain. On the other hand, the interaction sites of pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide dihy-
GN8 with the GPI-anchored PrPC on the cell surface could be drochloride); white solid; anal. calcd. for C25H34Cl2N4O2: C,
different from those with the free PrPC. However, a carboxy- 60.85; H, 6.94; Cl, 14.37; N, 11.35; O, 6.48. %Found: C, 60.83; H,
methyl moiety on the SPR sensor chip has a negative charge like 6.95; N, 11.35.
a phosphate moiety on the cell membrane. Because electrostatic
environments surrounding GN8 and prion on these surfaces are Recombinant Mouse PrP. The DNA of mouse PrP(23–231) was
similar, GN8 may also interact with the C-terminal domain of the amplified by PCR and cloned into the expression vector pET101/
GPI-anchored PrPC. D-TOPO (Invitrogen, Carlsbad, CA). As shown in SI Methods, the
We found here the effective anti-prion compound GN8, which 15N-labeled recombinant PrP for NMR measurements was ex-

specifically binds with the hot spots undergoing the slow fluc- pressed in Escherichia coli strain BL21 Star (DE3) (Invitrogen),
tuation on the time scale of microseconds to milliseconds and grown in 15N-labeled minimum medium Spectra9 (Spectra Gases,
exclusively interferes with the pathogenic conversion. According Branchburg, NJ), and purified by a Ni-chelating affinity chroma-
to the dynamics-based drug discovery strategy, we found ⬎20 tography method (46). Oxidization and refolding of the purified
compounds with any anti-prion activity, and the hit rate is ⬇10% protein (47) were performed in buffer containing 4 M urea at pH
at present (data not shown). However, no compound has been 8. A recombinant PrP sample for the surface plasmon resonance
more effective than GN8. For instance, we found a compound, sensorgram experiment was obtained by a similar procedure using
GN4 (see SI Table 1), whose structure is quite similar to GN8, a non-isotope-labeled LB medium instead of the Spectra9.
but the computer simulation suggested that the binding sites are
R156 and N159, which are quite close (2 aa). Thus, GN4 is Cell Culture and Antibodies. The immortalized mouse neuronal cell
expected to have less inhibitory effect on the global fluctuation line GT1-7 was cultured as described (20). GT1-7 cells stably
of PrPC, and indeed IC50 of GN4 was ⬎100 ␮M (data not shown). infected with Fukuoka-1, 22L, or Chandler/RML (designated
To potentially become of any use clinically, GN8 will need to GT⫹FK, GT⫹22L, or GT⫹Ch, respectively) were maintained for
clear many pharmacological hurdles; however, our basic principle more than a year in our laboratory. GT⫹BSE cells were infected ex
presented here constitutes a promising strategy with which to vivo in our laboratory with mouse-adapted BSE agent (a kind gift
approach the discovery of therapeutic compounds for TSE. Addi- from T. Yokoyama, National Institute of Animal Health, Tsukuba,
tionally, application of the dynamics-based drug discovery ap- Japan). Stock solutions of compounds were prepared fresh in 100%

11924 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0702671104 Kuwata et al.

DMSO at 100 mM and stored at 4°C. Before use, compounds were 10 mM, respectively. The backbone 1H and 15N chemical shifts
diluted with medium as indicated. Control cells were treated with for the GN8-bound protein were assigned by tracing the corre-
medium containing solvent alone (0.1%). Approximately 2 ⫻ 105 sponding peaks in 1H-15N heteronuclear single quantum coher-
cells were plated in each well of a six-well plate, and drug treatment ence spectra measured at various concentrations of GN8.
was started 15 h later. After 72 h of incubation, cells were lysed in
150 ␮l of 1⫻ Triton X-100/DOC lysis buffer (48), and samples Preparation of GN8 for Injection and in Vivo Test. The GN8 hydro-
normalized to 2 mg of protein per milliliter. Western blotting for chloride salt was dissolved in saline with 5% glucose and
PrPSc was done as described previously (48). Anti-mouse PrP sterilized by passing through a 0.2-␮m filter. The concentration
antisera (SS28) (49) and SAF32 antibody (SPI-BIO, Montigny le of the stock solution was adjusted to 10 mg/ml and kept at 4°C
Bretonneux, France) were used for PrPSc and PrPC, respectively, as until use. Ten percent FK-infected brain homogenate was inoc-
the primary antibody. The signals were visualized by ECL-plus ulated into right temporal lobes of 4-week-old male mice (ddY),
(Amersham, Buckinghamshire, U.K.) and scanned by using Fluor- followed by intraventricular infusion of GN8 using an osmotic
Chem (Alpha Innotech, San Leandro, CA). pump. Six or 10 weeks after inoculation, an osmotic pump was
inserted subcutaneously and the infusion cannula was implanted
NMR Measurements and Data Analysis. For NMR measurements, into the right ventricle. The pump was filled with GN8 (1.4
0.6 mg/ml 15N-uniformly labeled mouse PrP(23–231) was pre- mg/ml) or saline with 5% glucose. In a parallel experiment,
pared in 30 mM acetate-d3 buffer (pH 4.5) containing 1 mM pentosan polysulfate (Bene, Munich, Germany) was adminis-
NaN3, 4.5 ␮M 4-(2-aminoethyl)-benzenesulfonyl fluoride hydro- tered at 200 ␮g/kg per day by intraventricular or i.p. infusion. All
chloride, 20 ␮M EDTA, 0.4 ␮M Bestatin, 0.06 ␮M pepstatin, mice were carefully examined daily for neurological signs, and
0.06 ␮M E-64, and 1 nM sodium 2,2-dimethyl-2-silapentane-5- the incubation period was monitored. Survival data were statis-
sulfonate dissolved in 90% H2O/10% D2O. NMR spectra were tically evaluated according to Kaplan–Meier’s method using
recorded at 20.0°C on an Avance600 spectrometer (Bruker, StatMateIII (ATMS, Tokyo, Japan).
Rheinstetten, Germany) at Gifu University. The spectrometer In case of subcutaneous infusion of GN8, the concentration of
operates at 1H frequency of 600.13 MHz and 15N frequency of the stock solution was adjusted to 50 mg/ml. One percent

BIOCHEMISTRY
60.81 MHz. A 5-mm 1H inverse detection probe with triple-axis FK-infected brain homogenate was inoculated in the same way,
gradient coils was used for all measurements. 1H-15N hetero- and an osmotic pump including GN8 was inserted subcutane-
nuclear single quantum coherence spectra were acquired with ously at 9 weeks after inoculation.
2,048 complex points covering 9,600 Hz for 1H and 256 complex
points covering 1,200 Hz for 15N. NMR data were processed by Some compounds were kindly provided by the Drug Synthesis and
using the XWIN-NMR software package (Bruker) and Sparky Chemistry Branch, Developmental Therapeutic Program, Division of
(50). Resonance frequencies in these spectra were identified by Cancer Treatments and Diagnosis, National Cancer Institute. We thank
Ms. Sakiko Morishita and Mr. Yuki Matsui for technical help. K. Kuwata
using the chemical shift lists on mouse PrP(23–231) and PrP was supported in part by Grants-in-Aid for Scientific Research from the
(121–231) (51). For the chemical shift perturbation experiments, Ministry of Education, Culture, Sports, Science and Technology of Japan
aliquots of 2.5 ␮l of 0, 10, or 100 mM GN8 solutions in DMSO-d6 and grants from the Ministry of Health, Labour, and Welfare. This study
were added to 0.6 mg/ml 15N PrP(23–231) in a 5-mm-diameter was supported by the Program for Promotion of Fundamental Studies in
Shigemi microtube; the final concentration of GN8 is 0, 1, and Health Sciences of the National Institute of Biomedical Innovation.

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11926 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0702671104 Kuwata et al.