Synthesis and preliminary evaluation of peptidomimetic inhibitors of human β-secretase

Article abstract of DOI:10.1016/j.ejmech.2010.01.044

Based on the structure of OM99-2 and the X-ray crystal structure of its complex with β-secretase, a series of compounds containing the Leu*Ala hydroxyethylene isostere as a scissile bond substitution were designed. 31 compounds were synthesized and their

Full text of DOI:10.1016/j.ejmech.2010.01.044

European Journal of Medicinal Chemistry 45 (2010) 2089–2094
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Laboratory note
Synthesis and preliminary evaluation of peptidomimetic inhibitors
of human -secretase
b
a
Yan Niu a, Yuehua Wang b, Xiaomin Zou a, Xiaoming Yang a, Chao Ma a, Yang Lu¨ , Bo Zhou a,
,
Yue Yuan a, Guanhua Du b, Ping Xu a
*
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
b Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the structure of OM99-2 and the X-ray crystal structure of its complex with b-secretase, a series
Received 30 October 2008
Received in revised form
8 January 2010
Accepted 20 January 2010
Available online 28 January 2010
of compounds containing the Leu*Ala hydroxyethylene isostere as a scissile bond substitution were
designed. 31 compounds were synthesized and their -secretase inhibition activities were measured. It
was found that isobutyl group was a better R3 substitution as C-terminus in our target compounds, and
4-nitrobenzyl group was the best R2 side chain. With the aid of molecular modeling, the binding modes
b
of compounds 9 and 22 with b-secretase were compared. The result revealed a stronger bonding mode of
22 than 9. This explored that the optimal length of this series of peptidomimetic inhibitors was P3–P20.
The molecular weights of compounds with this length are around 600.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
b
-Secretase inhibitor
Hydroxyethylene isostere
CADD
Peptidomimetic
Synthesis
1. Introduction
Former reports have revealed that natural APP substrate sequence
of b-secretase is SEVKM/DAEFR while that of Swedish mutant type is
Alzheimer’s disease (AD) is a neurodegenerative disorder that
afflicts more and more people worldwide. Intensive research efforts
have focused on understanding the role of AD’s neuropathological
markers, plaques and tangles, in the disease process [1]. The
SEVNL/DAEFR [6,7]. And the hydroxyethylene transition-state iso-
stere has been proved to be a highly effective transition-state
analogue for the inhibition of aspartic proteases. Moreover, Ala is
a highly preferred residue in P10 position of peptide
b
-secretase
inhibitors [8]. So a hydroxyethylene isostere OM99-2 (Fig.1) of EVNL/
AAEFwasfound tobea potent -secretaseinhibitor[9]. And aseriesof
amyloid cascade hypothesis centers on the amyloid
which is the principal component of the amyloid plaques detected
in the brain of AD patients. Although the link between A and AD
b (Ab) peptide,
b
b
peptidomimetics of OM99-2 were reported later [10].
neuronal dysfunction is still controversial, conspicuous biochem-
ical, pathological and genetic evidence revealed that progressive
On the basis of these conclusions and the X-ray crystal structure
of the complex of b-secretase and its inhibitor OM99-2 [11], we
accumulation of the A
responsible for the neurodegeneration that occurs in AD [2].
Therefore, a strategy aimed at lowering the concentration of
b
peptide within the brain is critically
designed a series of compounds containing the Leu*Ala hydroxy-
ethylene isostere as a scissile bond substitution (Fig. 1). These
compounds have low molecular weights and overlap the P3–P20 of
OM99-2. This was a part of efforts to explore the optimal length of
peptidomimetic inhibitors. The compounds 1–31 were then
synthesized with different amino acid substitutions, mainly at P3
position, in order to maximize the R1 coverage (Table 1).
neurotoxic A
[3]. The dominant anti-amyloid (A
ted into two categories, increasing A
[4] or decreasing A production or aggregation. The latter approach
is based primarily on two proteolytic enzymes, - and -secretases,
that participate in the processing of the amyloid precursor protein
(APP) [5]. Our research project is targeted at -secretase, because it
b
represents an attractive way of clinical intervention
lowering) approaches are sor-
clearance by immunization
b
b
b
b
g
2. Chemistry
b
plays a crucial role in the rate-limiting step of the amyloid cascade.
We have reported the synthetic method of compound 9 as
a representative [12]. Scheme 1 showed the synthetic route of a key
intermediate lactone f.
* Corresponding author. Tel./fax: þ86 10 82801505.
E-mail address: pingxu@bjmu.edu.cn (P. Xu).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.044

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Y. Niu et al. / European Journal of Medicinal Chemistry 45 (2010) 2089–2094
Table 1
O
Structures of synthesized target compounds 1–31.
CNH2
O
OH
CH3
H
N
H
N
O
R2
OH
CH3
H
N
H
N
N
H
R1
N
H
R3
O
NH
O
O
O
O
NH
O
O
NH2
CO2H
HO2C
NH
CO2H
Compd
R1
R2
R3
1
OM99-2
O
R2
OH
CH3
O
H
N
H
N
R1
N
H
R3
2
3
O2N
O
structure template
Fig. 1. Structure of OM99-2 and template design.
Scheme 2 outlined the synthesis of compound 9 from lactone f. The
discussions on synthetic procedures of f had been presented therein
[12]. The methyl group was smoothly introduced at a-C of f in 80% yield
4
H3CO
using methyl iodide and lithium hexamethyldisilazide (LiHMDs,
Scheme 2). On a larger scale, the reaction duration should be prolonged
to 35–40 min instead of 30 min as reported [13], for exhaustion of the
starting materials. However, longer duration would also in the mean-
time lead to a dimethylated product (a white crystal, determined by 1H
OCH3
NMR and MS) in a small amount. The
as R configuration on the basis of NOE between
well as nonexistence of NOE between –OCH- and
hydrolysis of 2R,4S,5S-lactone h gave the hydroxyl acid derivative,
which was protected by tert-butyldimethylsilyl chloride (TBDMSCl) to
furnish i, followed by coupling with isobutylamine. The TBDMS group
was then removed to afford hydroxyl amide k as an amorphous white
solid, which proceeded from l to the model compound 9 through
a
-C of compound hwas identified
-CH3 and C4- -H, as
-CH–. Subsequent
5
6
7
a
a
a
O
O
classic peptide condensation using N-Boc-L-phenylalanine and then
O2N
3-(trifluoromethyl)cinnamic acid in turn. The reagents for condensa-
tion involved N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI) or N,N0-dicyclohexylcarbodiimide (DCC),
accompanied by 1-hydroxybenzotriazole (HOBt) and N,N0-diisopro-
pylethylamine (DIPEA) or N-methylmorpholine (NMM) in THF or DMF
at room temperature. The reaction duration time varied depending on
different acids used.
NO2
Cl
8
3. Bio-assay
Cl
The
-secretase substrate was used, which contains a highly fluo-
rescent group in one end and a quencher group in the other end,
also a -secretase cleavage site in the middle. The fluorescent group
can be efficiently quenched by resonance energy transfer to the
quencher group. When the substrate was cleaved by -secretase,
fluorescence will increase. Samples were added into 384-well black
plate first. Then -secretase (Recombinant Human BACE-1, R&D)
was diluted with 0.1 M sodium acetate buffer (pH 4.0) to final
concentration of 10 g/mL, and added into 384-well black plate.
b-secretase inhibition activity was assayed as following.
A
b
b
9
F3C
b
Cl
b
10
m
After shaking for 5 min, substrate (Fluorogenic Peptide Substrate
IV, R&D) was diluted with 0.1 M sodium acetate buffer (pH 4.0) to

Y. Niu et al. / European Journal of Medicinal Chemistry 45 (2010) 2089–2094
2091
Table 1 (continued)
Table 1 (continued)
Compd
R1
R2
R3
Compd
R1
R2
R3
NO2
NO2
H
O
O
N
11
O
O
22
H
N
O
12
13
23
24
O
O
NO2
H
N
O
NO2
NO2
H
N
O
14
F3C
O
25
26
27
H
N
15
O
O
O2N
O
O
NO2
H
N
H3CO
O2N
16
17
18
OCH3
NO2
NO2
28
29
O
O
Cl
NO2
NO2
Cl
Cl
NO2
19
20
21
30
31
O
O
OH
OH
H3CO
OH
final concentration of 10
plate. The total volume of reactive solution was 50
for 5 min, fluorescence was measured by excitation at 320 nm and
emission at 405 nm. F/min was expressed as the activity of
-secretase. If St represents the activity of standard and Sa
m
M, and added into the 384-well black
ml. After shaking
F3C
D
b

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Y. Niu et al. / European Journal of Medicinal Chemistry 45 (2010) 2089–2094
BocNH
OH
iii
ii
c
O
BocNH
BocNH
CHO
BocNH
BocNH
COOH
OH
OMe
v
i
iv
d
a
b
O
O
O
O
O
OEt
vi
BocHN
BocHN
+
e
f (61%)
g (14%)
Scheme 1. Synthetic route to intermediate lactone f. Reagents and conditions: (i) NaHCO3, CH3I, DMF; (ii) NaBH4, CaCl2, THF/EtOH; (iii) Py$SO3, TEA, DCM/DMSO; (iv) DIBAL,
toluene, 0 ꢂC, 10 min; (v) ethyl propiolate, LDA (n-BuLi and i-Pr2NH, ꢀ23 ꢂC in THF, 1 h), ꢀ78 ꢂC, N2, 4.5 h; (vi) H2/Pd, 60 psi, EtOAc, then reflux in toluene/acetic acid (97.5:2.5 v/v).
represents the activity of sample, the inhibition rate of sam-
-secretase inhibition activities for
compounds 1–31 are listed in Table 2.
group was more effective than the benzyl group, by comparing
compounds 14, 15 with 2, 9. The variation at P3 position (R1) of N-
terminus included phenyl vinyl, substituted phenyl, tert-butoxy,
Boc-amino acids with side chains of Leu, Ile and Val. The result
showed that compounds 1, 5, 6, 14, 15, 16, 18 and 22 were ten times
more potent than 2 and 9. How does the R1 structure affect the
inhibition activity? We have docked compound 9 into the active
ple ¼ [(St ꢀ Sa)/St] ꢁ 100%. The
b
4. Results and discussion
In our target compounds there were two C-terminal groups
(R3): isobutyl and benzyl. The compounds with isobutyl group
site of b-secretase structure (PDB code: 1FKN) using the docking
program GOLD [14]. Fig. 2 shows the docked conformation of 9. The
scaffold of 9 overlayed very well with the original ligand OM99-2,
which is in line with the computational work by Shen [10] except
that the N-terminus extended to S01 pocket due to its flexibility and
lacking of additional polar interactions with S03–S04 subsites of
1FKN. Benzyl group (R2) of 9 was accommodated in S2 pocket,
showed obviously more potent b-secretase inhibition activity than
that of benzyl group. This was in concordance with the result of
Ref. [10]. With regard to the P2 substitution (R2), three side chains
were selected as representatives. It was observed that the two
aromatic moieties (benzyl and 4-nitrobenzyl) were more active
than hydroxymethyl group. It was also seen that the 4-nitrobenzyl
O
O
OTBS CH3
O
O
i
ii
iii
BocHN
BocHN
BocHN
BocHN
OH
CH3
O
i
h
f
OTBS CH3
OH
CH3
H
N
H
v
iv
BocHN
N
O
O
j
k
O
OH
CH3
OH
CH3
H
N
H
N
H
N
H
N
vi
N
H
BocHN
O
O
O
O
F3C
9
l
Scheme 2. Synthesis of compound 9 from lactone f. Reagents and conditions: (i) CH3I, LiHMDS, ꢀ78 ꢂC; (ii) a. LiOH (1 N), THF; b. TBDMSCl, imidazole, DMF; (iii) isobutylamine, EDC,
HOBt, DIPEA, DMF/CH2Cl2; (iv) n-Bu4NþFꢀ, THF; (v) a. TFA/CH2Cl2; b. N-Boc-L-phenylalanine, EDC, HOBt, NMM, DMF; (vi) a. TFA/CH2Cl2; b. 4-(trifluoromethyl)cinnamic acid, EDC,
HOBt, NMM, DMF.

Y. Niu et al. / European Journal of Medicinal Chemistry 45 (2010) 2089–2094
2093
Table 2
-Secretase inhibition activities for compounds 1–31.
quite well with OM99-2, and the binding mode is also the same
with 9 in S1 and S2 subsites. So the docking results revealed
a stronger bonding mode of 22 than 9.
b
Compd
Inhibition rate, % (10
m
g/mL)
IC50, mM
1
2
11.20
1.47
6.93
15.73
5. Conclusion
3
4
5
6
7
8
9
22.83
14.66
ꢀ0.10
16.20
0.91
20.26
59.66
16.92
40.29
37.22
ꢀ4.43
0.13
–
–
Based on the structure of OM99-2 and the X-ray crystal struc-
ture of its complex with b-secretase, a series of compounds con-
4.67
8.07
–
–
taining the Leu*Ala hydroxyethylene isostere as a scissile bond
substitution were designed and synthesized. It was found that
isobutyl group was a better R3 substitution as C-terminus in our
target compounds, and 4-nitrobenzyl group was the best R2 side
chain. With the aid of molecular modeling, the binding modes of
19.54
–
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
–
compounds 9 and 22 with b-secretase were compared. The result
3.79
2.64
3.81
–
4.24
–
revealed a stronger bonding mode of 22 than 9. This explored that
the optimal length of this series of peptidomimetic inhibitors was
P3–P20. But the molecular weights of compounds with this length
are around 600, which are too high to be an ideal drug. So a phar-
macophore model has been produced from these compounds. That
model is being modified and tested in our lab. It will be used to
10.48
18.30
7.61
22.23
25.66
25.09
16.45
54.57
ꢀ2.37
ꢀ0.71
0.91
ꢀ1.56
ꢀ2.46
25.76
22.96
30.06
50.53
–
–
design non-peptide small molecular b-secretase inhibitors.
2.31
–
–
–
–
–
–
–
–
6. Experimental protocol
Unless otherwise mentioned, reagents were obtained commer-
cially and used without further purification. Petroleum ether used
here had a boiling point range from 60 to 90 ꢂC. Melting points
were taken with an X-4 apparatus and were uncorrected. IR (KBr),
1H NMR, MS, and elemental analysis data were taken with PERKIN-
ELMER983, INOVA-500, MDS SCIEX QSTAR, and FLASH EA 1112
instruments respectively.
–
The synthesis of compounds b–l followed the procedures in
Ref. [12].
forming no hydrogen bond with guanidyl group of Arg235. It is
supposed that if some polar substitutions were introduced onto R3
or R2 at proper position, additional hydrogen bonds might be
formed within these relavant subsites and potent inhibitors might
be achieved. The C-terminal 4-(trifluoromethyl)styryl group (R1) of
9 oriented in the gap between S3 and S4 subsites, and that evoked
us to change the rigid aromatic R1 into some flexible groups so that
it can enter either of the two subsites.
Compound 22 was also docked into the active site of 1FKN and
the result is shown in Fig. 3. We are surprised to find that the
binding mode of isobutyl group (R3) of 22 is different from that of 9.
The positions of the adjacent N atom and carbonyl group were
almost identical with OM99-2, which means an additional
hydrogen bond was formed between the substituted amino group
and Gly34 compared with 9. This hydrogen bond also made the
isobutyl group positioned into the hydrophobic S02 pocket, so that
more hydrophobic interaction was present than 9. As for the R1
moiety, the isobutyl group was found to extend to S4 subsite and
the tert-butyloxy carbonylamino go upside to form strong hydro-
phobic interaction with Lys107. The rest of the parts of 22 overlayed
6.1. General procedure for the synthesis of target
compounds (1–31)
Trifluoroacetic acid (TFA,1 mL) was added to a stirred solution of
amide l (65 mg, 0.13 mmol) in CH2Cl2. After 30 min, the solvent and
most of TFA were removed by evaporation. In another flask,
R1COOH (0.15 mmol), N-ethyl-N0-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDCI, 30 mg, 0.15 mmol) and 1-hydroxy-
benzotriazole (HOBt, 21 mg, 0.15 mmol) were dissolved in DMF
(4 mL), to which were added the above deprotected amine
(neutralized with N-methylmorpholine) and another N-methyl-
morpholine (0.1 mL, 0.3 mmol). The mixture was stirred at room
temperature for 48 h. After this period, the mixture was poured into
10 mL of aqueous NaHCO3 and extracted with EtOAc (30 mL ꢁ 3),
and then washed successively with 10% aqueous citric acid
(10 mL ꢁ 2), saturated aqueous NaHCO3 (10 mL ꢁ 2) and brine, and
dried over MgSO4. After evaporation of the solvent, the residue was
Fig. 2. The docked conformation of 9 (magenta) in comparison with OM99-2 (cyan) in the active site of 1FKN.

2094
Y. Niu et al. / European Journal of Medicinal Chemistry 45 (2010) 2089–2094
Fig. 3. The docked conformation of 22 (orange) in comparison with OM99-2 (cyan) in the active site of 1FKN.
dissolved in MeOH at 65 ꢂC, to which ether and petroleum ether was
added slowly. After 5 h at room temperature (lower temperature is
desirable), the product crystal was obtained. Recrystallization can be
repeated for several times. Synthesized target compounds 1–31
were characterized by melting point, MS, 1H NMR and elemental
analyses. Analyses indicated by the symbols of the elements (C, H, N)
were within ꢃ0.4% of the theoretical values.
Ph–C–CH–), 3.90–3.88 (m, 2H, –OH, –N–CHCO–), 3.60 (m, 1H,
–OCH–), 3.49–3.48 (m, 1H, –NCH–), 3.27–3.25 (m, 1H, PhCH2–),
3.16–3.00 (m, 3H, PhCH2–, –CON–CH2–), 2.53–2.52 (m, 1H, –C–
CHCO–), 1.80–1.73 (m, 1H, –CH–), 1.64–1.56 (m, 2H, 2-CH–), 1.44–
1.33 (m, 4H, 2-CH2–C–CO–), 1.33 (s, 9H, Boc), 1.33–1.21 (m, 2H,
–CH2–), 1.15–1.13 (d, 3H, J ¼ 7.0 Hz, CH3–), 0.94–0.90 (d, 12H,
J ¼ 7.0 Hz, 4CH3–), 0.86–0.84 (d, 6H, J ¼ 6.0 Hz, 2CH3–). MS (ESI)
calcd for C34H58N4O6 m/z: 619.44 [M þ Hþ], found 619.49.
Elemental data: calculated: C, 65.99; H, 9.45; N, 9.05; measured: C,
65.85; H, 9.77; N, 8.89.
6.1.1. [(2R,4S,5S)-5-[N-(3,4-dimethoxy)benzoyl-L-phenylalanyl-
amido]-4-hydroxy-2,7-dimethyloctal] N0-isobutyl amide (4)
White solid, yield 53%, m.p. 212–214 ꢂC. 1H NMR (DMSO-d6,
The spectra data of other compounds are given in
Supplementary information.
500 MHz):
d
¼ 8.43–8.41 (d, 1H, J ¼ 8.5 Hz, –CONH–), 7.65–7.63 (m,
1H, –CONH–), 7.47–7.45 (d, 1H, J ¼ 9.0 Hz, –CONH–), 7.41–7.39 (d,
1H, J ¼ 8.5 Hz, Ph(3,4-dimethoxy)-C6H–), 7.36–7.12 (m, 5H, Ph–),
7.32 (s, 1H, Ph(3,4-dimethoxy)-C2H–), 6.98–6.97 (d, 1H, J ¼ 8.5 Hz,
Ph(3,4-dimethoxy)-C5H–), 4.69–4.65 (m, 1H, –NCHCO–), 4.61–4.60
(d, 1H, J ¼ 5.5 Hz, –OCH–), 3.78–3.77 (s, 6H, 2CH3O–), 3.73 (m, 1H,
–NCH–), 3.14–3.10 (dd, 1H, J ¼ 4.5 Hz and 14.0 Hz, PhCH2–), 2.97–
2.92 (dd, 1H, J ¼ 10.5 Hz and 13.5 Hz, PhCH2–), 2.85–2.82 (t, 2H,
–CON–CH2–), 2.48–2.44 (m, 1H, –CHCO–), 1.69–1.61 (m, 1H, –N–C–
CH–), 1.60–1.55 (m, 1H, –CH–), 1.53–1.48 (m, 1H, –CH2–C–CO–),
1.41–1.35 (m, 1H, –CH2–C–CO–), 1.24–1.19 (m, 1H, –CH2–), 1.17–1.11
(m, 1H, –CH2–), 0.97–0.95 (d, 3H, J ¼ 7.0 Hz, –CH3), 0.83–0.80 (d,
12H, 4CH3–). MS (ESI) calcd for C32H47N3O6 m/z: 570.35 [M þ Hþ],
found 570.39. Elemental data: calculated: C, 67.46; H, 8.31; N, 7.38;
measured: C, 67.60; H, 8.58; N, 7.10.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant 30772650 and 20772008).
Appendix. Supplementary information
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2010.01.044.
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7.69–7.64 (m, 1H, –CONH–), 7.60 (d, 1H, J ¼ 9.5 Hz, –CONH–), 7.40
(d, 1H, J ¼ 16 Hz, CF3PhCH]), 7.29–7.14 (m, 5H, Ph–), 6.82 (d, 1H,
J ¼ 16 Hz, ]CHCO–), 4.71–4.66 (m, 1H, –NCHCO–), 4.58 (d, 1H,
J ¼ 5.5 Hz, –OCH–), 3.73 (m, 1H, –NCH–), 3.11–3.08 (dd, 1H,
J ¼ 4.5 Hz and 14 Hz, PhCH2–), 2.84–2.81 (m, 2H, –CON–CH2–),
2.80–2.75 (dd, 1H, J ¼ 10 Hz and 14 Hz, PhCH2–), 2.46 (m, 1H,
–CHCO–), 1.67–1.62 (m, 1H, –N–C–CH–), 1.60–1.55 (m, 1H, –CH–),
1.54–1.47 (m, 1H, –CH2–C–CO–), 1.38–1.32 (m, 1H, –CH2–C–CO–),
1.25–1.20 (m, 1H, –CH2–), 1.12–1.07 (m, 1H, –CH2–), 0.97 (d, 3H,
J ¼ 6.5 Hz, –CH3), 0.84–0.79 (d, 12H, 4CH3–). MS (ESI) calcd for
C33H44F3N3O4 m/z: 604.33 [M þ Hþ], found 604.41. Elemental data:
calculated: C, 65.65; H, 7.35; N, 6.96; measured: C, 65.65; H, 7.57;
N, 6.86.
6.1.3. [(2R,4S,5S)-5-(N-Boc-L-leucyl-L-phenylalanylamido)-4-hydroxy-
[12] X. Yang, X. Zou, Y. Fu, K. Mou, G. Fu, C. Ma, P. Xu, Synth. Commun. 37 (2007)
9–24.
[13] A.K. Ghosh, G. Bilcer, C. Harwood, K. Reiko, D. Shin, K.A. Hussain, L. Hong,
J.A. Loy, C. Nguyen, G. Koelsch, J. Ermolieff, J. Tang, J. Med. Chem. 44 (2001)
2865–2868.
2,7-dimethyloctal] N0-isobutyl amide (22)
White solid, yield 28.5%, m.p. 184–186 ꢂC. 1H NMR (CDCl3,
500 MHz):
d
¼ 7.31–7.19 (m, 5H, Ph–), 6.52–6.51 (d, 1H, J ¼ 5.0 Hz,
–CONH–), 6.42–6.41 (d, 1H, J ¼ 6.5 Hz, –CONH–), 6.15 (s, 1H,
–CONH–), 4.80–4.79 (d, 1H, J ¼ 4.5 Hz, BocNH–), 4.62–4.58 (m, 1H,
[14] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, J. Mol. Biol. 267 (1997)
727–748.