Template-constrained somatostatin analogues: a biphenyl linker induces a type-V' turn.

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Template-Constrained Somatostatin Analogues: A
Biphenyl Linker Induces a Type-V′ Turn
Richard P. Cheng,†Daniel J. Suich,†,§Hong Cheng,‡
Heinrich Roder,*,‡and William F. DeGrado*,†
Johnson Research Foundation
Department of Biochemistry and Biophysics
UniVersity of PennsylVania School of Medicine
Philadelphia, PennsylVania 19104-6059
Institute of Cancer Research
Fox Chase Cancer Center, 7701 Burholme AVenue
Philadelphia, PennsylVania 19111
ReceiVed July 11, 2001
Reverse turns are important for the folding and molecular
recognition of proteins and peptides.1Thus, there has been interest
in stabilizing various turns to provide conformational rigidity and
improved bioavailability, while retaining or even enhancing the
potency and selectivity of the parent peptide. Macrocyclization
has been an effective approach for restricting the conformation
of a turn sequence.1-5One of the simplest strategies involves
backbone cyclization1,3,5c,f,lwith the incorporation of hetero-
chiral sequences1,3a,bsuch as D-Pro-Pro3a,band peptoids.5fThis
concept has been expanded further by incorporating nonpeptidic
linkers4,5b,d,g,h,k,nsuch as dipeptide turn mimetics,6which provide
extra rigidification and reduced peptidic character. Although these
cyclic peptides are fairly rigid, receptor-induced conformational
change cannot be completely ruled out.3c,7Nevertheless, structures
of these cyclic peptides have been important for revealing
structure-activity relationships.2-4b,5b,d,g,h,k,n,7Previously, we in-
corporated two linkers into somatostatin analogues that elicit high
activity and subtype selectivity (DJS811 and DJS631).5bHerein,
we report the conformational analysis of these two somatostatin
analogues.
Somatostatin,8a,ba disulfide-linked 14-residue cyclic peptide,
is important for regulating hormone release (growth hormone,
glucagon, insulin, gastrin), and for neural transmission. Various
somatostatin receptor subtypes for mediating the different biologi-
cal activities have been identified.8c,dThe four central residues,
Phe-Trp-Lys-Thr, are essential for activity5nand appear to form
a ?-turn (probably type-I ?-turn1,9) in the native peptide. The
replacement of Trp with D-Trp results in peptides with enhanced
activity and tunable specificity profiles.5,8This D-Trp-Lys het-
erochiral sequence forms a type-II′ ?-turn,5,9which is more stable
than the type-I ?-turn conformation.1Streamlined cyclic somato-
statin analogues with a type-II′ ?-turn have been investigated by
the groups of Veber and Hirschman,5n,ovan Binst,5kGoodman,5f
Kessler,5gHruby,7aand others,5which have been recently
reviewed.5aThe conformational analyses of these somatostatin
analogues have provided the structural basis for bioactivity. In
the bioactive type-II′ turn conformation, the side chains of Lys
and Trp are in close proximity, which causes a characteristic
upfield shift of the Lys γ protons due to the ring current effect
of the Trp indole ring. More recently, there has also been
development of nonpeptidic compounds that exhibit somatostatin
activity and subtype selectivity.10In this communication, the
structural characterization of subtype-selective somatostatin ana-
* To whom correspondence should be addressed. Heinrich Roder,
email:h_roder@fccc.edu; William F. DeGrado, e-mail:
mail.med.upenn.edu.
†University of Pennsylvania School of Medicine.
‡Fox Chase Cancer Center.
§Current address: Chiron Corporation, M/S 4.5, 4560 Horton St., Em-
eryville, CA 94608.
(1) (a) Rose, G. D.; Gierasch, L. M.; Smith, J. A. AdV. Protein Chem.
1985, 37, 1. (b) Lewis, P. N.; Momany, F. A.; Scheraga, H. A. Biochim.
Biophys. Acta 1973, 303, 211.
(2) For recent examples of nonbackbone cyclization, see: (a) Brauer, A.
B. E.; Kelly, G.; McBride, J. D.; Cooke, R. M.; Matthews, S. J.; Leatherbarrow,
R. J. J. Mol. Biol. 2001, 306, 799. (b) Lombardi, A.; D’Auria, G.; Maglio,
O.; Nastri, F.; Quartara, L.; Pedone, C.; Pavone, V. J. Am. Chem. Soc. 1998,
120, 5879.
(3) For examples of backbone cyclization, see: (a) Favre, M.; Moehle,
K.; Jiang, L.; Pfeiffer, B.; Robinson, J. A. J. Am. Chem. Soc. 1999, 121, 2679.
(b) Bean, J. W.; Kopple, K. D.; Peishoff, C. E. J. Am. Chem. Soc. 1992, 114,
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(4) For recent examples of cyclic peptides with artificial linkers, see: (a)
Wang, Z.; Jin, S.; Feng, Y.; Burgess, K. Chem. Eur. J. 1999, 5, 3273. (c)
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Zubrzycka, M.; Janecki, T. J. Pept. Res. 2001, 58, 91. (b) Suich, D. J.; Mousa,
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Angew. Chem., Int. Ed. 2000, 38, 1223. (d) Souers, A. J.; Virgilio, A. A.;
Rosenquist, Å.; Fenuik, W.; Ellman, J. A. J. Am. Chem. Soc. 1999, 121, 1817.
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Ke ´ri, G. Y.; E Ärchegyi, J.; Horva ´th, A.; Mezo ˜, I.; Va ´ntus, T.; Balogh, A Ä .;
Vada ´sz, Z. S.; Bo ˜ko ˜nyi, G. Y.; Sepro ˜di, J.; Tepla ´n, I.; Czuka, O.; Tejeda, M.;
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Pharmacol. 1996, 50, 709. (k) Elseviers, M.; van der Auwera, L.; Pepermans,
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515. (l) Cutnell, J. D.; La Mar, G. N.; Dallas, J. L.; Hug, P.; Rink, H.; Rist,
G. Biochim. Biophys. Acta 1982, 700, 59. (m) Bauer, W.; Briner, U.; Doepfner,
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wdegrado@
(6) For recent reviews on turn mimetics, see: (a) Gillespie, P.; Cicariello,
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80).
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12710
J. Am. Chem. Soc. 2001, 123, 12710-12711
10.1021/ja0116932 CCC: $20.00© 2001 American Chemical Society
Published on Web 11/17/2001

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logues DJS811 and DJS631 reveals a novel conformation: the
first reported example of a type-V′ ?-turn.1,9
Sequence specific assignments11awere obtained for DJS811
and DJS631 based on TOCSY,11bDQF-COSY,11cECOSY,11dand
ROESY11espectra collected on water/D2O and D2O samples. For
both compounds, the upfield shift of the Lys γ protons indicative
of a bioactive turn conformation was observed (DJS811, 0.305
ppm; DJS631, 0.23 ppm). For DJS811, the Lys ? protons and
Val γ methyl groups were stereospecifically assigned. The
structures were calculated by simulated-annealing protocols11fwith
ROE-derived distance restraints.12The more potent compound
(DJS811) converged to a single conformation (Figure 1), while
the less bioactive compound (DJS631) appeared to adopt at least
six conformations. The multiple conformations for DJS631 were
evident in the two phenyl rings of the urea template. Although
all phenyl ring protons were individually assigned, multiple
conformations were necessary to account for all the corresponding
ROESY cross-peaks. Since DJS811 exhibits higher bioactivity
and a single converged conformation, suggesting the constraints
imposed by the linkers promote the bioactive conformation,
detailed conformational analysis of DJS811 was pursued.
For DJS811, 42 unique distance restraints were included in
the structure calculations (2 medium range, 11 sequential). DQF-
COSY spectra revealed average values for3JHNCRH, and ECOSY
spectra provided the3JR?values to give two side chain dihedral
restraints for two ? protons (one each for Val and Lys).12Thirty
two of the 50 structures converged to a single conformation
(Figure 1, Table 1), and the average pairwise heavy atom and
backbone13rmsd values were 0.82 ( 0.10 and 0.26 ( 0.07 Å,
respectively. The rigid backbone conformation may be attributed
to the biphenyl turn mimetic. Strikingly, the expected type-II′
?-turn was observed in only six of the 50 structures (for the D-Trp-
Lys heterochiral sequence), while the type-V′ ?-turn was observed
in 32 structures (Table 1). Both conformations are equally
consistent with the NMR-derived restraints, but the structures with
the type-V′ ?-turn represent lower-energy conformers than those
with the type-II′ ?-turn.12Additionally, the lack of strong
intramolecular hydrogen bonds for the amides in the type-V′
?-turn structures is more consistent with the high variable
temperature coefficients for all the amide protons (-∆δ/∆T > 5
ppb/K).12The type-V′ ?-turn has been mentioned only as a
possibility by Scheraga and co-workers,1bbut it has not yet been
reported in proteins or peptides.14Besides exhibiting this unex-
pected turn type, the Tyr adopts a left-handed conformation (φ
> 0), which is generally unfavorable for natural L-amino acids.
Attempts to force this residue to a more conventional conforma-
tion (φ < 0°) resulted in violations of NMR restraints. Thus, this
conformation may serve to stabilize the type-V′ turn or destabilize
the type-II′ turn. The unusual backbone conformations may be
due to the constraints imposed by the semirigid turn mimetic.
Nevertheless, the side chain conformations of D-Trp and Lys are
consistent with the established pharmacaphore for somatostatin
activity,5which explains the bioactivity. The combination of
bioactive side chain conformations with subtle changes in the
backbone structure may explain the high subtype selectivity for
DJS811.
These data illustrate how similar but nonidentical peptides can
adopt distinct backbone conformations that present nearly identical
constellations of amino acid side chains (Figure 2). The interplay
of the plasticity of the amide backbone with the rigidity of an
artificial linker allows tuning of the conformation and hence
receptor selectivity. In the pioneering development of somatostatin
analogues, the selectivity profile was altered by replacing the
type-I ?-turn with a heterochiral type-II′ ?-turn (Figure 2).5,8For
DJS811, a semirigid biphenyl linker induced a novel structure,
the type-V′ ?-turn,1,9and high receptor selectivity.5bThus,
introduction of semirigid linkers into cyclic peptides appears to
be a successful strategy for tuning conformational and biological
properties.
Acknowledgment. This work was supported by Grants from NSF
(CHE9634646 and the MRSEC program, DMR00-79909 to W.F.D.) and
NIH (GM-19664 to R.P.C.). The NMR facility at the Fox Chase Cancer
Center is supported by NIH (CA06927) and the Kresge Foundation.
Supporting Information Available: Experimental procedures, chemi-
cal shift assignments, and coupling constants (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA0116932
(11) (a) Wu ¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New
York, 1986. (b) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355. (c)
Bax, A.; Freeman, R. J. Magn. Reson. 1981, 44, 542. (d) Griesinger, C.;
Sørensen, O. W.; Ernst, R. R. J. Magn. Reson. 1987, 75, 474. (e) Bax, A.;
Davis, D. G. J. Magn. Reson. 1985, 63, 207. (f) Nilges, M.; Clore, G. M.;
Gronenborn, A. M. FEBS Lett. 1988, 239, 129.
(12) See Supporting Information.
(13) The backbone includes the biphenyl moiety.
(14) The type-V′ ?-turn is essentially a compound (classical γ)-(inverse
γ) turn (also referred to as γcγi(i, i + 1) or γc(i, i + 2) - γi(i, i + 2)), which
was not found in a recent survey by Guruprasad and co-workers: Guruprasad,
K.; Prasad, M. S.; Kumar, G. R. J. Pept. Res. 2000, 56, 250.
Figure 1. The conformation of DJS811 as determined by ROE-based
distance-restrained simulated-annealing. (Left) Superimposition of the 32
converged structures. (Right) Structure closest to the average structure.
Table 1.
Dihedral Angles for the Converged Structure of DJS811
residue
Tyr
D-Trp
Lys
Val
φ
ψ?1
72 ( 3
99 ( 7
-95 ( 5
-63 ( 6
136 ( 3
-93 ( 5
93 ( 2
117 ( 6
-175 ( 5
145 ( 5
-66 ( 6
179 ( 1
Figure 2. Ideal models of Ac-Ala1/D-Ala1-Ala2-NHMe in various turn
conformations. The C? atoms are colored in magenta and cyan for residues
1 and 2, respectively. The hydrogens are not shown for clarity except
for the CRH of residue 1 and all amide hydrogens.
Communications to the Editor J. Am. Chem. Soc., Vol. 123, No. 50, 2001 12711