Macrocyclic Modalities Combining Peptide Epitopes and Natural Product Fragments

Article abstract of DOI:10.1021/jacs.0c00269

"Hot loop" protein segments have variable structure and conformation and contribute crucially to protein-protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and-restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.

Full text of DOI:10.1021/jacs.0c00269

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Article
Macrocyclic Modalities Combining Peptide
Epitopes and Natural Product Fragments
Stéphanie M. Guéret, Sasikala Thavam, Rodrigo J. Carbajo, Marco Potowski,
Niklas Larsson, Göran Dahl, anita dellsén, Tom N. Grossmann,
Alleyn T. Plowright, Eric Valeur, Malin Lemurell, and Herbert Waldmann
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c00269 • Publication Date (Web): 14 Feb 2020
Downloaded from pubs.acs.org on February 16, 2020
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Macrocyclic Modalities Combining Peptide Epitopes and
Natural Product Fragments
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Stéphanie M. Guéret *, Sasikala Thavam , Rodrigo J. Carbajo , Marco Potowski ,
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Niklas Larsson , Göran Dahl , Anita Dellsén , Tom N. Grossmann , Alleyn T.
2,†
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Plowright , Eric Valeur , Malin Lemurell , and Herbert Waldmann *
(
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AstraZeneca-Max Planck Institute Satellite Unit, Department of Chemical Biology, Dortmund,
Germany.
(2)
Medicinal Chemistry, Research and Early Development, Cardiovascular, Renal and Metabolism
(
CVRM), BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
(3)
Max-Planck-Institute of Molecular Physiology, Department of Chemical Biology, Dortmund, Germany.
Chemistry, Oncology R&D, AstraZeneca, Cambridge, United Kingdom.
(4)
(5)
(6)
Faculty of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany.
Discovery Biology, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg,
Sweden.
(7)
Structure, Biophysics & Fragment Based Lead Generation, Discovery Sciences, BioPharmaceuticals
R&D, AstraZeneca, Gothenburg, Sweden.
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Mechanistic Biology & Profiling, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca,
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(9)
VU University Amsterdam, Department of Chemistry and Pharmaceutical Sciences, Amsterdam, The
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Netherlands.
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_
__________________________________________________________________________
ABSTRACT: “Hot loop” protein segments have variable structure and conformation
and contribute crucially to protein-protein interactions. We describe a new hot loop
mimicking modality, termed PepNats, in which natural product (NP)-inspired structures
are incorporated as conformation-determining and -restricting structural elements into
macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of
inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were
synthesized on solid support employing macrocyclization by imine formation and
subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived
from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel
and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that
binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors.
NP-inspired fragment absolute configuration determines the conformation of the
peptide part responsible for binding. These results demonstrate that combination of
NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop
mimics with conformationally constrained and biologically relevant structure.
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___________________________________________________________________________
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.
INTRODUCTION
For small molecule modulation of protein-protein interactions (PPIs) mediated by
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extended binding surfaces, new approaches and chemical modalities are in high
demand.2 , 3 Recently, loop segments composed of peptide sequences displaying
diverse structures and with their termini positioned in spatial proximity (“ loops”) were
identified as frequently occurring protein structural motifs, mediating numerous PPIs
(“hot loops”).4 , 5
For inhibition of PPIs mediated via hot loops, macrocyclic peptides have been
increasingly explored in recent years,6 , 7 , 8 and, in particular, disulfide bridges9 , 10 , 11
,
aromatic thioethers12 and alkyne linkers13 were established to connect amino acid
side chains in peptides.14 Mixed macrocycles have been reported to decorate peptide
sequences with aziridines,15 iminoborane phenyl units, oxadiazoles, heteroaryl
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scaffolds17 , 18 and aromatic moieties.7 , 8 , 16 , 19 , 20 In addition, in individual cases
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hybrid macrocycles which incorporate sp -configured stereocenters inspired by natural
product (NP) structure have recently been reported with the cyclization in general
performed in solution after solid phase peptide synthesis (SPPS) of precursors.21 , 22
Notably, flexible modification of the peptide moiety in the rapamycin macrocycle led to
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potent, isoform-specific and FKBP-dependent inhibitors of the equilibrate nucleoside
transporter, an activity that differs from the original Rapamycin target, FK506-binding
protein.23 New methods that give rapid and versatile synthetic access to such
macrocycles would offer novel opportunities to the modulation and study of challenging
PPIs and expand the tool box of available hybrid macrocycle.
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Hot loops can adopt diverse conformations such that application of established
techniques and small molecule classes for PPI modulator design is complicated or
even impeded.24 Thus, macrocycles are in high demand, in which the peptide
conformation can efficiently be installed or adjusted through non-peptidic units which
themselves may primarily modulate but not directly mediate binding.
Macrocycles combining peptidic and chiral non-peptidic structural elements, such as
25
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polyketide (e. g. the chondramides/jasplakinolides ) or biaryl motifs
(e.g.
the
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biphenomycins and arylomycins ), potently modulate PPIs. In these hybrid NPs, the
stereogenic character of both the amino acids and the non-peptidic units determines
the overall conformation.29 , 30 , 31 For example, in the case of the chondramides, the
polyketide unit may point away from the binding surface of their target, actin.
Nevertheless, modifications in the stereochemistry of the polyketide region force the
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macrocycle in a conformational manifold that leads to differing binding.25 This finding
suggests that hot loop mimics with adjustable conformation could be developed by
combination of peptidic epitopes derived from relevant “ loops” with chiral non-
peptidic units linking their C- and N-termini. Structure and configuration of these
stereogenic NP-inspired elements should be efficiently adjustable through asymmetric
synthesis.
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Beyond macrocyclic natural product-inspired hybrids, PPI modulators are frequently
derived from chiral natural products (NPs),32 and cheminformatic analysis indicates
that the properties of these NPs are conserved in and represented by NP-inspired
fragments and scaffolds, including stereogenic character.33 Therefore, we envisioned
combining NP-inspired structures with peptide sequences from hot loops to yield
macrocyclic Peptide-Natural product-inspired hybrids (“PepNats”) as a novel modality
to modulate PPIs (Figure 1). For efficient access to PepNats, both peptide and
stereoselective NP synthesis should ideally be carried out on solid support. However,
complexity and diversity generating transformations for NP-inspired scaffold synthesis
on solid support have been explored only for selected structures.34 , 35 , 36 , 37 , 38
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Successful combination of both stereoselective NP-inspired scaffold synthesis and
peptide synthesis was only reported in few cases and in solution.21 , 39 , 40
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Figure 1. Strategy for the development of Peptide-Natural product-inspired hybrids (PepNats). The
structure of the protein-protein interaction and the “hot loop” epitope are derived from the crystal
structure with PDB 3EMW. The amino acids of the peptide sequence of interest are represented by red
balls and the natural product-inspired scaffolds are depicted in blue and green.
Herein, we report the design, synthesis and biological investigation of structurally
diverse macrocyclic PepNat collections mimicking hot loop epitopes. Peptide synthesis
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on solid support followed by macrocyclization via imine formation enabled subsequent
pyrrolidine ring formation by means of stereoselective 1,3-dipolar cycloaddition on
resin. Investigation of the PepNats for hot loop mimicry of inducible nitric oxide
synthase (iNOS) and of the human agouti-related protein (AGRP), yielded novel potent
ligands for the SPRY domain-containing SOCS (suppressor of cytokine signaling) box
protein 2 (SPSB2) and selective ligands and agonists for the melanocortin (MC)
receptors respectively.
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RESULTS AND DISCUSSION
Solid phase synthesis strategy
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To establish a flexible synthesis, we envisioned a stereoselective 1,3-dipolar
cycloaddition reaction of azomethine ylides on solid support as the key step. The
azomethine ylides would be generated in situ on resin by deprotonation of
appropriately functionalized cyclic peptide imines, obtained by macrocyclization of
linear peptides through Schiff base formation (Figure 1). This cycloaddition reaction
has previously been employed for the highly stereoselective solution-41 , 42 , 43 and
solid-phase44 , 45 , 46 synthesis of different NP-inspired scaffolds containing multiple
stereogenic centers. It provides efficient and flexible access to fused- and spiro-
pyrrolidine NP-inspired structures from a common azomethine ylide by variation of the
dipolarophile with simultaneous establishment of up to four stereocenters. Recently,
imine formation followed by reductive amination has been employed before for peptide
cyclization in solution.39
Initially, a test peptide sequence (ALFPGF) 2 was assembled on commercially
available Rink Amide low loading resin and equipped with a glycine and a N-methyl
2
phenylalanine (Scheme 1, aa = Phe) dipeptide at the N-terminus as well as a 4-
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methyltrityl (Mtt)-protected lysine at the C-terminus. After Mtt deprotection, an aromatic
aldehyde was installed at the lysine side chain to afford peptide 3. Following the
removal of the Fmoc protecting group, a one pot sequence was developed on resin,
which consists of intramolecular cyclization through Schiff base 4 formation using
trimethyl orthoformate as the dehydrating agent (for imine formation screening
conditions, see Supplementary Table S1), azomethine ylide generation and 1,3-dipolar
cycloaddition. In the presence of lithium bromide, different dipolarophiles 6, 7, 10, 11
(Figure 2) were quantitatively converted to the desired cycloadducts (Supplementary
Table S2, entries 811). After release from the resin, removal of side chain protecting
groups and purification by reverse phase chromatography, the major diastereomer of
the desired cycloadducts A1A4 was obtained in overall yields of 8 to 14 % from the
starting unfunctionalized Rink Amide resin, through a total of six steps after the SPPS
linear precursor synthesis on resin (Supplementary Table S3, entries 14).
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Scheme 1. Synthesis of macrocyclic PepNats using the imine/cycloaddition strategy on solid support.
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NHFmoc
i) DCM/TFA/H2O
95:2:3)
ii) HATU, DIPEA
4-carboxybenzaldehyde MeN
DMF, rt, 2.5 h
NHFmoc
i) 20% piperidine
in DMF
ii) Et3N
CH(OCH3)/DMF
(2:1), rt, 2.5 h
(
O
O
MeN
O
O
O
O
SPPS
aa2
aa2
NHMtt
NH
O
Fmoc
O
O
n
H
N
n
H
N
HN
HN
epitope
N
aa1
epitope
N
H
aa1
H
O
O
O
1
2
3
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R1
EWG
R3
R1
EWG
R3
R2
R2
O
O
O
Me
N
LiBr, Et3N
THF, rt
6 to 48 h
O
O
N
N
TFA/H2O/TIS
(95:2.5:2.5)
N
NH
H
H
1
NH
NH
aa2
NMe
NMe
O
O
aa2
aa2
n
H
R1
O
O
EWG
R3
n
H
N
n
H
N
N
O
aa1
N
epitope
N
O
aa1
O
aa1
N
epitope
N
N
epitope
N
H
H
R2
H
H
H
H
O
O
O
O
O
O
NH2
4
5
PepNat-major
(2R, 3S, 4R, 5S)
-1
1
= Rink Amide resin (loading = 0.16 to 0.36 g.mol ); SPPS = Solid Phase Peptide Synthesis; epitope
peptide sequence inspired from hot loop epitope (for complete list of peptide epitope see
=
Supplementary Table S3); n = 1 to 4 carbon linker length; aa = amino acid (for details see Figure 3, 4
1
2
3
and Supplementary Table S3 and S4); R , R , R and EWG (Electron Withdrawing Group) are a
schematic representation of the dipolarophiles (Figure 2).
MeO
F
OMe
OMe
MeO
OMe
O
Me
O
O
O
O
O
MeN
N
N
N
N
2
CO Me
O
O
O
NMe
O
O
O
O
N
N
N
MeO2C
H
H
H
O
6
7
8
9
10
11
12
13
14
Figure 2. Structure of dipolarophiles (614) used in the 1,3-dipolar cycloaddition on solid support.
The influence of resin loading on the different steps of the synthesis was investigated
using commercially available Rink Amide low loading resin (loading 0.26 to 0.36
-1
mmol.g ) (Figure 3a, Table entries 1, 3, 5, 7, 8, 9, 10 and 12). In addition, lower loaded
-1
starting Rink Amide resin (loading = 0.19 to 0.16 mmol.g ) was obtained by capping
the resin with acetylated glycine (Figure 3a, Table entries 2, 4, 6, 11). The resin loading
1
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and related conversions during the SPPS and the aldehyde coupling (Figure 3a) were
determined by treatment of the Fmoc-protected related resin with 20% piperidine in
DMF followed by quantification by UV-Vis spectroscopy of the dibenzofulvene-
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-1
-
piperidine adduct at 301 nm maximum absorbance wavelength and 8021 l.mol .com
1
molar absorption coefficient using Lambert-Beer’s law for calculation (for details see
Supplementary Information, Section 3a). Overall, the SPPS of the linear peptide
precursors proceeded with ca. 50 to 60% conversion. Lower loading resin allowed a
slightly better synthesis efficiency of the desired linear precursor by SPPS (Figure 3a,
compare entries 2 and 1 with entries 11 and 10 respectively). After Mtt cleavage, the
coupling of the aromatic aldehyde to the unprotected lysine residue of the various linear
precursors proceeded with viable conversions from 64 to 94%. According to the
experimental loading of the functionalized aldehyde peptide precursor on resin, the
yield of the imine cyclization followed by cycloaddition was determined for the isolated
major diastereomers after purification by reverse phase chromatography. The use of
low functionalized resin did not improve the isolated yield for the imine/cycloaddition
final step to access the PepNats. For the longer and less demanding peptide
sequences (GNMeFALFPGFG and GNMeFDINNNKG Figure 3a, entries 16), the
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cycloaddition using maleimide dipolarophiles, 6 and 8 proceeded with viable isolated
yields (ca. 20% yield), independent of the initial loading of the starting resin. Lower
isolated yields for the cycloaddition step were observed when hindered dipolarophile
14 (Figure 3a, entry 7) or hindered peptide sequences such as RffN and Rff (Figure
1
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1
1
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3a, entries 812) were used. In these cases, the use of lower loading resin did not
improve the isolated yield of the major diastereomer after imine formation and
cycloaddition. In accordance with these observations and to allow rapid access to
various PepNats and modifications, the commercially available Rink Amide low loading
resin was used without further modification. Yields are reported as overall isolated
yields for the major diastereomers after reverse phase chromatography from the
starting resin.
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†
Figure 3. Scope of the imine/cycloaddition on solid support. Depicted as single capital letter code; D-
‡
amino acids are indicated by lower case letter. Depicted as major:minor isomers unless more than two
diastereomers were obtained; d.r. determined from the crude product by integration of the analytical- or
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optimized-RP-HPLC-MS profile at 210 nm (for details see the Supplementary Information). § Overall
isolated yield after preparative-RP-HPLC calculated from the loading of the starting unfunctionalized
#
rink amide resin. Purity of isolated PepNats determined by integration of the product peak of the HPLC
1
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0
profile (210 nm). conv. conversion was determined by integration of the product and starting material
(
SM) peaks using the analytical RP-HPLC profile at 210 nm. * reverse diastereoselectivity observed (for
details see Supporting Information). (a) Impact of the starting resin loading for each step of the strategy
SPPS, Mtt cleavage/aldehyde coupling and imine cyclization/cycloaddition reaction). The loading was
(
quantified by the UV absorbance of the piperidine-dibenzofulvene adduct after Fmoc deprotection of the
functionalized resin (for details see the Supplementary Information). (b) Ring size accessibility by
variation of the epitope length and carbon linker of the lysine unit. (c) Scope of the dipolarophiles (for
structures see Figure 2) used for the cycloaddition on resin. (d) Variation of N-methylated amino-acid 
to the natural product-inspired unit.
For the DINNN epitope peptide sequence, the synthesis proved robust and of wide
scope (Figure 3bd). The length of the amino acid sequence flanking the DINNN
epitope was successfully varied from nine to three amino acids (Figure 3b, top table).
Further structural variation was achieved by shortening the side chain of the amino
acid employed for aldehyde attachment to afford PepNats D1, D8D10 with viable
diastereoselectivity (Figure 3b, bottom table). Seven different PepNats bearing the
unique DINNN peptide sequence epitope were obtained by variation of the
dipolarophile used in the cycloaddition (Figure 3c). Moreover, the impact of an N-
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2
methylated amino acid (aa ) was investigated (Figure 3d). The stereoselectivity of the
cycloaddition increased with the size of the N-methylated amino acid next to the glycine
0
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employed for imine formation (e.g. compare D1 with D11) and N-alkylation was
2
beneficial (compare D1 with D14 and D16). A wide range of aa was tolerated including
an N-methylated -branched amino acid (PepNat D13).
Variation of amino acid structure (polar, hydrophobic and -branched) and peptide
epitope sequences (two to nine amino acids) as well as the dipolarophile (614, Figure
2
) yielded a diverse collection of 63 macrocyclic PepNats typically in multimilligram
amounts and with excellent purity (Figure 4, Supplementary Table S3 and S4). In total
5
1
6 different peptide loop epitope sequences mainly derived from PPIs were obtained
by the solid-support synthesis methodology to yield a wide range of ring sizes (3 to 12
amino acids).
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Figure 4. Top: Representation of Natural Product (NP)-inspired scaffolds contained in the PepNats.
Bottom: Illustrative examples of PepNats (major diastereomer) obtained through the imine/cycloaddition
synthesis on solid support. Selected examples (for a complete list see Supplementary Tables S3 and
*
†
S4). Depicted as single capital letter code with D-amino acids indicated by lower case letter. Depicted
as major:minor isomers unless more than two diastereomers were obtained; d.r. determined from the
crude product by integration of the analytical- or optimized-RP-HPLC-MS profile at 210 nm (for details
see Supplementary Information). ‡ Overall isolated yield after preparative-RP-HPLC from the starting
§
unfunctionalized resin. Purity of isolated PepNats determined by integration of the product peak of the
HPLC profile (210 nm). (a) Selected examples of NPs bearing succinimides and fused succinimide-
pyrrolidines. These NPs inspired the structure of the fused di-pyrrolidine PepNats obtained through the
cycloaddition in the presence of maleimide dipolarophiles 69 (for structures see Figure 2). (b) Selected
examples of pyrrolidine NPs which inspired the synthesis of the pyrrolidine-peptide macrocyclic
PepNats. (c) Selected examples of spirooxindole containing NPs and related representative structure
of the 3,3’-pyrrolidinyl-spirooxindole inspired PepNats obtained through the cycloaddition in the
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presence of arylidene oxindoles 1113 (for structures see Figure 2). (d) Representative structure of the
pyrrolidinyl-spirobarbiturate containing PepNat and selected example of spirobarbiturate derivatives.
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Using a variety of peptide epitopes inspired by hot loops, the NP-inspired unit of the
PepNats was readily changed by variation of the dipolarophiles used in the 1,3-dipolar
cycloaddition after intramolecular imine formation on resin (Figure 3c and 4). The
maleimide dipolarophiles 69 afforded fused di-pyrrolidine-peptide macrocycles (e.g.
A12, C1, H1, I1, L1, K1, P1, Figure 4a) which embody the heterocyclic scaffold
characteristic of NPs bearing succinimides47 , 48 and fused succinimide-pyrrolidine
analogues49 (Figure 4a), whereas use of dimethyl maleate 10 led to incorporation of
pyrrolidines (e.g. A3, D4, Figure 4b) into the hybrid macrocycle, reminiscent of the
underlying scaffolds of pyrrolidine alkaloids.50 The
arylidene
oxindoles
1113
delivered PepNats (e.g. C2, C3, D6, Figure 4c) combining a macrocyclic peptide
structure and a 3,3’-pyrrolidinyl-spirooxindole scaffold, inspired by the naturally
occurring spirooxindole alkaloids and pyrrolidine fused spirooxindole.51 , 52 , 53
Barbiturate-derived alkene dipolarophile 14 introduced additional diversity through
incorporation of the pyrrolidinyl-spirobarbiturate scaffold (Figure 4d)54 , 55 , 56 in
the
PepNat (e.g. D7, Figure 4d).
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With LiBr as catalyst, the 1,3-dipolar cycloaddition in the presence of N-substituted
maleimides typically yielded two endo cycloadducts in stereoisomer ratios of 51:49 to
1
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6:4, which could not be increased by using chiral Cu- or Ag-Fesulphos catalysts,57
indicating that the transformation is substrate-controlled. For arylidene-oxindoles, four
diastereomers were formed, probably due to E/Z-isomerization of the dipolarophiles or
ring opening/closure of the cycloadduct.58
For assignment of absolute configuration, enantiopure (2S, 3R, 4S, 5R)-configured
cycloadducts derived from N-Me- and N-Ph-maleimide dipolarophiles were
synthesized independently in solution, employing the chiral Cu-R-Fesulphos catalyst
complex and converted to diastereomerically pure PepNats H1-minor, K1-minor, D9-
minor and D10-minor, embodying GLGF-, RffN- and DINNN-epitope sequences
respectively (for synthesis of H1-minor and K1-minor see Supplementary Figure S1;
for choice of the epitope sequences, see below). Comparison of NMR spectra and
HPLC retention times revealed that in all cases, the (2R, 3S, 4R, 5S) diastereomer was
formed in excess (Supplementary Figures S2 and S3). Therefore, the 1,3-dipolar
cycloaddition on resin, by analogy to the solution reaction, proceeds via an endo-
transition state, steered here by the peptide moiety. Macrocyclic peptides and
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peptidomimetics adopt in general relatively rigid conformations,24 , 59 , 60 which
here
would lead to formation of E-configured -shaped azomethine ylides embedded in the
macrocycles. The dipolarophiles would then approach from the less hindered si face
of the dipole (Supplementary Figure S4). The configurations of the pyrrolidinyl
spirooxindole and spirobarbiturate cycloadducts were assigned by analogy.
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Conformational analysis
Analysis of the NMR spectra of the major (2R, 3S, 4R, 5S)-diastereomer of PepNats
H1-major and K1-major (Supplementary Information, Section 5) revealed that these
macrocyclic peptide hybrids adopt very different conformations in solution. Introduction
of a stereogenic NP-inspired fragment appears to lock the cyclic loop mimics into
relatively stable conformations, which should favor defined molecular interactions with
target proteins.
To determine the structure of the conformers present in solution, the conformational
space for each peptide was thoroughly explored using the Maestro Macrocycle
Sampling algorithm (OPLS3 force field; version 11.6.013, Schrödinger) with an energy
threshold of 25 Kcal/mol to allow for a full exploration of the rotation around the peptidic
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bonds. First, the resulting conformers were filtered to a reduced set of conformations
that matched key long-range nOes in the macrocycle. To avoid being too restrictive
and missing possible conformers complying with the NMR data, the filter for those
distances was set to an upper limit of 5.5 Å. Each of the conformers from the reduced
set was subjected to solvent explicit 10 ns MD simulations that were subsequently
clustered by RMSD. The most populated cluster for each conformer was taken as the
conformation which the molecule adopted most time in the dynamics run.61 The
average structure for each of the most populated clusters was extracted and a new
reduced set of conformations comprising each of the averaged structures was used
together with the experimental NMR restraints (nOes and J couplings) to find the best
fit via MSpin’s least squares algorithm (MSpin nOe Fitter algorithm, version 2.4.0-713;
MestReLab Research). Intramolecular H-bond information from sample exchange in
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CD OD was used for further refinement and introduced as distance restraints. The
3
clusters and the corresponding averaged structures for macrocycles H1-major and H1-
minor that showed the best agreement with the experimental NMR data are shown in
Figure 5. In addition, to the nOes and the intramolecular H-bonds, an extra indication
that the macrocycles are structured derives from the 3JNH-H values found for the backbone
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amides. Four of the amides in H1-major and three in H1-minor out of a total of six show
couplings deviating from the 7.5 Hz mean, the value normally interpreted as arising from free
mobility (see Supporting Information).62
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Notably, key NMR information shows major differences in the conformational space
explored by H1-major and H1-minor. For example, the fused di-pyrrolidine N-methyl
group in the major PepNat H1-major isomer embodying the GLGF sequence displayed
long-range nOe interactions with the peptide backbone (Supplementary Figure S5),
whereas only minor interactions in the form of long-range nOes with the leucine side
chain were detected for the minor isomer H1-minor (Supplementary Figure S6). In the
conformation of the major isomer, the N-methyl group of the NP points towards the
macrocycle peptidic core and comes close to the glycine and phenylalanine in the
transannular position (Figure 5, left). In the minor diastereomer, the N-methyl group
does not face the peptide backbone but is instead in proximity to the leucine (Figure 5,
right).
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Figure 5. NMR derived best-fit cluster and average structure for the fused di-pyrrolidine PepNat H1
major and minor diastereomers. Arrows indicate key long-range NMR nOe interactions with the N-methyl
group of the fused di-pyrrolidine unit. Additional long range nOe interactions were observed between
other regions of the peptides but are not shown for clarity. Dashed blue lines indicate intramolecular
hydrogen bonds observed in the NMR data between the peptide backbone and C=O groups of the fused
di-pyrrolidine unit.
NMR analysis of the PepNat K1-minor obtained through on resin cycloaddition
between the N-phenyl maleimide and the Schiff base incorporating the RffN epitope
revealed long range nOe cross peaks between the N-phenyl substituent of the fused
di-pyrrolidine unit and the Rff motif. In the case of K1-minor, the increased number of
aromatic rings in the molecule led to a large overlap of the NMR signals and an excess
of ambiguous nOe restraints that precluded a full conformational analysis of the
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structure (Supplementary Figure S7 and Supplementary Information, Section 6).
Similarly, the highly complex NMR spectra of K1-major prevented a full assignment of
the structure.
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These results indicate that the PepNat hot loop mimic collection is not only structurally
and stereochemically diverse, but also conformationally diverse. The different defined
solution conformations should enable tunable interactions with target proteins. Indeed,
the major and minor isomers displayed very different bioactivity (see below).
DINNN-PepNats bind to the SPSB2 protein
Hot loop mimicry was investigated using the iNOSSPSB2 protein-protein interaction
as a representative example. iNOS produces nitric oxide and plays key roles in the
63
immune system and defense against infections. SPSB2 is the adaptor protein in the
E3 ubiquitin ligase complex that ubiquitinates iNOS, targets it for proteasomal
degradation and therefore modulates its lifespan. The conserved DINNN type II -turn
loop motif of the N-terminal region of iNOS is the key binding epitope for the SPSB2
protein,63 , 64 , 65 and has been classified as a “hot loop”. Disulfide-, lactam-bridged
and simple aromatic non peptidic scaffolds have been employed to close the DINNN
epitope and led to cyclic peptides and peptidomimetics that confirm the importance of
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this loop for the iNOSSPSB2 interaction.7 , 11 , 66 Flexible incorporation of adjustable
NP-inspired scaffolds to close the DINNN motif has not yet been reported and would
readily give access to multiple single point modifications in both the peptidic unit and
the NP-inspired moiety to yield a structurally diverse library based on the DINNN
epitope for further study of the key interactions that modulate the iNOSSPSB2
interaction.
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Investigation of 17 PepNats, bearing the DINNN epitope for binding to human SPSB2,
revealed binding affinities from the low nanomolar to the micromolar range, as
measured by Surface Plasmon Resonance (SPR) (Table 1, entries 117).
In the N-methyl substituted fused di-pyrrolidine series (D1, D8D10, D12, D14),
variation of the N-methylated amino-acid next to the NP-inspired fragment and the
amino acid side chain employed for aldehyde introduction clearly influences target
affinity. PepNats D10-major (one carbon linker), D9-major (two carbon linker) and D1-
major (four carbons) displayed comparable K values of 72 nM, 66 nM and 33 nM
D
respectively (Table 1, entries 1, 2 and 4), whereas D8-major (three carbon linker)
bound with nine-fold lower affinity than D1-major (Table 1, entries 3 and 4). For the N-
methylated phenylalanine containing peptide sequence, the replacement of the methyl
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substituent in the fused di-pyrrolidine NP-inspired fragment by a phenyl group resulted
in seven-fold lower affinity with a K of 231 nM (PepNat D2-major; Table 1, entry 8).
D
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Variation of the N-methylated amino-acid next to the NP-inspired moiety from N-Me
phenylalanine to N-Me leucine or N-Me valine was beneficial for the affinity (e.g.
compare D1-major with D12-major or D13-major, and D2-major with D18-major; Table
1, entries 4, 6, 11 and 8, 14 respectively), and afforded cyclic PepNat SPSB2 binders
53
with potency comparable to the 13-mer truncated N-terminal iNOS linear peptide 22
(Table 1, entry 18) and with potency better than the linear DINNN epitope 23 (Table 1,
entry 16). Both linear reference peptides were synthesized and tested in the SPR
assay for comparison. The fused di-pyrrolidine-peptide macrocycle, D18-major,
showed 14- and 28-fold improved binding affinity compared to the disulfide bridged
9
analogue 24, or the related cyclic peptide 25, which were synthesized independently
as reference compounds and assessed in the SPR assay (Table 1, compare entry 13
with entries 20 and 21). To further demonstrate the ability of the NP-inspired unit to
fine tune the conformation of the peptide epitope and improve the binding affinity to the
hSPSB2 protein, the simple phenylmethanamine linker containing macrocycle 27 was
synthesized. The synthesis employed the same precursor and imine cyclization as
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described for the on resin imine/cycloaddition synthetic methodology described above,
but followed by reductive amination instead of cycloaddition. The non-constrained
macrocycle 27 bound hSPSB2 with 87 nM in affinity. For comparison the constrained
1
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3
PepNat D18-major which embodies sp stereocenters in the NP-inspired unit binds
hSPSB2 with 2 nM affinity (44-fold better affinity than 27).
PepNats D4-major and D7-major embodying the four carbon linker and N-Me
phenylalanine in combination with a pyrrolidine and a pyrrolidinyl-spirobarbiturate NP-
inspired scaffolds respectively, bound with low nanomolar affinity (Table 1, entries 16
and 15). The pyrrolidinyl-spirobarbiturate NP containing PepNat D7-major showed 3.8
nM affinity for the hSPSB2 and resulted in 4-fold and 46-fold higher affinity compared
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to the disulfide bridged peptide 24 or the ortho ether aromatic unit analogue 26
respectively (compare Table 1, entry 12 with Table 1, entries 17, 19).
Table 1. Binding affinities of the cyclic DINNN-PepNats for the hSPSB2 protein determined by SPR
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entry compound
structure*
K
D
(nM)†
1
2
3
4
5
6
7
8
9
1
1
1
D10-major
D9-major
D8-major
D1-major
D15-major
D12-major
D12-minor
D2-major
D3-major
D17-major
D13-major
D13-minor
D18-major
D18-minor
D7-major
D4-major
D4-minor
2253
[ I-FDINNNDap ]G
[ I-FDINNNDab ]G
[ I-FDINNNOrn ]G
[ I-FDINNNK ]G
[ I-fDINNNK ]G
[ I-LDINNNK ]G
[ I-LDINNNK ]G
[ II-FDINNNK ]G
[ III-FDINNNK ]G
[ II-FDINNNDap ]G
[ I-VDINNNK ]G
[ I-VDINNNK ]G
[ II-VDINNNK ]G
[ II-VDINNNK ]G
[ VII-FDINNNK ]G
[ IV-FDINNNK ]G
[ IV-FDINNNK ]G
Ac-KEEKDINNNVKKT
Ac-DINNN
72  16
66  8
282  93
33  15
> 10000
10  4
0
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0
127  9
231  43
309  39
1080  76
15  2
0
1
2
78  19
2.2  0.3
139  76
3.8  0.5
18  7
13
14
15
16
17
> 10000
7.1  1.5
237  34
16  4
18
19
239
20
249
[ CVDINNNC ]
21
22
23
2566
2614
[ WDINNNA ]
79  10
175  6
87  2
[ VIII-DINNN ]
27
[ IX-FDINNNK ]
*
[
] indicates cyclic structure; Dap = Diaminopropanoic acid; Dab =
Diaminobutanoic acid; Orn = Ornithine; Lower case letters indicate D-
†
amino acids; bold residues are N-methylated.
D
K values are
presented in nanomolar (nM) concentration as mean ± Standard
Error of the Mean (SEM) of three independent experiments using
Surface Plasmon Resonance (SPR) (for selected sensorgrams see
Supplementary Figure S8).
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Notably, the stereochemistry of the NP-inspired fragment seems to induce different
conformational constraints on the loop epitope which translates into very different
binding potency between the major vs. minor isomer. Thus, for PepNat D4, the major
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(2R, 3S, 4R, 5S) diastereomer showed nanomolar affinity for SPSB2 (K = 18 nM),
D
while the minor (2S, 3R, 4S, 5R) diastereomer did not bind (Table 1, entries 16 and
17). This observation was also made for the N-methyl fused di-pyrrolidine PepNat D13
and the N-phenyl fused di-pyrrolidine-DINNN macrocycle D18 with a 5-fold and 63-fold
loss of binding for the minor diastereomer respectively (Table 1, entries 11, 13 and 12,
14).
Further, it should be noted that linkers not incorporating stereogenic centers such as
disulfide bridged compound 24, ortho ether aromatic unit VI in peptidomimetic 26 and
phenylmethanamine VII in cyclic peptidomimetic 27, require separate and lengthy
syntheses (for synthesis details, see Supplementary Information Section 3, E). These
syntheses only afforded a single DINNN analogue that did not have improved affinity
for the protein of interest, hSPSB2. Further, these corresponding macrocycles did not
show increased potency compared to the full length linear precursor or other previously
reported cyclic DINNN containing peptides. In contrast, the flexible and rapid synthesis
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