Journal of the American Chemical Society
Article
higher intrinsic affinity to 14-3-3 but being less compatible
with the size of Trp+1 for optimal stabilization (Figure 3I).41
To better understand how structural changes in 13, 23, 27,
and 28 translated to different cooperativity, the compounds
were soaked into 14-3-3/Pin1 crystals. Analysis of the crystal
structures shows conformational changes at the composite
interface that potentially drive cooperative behavior. The 14-3-
3/Pin1/28 complex showed a conformational change in Asn42
side chain of 14-3-3 induced by the presence of the 2,4-
difluorophenyl ring of 28. Specifically, this induces a
conformational change in Asn42 of 14-3-3 facilitating a direct
hydrogen bond with Gln+3 of Pin1 (Figure 3H). Notably, this
interaction is absent in the crystal structures of 13 and 27
(Figure 3C, Figure S6). Additionally, we observed that the 4-
fluoro occupies a deep pocket formed by Cys38, Arg41, and
Phe119, thereby locking the orientation of the 2,4-difluor-
ophenyl ring. It was also observed that the indole side chain of
Trp+1 has an inverted conformation compared to 13 and 27
(Figure 3C,H, Figure S6). Notably, the 14-3-3/Pin1/23
complex shows two conformations for Trp+1, suggesting that
the side chain is not in the lowest energy state (Figure S5).
Furthermore, the alternative Trp+1 conformation induced by
28 allows the formation of water-mediated hydrogen bonds
between the indole moiety of Trp+1 and Gln+3 of Pin1 and
Asn42 and Ser45 of 14-3-3 (Figure 3H). These additional
contacts at the interface of the complex potentially explain the
improved cooperative behavior. We further hypothesize that
these Pin1 specific interactions will result in high selectivity of
these fragments toward the Pin1/14-3-3 complex.
Selectivity Screening of Covalent Fragments. Drug-
ging the hub protein 14-3-3 raises the challenge of selectivity.
We hypothesized that the high level of cooperative behavior
for the 14-3-3/Pin1_72/28 complex is a function of the unique
functionality and topology of the interface, specifically the +1
and +3 amino acid of Pin1_72 with the covalent fragment
(Figure 3H). We further rationalized that this cooperativity
would likely translate to high selectivity. To test this, fragments
13, 27, and 28 were screened against a panel of 13 peptides as
diverse representatives of 14-3-3 client proteins, differing in
size and hydrophobicity of the +1 amino acid (Figure 4A).
First, 14-3-3 interaction partners with polar amino acids in the
+1 position were investigated. C-Raf has a threonine in the +1
position, whereby the hydroxyl group sufficiently abolishes any
stabilizing effect of 13, 27, and 28. Glutamic acid, glutamine,
cysteine, or serine, in this position, as offered by the B-
Raf_729, TBC1D237, ERRγ_179, and Mypt1_ 472 peptides,
showed no significant stabilization with 13, 27, and 28. A polar
amino acid in the +1 position is not compatible with these
imine forming fragments. This is likely due to the direct
hydrogen bond possible between Lys122 of 14-3-3 and the
polar side chain of the +1 amino acid, coupled with the
repulsive behavior of a polar amino acid perpendicular to the
aromatic ring of benzaldehyde. Similar to polar +1 amino acids,
a C-terminal phosphorylation motif, as prototypical for ERα,
was also not responsive to fragment stabilization with 13, 27,
and 28. Again, salt bridge formation between Lys122 and the
carboxylic acid terminus of ERα is the most logical rationale.44
This is in contrast to the natural compound FCA which elicits
a 160-fold stabilization of the 14-3-3/ERα complex.
AS160/14-3-3γ complex was observed with any of the
fragments (13, 27, and 28), with SFs ranging from 1.2 to
2.7. The crystal structure of AS160 shows that the phenyl side
chain employs similar hydrophobic contacts with the roof of
14-3-3 as Trp+1 of Pin1_72 (Figure 4C). Unlike Pin1_72, the
C-terminus of AS160 engages Phe+1 in intramolecular
hydrophobic contacts with Pro+4 and Pro+5. The +1
phenylalanine likely cannot rearrange to allow fragment
binding. The Raptor/14-3-3γ binary complex proved to be
more responsive to fragment stabilization with 28 showing a
9.5-fold stabilization of the binary complex. No structural data
are available for the Raptor/14-3-3 interface.
The aldimine formation with Lys122 was first identified for
the p65/14-3-3 interaction, with p65, which contains an
isoleucine at the +1 position.34 Hence, small hydrophobic
residues could potentially form hydrophobic contacts with the
benzaldehyde scaffold. This was investigated by comparing the
effect of 13, 27, and 28 on 14-3-3 interaction partners with a
leucine (Abl1pT735), isoleucine (p65pS45), or valine
(CFTRpS753) at the +1 position. Fragments 13 and 28
elicited some stabilizing activity for all three interaction
partners with SF values ranging from 4.7 for 13 with p65 to
12.5 for 27 with CFTR. Fragment 27 induced no significant
complex stabilization. The B-Raf_365 peptide with an alanine
in the +1 position was not responsive to complex stabilization
by any of the imine-forming aldehydes. This is likely a result of
the topology formed by the C-terminus of B-Raf_365, which
creates a smaller binding pocket occluding the fragments.29
This demonstrates not only the functionality of the C-terminus
of the partner protein is important, but the topology of the
binding pocket formed by the interaction partner also dictates
binding (Figure 4D).
Remarkably, none of the fragments showed any significant
inhibiting effects on binary complex formation, indicating a
very low intrinsic affinity of the aldehyde fragments toward 14-
3-3 alone. This is exemplified by peptides B-Raf_365 (Ala+1),
ERRγ (Cys+1) and C-terminally truncated ERα (Val+1-
COOH) where the addition of 100 μM fragment resulted in
no change in KD value of the binary complex formation (Figure
S7A). We further investigated whether these fragments elicited
competitive behavior against non-stabilized interactions in a
dose-dependent manner, by titrating the fragment to a fixed
concentration of 14-3-3 and partner peptide (Figure S7B). B-
Raf_365, which has a hydrophobic residue in the +1 position
and is not stabilized by the fragments, showed no inhibition of
14-3-3/partner peptide complex formation at concentration of
≤1.5 mM of 27 or 28 (Figure S7C). Titration of 27 or 28 to a
complex of 14-3-3/ERRγ or 14-3-3/ERα, which forms
hydrogen bonds with Lys122, showed no competitive behavior
at concentration of ≤750 μM. Notably, 28 and 27 showed
moderate to low inhibition of ERα at a high micromolar
concentration (750−1500 μM), respectively. In regard to
ERRγ, inhibition of peptide binding was only observed at a
concentration of 1.5 mM for 28. This suggests that the
aldehyde fragments have a low intrinsic affinity toward 14-3-3
alone. This leads to a desirable, noncompetitive binding mode.
Soakings of 13 and 23 into p65/14-3-3σΔC complexes
provided an explanation of selectivity. The ternary complex
with p65/14-3-3σΔC/13 showed a distinct binding pose to the
fragments in comparison with Pin1_72/14-3-3σΔC. Specifi-
cally, the 2-phenyl ring of 13 and 23 points toward Ile+1 of
p65_45 (Figure 4E, Table S4). In this orientation, the Ile+1
makes hydrophobic contacts with both benzene rings of 13
Following the importance of the tryptophan for complex
stabilization of Pin1_72/14-3-3γ with the benzaldehydes, the
influence of phenylalanine (AS160) and tyrosine (Raptor) was
investigated (Figure 4A,B). No appreciable stabilization of the
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J. Am. Chem. Soc. 2021, 143, 8454−8464