Designer macrocyclic organo-peptide hybrids inhibit the interaction between p53 and HDM2/X by accommodating a functional α-helix

Article abstract of DOI:10.1039/c4cc01199f

We report the design of side-chain-to-tail linked organo-peptide hybrids incorporating an α-helical protein-binding motif. Using this strategy, macrocyclic inhibitors of the p53:HDM2 interaction displaying dual specificity against the HDMX homolog as well

Full text of DOI:10.1039/c4cc01199f

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Designer macrocyclic organo-peptide hybrids
inhibit the interaction between p53 and HDM2/X
by accommodating a functional a-helix†
Cite this: Chem. Commun., 2014,
50, 5027
Received 14th February 2014,
Accepted 18th March 2014
Jessica M. Smith, John R. Frost and Rudi Fasan*
DOI: 10.1039/c4cc01199f
www.rsc.org/chemcomm
We report the design of side-chain-to-tail linked organo-peptide
hybrids incorporating an a-helical protein-binding motif. Using this
strategy, macrocyclic inhibitors of the p53:HDM2 interaction dis-
playing dual specificity against the HDMX homolog as well as
increased proteolytic stability could be obtained.
The development of agents for selective modulation of protein–
protein interactions (PPIs) constitutes a prominent goal in drug
discovery and chemical biology.1 Since PPIs are often mediated
by well defined secondary structural elements, a promising strategy
in this area has involved the stabilization or mimicry of these motifs
via compact molecular scaffolds.2 Reflecting their abundance in
protein structures, a-helices are often encountered at the interface of
protein–protein complexes.3 Accordingly, a number of strategies
have been developed for stabilization of a-helical peptides,4
which include the use of hydrogen bond surrogates5 as well as
of a variety of inter-side-chain linkages such as disulfide,6
lactam,7 thioether8 or triazole9 bridges, ‘hydrocarbon staples’,10
and cysteine cross-linking moieties.11
We recently reported strategies for the synthesis of macrocyclic
organo-peptide hybrids (MOrPHs) via the chemo- and regioselective
ligation of bifunctional synthetic precursors to genetically encoded
precursor polypeptides (e.g. Fig. 1A).12 A key feature of this new class
of peptide-based macrocycles is their modular architecture, as given
by the diverse non-peptidic and peptidic moieties amenable to
Fig. 1 (A) MOrPH macrocyclization strategy and chemical structure of
synthetic precursors (box) investigated in this study. (B) Crystal structure of
HDM2:PMI complex (pdb 3EQS) and model of representative example
of designer a-helical MOrPH (i/i + 10 peptide cyclization with SP8).
HDM2/X are implicated in the negative regulation of p53
incorporation into these scaffolds.12a,c As part of ongoing studies activity and overexpression of these proteins has been linked to
directed at evaluating MOrPHs as disruptors of biomedically relevant several malignancies.13 While dual inhibition of HDM2/X has
PPIs, we were interested in assessing the potential of these macro- emerged as a most promising strategy for anticancer therapy,14
cyclic scaffolds to accommodate, and possibly, stabilize a functional small-molecule inhibitors of HDM2 typically fail to potently inter-
a-helical motif. In this work, we describe the successful implementa- fere with p53:HDMX interaction due to subtle differences in the
tion of this idea through the design and development of a-helical p53 binding clefts of these protein homologs.15 These limitations
MOrPHs that can effectively disrupt the interaction between the make the development of dual HDM2/X inhibitors a topic of
tumor suppressor p53 and the oncoproteins HDM2 and HDMX.
current interest.10c,16 HDM2 and HMDX bind to the N-terminal
transactivation domain of p53 (p5315–29), which upon complex
formation adopts a well defined a-helix.17 Thus, in addition
to its biomedical relevance, these structural features have made
the p53:HDM2 interaction an ideal test bed to probe strategies
for a-helix stabilization and mimicry.10c,11b,18
Department of Chemistry, University of Rochester, Rochester, NY, USA.
E-mail: fasan@chem.rochester.edu; Tel: +1-585-2733504
† Electronic supplementary information (ESI) available: Experimental and syn-
thetic procedures, additional inhibition curves. See DOI: 10.1039/c4cc01199f
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Table 1 Sequence and inhibitory activity of peptides
The starting point for the design of our MOrPH-based HDM2/
X-targeting inhibitors was a linear 12-mer peptide isolated via phage
display by Pazgier et al. (PMI: T1SFAEYWNLLSP12).19 PMI carries the
triad of cofacial i/i + 4/i + 7 amino acid residues known to be critical
for p53 interaction with HDM2/X17 (i.e. Phe3, Trp7, and Leu10
corresponding to Phe19, Trp23, and Leu26 in p53, respectively), but
inhibits these proteins with greater potency than a p53-derived
peptide (IC50: 30–40 nM vs. 200–300 nM, respectively).19 Upon
inspection of the PMI–HDM2 complex structure (Fig. 1B),19 two
solvent exposed residues, namely Thr1 and Glu5, were identified
as two equally viable side-chain attachment points for MOrPH
formation via substitution with p-acetyl-phenylalanine (pAcF) accord-
ing to our oxime/AMA-mediated cyclization method (Fig. 1A).12c
The C-terminal attachment site was chosen to lie after Ser11
(changed to Ala to promote a-helix formation), as Pro12 did not
appear to establish significant contacts with the HDM2 surface.19
Analysis of models of the resulting peptide sequences, namely PMI-2
(GTSFA(pAcF)YWNLLA) and PMI-3 (G(pAcF)SFAEYWNLLA), revealed
Name
Sequence
HDM2 IC50 (nM) HDMX IC50 (nM)
1 (p5315–29
)
920 Æ 65
1510 Æ 95
870 Æ 53
1200 Æ 110
7500 Æ 250
4100 Æ 190
2
3
4
5
1500 Æ 115
3500 Æ 95
10 000 Æ 400
ND
6
7
65 Æ 9
475 Æ 37
355 Æ 31
910 Æ 105
8
9
110 Æ 15
450 000
340 Æ 44
450 000
that the distances between pAcF side-chain keto group and the data supported the ability of designer MOrPHs 3 and 4 to accom-
C-terminal carbonyl group were about 13 and 16 Å, respectively. modate the target a-helical motif, a conclusion supported also by the
These distances matched the spacer distance (B13 Å) furnished by poor activity of 5. The latter indeed highlighted the deleterious effect
one of our previously described synthetic linkers, called SP612c of a mismatch between the length of the synthetic linker and the
(Fig. 1A), based on an energy-minimized model of the compound target side-chainÁÁÁC-terminus bridging distance as anticipated. To
(Fig. S1, ESI†). SP6 was thus selected as a first candidate for our disappointment, however, both SP6- and SP8-based macrocycles
macrocyclization of the target peptide sequences PMI-2 and PMI-3. were weaker inhibitors of HDM2/X compared to the wild-type p53
To examine the influence of the non-peptidic linker structure on sequence (Table 1). This result can be rationalized based on the
the functional properties of the resulting MOrPHs, a second negative effect of replacing Glu5 with pAcF as required for macro-
linker reagent, SP8 (Fig. 1A), was prepared (see ESI† for details). cyclization. Indeed, in the crystal structure of the HDM2:PMI
SP8 satisfies the aforementioned distance requirements (Fig. S1, complex, Glu5 is found to form a hydrogen bond network with
ESI†), but has higher flexibility compared to SP6 due to replace- the neighboring Ser2 (Fig. S4, ESI†), which is likely to contribute
ment of the triazole unit with an alkyl chain.
to a-helix stabilization.16 This conclusion is supported also by
According to these design principles, macrocycles 3–5 and the much higher inhibitory activity of the linear peptide 6 (vs. 2),
7–9 were prepared via cyclization of PMI-2 or PMI-3 target in which the Ser2–Glu5 pair is preserved (Table 1).
sequences with SP6 or SP8 (see ESI† for details). 7–9 thus
To our delight, the i/i + 10 macrocycles 7 and 8 exhibited
feature an i/i + 10 side-chain-to-backbone connectivity, whereas in significantly improved ability to disrupt p53 interaction with
3–5 the non-peptidic moiety bridges the i and i + 6 residue. As HMD2/X as compared to 3 and 4 (Fig. 2A and Fig. S3, ESI†).
controls, the same two peptide sequences were cyclized in the A notable effect of the type of synthetic linker on the binding
presence of the shorter reagent SP4 (Fig. 1A). Since the spacing properties of the corresponding MOrPH was also apparent.
distance provided by SP4 (B8 Å, Fig. S1, ESI†) represents a Notably, the SP4-containing 9 was found to possess negligible
mismatch with the target ones (13–16 Å), the resulting macrocycles inhibitory activity against HDM2 or HDMX (IC50 4 50 mM),
(5 and 9) were intended to serve as negative control designs.
confirming that cyclization via the ‘mismatching’ SP4 strongly
The ability of the designed macrocycles to disrupt the disfavored adoption of the bioactive a-helical conformation by
p53:HDM2/X interaction was assessed using a surface plasmon the embedded PMI-3 peptide sequence. In stark contrast, much higher
resonance (SPR) inhibition assay (Fig. S2, ESI†). Herein, bio- inhibitory activity was observed in the presence of the ‘distance-
tinylated p53(15–29) was immobilized on a streptavidin-coated bio- matching’ SP6, leading to a compound with sub-micromolar
sensor chip and increasing concentrations of inhibitors were added IC50 values for both protein homologues (Table 1). Interest-
to a fixed concentration of HDM2 or HDMX. Using this assay, half- ingly, the simple replacement of the triazole unit in 7 with the
maximal inhibitory concentrations (IC50) were determined for the alkyl chain in 8 led to a significant further improvement of
i/i + 6 macrocycles 3–5 and compared to those obtained for the inhibitory activity (3- to 4-fold) against both HDM2 (IC50: 110 vs.
corresponding acyclic 2 and for a linear peptide (1) corresponding 475 nM) and HDMX (IC50: 340 vs. 910 nM). Intriguingly, the
to the Hdm2/X-binding domain in p53. Gratifyingly, these studies nature of the linker was found to have an effect also on the
revealed that both 3 and 4 possess improved inhibitory activity as selectivity of the compounds against the two protein homologs.
compared to the acyclic counterpart 2 (Fig. S3, ESI†), exhibiting an Indeed, while the unconstrained peptide 6 has stronger pre-
approximately 2-fold lower IC50 for HDMX (4) or for both HMD2 ference for HDM2 over HDMX, the macrocyclic counterparts,
and HDMX (3) (Table 1). In contrast, SP4-based macrocycle 5 and in particular 7, behave more as dual, equipotent inhibitors
showed very weak inhibition (IC50 E 10 mM). Thus, these initial (IC50(HDMX)/IC50(HDM2) = 5.5 vs. 1.9, respectively). Overall, these
5028 | Chem. Commun., 2014, 50, 5027--5030
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linker moiety with the protein surface.16 Nevertheless, these
experiments proved that a functional a-helical motif can be
accommodated, and to some extent stabilized within the MOrPH
scaffolds, thereby providing a proof-of-principle validation of the
design strategy outlined in Fig. 1B.
A potential benefit deriving from peptide macrocyclization is an
enhancement in proteolytic stability. Despite its high potency
in vitro, the linear peptide PMI was indeed found to be ineffective
in cell-based assays in part due to rapid proteolysis.19 To assess
this aspect, macrocycles 7 and 8, along with the linear peptide 6,
were incubated in the presence of chymotrypsin (Fig. 2C). Not
surprisingly, 6 was found to undergo rapid proteolytic degradation,
with the original peptide becoming undetectable after only
30 minutes. In contrast, the macrocyclic peptides 7 and 8 survived
up to 3 and 4 hour incubation with the protease, respectively,
exhibiting a 10- to 15-fold longer half-life compared to the acyclic
counterpart. These data clearly showed the beneficial effect of the
intramolecular linkage in imparting these compounds with
increased resistance against proteolysis. It was also interesting to
note how the linker SP6 provided superior performance in
terms of both a-helix stabilization and proteolytic resistance
as compared to SP8, which may be linked to the reduced
conformational flexibility of the former over the latter.
In summary, we have described the rational design of macro-
cyclic organo-peptide hybrids that can effectively accommodate
and, to a certain extent, stabilize an a-helical protein binding motif.
While a common approach in the area of a-helix stabilization has
involved the use of inter-side-chain covalent linkages,4 this work
represents, to the best of our knowledge, the first example of
exploiting side-chain-to-tail peptide cyclization for this purpose.
Using this strategy, submicromolar inhibitors of the p53:HDM2
interaction which display dual specificity against the HDMX
isoform as well as increased proteolytic stability were obtained.
Another intriguing aspect concerns the influence of the non-
peptidic moiety in modulating the functional, conformational,
and stability properties of these a-helical MOrPHs. These
Fig. 2 Characterization data for representative compounds of Table 1.
(A) SPR-based inhibition curves corresponding to disruption of HDM2/p53
interaction; (B) circular dichroism spectra measured in phosphate buffer
(pH 7.0) with 40% TFE; (C) proteolytic stability tests in the presence of
chymotrypsin (1.0 mg mLÀ1) at room temperature.
studies led to the identification of macrocyclic inhibitors of the findings lay the ground for future efforts directed at leveraging
p53:HDM2/X interaction with much improved inhibitory activ- this feature to further optimize these compounds and exploring
ity compared to the wild-type p53 sequence, with the best the potential of the present approach toward disrupting other
compound, 8, exhibiting a 8- and 3.5-fold lower IC50 value in a-helix-mediated protein–protein interactions.
the presence of HDM2 and HDMX, respectively.
This work was supported by the U.S. National Science
To examine the impact of macrocyclization on the peptide Foundation grant CHE-1112342. J.M.S. acknowledges the NSF
conformational properties, circular dichroism (CD) analyses GRF program for financial support. MS instrumentation
were performed on the most potent compounds 7 and 8 as was supported by the U.S. National Science Foundation grants
well as on the linear peptide 6 as a control (Fig. 2B). Peptide 6 CHE-0840410 and CHE-0946653.
was found to display minima at 222 nm and 208 nm, which is
consistent with the presence of an a-helical conformation. The
Notes and references
a-helical content of the peptide was estimated to be about 31%.
1 (a) J. A. Wells and C. L. McClendon, Nature, 2007, 450, 1001;
Cyclization of this sequence with SP6 (7) produced an increase
(b) M. C. Smith and J. E. Gestwicki, Expert Rev. Mol. Med., 2012,
in a-helicity (40%), whereas 8 showed a reduction in the
a-helical content of the embedded peptide sequence (21%).
The lack of a strict correlation between a-helicity and in vitro
inhibitory activity has been observed for other types of
p53:HDM2 inhibitors10c,11b and it is not entirely unexpected
considering that additional factors can affect the binding proper-
ties of these compounds, including potential interactions of the
14, e16.
2 (a) J. A. Robinson, S. Demarco, F. Gombert, K. Moehle and
D. Obrecht, Drug Discovery Today, 2008, 13, 944; (b) L. R. Whitby
and D. L. Boger, Acc. Chem. Res., 2012, 45, 1698.
3 A. L. Jochim and P. S. Arora, Mol. Biosyst., 2009, 5, 924.
4 L. K. Henchey, A. L. Jochim and P. S. Arora, Curr. Opin. Chem. Biol.,
2008, 12, 692.
5 D. Wang, W. Liao and P. S. Arora, Angew. Chem., Int. Ed., 2005,
44, 6525.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 5027--5030 | 5029

View Article Online
Communication
ChemComm
6 D. Y. Jackson, D. S. King, J. Chmielewski, S. Singh and P. G. Schultz, 14 (a) B. Hu, D. M. Gilkes, B. Farooqi, S. M. Sebti and J. Chen, J. Biol.
J. Am. Chem. Soc., 1991, 113, 9391.
Chem., 2006, 281, 33030; (b) M. Wade, E. T. Wong, M. Tang,
J. M. Stommel and G. M. Wahl, J. Biol. Chem., 2006, 281, 33036.
15 G. M. Popowicz, A. Czarna, U. Rothweiler, A. Szwagierczak,
M. Krajewski, L. Weber and T. A. Holak, Cell Cycle, 2007, 6, 2386.
7 G. Osapay and J. W. Taylor, J. Am. Chem. Soc., 1992, 114, 6966.
8 F. M. Brunel and P. E. Dawson, Chem. Commun., 2005, 2552.
9 (a) M. Scrima, A. Le Chevalier-Isaad, P. Rovero, A. M. Papini,
M. Chorev and A. M. D’Ursi, Eur. J. Org. Chem., 2010, 446; 16 C. J. Brown, S. T. Quah, J. Jong, A. M. Goh, P. C. Chiam, K. H. Khoo,
(b) S. A. Kawamoto, A. Coleska, X. Ran, H. Yi, C. Y. Yang and
S. Wang, J. Med. Chem., 2012, 55, 1137.
M. L. Choong, M. A. Lee, L. Yurlova, K. Zolghadr, T. L. Joseph,
C. S. Verma and D. P. Lane, ACS Chem. Biol., 2013, 8, 506.
10 (a) H. E. Blackwell and R. H. Grubbs, Angew. Chem., Int. Ed., 1998, 17 (a) P. H. Kussie, S. Gorina, V. Marechal, B. Elenbaas, J. Moreau,
37, 3281; (b) C. E. Schafmeister, J. Po and G. L. Verdine, J. Am. Chem.
Soc., 2000, 122, 5891; (c) F. Bernal, M. Wade, M. Godes, T. N. Davis,
A. J. Levine and N. P. Pavletich, Science, 1996, 274, 948;
(b) G. M. Popowicz, A. Czarna and T. A. Holak, Cell Cycle, 2008, 7, 2441.
D. G. Whitehead, A. L. Kung, G. M. Wahl and L. D. Walensky, Cancer 18 (a) R. Fasan, R. L. Dias, K. Moehle, O. Zerbe, J. W. Vrijbloed, D. Obrecht
Cell, 2010, 18, 411.
and J. A. Robinson, Angew. Chem., Int. Ed., 2004, 43, 2109; (b) J. A. Kritzer,
J. D. Lear, M. E. Hodsdon and A. Schepartz, J. Am. Chem. Soc., 2004,
126, 9468; (c) H. Yin, G. I. Lee, H. S. Park, G. A. Payne, J. M. Rodriguez,
S. M. Sebti and A. D. Hamilton, Angew. Chem., Int. Ed., 2005, 44, 2704;
(d) R. Fasan, R. L. Dias, K. Moehle, O. Zerbe, D. Obrecht, P. R. Mittl,
M. G. Grutter and J. A. Robinson, ChemBioChem, 2006, 7, 515;
(e) T. Hara, S. R. Durell, M. C. Myers and D. H. Appella, J. Am. Chem.
Soc., 2006, 128, 1995; ( f ) J. K. Murray and S. H. Gellman, Biopolymers,
2007, 88, 657; (g) C. Li, M. Liu, J. Monbo, G. Zou, W. Yuan, D. Zella,
W. Y. Lu and W. Lu, J. Am. Chem. Soc., 2008, 130, 13546;
(h) L. K. Henchey, J. R. Porter, I. Ghosh and P. S. Arora, ChemBioChem,
2010, 11, 2104.
11 (a) F. Z. Zhang, O. Sadovski, S. J. Xin and G. A. Woolley, J. Am. Chem.
Soc., 2007, 129, 14154; (b) A. Muppidi, Z. Wang, X. Li, J. Chen and
Q. Lin, Chem. Commun., 2011, 47, 9396; (c) H. Jo, N. Meinhardt,
Y. B. Wu, S. Kulkarni, X. Z. Hu, K. E. Low, P. L. Davies,
W. F. DeGrado and D. C. Greenbaum, J. Am. Chem. Soc., 2012,
134, 17704; (d) A. M. Spokoyny, Y. Zou, J. J. Ling, H. Yu, Y. S. Lin and
B. L. Pentelute, J. Am. Chem. Soc., 2013, 135, 5946.
12 (a) J. M. Smith, F. Vitali, S. A. Archer and R. Fasan, Angew. Chem., Int.
Ed., 2011, 50, 5075; (b) M. Satyanarayana, F. Vitali, J. R. Frost and
R. Fasan, Chem. Commun., 2012, 48, 1461; (c) J. R. Frost, F. Vitali,
N. T. Jacob, M. D. Brown and R. Fasan, ChemBioChem, 2013, 14, 147.
13 (a) J. C. Marine, M. A. Dyer and A. G. Jochemsen, J. Cell Sci., 2007, 19 M. Pazgier, M. Liu, G. Zou, W. Yuan, C. Li, J. Li, J. Monbo, D. Zella,
120, 371; (b) G. M. Wahl and M. Wade, Mol. Cancer Res., 2009, 7, 1.
S. G. Tarasov and W. Lu, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 4665.
5030 | Chem. Commun., 2014, 50, 5027--5030
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