Novel pyrrolopyrimidine-based α-helix mimetics: Cell-permeable inhibitors of protein-protein interactions

Article abstract of DOI:10.1021/ja108230s

There is considerable interest in developing non-peptidic, small-molecule α-helix mimetics to disrupt α-helix-mediated protein-protein interactions. Herein, we report the design of a novel pyrrolopyrimidine-based scaffold for such α-helix mimetics with increased conformational rigidity. We also developed a facile solid-phase synthetic route that is amenable to divergent synthesis of a large library. Using a fluorescence polarization-based assay, we identified cell-permeable, dual MDMX/MDM2 inhibitors, demonstrating that the designed molecules can act as a-helix mimetics.

Full text of DOI:10.1021/ja108230s

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Novel Pyrrolopyrimidine-Based R-Helix Mimetics: Cell-Permeable
Inhibitors of Protein-Protein Interactions
Ji Hoon Lee,† Qi Zhang,† Sunhwan Jo,‡ Sergio C. Chai,† Misook Oh,† Wonpil Im,‡ Hua Lu,*,† and
Hyun-Suk Lim*,†
†Department of Biochemistry and Molecular Biology, and Indiana University Simon Cancer Center, Indiana University School
of Medicine, Indianapolis, Indiana 46202, United States
‡Department of Molecular Biosciences and Center for Bioinformatics, The University of Kansas, 2030 Becker Drive, Lawrence,
Kansas 66047, United States
S Supporting Information
b
ABSTRACT: There is considerable interest in developing
non-peptidic, small-molecule R-helix mimetics to disrupt
R-helix-mediated protein-protein interactions. Herein, we
report the design of a novel pyrrolopyrimidine-based scaf-
fold for such R-helix mimetics with increased conforma-
tional rigidity. We also developed a facile solid-phase
synthetic route that is amenable to divergent synthesis of
a large library. Using a fluorescence polarization-based assay,
Figure 1. (A) R-Helix with i, iþ4, and iþ7 side-chain positions. (B)
we identified cell-permeable, dual MDMX/MDM2 inhibi-
tors, demonstrating that the designed molecules can act as
R-helix mimetics.
R-Helix mimetics with terphenyl 1 and terephthalamide 2 scaffolds. (C)
Pyrrolopyrimidine-based scaffold 3 and its energy-minimized conforma-
tion (R1, R2, R3 = CH3).
with a hypothesis that the bicyclic ring in 3 could replace a
pseudo-bicyclic structure formed by an intramolecular hydrogen
bond in the terephthalamide 2. Thus, the planar heterocyclic frame-
work could provide a pre-organized structure with increased
conformational rigidity. An energy-minimization study suggests
that three functional groups on R1, R2, and R3 of this scaffold can
mimic the spatial orientation of the side chains of i, iþ3 or iþ4,
and iþ7 amino acids in an R-helix (Figure 1A,C). In addition, the
pyrrolopyrimidine template is expected to possess favorable
physical properties, including water solubility and cell perme-
ability, similar to its structurally related purine and indole
scaffolds, which are frequently used as privileged scaffolds in
biology and medicine.5
For the preparation of the designed compound 3, we developed
a straightforward solid-phase synthetic strategy by modifying a
solution-phase synthesis of pyrrolopyrimidines (Scheme 1).6 First,
we utilized peptoid synthesis conditions to introduce two func-
tionalities (R1 and R2). This efficient sub-monomer route7
includes bromoacetylation of amine group on Rink amide MBHA
resin, followed by displacement of the bromide with primary
amines. After repeating the same procedure, the resulting dimeric
peptoids 6 were then coupled with 4,6-dichloro-2-(methylthio)-
pyrimidine-5-carbaldehyde to afford aldehydes 7 in nearly quanti-
tative yield in five steps (Figure S1). Treatment of the aldehydes 7
with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF and
MeOH provided pyrrolopyrimidines 8 through concomitant
cyclization and dimethylamination.8 The thioether group was
oxidized by m-chloroperbenzoic acid (mCPBA) to give a sulfone,
R-Helices represent one of the most common protein sec-
ondary structures and are involved in various protein-protein
interactions (PPIs). In many PPIs, short helical peptides play an
important role as a recognition motif, where side chains at i, iþ3
or iþ4, and iþ7 positions often become a critical determinant for
PPIs (Figure 1A).1 Since R-helix-mediated PPIs are involved in a
wide array of cellular signaling pathways, inhibitors of these
interactions could be promising therapeutic targets. While pep-
tide- and peptidomimetic-based approaches have shown success-
ful applications toward such targets,2 non-peptidic, small
molecules have advantages in terms of desirable bioavailability
and cell permeability. Thus, there is considerable interest in
developing non-peptidic, small-molecule R-helix mimetics that
can disrupt such PPIs. Hamilton and co-workers demonstrated
that rationally designed terphenyl 1 and similar scaffolds can
serve as R-helix mimetics (Figure 1B).1e Following their pioneer-
ing work, a number of terphenyl-inspired structures have been
reported.1e,3 While some of these compounds have been shown
to effectively disrupt certain PPIs, there are several important
drawbacks associated with terphenyl-related structures, such as
low aqueous solubility, relatively flexible scaffold structure, and
long synthetic routes. Herein, we report design of a novel class of
small-molecule R-helix mimetics, development of a facile solid-
phase synthetic pathway, and a subsequent high-throughput
screen, which led to the identification of potent inhibitors that
disrupt the interaction between p53 and MDMX/MDM2.
On the basis of Hamilton's terephthalamide 2 (Figure 1B),4
we designed a pyrrolopyrimidine-based scaffold 3 (Figure 1C)
Received: September 12, 2010
Published: December 20, 2010
r
2010 American Chemical Society
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dx.doi.org/10.1021/ja108230s J. Am. Chem. Soc. 2011, 133, 676–679
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Scheme 1. Solid-Phase Synthesisa
a Reagents and conditions: (a) BrCH2CO2H, DIC, DMF, rt; (b) RNH2,
DMF, rt; (c) 4,6-dichloro-2-(methylthio)pyrimidine-5-carbaldehyde,
DIEA/DMF, rt; (d) DBU/DMF-MeOH, 90 °C; (e) mCPBA, NaH-
CO3, DCM, rt; (f) R3NH2, DIEA/NMP, 170 °C; (g) TFA/DCM, rt.
which was subsequently substituted with various amines. The
resin-bound trisubstituted bicyclic product 3 was cleaved with
trifluoroacetic acid (TFA). Crude products were analyzed for
purity and identity by LC-MS. To investigate the efficiency of our
synthetic pathway, a series of compounds 3 were synthesized,
employing a variety of different amines. Synthesis with most tested
amines resulted in >80% purity of the final products 3, as
determined by LC-MS (Figure S1), which is sufficiently pure for
biological testing without further purification. The most common
byproduct (<5%) in the synthesis was the 4-methoxy-substituted
product (instead of the N,N-dimethylamino group) obtained
during the cyclization step. Those amines that resulted in low
purity (<70%) of final products were excluded in library synthesis.
Compared to the relatively lengthy, linear synthesis of most
terphenyl-related structures, our synthetic route is simple and
divergent and thus amenable to library synthesis. Notably, since
many structurally diverse primary amines are commercially avail-
able, a large combinatorial library is readily accessible.
The efficacy of this scaffold as R-helix mimetics was assessed
by monitoring its ability to disrupt the p53-MDMX interaction.
MDM2 and its homologue MDMX bind to the tumor suppressor
p53 and regulate its stability and activity. The interactions are
mediated mainly by three key residues (Phe19, Trp23, and
Leu26) of p53 and the hydrophobic pocket in MDMX and
MDM2 (Figure 2A).2a,b,9 Despite the similarity in the p53
recognition surface of MDMX and MDM2, it proved to be
challenging to identify MDMX inhibitors, whereas a number of
MDM2 inhibitors have been reported.10 Indeed, inhibitors
developed to target MDM2 show significantly less affinity to
MDMX,9,11 and there is only one report of an MDMX-specific
small-molecule inhibitor.12 Given that MDMX is overexpressed
in many cancers and functions as a major regulator of p53 activity
(both independently of and synergistically with MDM2), the
development of MDMX inhibitors (either MDMX-specific or
dual MDMX/MDM2 inhibitors) is highly desirable but still
remains challenging.11,13
Figure 2. (A) Crystal structure of MDMX in complex with a p53
peptide (PDB entry: 2Z5S). (B) Overlay of compound 3 (R1, R2, R3 =
CH3) and an MDMX-bound p53 peptide. (C) Structures of the active
compounds (3a and 3b) and an inactive compound (NC-1).
average purity of the crude final products was 83%, indicating the
robustness of the synthetic route. The library was used for
screening without further purification. A computational model-
ing showed that the scaffold could mimic MDMX-bound p53
peptide (Figure 2B and Figure S3). The library molecules were
screened at ∼40 μM concentration for the ability to displace a
Rhodamine-labeled 15-mer p53 peptide from MDMX protein by
a fluorescence polarization (FP) assay (Figure S4; see Support-
ing Information for details). From the screen, we identified the
two most active compounds, 3a and 3b (Figure 2C), which were
resynthesized and purified by HPLC for further studies. The
overall yields for the synthesis of 3a and 3b were 26% and 40%,
respectively, based on the loading level of the resin (Figure S5).
As expected, these compounds are highly soluble in aqueous
solution.14,15
We assessed the binding affinities of the selected compounds
using the same FP-based competitive assays. A 15-mer peptide
derived from p53 and a known MDM2 inhibitor, MI-63,16 were
used as controls. As shown in Figure 3A, 3a and 3b effectively
inhibited the p53-MDMX binding with Ki = 0.62 and 0.45 μM,
respectively, which were comparable with that of a 15-mer p53
peptide (Ki = 0.8 μM). We then tested whether they could bind to
MDM2. Interestingly, they were found to inhibit the p53-MDM2
interaction as well, with Ki = 0.62 and 0.84 μM, respectively, similar
to the binding affinities for MDMX (Figure 3B,C). These results
suggest that the compounds act as dual inhibitors of MDMX- and
MDM2-p53 interactions.
Next we investigated the cellular activity of the inhibitors,
since the compounds were expected to be cell-permeable, as indi-
cated above. To test this, we examined the effect of the inhibitors
on cellular levels of p53 and the cyclin-dependent kinase
inhibitor p21, a major transcriptional target of p53. Human lung
cancer H460 cells expressing wild-type p53 were incubated with
3a, 3b, DMSO, or NC-1 (as a negative control), and cell lysates
were analyzed by Western blot to monitor p53 and p21 levels.
Indeed, treatment of 3a and 3b led to the increase of p53 and p21
levels in a dose-dependent manner, whereas DMSO and NC-1
had no effects (Figure 3D and Figure S6). This result clearly
To search for such inhibitors, we constructed a 900-member
library of compound 3 following Scheme 1. This was accom-
plished by a manual parallel synthesis (see Supporting Informa-
tion for details). Primary amines containing hydrophobic groups
(aromatic or aliphatic, shown in Figure S2) were selected with
the intent of mimicking the side chains of the three amino acids of
p53 (Phe, Trp, and Leu). For quality control, 90 compounds
(10%) were randomly chosen from the library, and their purity
and identity were verified by LC-MS. As shown in Table S1, the
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Journal of the American Chemical Society
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initial hit compounds, further studies are needed. These include
biological evaluation of the inhibitors to elucidate the mode of
mechanism as dual inhibitors, investigation of the selectivity of the
compounds among R-helix-recognizing proteins, and X-ray and
computational studies to better understand the molecular basis for
the binding. Experiments along these lines are underway.
In summary, we have described the design of a pyrrolopyr-
imidine-based scaffold 3 as a novel class of R-helix mimetics and the
development of a facile solid-phase synthetic route (Scheme 1). In
addition, we have identified potent, dual inhibitors of MDMX- and
MDM2-p53 interactions through high-throughput screening, high-
lighting that they can serve as R-helix mimetics (Figures 2-4). Of
note, our compounds have several important advantages compared
to most terphenyl-related structures, which include increased con-
formational rigidity, improved aqueous solubility, excellent cell per-
meability, and ease of synthesis. Importantly, our divergent solid-
phase synthesis is amenable to the construction of large libraries and
subsequent high-throughput screening. Taken together, we believe
that our pyrrolopyrimidine-based scaffold, along with the efficient
solid-phase synthesis, will serve as a useful tool to discover inhibitors
of many R-helix-mediated PPIs.
Figure 3. Inhibition curves for Rhodamine-labeled p53 peptide
(SQETFSDLWKLLPEN-NH-Rhodamine) binding to human MDMX
(amino acids 1-137) (A) and human MDM2 (amino acids 1-138) (B)
a
by fluorescence polarization. (C) Ki values of inhibitors. Not deter-
mined. bUnlabeled 15-mer p53 peptide (SQETFSDLWKLLPEN).
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed experimental proce-
b
dures, LC-MS spectra, and complete refs 6 and 11. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
limhyun@iupui.edu; hualu@iupui.edu
Figure 4. p53 activation by 3a. (A) Human lung cancer H460 cells
expressing wild-type p53 were treated with DMSO or the indicated
concentrations of 3a for 12 h. Lysates were subjected to Western blot
analysis. (B) H460 cells with wild-type 53 and human lung cancer
H1299 cells with deleted p53 were exposed to DMSO, NC-1 (20 μM),
3a (20 μM), or MI-63 (20 μM) for 24 h. Caspase activity was analyzed
by Caspase-Glo 3/7 assay kit (Promega).
’ ACKNOWLEDGMENT
We are grateful to Prof. Zhong-Yin Zhang (Indiana Uni-
versity) for critical reading of the manuscript, Prof. Qizhuang
Ye (Indiana University) for providing access to a liquid handling
system, and Dr. Jaeki Min (St. Jude Children's Research Hos-
pital) for technical assistance. H.L. was supported by NIH-NCI
grants CA127724, CA095441, and CA129828. This research was
also supported in part by NSF OCI-0503992 through TeraGrid
resources provided by Purdue University.
demonstrates that the compounds are not only cell-permeable
but also able to induce p53 level and activity in cells.
Activation of p53 by the MDMX/MDM2 inhibitors would
result in cell cycle arrest and apoptosis. We examined the ability
of the inhibitors to induce apoptosis by monitoring the effect of
3a on caspase activity. H460 cells with wild-type p53 and human
lung cancer H1299 cells with deleted p53 were exposed to
DMSO, NC-1, 3a, or MI-63 (a positive control) for 24 h, and
caspase activity was measured by Caspase-Glo 3/7 assay kit (see
Supporting Information). As shown in Figure 4, 3a and MI-63
induced caspase 3/7 activity (about 3- and 5-fold, respectively) in
H460 cells, while they had no effect on H1299 cells. This result
indicates that 3a triggers apoptosis through a p53-dependent
pathway by binding to MDMX/MDM2 and inhibiting their
function toward p53.
While unnatural oligomer-based dual MDMX/MDM2 inhibitors
such as β-peptides17 and N-acylpolyamines18 have been reported,
our compounds would have advantages as cell-permeable, small
“drug-like” molecules. These dual-specific inhibitors should have a
unique, but overlapping, mechanism with current MDM2 inhibitors
and thus could provide a promising starting point for the develop-
ment of a novel class of anti-cancer agents. Because the inhibitors are
’ REFERENCES
(1) (a) Davis, J. M.; Tsou, L. K.; Hamilton, A. D. Chem. Soc. Rev.
2007, 36, 326. (b) Hershberger, S. J.; Lee, S. G.; Chmielewski, J. Curr.
Top. Med. Chem. 2007, 7, 928. (c) Wilson, A. J. Chem. Soc. Rev. 2009, 38,
3289. (d) Jochim, A. L.; Arora, P. S. Mol. Biosyst. 2009, 5, 924.
(e) Cummings, C. G.; Hamilton, A. D. Curr. Opin. Chem. Biol. 2010,
14, 341.
(2) (a) Pazgier, M.; Liu, M.; Zou, G.; Yuan, W.; Li, C.; Li, C.; Li, J.;
Monbo, J.; Zella, D.; Tarasov, S. G.; Lu, W. Proc. Natl. Acad. Sci. U.S.A.
2009, 106, 4665. (b) Phan, J.; Li, Z.; Kasprzak, A.; Li, B.; Sebti, S.;
Guida, W.; Sch€onbrunn, E.; Chen, J. J. Biol. Chem. 2010, 285, 2174.
(c) Walensky, L. D.; Kung, A. L.; Escher, I.; Malia, T. J.; Barbuto, S.;
Wright, R. D.; Wagner, G.; Verdine, G. L.; Korsmeyer, S. J. Science 2004,
305, 1466. (d) Fasan, R.; Dias, R. L.; Moehle, K.; Zerbe, O.; Vrijbloed,
J. W.; Obrecht, D.; Robinson, J. A. Angew. Chem., Int. Ed. 2004, 43, 2109.
(e) Wang, D.; Liao, W.; Arora, P. S. Angew. Chem., Int. Ed. 2005, 44,
6525. (f) Hara, T.; Durell, S. R.; Myers, M. C.; Appella, D. H. J. Am.
Chem. Soc. 2006, 128, 1995. (g) Sadowsky, J. D.; Fairlie, W. D.; Hadley,
E. B.; Lee, H. S.; Umezawa, N.; Nikolovska-Coleska, Z.; Wang, S.;
678
dx.doi.org/10.1021/ja108230s |J. Am. Chem. Soc. 2011, 133, 676–679

Journal of the American Chemical Society
COMMUNICATION
Huang, D. C.; Tomita, Y.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129,
139. (h) Bautista, A. D.; Appelbaum, J. S.; Craig, C. J.; Michel, J.;
Schepartz, A. J. Am. Chem. Soc. 2010, 132, 2904.
(3) (a) Lu, F.; Chi, S. W.; Kim, D. H.; Han, K. H.; Kuntz, I. D.; Guy,
R. K. J. Comb. Chem. 2006, 8, 315. (b) Volonterio, A.; Moisan, L.; Rebek,
J., Jr. Org. Lett. 2007, 9, 3733. (c) Maity, P.; Konig, B. Org. Lett. 2008, 10,
1473. (d) Plante, J. P.; Burnley, T.; Malkova, B.; Webb, M. E.; Warriner,
S. L.; Edwards, T. A.; Wilson, A. J. Chem. Commun 2009, 5091.
(e) Shaginian, A.; Whitby, L. R.; Hong, S.; Hwang, I.; Farooqi, B;
Searcey, M.; Chen, J.; Vogt, P. K.; Boger, D. J. Am. Chem. Soc. 2009, 131,
5564. (f) Marimganti, S.; Cheemala, M. N.; Ahn, J.-M. Org. Lett. 2009,
11, 4418. (g) Tosovska, P.; Arora, P. S. Org. Lett. 2010, 12, 1588.
(h) Bourne, G. T.; Kuster, D. J.; Marshall, G. R. Chemistry 2010, 16,
8439.
(4) Yin, H.; Lee, G. I.; Sedey, K. A.; Rodriguez, J. M.; Wang, H. G.;
Sebti, S. M.; Hamilton, A. D. J. Am. Chem. Soc. 2005, 127, 5463.
(5) Welsch, M. E.; Snyder, S. A.; Stockwell, B. R. Curr. Opin. Chem.
Biol. 2010, 14, 347.
(6) Clark, M. P.; et al. Bioorg. Med. Chem. Lett. 2007, 17, 1250.
(7) Figliozzi, G. M.; Goldsmith, R.; Ng, S. C.; Banville, S. C.;
Zuckermann, R. N. Methods Enzymol. 1996, 267, 437.
(8) Agarwal, A.; Chauhan, P. M. S. Synth. Commun. 2004, 34, 2925.
(9) Czarna, A.; Popowicz, G. M.; Pecak, A.; Wolf, S.; Dubin, G.;
Holak, T. A. Cell Cycle 2009, 8, 1176.
(10) (a) Murray, J. K.; Gellman, S. H. Biopolymers 2007, 88, 657.
(b) D€omling, A. Curr. Opin. Chem. Biol. 2008, 12, 281. (c) Shangary, S.;
Wang, S. Annu. Rev. Pharmacol. Toxicol. 2009, 49, 223.
(11) Laurie, N. A.; et al. Nature 2006, 444, 61.
(12) Reed, D.; Shen, Y.; Shelat, A. A.; Arnold, L. A.; Ferreira, A. M.;
Zhu, F.; Mills, N.; Smithson, D. C.; Regni, C. A.; Bashford, D.; Cicero,
S. A.; Schulman, B. A.; Jochemsen, A. G.; Guy, R. K.; Dyer, M. A. J. Biol.
Chem. 2010, 285, 10786.
(13) (a) Toledo, F.; Wahl, G. M. Nat. Rev. Cancer 2006, 6, 909.
(b) Marine, J. C.; Dyer, M. A.; Jochemsen, A. G. J. Cell Sci. 2006, 120,
371.
(14) Solubilities of 3a and 3b in water and phosphate buffer saline
(pH 7.4) were >150 μg/mL, which is above the desirable water solubility
(100 μg/mL) for favorable oral absorption of drugs.15
(15) Muller, C. E. Chem. Biodiv. 2009, 6, 2071.
(16) Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Wang, G.; Qiu, S.;
Shangary, S.; Gao, W.; Qin, D.; Stuckey, J.; Krajewski, K.; Roller, P. P.;
Wang, S. J. Med. Chem. 2006, 49, 3432.
(17) Michel, J.; Harker, E. A.; Tirado-Rives, J.; Jorgensen, W. L.;
Schepartz, A. J. Am. Chem. Soc. 2009, 131, 6356.
(18) Hayashi, R.; Wang, D.; Hara, T.; Iera, J. A.; Durell, S. R.;
Appella, D. H. Bioorg. Med. Chem. 2009, 17, 7884.
679
dx.doi.org/10.1021/ja108230s |J. Am. Chem. Soc. 2011, 133, 676–679