Towards more drug-like proteomimetics: Two-faced, synthetic α-helix mimetics based on a purine scaffold

Article abstract of DOI:10.1039/c5ob00478k

Mimicry of two faces of an α-helix might yield more potent and more selective inhibitors of aberrant, helix-mediated protein-protein interactions (PPI). Herein, we demonstrate that a 2,6,9-tri-substituted purine is capable of disrupting the Mcl-1-Bak-BH3 PPI through effective mimicry of key residues on opposing faces of the Bak-BH3 α-helix.

Full text of DOI:10.1039/c5ob00478k

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Towards more drug-like proteomimetics:
two-faced, synthetic α-helix mimetics
based on a purine scaffold†
Cite this: DOI: 10.1039/c5ob00478k
Received 11th March 2015,
Accepted 9th July 2015
M. E. Lanning,a P. T. Wilder,b,c H. Bailey,d B. Drennen,a M. Cavalier,b,c L. Chen,a
J. L. Yap,a M. Rajea and S. Fletcher*a,c
DOI: 10.1039/c5ob00478k
www.rsc.org/obc
Mimicry of two faces of an α-helix might yield more potent and teins.16 Most typically, the i, i + 3/4 and i + 7 hydrophobic side
more selective inhibitors of aberrant, helix-mediated protein– chains are emulated.12–15 A potential caveat with this strategy
protein interactions (PPI). Herein, we demonstrate that a 2,6,9-tri- is that the resulting molecules are rather hydrophobic, particu-
substituted purine is capable of disrupting the Mcl-1-Bak-BH3 PPI larly the pioneering terphenyls, with clog P values often
through effective mimicry of key residues on opposing faces of the around 5 or higher, which might result in poor solubilities
Bak-BH3 α-helix.
and off-target effects.17 Also, the molecular weights (MW) of
these compounds are commonly greater than 500. Thus, in at
least two ways, some of the original helix mimetics contravene
Lipinksi’s rules. The mimicry of multiple faces of an α-helix is
expected to afford more potent agents that can better discrimi-
nate between protein surfaces through a richer recognition
profile and may, therefore, provide a source of selectivity.
Accordingly, towards the discovery of novel and potent Mcl-1
inhibitors, we recently described amphipathic α-helix
mimetics based on a 1,2-diphenylacetylene scaffold.18 Other
groups have also developed two-faced, synthetic α-helix
mimetics,19–22 although all of these designs suffer from
lengthy syntheses.18–22 As part of our group’s continued inter-
est in disrupting helix-mediated PPIs with small-molecule
α-helix mimetics, we sought to discover more drug-like (MW <
500, clog P < 5) agents with therapeutic utility in which
mimicry of two faces is once more accomplished, and in which
the target molecules may be more readily accessed. At the
same time, it is acknowledged that mimicry of just one face of
an α-helix might afford inhibitors that exhibit polypharmacolo-
gies,23,24 hitting multiple targets as a more effective strategy to
kill cancer cells, for example. In this latter scenario, a con-
venient means of installing a solubilizing group would be of
utility in a location that has limited interference with the PPI,
such as the opposing face to that emulated at the protein–
protein interface. This would complement work by Rebek,
Hamilton and Wilson who have described helix mimetics with
a “wet” edge.25–27
Protein–protein interactions (PPIs) participate in pivotal roles
in cell signaling pathways, and their dysregulations can lead to
a wide range of pathologies, including cancer, cardiovascular
and neurodegenerative diseases.1–4 In addition, PPIs featuring
exogenous proteins are involved in the transmission of
infectious diseases, such as HIV.5 Given the potential health
benefits, it is no surprise that the disruption of PPIs by syn-
thetic agents has rapidly become a major goal in contemporary
medicinal chemistry.6–9 The intensity with which these biologi-
cal interfaces are being interrogated is likely fuelled by the
various successful approaches reported over the last decade,
quashing the pessimism that this is too formidable a goal. In
particular, there have been exciting advances in the realm of
helix-mediated PPIs; the most notable examples of which are
ABT-73710 and the Nutlins,11 which are low-molecular-weight,
nanomolar inhibitors of the oncoproteins Bcl-xL and HDM2,
respectively.
One strategy to disrupt helix-mediated PPIs is through
synthetic α-helix mimetics.12–15 Traditionally, helix mimetics
reproduce key functionality located on only one face of an
α-helix, even though many α-helices that engage in PPIs utilize
multiple faces to accomplish recognition of their target pro-
aDepartment of Pharmaceutical Sciences, University of Maryland School of
Pharmacy, 20 N. Pine St., Baltimore, MD 21201, USA.
E-mail: sfletcher@rx.umaryland.edu
bDepartment of Biochemistry, University of Maryland School of Medicine,
Lim and colleagues have described single-faced helix
mimetics based on a rigid pyrrolopyrimidine scaffold wherein
side chains are projected from the 2, 6 and 7 positions.28 Their
molecules proved potent disrupters of the MDM2–p53 PPI,
indicating that a flexible scaffold is not crucial for effective
helix mimicry. We hypothesized that a related 2,6,9-tri-substi-
22 S. Greene St., Baltimore, MD 21201, USA
cUniversity of Maryland Greenebaum Cancer Center, 22 S. Greene St., Baltimore,
MD 21201, USA
dBryn Mawr School, 109 W. Melrose Ave., Baltimore, MD 21210, USA
†Electronic supplementary information (ESI) available: Synthetic procedures
and fluorescence anisotropy competition assays. See DOI: 10.1039/c5ob00478k
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To test our hypothesis, we designed molecules to inhibit
the anti-apoptotic Bcl-2 protein Mcl-1, which, along with its
relative Bcl-xL, is overexpressed in a range of cancers, including
pancreatic cancer and acute myeloid leukaemia.29,30 Mcl-1
seizes the BH3 α-helical domains of pro-apoptotic Bcl-2 family
members, including Bak and Bim, through a hydrophobic
crevice on its surface.29 Under normal conditions, the levels of
anti- and pro-apoptotic Bcl-2 proteins are tightly regulated to
maintain a healthy population of cells but in many cancers,
the high levels of Mcl-1 and other anti-apoptotic proteins
results in the neutralization of the pro-apoptotic proteins
and, thus, tumorigenesis.29 Akin to the successful inhibition
of Bcl-xL and HDM2,11,12,31,32 it has been proposed that low-
molecular-weight ligands that can mimic these BH3 α-helices
should compete with Mcl-1 binding, freeing up the pro-
apoptotic proteins to initiate the intrinsic apoptosis pathway,
and, thereby, destroy the cancer cell.33,34
Fig. 1 Substitution of the 2, 6 and 9 positions of the purine scaffold
putatively permits mimicry of two faces of an α-helix. Generic examples
illustrated are amphipathic helix mimetics, but other two-faced helix
mimetics may be prepared similarly.
Alanine scanning mutagenesis has revealed that the key
side chains of the Bak-BH3 α-helix that engage the anti-
apoptotic Bcl-2 proteins include Val74, Leu78 (i), Ile81 (i + 3),
Ile85 (i + 7), on one face, which bind the p1, p2, p3 and p4
pockets, respectively, and Asp83 (i + 5) on the other face,
which binds Arg139 of Bcl-xL and Arg263 of Mcl-1.29,35 Since
potent inhibitors have been fashioned by mimicking only
three of the four hydrophobic residues,12–15 we designed
amphipathic α-helix mimetics that reproduced the functional-
ity of only Leu78, Ile81 and Ile85 on the hydrophobic face and
Asp83 on the opposing face; these residues are highlighted in
Fig. 2A. In Fig. 2B, the corresponding purine-based synthetic
α-helix mimetic of the helix in Fig. 2A is given. Fig. 2C illus-
trates the Bak-BH3 α-helix overlaid with an MM2 energy-mini-
mized conformation of the helix mimetic in Fig. 2B; very good
mimicry of the key side chains is observed. Fig. 2D describes
our simplified, two-faced α-helix mimetic design, wherein R1
tuted purine scaffold would permit mimicry of the i and i + 3/4
side chains on one face of the helix, and the i + 2 side chain
(or simply a solubilizing group) on the opposing face, as
depicted in Fig. 1A. Mimicry of opposing faces of one turn of
an α-helix in this way might afford potent and selective inhibi-
tors that exhibit lower-molecular-weights than the terphenyls
and their counterparts. Additionally, the purine scaffold is
more hydrophilic than the terphenyl scaffold, which would
serve to lower the clog P of the helix mimetic. Finally, elabo-
ration of functionality introduced at the 2-position, such as a
carbamate or a sulfonamide, might allow suitable emulation
of both the i + 3/4 and the i + 7 side chains from the same
position (Fig. 1B).
Fig. 2 A. Key residues of the Bak-BH3 α-helix. B.
A purine-based α-helix mimetic designed to imitate Leu78, Ile81, Ile85 and
Asp83. C. Superimposition of an MM2 energy-minimized conformation of the molecule in B. On the Bak-BH3 α-helix. D. Simplified Bak-BH3 α-helix
mimetic where R1 and R2 are proposed to imitate the i + 7 and i + 3 side chains, respectively, whilst the Asp83 mimetic is fixed as CH2COOH, and the
Leu78 mimetic constrained as tert-butyl.
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Scheme 1 (a) Boc2O, cat. DMAP, DMSO, 0 °C to RT; (b) NaH, THF, 0 °C
to RT; (c) R1OH, DIAD, PPh3, THF, RT; (d) R2OH, DIAD, PPh3, THF, 35 °C;
(e) glycine, K2CO3, iPrOH, reflux.
Fig. 3 FITC-BAK71–89 was competed off Mcl-1172–327 with 5db giving an
estimated IC50 of 72.3 21.2 μM. Data are an average of biological tripli-
cates and errors are standard deviations. mA = milli-anisotropy. An
absolute anisotropy of 40 mA indicates complete competition. For
further details, please see the ESI.†
and R2 are intended to mimic the i + 7 and i + 3 side chains,
respectively, and, to expedite compound synthesis, we fixed
the Asp83 mimetic (i + 5) as CH2COOH, and the carbamate
group as a Boc group. This last constraint was anticipated to
result in sub-optimal mimicry of Leu78 (i) but would greatly
facilitate the synthetic chemistry.
disrupt the Mcl-1–Bak-BH3 PPI using a fluorescence an-
isotropy competition assay with a fluorescein-labeled Bak-BH3
peptide GQVGRQLAIIGDDINR, “FITC-Bak71–89
”
(ESI†).37
Unfortunately, only one compound, 5db, demonstrated
appreciable activity with an estimated IC50 of 72.3 μM (Fig. 3).
Notably, the construction of a sufficiently hydrophobic face
was required since there is a seven-fold variation in the esti-
mated IC50 values for 5db (R2 = benzyl: IC50 = 72.3 μM) and
5dc (R2 = isobutyl: IC50 = 472 μM), and no activity for 5aa (R1 =
R2 = isopropyl) or compound 7. Furthermore, deletion of the
Boc group (6db) abrogated binding. Also of particular signifi-
cance, the carboxylic acid at the i + 5 position was required for
binding to Mcl-1 (4db exhibited no inhibitory activity). Taken
together, these data demonstrate that the appropriate functionali-
zation of both faces of a purine scaffold furnished synthetic
agents that are capable of recognition of Mcl-1. Although the
MW of 5db is just over 500, its clog D at pH 7.4 (calculated
using ACD Labs) is 2.56, which is considerably lower than
those of analogous terphenyl-based helix mimetics (clog Ds
7.22 (mono-acid), 5.27 (di-acid)). We anticipate that a struc-
ture–activity relationship (SAR) analysis of 5db will identify
more potent Mcl-1 inhibitors that conform to Lipinksi’s rules.
More generally, these findings validate purine scaffolds as suit-
able frameworks to achieve two-faced, α-helix mimicry.
We have previously shown that N2-Boc-2-amino-6-chloro-
purine (2) can be regioselectively alkylated at the N9 position
under standard Mitsunobu conditions.36 NB: In the prepa-
ration of 2, NaH as a dispersion in mineral oil gave capricious
results, whereas dry NaH consistently drove complete Boc
transfer from the N9 to the N2 position. Therefore, as shown in
Scheme 1, to ensure projection of the R1 (the i + 7 side chain
of the Bak-BH3 α-helix) and CH2COOH (i + 5) groups from
opposite faces of the helix mimetic, 2 was condensed with
alcohols R1OH in the presence of DIAD and PPh3. The result-
ing N9-alkylated products 3 were subjected to a second round
of Mitsunobu chemistry using modified conditions (2.5 equiv.
of R2OH, PPh3 and DIAD, and gentle warming) to introduce
the R2 group (i + 3 mimetic). Finally, an SNAr reaction with
glycine installed the i + 5 mimetic on the opposite face of the
scaffold, providing the target molecules 5 in five simple steps.
Accordingly,
a small library of amphipathic α-helix
mimetics based on 5 was rapidly developed (Scheme 2) and
then each library member was analyzed for its ability to
The inhibitory profile of 5db was investigated further by
interrogating the HDM2–p53 and Bcl-xL–Bak-BH3 PPIs using
similar fluorescence anisotropy competition assays. In the
latter case, the same Bak-BH3 peptide from the Mcl-1 experi-
ments was utilized, whilst in the HDM2 experiments, a
TAMRA-labeled p53 peptide SQETFSDLWKLLPEN was
employed (ESI†). Although no inhibition of HDM2 was
observed, 5db weakly inhibited Bcl-xL with an estimated IC50
of 201 μM. Whilst the p53 helical peptide that binds HDM2
also presents a similar recognition pattern of amino acids
(Phe19, Trp23, Leu26 on one face, and Asp21 on the other
face),38 the naphthyl group (a good mimetic of Trp) of 5db, is
at a terminus of the helix mimetic and so is improperly posi-
Scheme 2 Purines prepared in this study.
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pocket may explain the observed two-fold selectivity for Mcl-1
over Bcl-xL since occupation of this pocket appears to be a
determinant in Mcl-1 specificity.33 In closing, we would like to
note that our work complements other research in the field of
inhibiting helix-mediated PPIs. Particularly, occupation of the
p2 pocket by the naphthylmethyl group of 5db possibly
explains the inverse selectivity of our purine-based helix
mimetics for Mcl-1 over HDM2 for previously reported helix
mimetics.39 Also,
a recently disclosed phenyl-piperazine-
triazine-based helix mimetic demonstrates significantly
greater selectivity for Mcl-1 over Bcl-xL than that which we
observed with our purines, although it is unclear if the groups
mimicking the i, i + 3 and i + 7 side chains and/or the scaffold
itself are responsible for this observation since only one com-
pound was analysed for selectivity.40
Fig. 4 GOLD docking of 5db to Mcl-1 (PDB ID: 2PQK). Coloured by
atom type: grey = carbon, red = oxygen; blue = nitrogen; yellow =
sulfur). A. Superimposition of 5db (yellow, coloured by atom type) with
the Bim-BH3 α-helix (green, key side chains shown in stick format,
labelled in green). B. Predicted binding mode of 5db from A with BH3
α-helix removed: Arg263 and p1–p4 subpockets on Mcl-1 labelled in
black.
Conclusions
In conclusion, 2,6,9-trisubstituted purines were proposed and
then validated as two-faced, α-helix mimetics, permitting
reasonable mimicry of the i, i + 3 and i + 7 side chains on one
tioned to emulate Trp23. Moreover, Asp21 does not play a func- face and i + 5 on the opposing face of the Bak-BH3 α-helix.
tional role in binding HDM2. Rather, the carboxylic acid of Alternatively, functionalization at the i + 5 position may simply
Asp21 forms a hydrogen bond with the hydroxyl of Thr18 be applied to install a solubilizing group opposite the deco-
within the p53 peptide to ensure it folds into the correct rated face. Computational modelling indicated that the non-
helical conformation to permit recognition of HDM2.38 Thus, flexible tert-butyl group incorporated to facilitate the synthetic
appropriately-functionalized two-faced synthetic α-helix chemistry may have compromised helix mimicry somewhat;
mimetics may, indeed, provide a source of selectivity over their more flexible carbamates and sulfonamides at the N2 position
single-faced counterparts.
are predicted to remedy this undesired outcome. In the course
To gain a better appreciation of the likely binding mode of of this research, a novel inhibitor of Mcl-1 was discovered.
5db, as well as a possible explanation to the approximate Efforts in our laboratory are now underway to optimize this
3-fold selectivity for Mcl-1 over Bcl-xL, we performed molecular compound through an SAR campaign, a goal that will be
docking simulations with GOLD. For this purpose, we used the directly facilitated by the short and simple synthesis.
crystal structure of Mcl-1 bound to the Bim-BH3 α-helix, which
has a similar pattern of hydrophobic residues to Bak (Ile58,
Leu62 (i), Ile65 (i + 3) and Phe69 (i + 7) on one face and Asp67
(i + 5) on the other (PDB ID: 2PQK)). For modelling experi-
Acknowledgements
ments, the Bim-BH3 α-helix was first extracted to reveal the We thank the University of Maryland School of Pharmacy (SF)
hydrophobic crevice binding site on the surface of Mcl-1. A and the National Institutes of Health Grant #T32GM066706
high-scoring docking solution is given in Fig. 4A with 5db (MEL) for financially supporting this research.
shown in yellow, which is superimposed on the Bim-BH3
α-helix illustrated in green; key side chains of the helix are
shown in stick format. It should be noted that the orientation
of the molecule is anti-parallel to the original design. This
Notes and references
finding may be due to the binding mode being driven by occu-
pation of the p2 pocket by the large naphthylmethyl group,
which, along with the salt bridge interaction with Arg263, is
seen more clearly in Fig. 4B. The side chains of Leu62 (Leu78
in Bak), Ile65 (Ile81) and Asp67 (Asp83) overlapped well with
the respective functionalities in 5db. As expected, the tert-butyl
of the Boc group inadequately simulated the spatial orien-
tation of the side chain of Phe69 (Ile85 in Bak, originally
intended to mimic Leu78), clearly providing a focal point for
the design of second-generation compounds. Interestingly, the
accommodation of the naphthylmethyl moiety in the p2
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