Bcl-XL-templated assembly of its own protein-protein interaction modulator from fragments decorated with thio acids and sulfonyl azides

Article abstract of DOI:10.1021/ja802683u

Protein-protein interactions have key importance in various biological processes and modulation of particular protein-protein interactions has been shown to have therapeutic effects. However, disrupting or modulating protein-protein interactions with low-molecular-weight compounds is extremely difficult due to the lack of deep binding pockets on protein surfaces. Herein we describe the development of an unprecedented lead synthesis and discovery method that generates only biologically active compounds from a library of reactive fragments. Using the protein Bcl-X_L, a central regulator of programmed cell death, we demonstrated that an amidation reaction between thio acids and sulfonyl azides is applicable for Bcl-X_L-templated assembly of inhibitory compounds. We have demonstrated for the first time that kinetic target-guided synthesis can be applied not only on enzymatic targets but also for the discovery of small molecules modulating protein-protein interactions. Copyright

Full text of DOI:10.1021/ja802683u

Published on Web 09/24/2008
Bcl-XL-Templated Assembly of Its Own Protein-Protein Interaction Modulator
from Fragments Decorated with Thio Acids and Sulfonyl Azides
Xiangdong Hu,† Jiazhi Sun,‡ Hong-Gang Wang,‡ and Roman Manetsch*,†
Department of Chemistry, UniVersity of South Florida, CHE205, 4202 East Fowler AVenue, Tampa, Florida 33620
and, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia DriVe, Tampa, Florida 33612
Received April 11, 2008; E-mail: manetsch@cas.usf.edu
Scheme 1. Bcl-XL-Templated Assembly of Acylsulfonamide
Protein-protein interactions are central to many biological
SZ4TA2 from Fragments Decorated with Thio Acid or Sulfonyl
Azide Functionalities
processes and hence represent a large and important class of
potential targets for human therapeutics.1 Recent discovery of a
variety of low-molecular-weight compounds interfering with
protein-protein complexes launches and validates viable routes for
a large number of new therapies.2 However, disrupting or modulat-
ing protein-protein interactions with low-molecular-weight com-
pounds remains challenging. Most protein-protein interfaces lack
deep pockets that might provide binding sites for small molecules,
and they are composed of two relatively large protein surfaces that
are complementary with respect to shape and electrostatics.3
Moreover, the adaptive and flexible nature of amino acid residues
on protein surfaces creates additional challenges for lead compound
design and discovery.4
The Bcl-2 family consists of both anti- and proapoptotic
members, which are central regulators of programmed cell death.5
The antiapoptotic proteins like Bcl-2, Bcl-XL, and Mcl-1 het-
for the discovery of enzyme inhibitors but not for protein-protein
interaction modulators (PPIMs). Based on studies by Abbott
laboratories, we investigated whether a kinetic TGS approach is a
viable route for the discovery of PPIMs targeting the antiapoptotic
protein Bcl-XL.
erodimerize with the proapoptotic constituents, which include the
multidomain molecules Bax and Bak, and BH3-only proteins such
as Bim, Bad, Bid, Noxa, or Puma, through the conserved BH3
domain.5 The relative ratios of pro- and antiapoptotic Bcl-2 family
proteins determine the ultimate sensitivity or resistance of cells to
a wide variety of apoptotic signals.6 Meanwhile, small molecules
have been reported to modulate the extent of heterodimerization
between anti- and proapoptotic Bcl-2 family members inducing
apoptosis in cancer cells.5 Using a combination of SAR by NMR
screening, parallel synthesis, and structure-guided lead design,
Abbott laboratories developed ABT-737 and a large series of
analogues displaying inhibition constants in the nanomolar or
subnanomolar range.7
Herein, we report our progress towards the development of lead
synthesis and discovery methods that generate only biologically
active compounds targeting protein-protein interactions. In the past
decade, various fragment-based lead discovery approaches have
been reported in which the biological target is actively engaged in
the assembly of its own multidentate inhibitor from a pool of smaller
reactive fragments. These approaches can roughly be divided into
three different categories: (1) dynamic combinatorial chemistry
(DCC),8 (2) catalyst/reagent-accelerated target-supported assembly,9
and (3) kinetic target-guided synthesis (TGS).10 While DCC utilizes
reversible reactions to create a library of all possible heterodimeric
compounds, kinetic TGS approaches employ irreversible reactions
generating only the biologically active bidentate members of the
entire virtual library. In situ click chemistry,10c in particular, has
been shown to produce potent triazole inhibitors of the enzymes
acetylcholine esterase,11 carbonic anhydrase,12 and HIV
protease.13So far, kinetic TGS approaches have been applied only
The reaction combining two fragments in the presence of a
biological template into a larger molecule plays a crucial role for
kinetic TGS. For example, the slow nature and the bio-orthogonality
of the Huisgen cycloaddition, the compatibility of the reactants with
water, and the stability of the triazole products are distinct
characteristics of in situ click chemistry.10c Recently, Williams and
co-workers developed an amidation reaction displaying a reactivity
profile suitable for kinetic TGS applications.14 This particular
amidation reaction between thio acids 1 and sulfonyl azides 2 to
give corresponding acylsulfonamides 3 is effective at room tem-
perature in both organic and aqueous solvents (Scheme 1A).
To probe if this amidation reaction is suited for kinetic TGS
applications targeting protein-protein interactions, building blocks,
some of which are structurally related to fragments of ABT-737,
were decorated with sulfonyl azide or thio acid groups (Scheme
1B). Thio acids TA1-TA3 and sulfonyl azides SZ1-SZ6 were
incubated as binary mixtures in the presence of buffer solution
containing Bcl-XL for 6 h at 38 °C. As a control, 18 identical binary
building block combinations were kept under the same incubation
conditions in buffer without Bcl-XL. All incubations were then
analyzed by HPLC with product detection by electrospray ionization
in the positive selected ion mode (LC/MS-SIM).11b From all the
screened samples, only one incubation sample led to an increased
amount of acylsulfonamide SZ4TA27e in the Bcl-XL-containing
sample compared to the incubation without the protein. We
synthesized this compound for hit validation.15 Comparison of the
LC/MS-SIM traces of the Bcl-XL incubation mixture with the trace
of the corresponding synthesized SZ4TA2 clearly confirmed that
† University of South Florida.
‡ H. Lee Moffitt Cancer Center and Research Institute.
9
13820 J. AM. CHEM. SOC. 2008, 130, 13820–13821
10.1021/ja802683u CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
together, these results indicate that the hit compound SZ4TA2
identified through the kinetic TGS screening is indeed a respectable
ligand of the biological target, which underscores the utility of
kinetic TGS as a valuable approach to PPIM discovery and
optimization.
In a proof-of-concept study, we have shown for the first time
that kinetic TGS can be applied not only for enzymatic targets but
also for protein-protein interaction disruption by using the recently
reported amidation reaction between sulfonyl azides and thio acids.
In the future, we will study in great detail the kinetics and
mechanism of the kinetic TGS approach. Additionally, we will
investigate the scope and limitations of the herein reported lead
discovery and optimization method targeting other protein-protein
interactions related to various diseases.
Figure 1. Hit identification of acylsulfonamide SZ4TA2 by LC/MS-SIM.
(A) Incubation of SZ4 and TA2 in buffer without Bcl-XL. (B) Bcl-XL-
templated reaction after 6 h of incubation. (C) Synthesized SZ4TA2 as
reference. (D) Suppression of Bcl-XL-templated reaction by Bak BH3
peptide. (E) Bcl-XL-templated incubation in presence of mutant Bak BH3
peptide. (F) Suppression of Bcl-XL-templated reaction by Bim BH3 peptide.
(G) Bcl-XL-templated incubation in presence of mutant Bim BH3 peptide.
Acknowledgment. We are grateful to the James and Esther King
Biomedical Research Program (NIR Grant 07KN-08 to R.M.) and
the National Cancer Institute, National Institutes of Health (Grant
P01CA118210 to H-G.W.) for financial support.
Supporting Information Available: Synthetic procedures, LC/MS-
SIM traces, and determination of IC50 values. This material is available
free of charge via the Internet at http://pubs.acs.org.
Bcl-XL templates the formation of the characterized hit compound
(Figure 1A-C).
To assess whether the Bcl-XL-templated reactions occur at the
BH3 binding pocket or randomly elsewhere on the protein surface,
control experiments were performed in which the reactive fragments
SZ4 and TA2 were incubated with Bcl-XL and various proapoptotic
BH3-containing peptides.15 Bak BH3 and Bim BH3 peptides bind
to Bcl-XL through their BH3 domain and theoretically compete with
the reactive building blocks for binding during these incubations.
In comparison, their mutants exhibit lower affinity toward Bcl-XL
and hence do not suppress the Bcl-XL-templated acylsulfonamide
formation to the same extent. Comparison of the LC/MS-SIM traces
(Figure 1D-G) between the Bcl-XL incubations with and without
these peptides suggests that the generation of SZ4TA2 occurs at
the BH3 binding site on Bcl-XL.
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An advantage of kinetic TGS approaches is the increased
throughput screening capability with incubations containing more
than two reactive building blocks. Experiments were undertaken
to test whether our amidation kinetic TGS screening can be
performed as incubations containing more than two complimentary
reacting building blocks. Best results were obtained with reactions
containing one thio acid and six sulfonyl azides.15 Samples of Bcl-
XL with all 9 reactive building blocks at the same time failed at
giving clear results. Although the multicomponent screening of the
entire library failed, these experiments prove that the amidation
kinetic TGS approach can be tested in an enhanced screening
throughput.
To investigate the ability of the hit compound to disrupt the
interaction between Bcl-XL and Bak, we performed the well-
established fluorescence polarization competition assay using Bcl-
XL and fluorescein-labeled Bak BH3 peptide.16 Abbott laboratories
reported that SZ4TA2 is a good Bcl-XL PPIM with a Ki constant
of 19 nM, as determined by a competitive fluorescence polarization
assay using a fluorescein-labeled Bad-BH3 peptide.7e Consistently,
compound SZ4TA2 is validated again as a Bcl-XL inhibitor with
an IC50 constant of 78.8 nM by our assay.15 To compare the activity
of acylsulfonamides not assembled by Bcl-XL with that of the hit
compound SZ4TA2, compounds SZ2TA1, SZ2TA2, SZ2TA3,
SZ4TA1, SZ5TA1, and SZ5TA2 have been synthesized and tested
for their binding to Bcl-XL. The IC50 values of these compounds
have been determined to be 5 µM or higher.15 Finally, we also
determined the IC50 constants for the corresponding reactive
building blocks SZ4 and TA2 to be higher than 100 µM. Taken
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(15) Please see Supporting Information for details.
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