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Kinetic Target-Guided Synthesis (TGS) and in situ click chemistry are among unconventional discovery strategies having the potential to streamline the development of protein-protein interaction modulators (PPIMs). In kinetic TGS and in situ click chemistry, the target is directly involved in the assembly of its own potent, bidentate ligand from a pool of reactive fragments. Herein, we report the use and validation of kinetic TGS based on the sulfo-click reaction between thio acids and sulfonyl azides as a screening and synthesis platform for the identification of high-quality PPIMs. Starting from a randomly designed library consisting of 9 thio acids and 9 sulfonyl azides leading to 81 potential acylsulfonamides, the target protein, Bcl-XL selectively assembled four PPIMs, acylsulfonamides SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5, which have been shown to modulate Bcl-XL/BH3 interactions. To further investigate the Bcl-XL templation effect, control experiments were carried out using two mutants of Bcl-XL. In one mutant, phenylalanine Phe131 and aspartic acid Asp133, which are critical for the BH3 domain binding, have been substituted by alanines, while arginine Arg139, a residue identified to play a crucial role in the binding of ABT-737, a BH3 mimetic, has been replaced by an alanine in the other mutant. Incubation of these mutants with the reactive fragments and subsequent LC/MS-SIM analysis confirmed that these building block combinations yield the corresponding acylsulfonamides at the BH3 binding site, the actual “hot spot” of Bcl-XL. These results validate kinetic TGS using the sulfo-click reaction as a valuable tool for the straightforward identification of high-quality PPIMs.
Protein-protein interactions (PPIs) are central to a large number of vital biological processes and thus represent attractive targets for the development of novel therapies for a variety of diseases.(1-4) Although scientists recognized the tremendous potential in targeting PPIs over the last two decades, the development of small molecules, which specifically modulate or disrupt a particular PPI, remains a challenging and risky undertaking.(1) Commonly, protein-protein interfaces are large and flat, and they lack deep cavities that might serve as good binding sites for small molecules.(5, 6) Moreover, amino acids at the interfaces of PPIs are flexible and thus pose challenges at conducting computer-guided compound design.(7-9)
Although protein-protein interfaces bury 500–3000 Å2 of total surface area, which exceeds the potential binding area of low-molecular-weight compounds,(10, 11) Wells and co-workers demonstrated that only a fraction of the amino acid residues at the protein-protein interface contributes to the major portion of the binding free energy.(12-14) These key amino acids, defined as recognition patches or hot spots, therefore provide the theoretical and experimental evidence that PPIs can be disrupted or modulated by low-molecular-weight compounds. In the last 15 years, numerous approaches have been developed for the discovery of small molecules modulating or disrupting PPIs. Often, small molecule design is aimed at mimicking a peptide or a protein secondary structure in a truncated form.(15, 16) Alternatively, fragment-based drug discovery strategies using biomolecular NMR, X-ray crystallography, or surface plasmon resonance (SPR) lead to the identification of fragments with good ligand efficiencies, which are further developed into potent protein-protein interaction modulators (PPIMs). Herein we report the expansion and utilization of kinetic Target-Guided Synthesis (TGS) as a screening platform for the identification of PPIMs.
In the last two decades, several TGS approaches have been described, in which the target biomolecule assembles its inhibitory ligand from a collection of reactive fragments. Depending on the nature of the assembly step, TGS approaches can be classified into (a) dynamic combinatorial chemistry (DCC), (b) reagent-accelerated TGS, and (c) kinetic TGS.(17-20) In dynamic combinatorial chemistry, the assembly process is reversible, whereas reagent-accelerated TGS uses building blocks, which combine in an irreversible fashion only in presence of an external reagent or a catalyst upon binding to the biological target. In kinetic TGS, a biological target accelerates the irreversible covalent bond formation only between complementary reacting fragments binding to adjacent binding sites of the target (Figure 1A). Kinetic TGS(16) and in situ click chemistry(17, 18) have been exclusively applied for the identification of inhibitors of enzymatic targets with well defined binding pockets. In a recent proof-of-concept study with the anti-apoptotic protein Bcl-XL as the biological target, we demonstrated that kinetic TGS can also be used for the “rediscovery” of a PPIM previously reported by the Abbott Laboratories starting from smaller fragments bearing a thio acid or a sulfonyl azide functional group.(20) Williams and coworkers described that the amidation reaction between thio acids and sulfonyl azides,(21, 22) which in the meantime has been named as the sulfo-click reaction,(23) proceeds in aqueous media.
The proteins of the Bcl-2 family have been validated as attractive PPI targets for cancer therapy.(24) The Bcl-2 family of proteins, which consists of both anti- and pro-apoptotic molecules, plays a pivotal role in the regulation of the intrinsic pathway of apoptosis. The anti-apoptotic Bcl-2 family proteins Bcl-2, Bcl-XL, and Mcl-1 inhibit the release of certain pro-apoptotic factors from mitochondria. In contrast, pro-apoptotic Bcl-2 family members, which can be further separated into two subgroups, the multidomain BH1–3 proteins (i.e., Bax and Bak) and the BH3-only proteins (e.g., Bad, Bim, and Noxa), induce the release of mitochondrial apoptogenic molecules into the cytosol.(25, 26) Evidence has been accumulated that the majority of human cancers overexpress the pro-survival Bcl-2 family proteins, which not only contribute to cancer progression by preventing normal cell turnover, but also render cancer cells resistant to current cancer treatments.(27, 28) Although there is a controversy over how anti-apoptotic Bcl-2 family proteins function,(29, 30) it is generally accepted that apoptosis is initiated by the binding of pro-apoptotic BH3-only proteins to anti-apoptotic Bcl-2 family molecules in cancer cells. These interactions are mediated by the insertion of the BH3 domain of pro-death proteins into the hydrophobic groove on the surface of anti-apoptotic proteins Bcl-2, Bcl-XL, or Mcl-1.(31, 32) Therefore, small molecules that mimic the BH3 domains of pro-apoptotic Bcl-2 family proteins have potential as anti-cancer therapeutics.
Previously, Abbott Laboratories developed acylsulfonamide 1, ABT-737, ABT-263, and other structurally related acylsulfonamides, which efficiently disrupt Bcl-XL-Bad interaction (Figure 1B).(33-35) Based on these reports, we designed reactive fragments structurally related to ABT-737 and ABT-263 (SZ1–SZ6 and TA1–TA3), and incubated these as binary fragment mixtures in presence of Bcl-XL (Figure 1C). Analysis of each incubation sample by liquid chromatography combined with mass spectrometry detection in the selected ion mode (LC/MS-SIM) showed that of all 18 possible products only compound SZ4TA2, which was developed by Abbott Laboratories, has been detected. In comparison, incubations of fragments in the absence of Bcl-XL or in presence of Bcl-XL and various BH3-containing peptides failed to yield detectable amounts of acylsulfonamide products. In addition, IC50 inhibitory constants in the nM range have been determined for SZ4TA2, while their corresponding thio acid or sulfonyl azide fragments did not show any inhibition up to 100 μM concentrations.
Herein, we successfully employed and validated the sulfo-click kinetic TGS approach as a straightforward yet reliable PPIM screening platform for the identification of Bcl-XL-protein modulators. The design of kinetic TGS incubations with wildtype and mutant Bcl-XL proteins provided an additional layer of confirmatory experiments for the delivery of high-quality PPIMs. Furthermore, experimental evidence has been accumulated indicating that kinetic TGS is a PPIM screening and synthesis method generating only active compounds.
The proof-of-concept study motivated us to investigate whether kinetic TGS is also successful at generating hit compounds which have not been previously reported. Two sublibraries of reactive fragments, one consisting of thio acids and the other of sulfonyl azides, have been synthesized. The thio acids were generated from the corresponding acid chlorides and sodium hydrosulfide, while the sulfonyl azides were prepared by alkylation of amines with 4-(bromomethyl)benzenesulfonyl azide (Figure 2A-C). A selection of acylsulfonamides has been synthesized mainly by: a) EDCI coupling of corresponding carboxylic acids and sulfonamides, or b) the previsouly reported reaction between sulfonyl azides and selenocarboxylates which were generated from corresponding carboxylic acids and the selenating reagent, LiAlHSeH (Figure 2D).(36)
The majority of the reactive fragments have been randomly selected, while a small fraction of the reactive fragments has been designed to be structurally related to ABT-737 or ABT-263. Eighty one binary mixtures containing one thio acid (TA1–TA9) and one sulfonyl azide (SZ1–SZ9) were incubated with the target protein Bcl-XL for 6 hours at 37 °C (Figure 3). In parallel, identical binary fragment mixtures were incubated in buffer without Bcl-XL. Similar to in situ click chemistry,(17, 18) all incubations were directly subjected to HPLC analysis with acylsulfonamide product detection by electrospray ionization in the positive selected ion mode (LC/MS-SIM).(37) Comparison of the LC/MS-SIM traces of identical fragment combinations with or without protein Bcl-XL, led to the identification of the previously reported fragment combination SZ4TA2(20) and three new combinations SZ7TA2, SZ9TA1, and SZ9TA5 with increased amounts of acylsulfonamide products in the incubations containing Bcl-XL (Figure 4A-B and supporting information).
Prior to synthesizing the new TGS hit compounds SZ7TA2, SZ9TA1, and SZ9TA5, control incubations with wildtype and mutant pro-apoptotic Bim BH3 peptides were conducted to assess whether the hit combinations assemble at the targeted binding sites of Bcl-XL or randomly elsewhere on the protein surface (Figure 4C-D and supporting information). These control experiments with Bak BH3 peptide have been previously introduced to confirm the kinetic TGS assembly of compound SZ4TA2.(20) Wildtype Bim BH3 peptide (Bim sequence CEIWIAQELRRIGDEFNAYYAR), the natural Bcl-XL ligand, outcompetes the reactive fragments for binding at the BH3 binding site of Bcl-XL and thus suppresses the Bcl-XL-templated assembly of acylsulfonamides SZ7TA2, SZ9TA1, and SZ9TA5. Contrarily, mutant of the Bim BH3 peptide (mutant Bim sequence CEIWIAQEARRIGAEFNAYYAR) exhibits low affinity towards Bcl-XL and therefore does not significantly affect the Bcl-XL-templated assembly of SZ7TA2, SZ9TA1, and SZ9TA5. Since these co-incubations with wildtype and mutant BH3 peptides strongly suggest that the formation of acylsulfonamides SZ7TA2, SZ9TA1, and SZ9TA5 takes place at the BH3 binding site of Bcl-XL, compounds SZ7TA2, SZ9TA1, and SZ9TA5 have been synthesized and subjected to LC/MS-SIM analysis. Comparison of the LC/MS-SIM traces of the Bcl-XL-templated reactions with the ones of the synthetic compounds clearly confirmed that Bcl-XL templates the formation of hit compounds SZ7TA2, SZ9TA1, and SZ9TA5 (Figure 4E and supporting information).
Experiments were designed, in which mutated Bcl-XL proteins are incubated with reactive fragments. Alterations of the BH3 binding site directly affect the binding of reactive fragments SZ4, SZ7, SZ9, TA1, TA2, and TA5 to the protein, which in turn will influence the rate of the protein-templated acylsulfonamide formation. The purpose of these mutant Bcl-XL proteins is to expand the repertoire of controls with Bim BH3 peptides with complementary experiments indicating whether the TGS reaction occurs with the help of the target protein Bcl-XL and specifically at the binding site of interest. The known mutant of Bcl-XL, in which phenylalanine Phe131 and aspartic acid Asp133 have been substituted by alanines, has been prepared since it fails at interacting with Bak or Bim BH3 peptides.(38) In addition, a second mutant Bcl-XL has been prepared, in which arginine Arg139 has been replaced by alanine. Arginine Arg139 has been identified to be a key residue interacting with ABT-737 and analogues thereof.(33) As a proof-of-concept, incubations of the mutant Bcl-XL with building blocks SZ4 and TA2 were first undertaken at various reactive fragment concentrations (Figures 5,,66 and supporting information). In comparison to the incubation with wildtype Bcl-XL, a reduction in the templation activity by approximately 40% or more has been observed in both mutant Bcl-XL-templated reactions (Table 1). This observation can be explained by closer examination of a reported NMR-structure of Bcl-XL complexed with acylsulfonamide 1, whose structure is closely related to the kinetic TGS product SZ4TA2.(33) Comparison of the location of Phe131 and Asp133 with the position of compound 1 in the wildtype Bcl-XL binding site reveals that the residues Phe131 and Asp133, although important for the binding to Bak or Bim BH3 peptides, are relatively distant from the acylsulfonamide 1, while Arg139 appears to be closer to compound 1. Surprisingly, mutant R139ABcl-XL displays a slightly increased templation reaction in comparison to F131A,D133ABcl-XL. Conformational changes induced by seemingly distant amino acid residues are difficult to trace and may probably influence the templation effect observed during the incubations with wildtype and mutant Bcl-XL proteins.
For TGS hit combinations SZ7TA2, SZ9TA1, and SZ9TA5, confirmatory experiments have been conducted with single mutant R139ABcl-XL only, since the preparation of double mutant F131A,D133ABcl-XL has been cumbersome. Similar to the incubations of fragments SZ4 and TA2, experiments with the mutant protein leading to acylsulfonamides SZ7TA2, SZ9TA1, and SZ9TA5 displayed a reduction in acylsulfonamide formation compared to the incubations with wildtype Bcl-XL. These experiments suggest that the acylsulfonamide genesis occurs in proximity to key amino acid residue Arg139.
The kinetic TGS hits were subjected to dose response studies to obtain IC50s and to investigate if the hit compounds are also modulating or disrupting the interaction between Bcl-XL and a native BH3 peptide ligand. Previously, Abbott Laboratories determined by their assay, that SZ4TA2 is a good PPIM with a Ki constant of 19 nM.(34, 35) Abbott determined the dissociation constants by a competitive fluorescence polarization assay using a fluorescein-labeled Bad-BH3 peptide. In order to precisely compare the inhibitory properties of our kinetic TGS hits with the compounds reported by Abbott, we decided to perform binding studies by a fluorescence polarization assay implemented in our laboratories, which uses GST-Bcl-XL and fluorescein-labeled Bak-BH3 peptide. Consistently, compound SZ4TA2 has been validated by our assay as a Bcl-XL inhibitor against Bak-BH3 with an IC50 constant of 106 nM (Table 2). Kinetic TGS hit compounds SZ7TA2, SZ9TA1, and SZ9TA5 showed IC50s in the low μM range (Figure 3 and supporting information). Taken together, these results indicate that the hit compounds SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5 identified through the kinetic TGS screening are indeed respectable ligands of the biological target, which underscores the utility of kinetic TGS as a valuable approach to PPIM discovery.
To assess whether the kinetic TGS hits are more potent than acylsulfonamides, which were not identified in the kinetic TGS screening, 33 randomly selected acylsulfonamides were synthesized. All compounds, as well as TGS hit compounds SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5 were tested at a 50 μM concentration for PPI disruption in the Bcl-XL/Bak-BH3 fluorescence polarization assay. The 37 acylsulfonamides tested corresponds to 45.7% of the 81 member library. Strikingly, the four kinetic TGS hits were the most potent compounds tested, disrupting the Bcl-XL/BH3 interaction with 60% inhibition or more, while the randomly selected acylsulfonamides demonstrated an average of 15% inhibition (Table 3). Only four of the 33 randomly selected acylsulfonamides demonstrated moderate inhibition (35–45%). In contrast, all reactive fragments SZ1–SZ9 and TA1–TA9 have been tested in the fluorescence polarization assay at 100 μM concentration and less than 5% inhibition was detected. These measurements indicate that the dissociation constants for the corresponding reactive building blocks SZ1–SZ9 and TA1–TA9 have to be higher than 100 μM. These important results suggest that the amidation reaction between thio acids and sulfonyl azides is suitable for kinetic TGS using building blocks displaying weak binding affinities. In addition, this study strongly suggests that the kinetic TGS screening identified the more active members of the library of potential acylsulfonamides arising from reactive fragments SZ1–SZ9 and TA1–TA9.
Generally, cell-permeable small modulators of PPIs have been considered to be desirable tools with great implications for drug discovery and development.(3, 4) Nevertheless, reliable yet straightforward techniques or approaches for the development of potent and effective PPIMs are currently unavailable. Over the past 15 years, a variety of fragment-based lead discovery approaches have been developed and successfully applied for the development of potent PPIMs.(39-41) These approaches are commonly based on the detection of fragments binding to the target protein followed by the study of their binding to the protein target at atomic level resolution using X-ray crystallography or NMR spectroscopy. The initial hits are further optimized via fragment growing, in which fragments are extended into identified binding sites step-by-step, or via fragment linking, in which fragments identified to bind to adjacent binding sites are covalently linked together.(41-44) Even though fragment-based lead discovery strategies have been very successful for the development of PPIMs, they are mainly limited by two constraints. Detection and quantification of fragment binding requires specially designed methodology due to the weak binding typically observed for fragments. Furthermore, the optimization of fragments into potent and selective compounds is not straightforward and not rapidly achievable, even though structural information is available.(43, 45) For example, though high quality NMR structures were available, the development of Bcl-XL PPIMs by Abbott(33, 34) required several design iterations, and the preparation and testing of more than thousand compounds in order to yield ABT-737 and ABT-263.(46) Furthermore, of the very first design consisting of 21 different structures containing the structural motifs of the initial fragments identified by NMR, most compounds bound to Bcl-XL with a dissociation constant greater than 10 μM.(35) Thus, though the hit compounds SZ7TA2, SZ9TA1, and SZ9TA5 display IC50 constants of 28 to 37 μM in the Bak-BH3 fluorescence polarization assay, the herein reported kinetic TGS approach suggests that the high-quality PPIMs will be identified early on in the screening process. This outcome is consistent with previously reported kinetic TGS studies, in which the enzyme carbonic anhydrase II preferably accelerates the formation of the better inhibitory compounds from a pool of reactive fragments.(47, 48) Other kinetic TGS examples using exclusively in situ click chemistry also suggest that the triazoles generated in the protein-templated reactions are the more potent inhibitors.(37, 48-54)
Recently, fragment-based discovery strategies have been reported which involve the protein target directly to select and assemble its own inhibitory compounds from a pool of reactive fragments. These approaches, also termed as in situ click chemistry or kinetic TGS approaches,(16, 18) were conceptually described in the 1980s(55) and are still relatively unexplored compared to dynamic combinatorial chemistry. Thus far, kinetic TGS has mainly been applied to the identification of potent enzyme inhibitors. Nevertheless, the herein reported kinetic TGS offers an attractive approach to PPIM lead discovery because it allows the protein to select and combine building blocks that fit best into its binding sites, thus assembling larger compounds.(16, 18) The screening method can be as simple as determining whether or not the PPIM product has been formed in a given test mixture. This is especially advantageous over a conventional high-throughput screening of difficult targets such as protein interfaces requiring cumbersome and time-consuming experiments to confirm whether screening hits are true or false positives.
Finally, considering that the flexible nature of protein interfaces complicates the development of PPIMs by conventional means, kinetic TGS has the potential to target the protein in a conformation, which is short-lived, undetectable or easily missed with present techniques. A small number of in situ click chemistry approaches targeting enzymatic systems lead to the identification of triazole inhibitors stabilizing the protein in an unprecedented and less abundant conformation.(56-58) Thus, we speculate that the herein reported sulfo-click chemistry kinetic TGS approach provides medicinal chemists a straightforward search strategy to stabilize conformations of dynamic protein targets such as PPIs.
Herein, we demonstrate that the sulfo-click kinetic TGS approach exhibits great promise in fragment-based PPIM discovery since it combines synthesis and screening of libraries of low-molecular-weight PPIMs into a single step. Samples containing the protein target Bcl-XL and reacting fragments leading to 81 structurally different acylsulfonamides have been incubated and analyzed by LC/MS-SIM for acylsulfonamide formation. Of the 81 possible fragment combinations, only combinations SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5 yielded acylsulfonamides in the Bcl-XL-templated reactions. Control incubations with the four hit fragment combinations, in which the BH3 binding site of the wildtype Bcl-XL has been competitively occupied by a Bim BH3 peptide, generated decreased amounts of acylsulfonamides. Furthermore, control incubations with mutants R139ABcl-XL or F131A,D133ABcl-XL, in which amino acid residues at the BH3 binding site have been replaced by alanines, also failed at generating the hit acylsulfonamides suggesting that the protein-templated assembly of SZ4TA2, SZ7TA2, SZ9TA1, and SZ9TA5 occurs at the desired BH3 binding site of Bcl-XL. Subsequent testing of synthesized kinetic TGS hit acylsulfonamides in a fluorescence-based competitive binding assay demonstrated that the kinetic TGS hit compounds indeed display PPIM activity. These findings have been supported by a set of 33 additional acylsulfonamides randomly selected from the 81-member library, which have been shown to fail at demonstrating potent PPIM activity in the fluorescence-based competitive binding assay. These results provide a general test case for the sulfo-click kinetic TGS approach to generate hits targeting the proteins of the Bcl-2 family and further validate the kinetic TGS approach to be suitable for PPIM discovery. In contrast to conventional screening approaches, experimental data suggests that PPIM screening via kinetic TGS reduces the number of false positives, cutting down the number of screening hits to be validated in confirmatory assays. We speculate that the herein reported PPIM discovery strategy for the family of the Bcl-2 proteins is general and can easily be implemented to lead development targeting other PPIs such as MDM2/p53, IAP/caspase, and others.(1, 4, 59)
The synthesis of reactive fragments and acylsulfonamides has been reported in the supporting information.
The protocols have been reported in the supporting information.
In a 96-well plate, one thio acid building block (1 μL of a 2 mM solution in methanol) and one sulfonyl azide building block (1 μL of a 2 mM solution in methanol) were added to a solution of Bcl-XL (98 μL of a 2 μM Bcl-XL solution in buffer (58 mM Na2HPO4, 17 mM NaH2PO4, 68 mM NaCl, 1 mM NaN3, pH = 7.40)). The 96-well plate was sealed and incubated at 37 °C for six hours. The incubation samples were then subjected to liquid chromatography combined with mass spectrometry analysis in the selected ion mode (LC/MS-SIM, Zorbax SB-C18 preceded by a Phenomenex C18 guard column, electrospray ionization and mass spectrometric detection in the positive selected ion mode, tuned to the expected molecular mass of the product). The TGS hit compound was identified by the mass and the retention time. As a control, identical building block combinations were incubated in buffer without Bcl-XL and subjected to LC/MS-SIM analysis. Comparison of the LC/MS-SIM chromatograms of these control incubations with the chromatograms of the Bcl-XL containing incubations allows us to determine whether the protein is templating the corresponding amidation reaction. Furthermore, synthetically prepared acylsulfonamide was subjected to LC/MS-SIM analysis and the retention time was compared with the one identified in the Bcl-XL containing incubation.
The detailed protocol to conduct fluorescence polarization-based competitive binding assays has been previously reported.(20)
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.