Dual Bcl-XL /Bcl-2 inhibitors discovered from DNA-encoded libraries using a fragment pairing strategy

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Article

Reference

Dual Bcl-XL /Bcl-2 inhibitors discovered from DNA-encoded libraries using a fragment pairing strategy

DAGUER, Jean Pierre, et al.

Abstract

A dual Bcl-XL / Bcl-2 inhibitor was discovered from DNA-encoded libraries using a two steps process. In the first step, DNA was used to pair PNA-encoded fragments exploring > 250 000 combinations. In the second step, a focused library combining the selected fragments with linkers of different lengths and geometries led to the identification of tight binding adducts that were further investigated for their selective target engagement in pull-down assays, for their affinity by SPR, and their selectivity in a cytotoxicity assay. The best compound showed comparable cellular activity to venetoclax, the first-in-class therapeutic targeting Bcl-2.

DAGUER, Jean Pierre, et al . Dual Bcl-XL /Bcl-2 inhibitors discovered from DNA-encoded libraries using a fragment pairing strategy. Bioorganic & Medicinal Chemistry , 2021, vol. 44, no. 116282

DOI : 10.1016/j.bmc.2021.116282

Available at:

http://archive-ouverte.unige.ch/unige:152761

Disclaimer: layout of this document may differ from the published version.

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Available online 24 June 2021

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Jean-Pierre Daguer, Arthur Gonse, Yevhenii Shchukin, Lluc Farrera-Soler, Sofia Barluenga, Nicolas Winssinger

^*

Department of Organic Chemistry, Faculty of Sciences, NCCR Chemical Biology, University of Geneva, 1211 Geneva, Switzerland

A R T I C L E I N F O Keywords:

Peptide Nucleic Acid (PNA) Bcl-XL

Bcl-2

DNA-encoded library Fragment based drug discovery

A B S T R A C T

A dual Bcl-XL / Bcl-2 inhibitor was discovered from DNA-encoded libraries using a two steps process. In the first step, DNA was used to pair PNA-encoded fragments exploring >250 000 combinations. In the second step, a focused library combining the selected fragments with linkers of different lengths and geometries led to the identification of tight binding adducts that were further investigated for their selective target engagement in pull- down assays, for their affinity by SPR, and their selectivity in a cytotoxicity assay. The best compound showed comparable cellular activity to venetoclax, the first-in-class therapeutic targeting Bcl-2.

1. Introduction

DNA-encoded libraries have emerged as a powerful approach for hit identification, a critical step in drug discovery and chemical biology.^1-3 While DNA-encoding offers the opportunity to screen large libraries (>10⁹), libraries prepared with a single chemistry sequence will inher- ently be redundant, mitigating the benefits of large numbers.^4,5Ideally, screens would be performed with diverse libraries from different chemistries, as is the case for many historical libraries in HTS screening collections. An alternative approach is the use of fragment libraries.⁶ This offers the opportunity to interrogate the virtual diversity of connection between the fragments before investing the synthetic effort of making every permutation. Several DNA-encoded library designs have been engineered to combine fragments and explore the benefits of the synergy of interactions.^7-18It is now well established that significant gains in affinity can be achieved by pairing fragments through hybridization.^19-25However, identifying the optimal strategy to link the fragments to translate the supramolecular fragment pair into a covalent adduct is a trial and error process that can be tedious. PNA-encoded libraries are prepared by standard SPPS, which dramatically facilitates library preparation by split and mix synthesis. We had shown that identifying the optimal linking strategy for a collection of fragment pairs could be addressed combinatorially with a PNA-encoded focused library (Figure 1A).^15,26Herein, we applied this workflow to Bcl-XL, a key regulator of apoptosis for which inhibitors have been identified by NMR- guided fragment linking approach²⁷^–³⁰and DNA-encoded library^.31

Apoptosis is a highly regulated process critical in development and normal tissue homeostasis, where DNA damage or cellular stress leads to cell death.^32-34Dysregulation in this pathway is frequently observed during oncogenesis or in response to anti-cancer therapy with tumors acquiring anti-apoptotic defense mechanisms.^.35-37 Several pro- apoptotic proteins regulate the pathway (e.g., Bid, Bim, Puma) interacting with anti-apoptotic partners (e.g., Bcl-2, Bcl-XL, Mcl-1), which sequester the pro-apoptotic proteins from interacting with the effectors (Bak and Bax).³⁸The interaction of a pro-apoptotic protein with an effector leads to oligomerization and permeabilization of mitochondria, releasing cytochrome c and Smac, which trigger the apoptotic cascade (Figure 1B). Thus, molecules that can bind to anti-apoptotic proteins and outcompete their interactions with pro-apoptotic proteins are of interest in cancer treatment.^39-42Despite the challenge of inhibiting a protein–protein interaction, successful clinical candidates have been developed, demonstrating that it is possible. However, there remains a debate regarding which anti-apoptotic protein should be targeted; Bcl-2 inhibition is sufficient in lymphoid malignancies,⁴³while Bcl-XL inhibition is more effective in solid tumors.⁴⁴The selectivity also impacts side effects, and Bcl-XL inhibition has been observed to result in platelet death while Bcl-2 inhibition leads to neutropenia.⁴⁵Furthermore, the emergence of resistance to venetoclax,⁴⁶the first-in-class therapeutic demands second-generation therapeutics. ^47–49

* Corresponding author.

E-mail address: nicolas.winssinger@unige.ch (N. Winssinger).

https://doi.org/10.1016/j.bmc.2021.116282

Received 24 March 2021; Received in revised form 9 June 2021; Accepted 16 June 2021

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Bioorganic & Medicinal Chemistry 44 (2021) 116282

2 2. Results and discussion

PNA-encoded libraries are synthesized by traditional solid-phase peptide synthesis (SPPS) with “split and mix” cycles to introduce diversity in a combinatorial fashion.^50,51This technique has been used to prepare libraries of peptides^,52-59glycans,8,60-61,62 heterocycles, and bioactive fragment libraries.9,13,15,17,26,63-64,65 The simplicity of execu- tion of this chemistry makes the synthesis of focused libraries highly accessible. These PNA-encoded libraries can be screened in solution or on a microarray by displaying the libraries onto commercially available DNA arrays (Figure 2), which allows for a fast and straightforward read-

out.⁵¹While microarrays are limited in the number of compounds that can be screened (10⁶), the results can be obtained in less than a day. To broaden the molecular diversity, it is also possible to use the DNA as a template for the non-covalent combinatorial assembly of PNA encoded fragment pairs.

2.1. Bcl-XL microarray-based screening

Using this technology, we engaged in finding new binders to a pro- totypical anti-apoptotic protein, Bcl-XL. The screens are performed by incubating the microarray with the His-tagged protein for 1 h, followed Fig. 1. A) General scheme for the fragment selection by initially paring them by hybridization to a DNA microarray followed by a focused library that covalently combines the best fragments combinatorally with diverse linkers; B) Schematic representation of B-cell lymphoma 2 (Bcl-2) family members interactions on the mitochondria membrane. Bid =BH3 interacting-domain death agonist, Bim =Bcl-2-like protein 11, Puma =p53 upregulated modulator of apoptosis, BAX =Bcl-2- like protein 4, BAK =Bcl-2 homologous antagonist/killer, Bcl-XL =B-cell lymphoma-extra-large, Bcl-2 =B-cell lymphoma 2, Mcl-1 =myeloid leukemia cell dif- ferentiation protein, Smac =second mitochondria-derived activator caspase, MOMP =mitochondrial outer membrane permeabilization.

Fig. 2. Bcl-XL microarray-based screening by PNA/DNA hybridization of fragment pairs combinations. Two selected examples of heat map fluorescence intensities (Cy5 channel) for the screen of 50 nM Bcl-XL with PNA encoded libraries of fragments. A) sulfomimetics fragments and triazole fragments , B) natural products and heterocycles fragments, hybridized into DNA microarrays forming combinations of non-covalently attached fragments pairs. BK =background. The heat maps have been normalized over the corresponding background for comparison purposes.

J.-P. Daguer et al.

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by detection with a fluorescently labeled anti-His tag MAb. We first screened a recombinant Bcl-XL protein against libraries of fragment pairs (250 000 combinations in total), from which two sub-libraries of fragments emerged as rich in binders. The first library is composed pre- dominantly of fragments containing sulfonamides which was comforting given that this is a key structural motif in venetoclax and its analogs. The second is composed of diverse heterocycles. The combination of these two sub-libraries represents 62 500 pairs, and the heatmap of the microarray screen is shown in Figure 2 and Fig S1. A particular sulfonamide fragment’s fitness appears as a horizontal line, while a specific heterocycle fragment appears as a vertical line. There is a high intrinsic redundancy since each fragment is present multiple times in a different pair. It is interesting to note that the intensity is often higher at the intersection of two fit fragments indicating a potential synergy between them. However, identifying the best linking strategy is not trivial⁶⁶and is best addressed by a combinatorial approach that uses a focused library of the selected fragments with a different combination of linker lengths

and geometries. This focused library used ten different linkers (Figure 3A) to pair the 10x100 fragments (10 000 compounds).

Following this strategy with Bcl-XL led to the identification of a given combination that stood out (Figure 3B). The array is such that each heterocycle is a vertical line, and each horizontal line is a linker- sulfonamide or phenol combination. Strikingly, the linker’s nature between the two fragments has a significant influence, and thus particular fragment pairs are observed with a specific linker, whereas different fragment pairs are observed with others. Amongst the ten possible linkers, there is a straightforward selection for particular geometries and lengths concerning a fragment pair. For instance, the linker L7 with cyclopropane is present amongst three hits, while two linkers of comparable length but containing a cyclohexane with different geometries (L4, L9) were not showing notable binding with the same fragment pairs. The identified compounds were then synthesized off-oligo and as biotin conjugates to confirm and characterize Bcl-XL binding (Figure 3C).

Fig. 3.A) Linkers used in the focused library synthesis; B)100 ×100 heat map of fluorescence intensity (Cy5 channel) for the screen of 300 nM Bcl-XL with a 10,000 membered DNA/PNA encoded focused library, where codon A ×Linker is on the horizontal axis and codon B on the vertical axis. Selected compounds have been circled. Each of the 10 000 compounds is spotted 4 times and the result represents the average fluorescence; C) Chemical structure of selected-compounds synthesized off-oligo and as biotin conjugates for hit validation. BK =background. The heat maps have been normalized over the corresponding background for comparison purposes.

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4 2.2. Hit validation and binding characterization

The biotinylated compounds were captured on Streptavidin magnetic beads to evaluate their capacity to pull-down Bcl-XL. Quantifica- tion after SDS-PAGE analysis showed how the selected compounds were able to bind the recombinant protein (Figure 4A and Figure S2 for full images of the gel). To understand if these interactions could be main- tained in the complex environment of a whole proteome, we then

premixed recombinant Bcl-XL with HeLa cell lysate and repeated the pull-down. As shown on the SDS-PAGE western blot (Figure 4B, Figure S3 for whole gel images of the gel), under these conditions, compounds 80–51 and 80–28 were still able to bind to Bcl-XL; however, the others showed a dramatic reduction in their capacity to pull down Bcl-XL, suggesting that other promiscuous interactions out-competed the desired binding. Additionally, we confirmed the synergistic effect of pairing building blocks 80 and 28 by comparing the pull-down of Fig. 4. Hit confirmation and characterization of binding. A) Pull-down of Bcl-XL with immobilized biotinylated compounds (silver stain). B) Western-Blot performed with the anti-His monoclonal antibody on the pulldown of Bcl-XL 0.2 µM spiked on 10 µg of HeLa cell lysate. C) Pull-down comparing compound 80–28 and corresponding building blocks 80 and 28 (silver stain). D) Competitive pull-down quantification of the retention of Bcl-XL with 80–28 and venetoclax (ABT-199). Graph normalized to empty beads.

Fig. 5. Selectivity and affinity measurements. A) Pull-down of Mcl-1, Bcl-2, Bcl-XL with immobilized 80–28; B) SPR kinetics measurements of biotinylated 80–28 binding to Bcl-XL, Bcl-2 and Mcl-1; C) Kinetic and thermodynamic parameters of 80–28 interacting with Bcl-2 family members. BCL family proteins were diluted on HBS-EP buffer and injected over the 80–28-functionalized surface at 0.5 µM, 0.25 µM, 0.125 µM and 0.062 µM.

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individual fragments with the covalently linked compound (80–28).

Neither of the individual fragments showed a detectable target pull- down (Figure 4C and Figure S4 for full images of the gel). To characterize the interaction of 80–28 to Bcl-XL, we also performed the protein’s pull-down in competition with the FDA-approved ABT199 (venetoclax).^46,67Venetoclax was developed as a BH3 mimetic drug with specificity for Bcl-2 but also bind to Bcl-XL. As shown in figure 4D, the binding of Bcl-XL to 80–28 is out-competed in a concentration depen- dent manner by venetoclax, suggesting that both compounds are competing for the same pocket or that there is a steric clash in their interactions.

2.3. Affinity measurements to Bcl-XL and related proteins

Considering the homology in the BH domain of Bcl-2-related proteins involved in apoptotic regulation, we decided to assess the binding of 80–28 not only to Bcl-XL but also to Mcl-1 and Bcl-2. Thus, we performed simultaneous pull-downs with the three recombinant proteins showing that 80–28 retains all of them (Figure 5A). While the expressed protein Mcl-1 was obtained with a truncated proteolytic product, it is noteworthy that the major truncated fragment of Mcl-1 is not pulled down. Using Surface Plasmon Resonance (SPR), we measured 80–28 affinities. Biotinylated 80–28 was captured on a SA sensor chip and, serial dilutions of the protein at 0.5 µM, 0.25 µM, 0.125 µM, and 0.062 µM were injected over the functionalized surface. The measured KD for

grown in the presence of 80–28 or venetoclax (ABT 199) during 72 h of drug exposure, after which we measured the cell survival. Gratifyingly, compound 80–28 showed comparable activity to venetoclax against K562 with an EC50 value of 1.7 µM and 2.1 µM, respectively (Figure 7).

The same experiment was run on U251 MG, a cell line not dependent on the Bcl family, where 80–28 showed no cytotoxicity (Figure S5).

3. Conclusion

The work reported herein highlights the potential of using hybridization to pair fragments as an entry in selecting structures to be used in a focused DEL library covalently pairing them through diverse covalent linkers. The benefit of this two-step process is to hone on a productive diversity space, which can be enriched in the focused library with close analogs of selected fragments. The fact that fragments are tethered to a large tag may preclude some binding orientation but greatly facilitates the covalent connection of the fragments by providing a suitably reactive functional group and a vector that is sterically unhindered. Simi- larly, screening of DELs provides a synthetically tractable handle for biotin conjugation, which facilitates biophysical characterization. The robustness of this workflow is demonstrated with a challenging case study, inhibition of protein–protein interaction. The best inhibitor identified (80–28) showed good affinity to Bcl-2 and Bcl-XL (21 nM and 96 nM, respectively), with slow dissociation rates (2.48 x10^-3M⁻¹s⁻¹).

Importantly, it pulled down Bcl-XL from crude lysates. Competition with known binders indicates this ligand mimics the BH domain in its interaction with Bcl-XL. In a cytotoxicity assay, it compared favorably to venetoclax, an FDA-approved therapeutic.

Fig. 6. SPR competition experiment with native Puma and Bim BH3 25 and 26mer peptides, respectively. A streptavidin sensor chip functionalized with 2800 RU of biotinylated 80–28 was flowed with 0.5 µM Bcl-XL (red curve) and the same concentration of Bcl-XL premixed with 1.0 µM of Puma or Bim peptide (green and blue curves respectively). Puma =p53 upregulated modulator of apoptosis, Bim =Bcl-2 interacting mediator of cell death.

Fig. 7. Cell viability MTT assay. 10⁴K562 cells were seeded on 96 well plates and incubated with variable concentration of ABT199 (venetoclax) or 80–28 for 72 h.

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6 4. Methods

4.1. PNA / DNA screening

The PNA library was hybridized on an Agilent DNA microarray slide (designs 055112–105 000 spots or 048196–44 000 spots) on which the DNA sequences complementary to the 14mer PNA tags encoding our library members are arrayed. The PNA tagged library was diluted at 5 µM in the hybridization buffer (1X – 1.2 M lithium chloride; 0.3 M Li- MES; 0.012 M EDTA; 3% Li-DS; 5% Triton X100), complemented with 10 µg/ml salmon sperm DNA. The hybridization was carried out for 18 h at 60 ^◦C. The hybridized slide was washed twice on 2x SSC, 0.1% SDS buffer for 5 min, 0.2x SSC, 0.1% SDS for 5 more min. at room temperature, briefly rinsed with mQ water and dried.

Recombinant protein Bcl-X_LHis-tag purified (ACRO, cat-BCI-H5120) was diluted to 100 nM binding buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.005% Tween 20). The protein was incubated for one hour at room temperature on the hybridized slides, and then the non-bound protein was washed by soaking the slide on 200 ml of the same buffer.

The presence of the protein on the microarray features was detected by immunostaining the slide. Anti His antibody DyLigth ^Tm649 conjugated (Rockland Immunogenics Cat# 200 343 382) diluted 1/5000 in PBS-T, 0.5% BSA. The slide was incubated with the diluted antibody for 20 min at room temperature, PBS-T washed, dried, and scanned on Genepix 4100A Personal Scanner. The scanned array was quantified using Gen- epixPro7 Software (Molecular Devices) and the median fluorescence corresponding to each feature was used to create the represented heat maps onin Microsoft Excel.

4.2. Pulldown

Selected hits identified from the screening were resynthesized with or without a biotin handle to proceed to validation and characterization of binding. (see SI)

Streptavidin coated magnetic beads Dynal (Dynabeads Thermo Fischer ref: 11206D) were functionalized with the selected biotinylated compounds. 20 µl magnetic beads were PBS-T washed 3 times and resuspended on 20 µl of 100 µM biotinylated compounds diluted on PBS- T. The beads were functionalized 30 min. with gentle agitation and washed with PBS-T.

Then the functionalized beads were equilibrated with HBS-EP (binding buffer).

Bcl-XL-, Bcl-2, or Mcl-1 were diluted on HBSEP to the desired concentration, and the functionalized magnetic beads were resuspended with the Bcl protein solution. The beads were then incubated with gentle rotation for 1 h, washed three times with binding buffer, and retained protein was heat eluted on SDS buffer, 95 ^◦C for 5 min. The samples were directly loaded and separated on SDS PAGE electrophoresis. Western blot of Bcl-XL- was done using mAB antiBcl-XL (cat-SC8392).

4.3. SPR

Biotinylated 80–28 was diluted on HBS-EP buffer and injected as recommended by the manufacturer to functionalize a flow cell on a SA Sensorchip Biacore T200. 2800 RU of biotinylated 80–28 were immobilized and after equilibration the Bcl proteins were injected diluted on running buffer HBS-EP at different concentrations (0.5 µM, 0.25 µM, 0.125 µM 0.062 µM. Association 120 s and dissociation 240 s. All runs were performed at 30 µl/min at 25 ^◦C. After each cycle the surface was regenerated with a 5 s pulse of 10 mM NaOH. Kd estimations were done using Bia Evaluation software.

4.4. Cell viability

Growth inhibition was determined by an MTT assay carried on K562 cell line. Cells were seeded (10⁴cells per well) on 96 well tissue culture

plates (Corning 353219) on 1x IMDM (Gibco) complemented with 10%

Fetal Calf Serum (Sigma Aldrich F4135), 5% glutamine and 1X PenStrep.

The cells were incubated overnight on a cell incubator at 37 ^◦C (5%

CO2). The following morning the selected compounds were serially diluted on DMEM and added to the live cells. After 48 h of incubation, cell mass was measured by adding MTT (Thiazolyl blue tetrazolium bromide, Sigma Aldrich M5655) on sterile PBS, 0.3 mg/ml final concentration followed by incubation for 4 h at 37 ^◦C. Formazan crystals were solubilized by addition of 100 µl per well of 10% SDS, 0.01 N HCl and gentle agitation for 4 h. Once the Formazan crystals completely solubilized, absorbance at 560 nm and 690 nm was recorded on a Spectramax M5 (Molecular Devices) plate reader. The IC50 corresponding to the tested compounds was obtained by plotting the Abs 560/690 against compound concentration and calculated using Graph- pad Prism 6 software analysis. The IC₅₀values presented are represen- tative of at least three independent experiments.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Swiss National Science Foundation (188406&169141) and the NCCR in Chemical Biology (185898). We thank Prof. Jean-Claude Martinou for reagents.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bmc.2021.116282. The raw data has been deposited on Zenodo (https://zenodo.org).

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