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Targeting lipid–protein interaction to treat Syk-mediated acute myeloid leukemia

Nature Chemical Biology volume 19, pages 239–250 (2023)Cite this article

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Abstract

Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid–SH2 domain interactions. Here we report development of new nonlipidic small molecule inhibitors of the lipid–SH2 domain interaction that block the cellular activity of their host proteins. As a pilot study, we evaluated the efficacy of lipid–SH2 domain interaction inhibitors for spleen tyrosine kinase (Syk), which is implicated in hematopoietic malignancies, including acute myeloid leukemia (AML). An optimized inhibitor (WC36) specifically and potently suppressed oncogenic activities of Syk in AML cell lines and patient-derived AML cells. Unlike ATP-competitive Syk inhibitors, WC36 was refractory to de novo and acquired drug resistance due to its ability to block not only the Syk kinase activity, but also its noncatalytic scaffolding function that is linked to drug resistance. Collectively, our study shows that targeting lipid–protein interaction is a powerful approach to developing new small molecule drugs.

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Fig. 1: The membrane-binding mechanisms of SH2 domains.
Fig. 2: Small molecule inhibition of the cellular activity of Syk, BLNK and PLCγ2.
Fig. 3: Inhibition of the signaling activity and proliferation of MV4-11 AML cells by Syk-cSH2 inhibitors.
Fig. 4: Suppression of HL-60 and patient-derived AML cells by Syk-cSH2 inhibitors.
Fig. 5: Signaling function of Syk in entospletinib-resistant MV4-11 cells determined by genetic modulation.
Fig. 6: Cellular target validation of WC36.

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Data availability

All described data are contained within the paper and its Supplementary Information files. The raw data will be shared upon request to the corresponding author. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (grant nos. R35GM122530 to W.C., GM 120281 to V.G., P41-GM104601 and R01-GM123455 to E.T. and R01-NS114413 to S.C.). I.K. acknowledges University of Illinois Cancer Center’s 2020 Pilot Program (grant no. 2020-PP-01). E.T. thanks Blue Waters at National Center for Supercomputing Applications for providing computing resources and Extreme Science and Engineering Discovery Environment (grant no. MCA06N060). E.T. is also grateful to the School of Chemical Sciences Scientific Software Program for access to the Schrödinger Suite.

Author information

Author notes

  1. Shashank Pant

    Present address: Loxo Oncology @ Lilly, Louisville, CO, USA

  2. Yusi Hu

    Present address: College of Chemistry and School of Medicine, Nankai University, Tianjin, P. R. China

  3. These authors contributed equally: Indira Singaram, Ashutosh Sharma.

Authors and Affiliations

  1. Department of Chemistry, University of Illinois Chicago (UIC), Chicago, IL, USA

    Indira Singaram, Ashutosh Sharma, Melissa Pergande, Pawanthi Buwaneka, Yusi Hu, Stephanie Cologna & Wonhwa Cho

  2. NIH Center for Macromolecular Modeling and Bioinformatics, Beckman Institute for Advanced Science and Technology, Department of Biochemistry, Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Shashank Pant, Muyun Lihan & Emad Tajkhorshid

  3. Department of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Republic of Korea

    Mi-Jeong Park

  4. Department of Medicine, University of Illinois Chicago, Chicago, IL, USA

    Nadim Mahmud & Irum Khan

  5. Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea

    You-Me Kim

  6. Department of Chemistry, University of Texas at Dallas, Dallas, TX, USA

    Vladimir Gevorgyan

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Contributions

A.S. and I.S. carried out cell studies on Syk and Syk inhibitors. A.S. performed synthesis, screening, optimization and characterization of inhibitors. S.P., M.L. and E.T. performed computational analysis. M.-J.P. and P.B. performed SPR studies on Syk-SH2 and BLNK-SH2, respectively. Y.H. contributed to initial Syk inhibitor screening. N.M. and I.K. collected and performed experiments with the clinical samples. M.P. and S.C. performed MS analysis. V.G. constructed a small molecule library. Y.-M.K. participated in BCR signaling analysis. W.C. conceived and supervised the work and wrote the paper.

Corresponding author

Correspondence to Wonhwa Cho.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. S.P. is currently an employee of Loxo Oncology @ Lilly and is a shareholder of stock in Eli Lilly and Co.

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Nature Chemical Biology thanks Thomas Smithgall and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 PIP3-dependent membrane binding of Syk-cSH2.

a. A representative simulation box for the HMMM membrane binding simulations of Syk-cSH2, which is initially placed at least 10 Å away from the membrane surface. 1,2-dihexanoyl derivatives of phosphatidylcholine (PC) and phosphatidylserine (PS) are shown in gray and purple lines, respectively, and PIP3 (6 in each leaflet) in space-filling representation (carbon, oxygen, and phosphorous atoms in yellow, red and tan, respectively). The hydrophobic core of the membrane is filled with an organic solvent (DCLE), shown in transparent surface representation. Bulk water molecules and ions are not shown for clarity. b. Ensemble-averaged residue profile of the Syk-cSH2, calculated during last the 50 ns of HMMM simulations. The gray bars denote the standard deviation in the residue distances. The pY-interacting residue (R195) is highlighted in black star. The representative PIP3-interacting conformations captured at the end of membrane binding simulations are shown at the bottom panel. c. Probability histogram of the number of PIP3 within 5 Å of Syk-cSH2, calculated over last 50 ns of all the HMMM membrane binding simulations, highlights that the protein can simultaneously interact with multiple PIP3 interacting which might help in its effective recruitment. d. Histograms of the protein residues in contact (within 3.5 Å) with the 3’, 4’ and 5’ phosphate moieties (P3 (red), P4 (green), and P5 (blue)) of the PIP3 headgroup are calculated over last 50 ns of all the HMMM membrane binding simulations. Notice that K220 and K222 make contact primarily with P3 whereas H163 and K165 interact with both P3 and P4. Many other residues primarily interact with P5. e. All the residues that form contacts with PIP3 for more than 10% of the time are highlighted and those that make the closest contact with PIP3 are labeled. All the basic residues are colored in blue and polar residues in green. f. Enlarged view of interaction between K172 of the Syk-cSH2 domain and the P4 and P5 moieties of PIP3. g. A schematic representation of predicted hydrogen bonds in this binding mode. H-bonds are shown as arrows. Notice that K172 interacts with both P4 and P5, but not with P3. It is thus expected to be involved in non-specific interaction with non-3’ P-containing PtdInsPs, such as PI(4,5)P2. h. Determination of Kd for binding of Syk-cSH2 WT (blue) and mutants (K220A/K222A (red), K172A (green), and R195A (orange) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)/PIP3 (77:20:3) large unilamellar vesicles (LUVs). The protein concentration was varied from 0 to 500 nM. Each data is average ± SD from 3 independent measurements.

Extended Data Fig. 2 Role of Syk-cSH2-PIP3 binding in plasma membrane translocation of Syk.

a. Subcellular localization of EGFP-Syk-WT and a PIP3 sensor, mCherry-eMyoX-tPH co-expressed in Syk-null DT40 cells after IgM stimulation. b. Subcellular localization of EGFP-Syk-WT and mCherry-eMyoX-tPH expressed in Syk-null DT40 cells before IgM stimulation. c. The effect of depleting cellular PIP3 on IgM-stimulated membrane translocation of EGFP-Syk-WT and mCherry-eMyoX-tPH was measured by treating cells with a Class I PI3K inhibitor, GDC-0941 (10 nM, overnight). d. Subcellular localization of EGFP-Syk-K220A/K222A and mCherry-eMyoX-tPH expressed in Syk-null DT40 cells after IgM stimulation. IgM stimulation was performed by dropping cells on the IgM-coated surface (10 ng/ml) for 10 min. r values indicate Pearson’s correlation coefficients (average ± SD from 3 independent measurements). r was not determined (ND) for Extended Data Fig. 2c because localization of neither EGFP-Syk-WT nor mCherry-eMyoX-tPH was observed in the plasma membrane. Scale bars indicate 2.5 μm. e. IgM-stimulated (10 ng/ml for 10 min) Ca2+ release in Syk-null DT40 cells (black line) stably transfected with Syk-WT (blue line) and Syk-K220A/K222A (red line). Ca2+ levels were ratiometrically (F340/F380: the ratio of fluorescence emission intensity at 380 nm to that at 340 nm) estimated using Fura-2. Each data is a representative curve of 3 independent measurements. Inset: expression levels of transfected Syk-WT and K220A/K222A were comparable as seen in the similar density of immunoblot bands. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a gel loading control. The image is representative of 3 independent measurements.

Source data

Extended Data Fig. 3 PIP3-dependent membrane binding of BLNK-SH2.

a. A representative simulation box for the HMMM membrane binding simulations of BLNK-SH2, which is initially placed at least 10 Å away from the membrane surface. See Extended Data Fig. 1 for details. b. A predicted mode of binding of BLNK-SH2 to a PIP3 molecule in the membrane. Left panel: a PIP3 molecule (C and O, N atoms are shown in yellow, red and blue, respectively) and BLNK-SH2 residues (C and O, N atoms are shown in white, red and blue, respectively) interacting with it are shown in stick presentation and labeled. Gray circles indicate PC and PS molecules. This representative structure is one of multiple low energy conformations obtained from the simulation and docking and not all of residues predicted for PIP3 binding are shown. Right panel: the structure is rotated 90o for the top of the protein surface to face the membrane. The protein is shown in surface representation to better illustrate the shape of the primary lipid binging site. c. Superimposition of PIP3 and VG594 docked into the primary lipid binding groove of BLNK-SH2 in the membrane environment. In its lowest energy binding mode, VG594 binds deeper to the groove than the PIP3 headgroup, leading to tight binding and effective inhibition of PIP3 binding. d. Determination of Kd for binding of BLNK-SH2 WT (circles) and K411A/K412A/K413A (triangles) to POPC/POPS/PIP3 (77:20:3) LUVs. The protein concentration was varied from 0 to 500 nM. Each data is average ± SD from 3 independent measurements. e. Selectivity of BLNK-SH2 WT (red) and K411A/K412A/K413A (blue) for POPC/POPS/ PIP3 (77:20:3) (solid lines) over POPC/POPS/PI(4,5)P2 (PIP2) (77:20:3) (broken lines) vesicles determined by SPR analysis. The protein concentration was 100 nM.

Extended Data Fig. 4 PIP3-dependent membrane binding of PLCγ2-cSH2.

a. A representative simulation box for the HMMM membrane binding simulations of PLCγ2-cSH2, which is initially placed at least 10 Å away from the membrane surface. See Extended Data Fig. 1 for details. b. A predicted mode of binding of PLCγ2-cSH2 to a PIP3 molecule in the membrane. Left panel: a PLCγ2-cSH2 residues (C and O, N atoms are shown in white, red and blue, respectively) interacting with a PIP3 molecule (C and O, N atoms are shown in yellow, red and blue, respectively) are shown in stick presentation and labeled. Gray circles indicate PC and PS molecules. This representative structure is one of multiple low energy conformations obtained from the simulation and docking and not all of residues predicted for PIP3 binding are shown. Right panel: the structure is rotated 90° for the top of the protein surface to face the membrane. The protein is shown in surface representation to better illustrate the shape of the primary lipid binging site. c. Superimposition of PIP3 and VG370 docked into the primary lipid binding groove of PLCγ2-cSH2 in the membrane environment. In its lowest energy binding mode, VG370 binds deeper to the groove than the PIP3 headgroup, leading to tight binding and effective inhibition of PIP3 binding. d. Determination of Kd for binding of PLCγ2-cSH2 WT (circles) and K727A/K728A (triangles) to POPC/POPS/PIP3 (77:20:3) LUVs. The protein concentration was varied from 0 to 500 nM. Each data is average ± SD from 3 independent measurements. (e) Selectivity of PLCγ2-cSH2 WT (red) and K727A/K728A (blue) for POPC/POPS/PIP3 (77:20:3) (solid lines) over POPC/POPS/PI(4,5)P2 (PIP2) (77:20:3) (broken lines) vesicles determined by SPR analysis. The protein concentration was 100 nM.

Extended Data Fig. 5 Efficacy and specificity of Syk inhibitors.

a. Cellular efficacy of WC35, WC36, and WC38 was determined by dose-dependent inhibition of Syk phosphorylation in Raji B cells. b. Imax and IC50 values (see Supplementary Table 3) were determined by nonlinear least-squares analysis of data using the equation: I = Imax / (1 + IC50 / [inhibitor]) where I indicates %inhibition. %inhibition was calculated by 100 x (relative decrease in pSyk/(total Syk) at a given inhibitor concentration ([inhibitor]) compared to that without an inhibitor. Each data represents average ± SD from 3 independent measurements. c. Specificity of WC35, WC36, WC38, and entospletinib (ENTO) was determined by measuring the inhibition of ZAP70 phosphorylation by these molecules (5 μM overnight) in Jurkat T cells. Jurkat T cells were stimulated by OKT3. Each gel image is a representative of 3 independent experiments.

Source data

Extended Data Fig. 6 Proposed mechanisms of Syk signaling, inhibition, and resistance in AML cells.

a. In the resting state, Syk is autoinhibited by intramolecular tethering by interdomain A (ID-A; dark red) and B (ID-B) which occludes the kinase (Kin) domain. b. IgG2-activated FcγRI causes the associated ITAM motif to be doubly phosphorylated (pY2-ITAM) by a Src family kinase, creating a docking site for Syk. c. Recruitment of Syk to the activated receptor requires coincident Syk-cSH2-PIP3 binding and Syk-n/cSH2-pY2-ITAM binding, which leads to conformational changes and activation of Syk. d. WC36 specifically blocks this process by inhibiting Syk-cSH2-PIP3 binding while entospletinib cannot. e. Once activated by the receptor, Syk typically undergoes autophosphorylation (pY: green) of ID-A and ID-B for sustained activation and functions as both a kinase and a scaffolding protein. As a kinase, Syk phosphorylates a constitutively active FLT3 mutant (for example, FLT3-ITD), which is essential for AML cell proliferation. Activated Syk also serves as a scaffolding protein for FLT3, RAS-RAF-MEK-ERK and STA3/5 either directly or via an adaptor protein (for example, X = KSR). f. Entospletinib can block the kinase activity of Syk but cannot inhibit the scaffolding function, allowing the AML cells to develop acquired resistance mechanism that bypasses the Syk kinase activity. WC36 does not allow any type of resistance because it blocks all functions of Syk at the source.

Extended Data Fig. 7 Target validation of WC36.

a. Design of mutants that may potentially inhibit WC36 binding without affecting PIP3 binding. In the model structures of Syk-cSH2-PIP3 and Syk-cSH2-WC36 complexes (superimposed here as in Fig. 2d), the side chain of E164 forms a hydrogen bond (the red dotted line) with the N-H group of the imidazole moiety of WC36 but is remote from the PIP3 head group. We thus reasoned that the mutation of E164 might selectively suppress WC36 binding without impacting PIP3 binding. The blue dotted line indicates the location of the membrane surface. b. Membrane binding activity of EGFP-Syk-cSH2-WT (blue), -E164D (red), -E164A (purple), -E164Q (green), and -E164K (orange) was measured by the fluorescence quenching assay using POPC/POPS/PIP3/dabsyl-PE (67:20:3:10 in mol%) LUVs. The quenching of the EGF fluorescence by dabsyl-PE (△F) of was determined as a function of the total lipid concentration in the vesicles. E164D showed essentially the same vesicle affinity as WT. The protein concentration was kept at 25 nM. c. Inhibition of membrane binding activity of EGFP-Syk-cSH2-WT (blue), -E164D (red), -E164A (purple), -E164Q (green), and -E164K (orange) by WC36. 25 nM of Syk-cSH2 proteins and 50 μM POPC/POPS/PIP3/dabsyl-PE (67:20:3:10 in mol%) LUVs were employed. % inhibition ((△F / △Fmax) x 100)) was plotted as the function of the WC36 concentration where △F and △Fmax indicate fluorescence quenching in the presence of and absence of WC36, respectively. Neither E164D nor W164Q was inhibited by WC36 whereas WT was. For Expended Data Fig. 7b, d, each data represents average ± SD from 3 independent measurements. d. The structure of WC36B.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7, Tables 1–5, Notes 1 and 2 and Data files 1 and 2.

Reporting Summary

Supplementary Data 1

Proteins captured by WC36B and biotin were subject to identification by data-dependent mass spectrometry analysis. Raw data was searched against the Swiss Protein Homosapien database using the Proteome Discoverer software using the Sequest HT algorithm. Shown herein are proteins identified in the capture by WC36B and biotin. In this analysis, Syk as only detected in the WC36B fluorophore capture. Peptide sequences for the 28 unique peptides (47% sequence coverage) for Syk are also included. In our approach, the more intense a peptide, the more it is selected for fragmentation (peptide spectral matches, PSM). Although peptides may have different ionization efficiencies, PSM values can be in general related to the abundance of proteins in the capture. In the WC36B-captured proteins, Syk has 219 spectra whereas the next highest one is 79, 71, 61, 15, 11 and many at only 2 PSMs. Many of these proteins were also identified in the biotin capture, whereas not a single peptide was identified for Syk in the biotin capture. Collectively, these results show Syk is the major protein species captured by WC36B.

Source data

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots.

Source Data Fig. 4

Unprocessed western blots.

Source Data Fig. 5

Unprocessed western blots.

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed western blots.

Source Data Extended Data Fig. 5

Unprocessed western blots.

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Singaram, I., Sharma, A., Pant, S. et al. Targeting lipid–protein interaction to treat Syk-mediated acute myeloid leukemia. Nat Chem Biol 19, 239–250 (2023). https://doi.org/10.1038/s41589-022-01150-z

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