Fig 1: Selectivity of C28 toward LMTK3.(A) Selectivity profile of C28 (1 μM) against 140 kinases using radioactive filter binding assay. The data are displayed as percent activity remaining of assay duplicates with an SD. Only kinases with >50% decrease in their activity are shown. The relative IC50 values are also presented. (B) Treespot interaction map depicting the kinome phylogenetic grouping, with kinases interacting with C28 (5 μM) represented as red circles. The larger the diameter of the circle, the higher the C28 binding affinity to the respective kinase. Kinases whose binding was inhibited by C28 to less than 10% of the control (DMSO) are shown. Lower numbers indicate most probable hits to bind to C28. Overlapping kinases identified in assays (radioactive filter binding and site-directed competition binding) are highlighted in yellow. (C) Western blots of LMTK3 and ERα in BC cell lines treated with increasing concentrations of C28 at different time points. Mean densitometry values of three independent experiments were calculated using ImageJ. GADPH, glyceraldehyde 3-phosphate dehydrogenase. (D) Effects of C28 on LMTK3 protein half-life in MDA-MB-231 cells. Cells were treated with CHX (cycloheximide) (100 μg/ml) and 10 μΜ C28 (or DMSO) for different time points. The relative LMTK3 protein levels (−/+ C28) were calculated and plotted against the time of treatment with CHX. (E) Western blots of total and phospho-HSP27 (normalized versus total) in MCF7, T47D, and MDA-MB-231 cell lines treated with 10 μΜ C28 for different time points. Mean densitometry values of three independent experiments are shown. (F) Western blots of total and phospho-HSP27 (normalized versus total) in BC cell lines treated with either control or LMTK3 small interfering RNAs (siRNAs) for 72 hours. Mean densitometry values of three independent experiments are shown. (G) Western blots of different kinases in BC cell lines treated with C28 (10 μΜ) at different time points.
Fig 2: Identification of C28 as a potent inhibitor against LMTK3.(A) Top: Experimental pipeline to identify LMTK3 inhibitors. Middle: HTRF data showing coverage of different inhibitors per active cluster. Green stars are the 868 compounds chosen for further confirmation, showing >50% mean inhibition (blue crosses are nonselected compounds). Selections were biased toward higher potency, sensible calculated physicochemical properties, and structural coverage within each cluster. Bottom: HTRF data showing the range of IC50 values and purity of the top 160 compounds. (B) The IC50 value for C28 against LMTK3cat was determined by in vitro kinase assays. (C) EC50 values for C28 in FDCP1 and FDCP1-LMTK3 cell lines. Error bars represent the means ± SD from three independent experiments. (D) Table summarizing the IC50 and EC50 values of the top 38 compounds. (E) Chemical structure of C28. (F) Characteristic thermal denaturation curves of LMTK3 (black) and LMTK3/C28 complex (red) as monitored by DSF and (G) CD spectroscopy, indicating the increased protein thermodynamic stability upon ligand binding. Tm values from DSF were determined from the maximum in the first derivative of the fluorescence with respect to the temperature, or the midpoint in the transition region by fitting a Boltzmann sigmoidal to the CD data. Experiments were performed in triplicate. DMSO, dimethyl sulfoxide; OD, optical density; RFU, relative fluorescence units. (H) Kinetic analysis of HSP27 phosphorylation by LMTK3 in the absence or presence of C28. Kinetic parameters were determined from nonlinear regression fit of the initial reaction rates as a function of HSP27 concentration to the Michaelis-Menten equation using Prism 8. (I) Kinetic analysis as a function of ATP concentration for 0.6 μM HSP27 substrate, in the absence or presence of C28. Kinetic parameters were determined from nonlinear regression fit of the initial reaction rates as a function of ATP concentration to the Michaelis-Menten equation using Prism 8.
Fig 3: Defining the LMTK3 consensus phosphorylation motif and identifying HSP27 as an LMTK3 substrate.(A) A spatially 198 components arrayed PSPL was subjected to in vitro phosphorylation with active LMTK3cat. A representative image of the average log2 values of two independent experiments is shown. (B) Scaled-sequence PhosphoLogo representation of the LMTK3 consensus phosphorylation motif. The size of the letter is proportional to the signal for the corresponding amino acid at the indicated position. (C) In vitro kinase assays using wild-type (WT) LMTK3cat as source of enzyme activity and peptide variants with individual amino acid substitutions at different positions. Data shown are the average of two separate experiments (±SEM). (D) Top: In vitro kinase assay using recombinant HSP27 as a substrate and WT LMTK3cat or kinase-dead (KD) LMTK3 mutant (LMTK3cat-KD) as source of enzyme activity. Bottom: Time course in vitro kinase assay using recombinant HSP27 and LMTK3cat. (E) Schematic representation of SILAC proteomic experiment. Western blotting analysis of LMTK3 and FLAG-LMTK3 protein levels showing the transient overexpression of full-length pCMV6-LMTK3 (FLAG-tag) plasmid. m/z, mass/charge ratio. (F) Volcano scatter plot showing the log2 “normalized ratios” (H/L) against log10 “intensity” (H+L) for each characterized phosphorylated protein (phosphopeptide) following overexpression of WT-LMTK3 in MCF7 cells. Proteins are displayed in circles based on P values from significant B test. Red, P < 0.001; yellow, 0.001 > P < 0.01; green, 0.01 > P < 0.05; blue, P > 0.05.
Fig 4: Oral GGA treatment redistributes HSPB5 and HSPB1 from the cytosol to the myofilaments in the left ventricle of obese ZSF1 rats. (a and b) HSPB5 and HSPB1 levels were similar between myocardia of all groups in cytosolic fraction. (c and d) myofilament levels of HSPB5 and HSPB1 were higher in the myocardium of obese ZSF1 rats treated with GGA when compared to vehicle‐treated obese and lean ZSF1 rats. (e and f) There was a redistribution of HSPB5 and HSPB1 to the myofilaments (myofilament expression levels relative to cytosolic expression levels) in the GGA‐treated obese ZSF1 rats compared to lean and vehicle‐treated obese ZSF1 rats, although this was particularly strong for HSPB1. Data are expressed as mean ± SEM, n = 4–5 per group. A one‐way ANOVA with a Bonferroni's post hoc test was utilized to assess differences between the groups. (g and h) Representative images of confocal laser microscopy for HSPB5 (upper panel) and HSPB1 (lower panel) in LV sections from lean, obese and obese ZSF1 rats treated with GGA (200 mg/kg/day). Immunohistochemical visualization of cell membranes (WGA; green), nuclei (DAPI; blue), and HSPB5/HSPB1 (red) was achieved with confocal laser microscopy. Both HSPB5 and HSPB1 immunostaining primarily occurred in the vicinity of the myofilaments for all rats. Greater HSPB5 immunostaining in the vicinity of the myofilaments was observed in both vehicle‐treated and GGA‐treated obese ZSF1 rats, whereas prominent elevation of HSPB1 immunostaining was noted in the GGA‐treated obese ZSF1 rats. n = 4 per group.
Fig 5: Recombinant human HSPB5 (0.01 mg/mL) and human HSPB1 (0.01 mg/mL) reduce stiffness of permeabilized cardiomyocytes of obese ZSF1 rats and abrogate the effect of oral GGA treatment. Stiffness was measured as the passive force (Fpassive). (a and b) SL‐Fpassive curves after HSP treatment for lean, vehicle‐treated obese and GGA‐treated obese ZSF1 rats after incubation with recombinant human HSPB5 (a) or human HSPB1 (b). A second order polynomial (quadratic function) was used for curve fitting. For comparison, the Fpassive curve of cardiomyocytes isolated from vehicle‐treated obese ZSF1 rats (dashed line, triangles) and the Fpassive curve of cardiomyocytes isolated from lean rats are shown in panels a and b. (c–f) comparison of HSP effects on Fpassive of cardiomyocytes in vehicle‐ and GGA‐treated rats. Data are expressed as mean ± SD. ‡ p < 0.0001, † p = 0.0001 and *p < 0.05 vs. baseline of the same group. N = 4–5 per group, with four cardiomyocytes tested per rat. Effects of recombinant sHSPs were established using a two‐way ANOVA with a Bonferroni's post hoc test to correct for differences between the groups.
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