Extended Data Fig. 9: The polycationic polyamine analogue BENSpm allows the distinction of conventional polyamine activities and metabolic shielding.
From: Polyamine-dependent metabolic shielding regulates alternative splicing
a, Potential of mean force (PMF) profiles for the binding free energy calculations of BENSpm with SF3A3 by umbrella sampling simulations. b, Kernel density estimates of salt bridge distances involving BENSpm and seven selected glutamic residues flanking the phosphorylatable positions Ser365, Ser367, and Ser369. A maximum in the 3─3.6 Å range is indicative of the presence of dynamic salt bridges with BENSpm. c, Snapshot from a MD simulation (t = 10 ns) of the SF3A3:CK1 complex in the presence of BENSpm (in green). ATP: magenta balls-and-sticks; Mg2+: green sphere; phosphorylatable residues (Ser365, Ser367, Ser369): yellow sticks; CK1 catalytic residues (Asp157, Asp136): pink sticks. Non-polar hydrogens omitted. d, Selected region of the overlaid 1H-1H-TOCSY NMR spectra of SRRM1 fragment (as a negative control) in the absence (black) or presence (from blue to yellow) of increasing amounts of BENSpm (top) and their quantification (bottom). One spectrum recorded along the titration is not shown for the sake of clarity. The line is fitting to a 1:1 binding model and the constant dissociation is indicated with the standard error. e, LC-MS quantification of total polyamines (left) and [¹³C]-methionine incorporation (right) in DU145 cells treated ± SAM486A (1 µM) + DFMO (50 µM) and ± BENSpm (10 µM). f–i, Foci formation and growth assays in DU145 cells treated with SAM486A (1 µM) + DFMO (50 µM) ± BENSpm (10 µM) or spermine (10 µM). Representative colony formation (f); quantification: colony number (left), crystal violet absorbance (right) (g); representative growth assay by crystal violet staining (h); quantification of growth experiments (i). Abbreviations: S: SAM486A, D: DFMO, ppm: parts per million, CSP: chemical shift perturbation. ND: non-detectable. Statistics: One-tailed paired student’s t-test for metabolomics (e). One-tailed one-sample t-test for quantifications in (g,i). *p < 0.05, **p < 0.01, ***p < 0.001. Dollar signs: Two-tailed paired t-test comparing S + D vs S + D+BENSpm or S + D+Spm (g, i). $ p < 0.05, $$ p < 0.01. Error bars: Mean ± S.E.M. n= independent biological replicates (e, f, g, h, i: 4).
