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. 2021 Jun 30;143(25):9297-9302.
doi: 10.1021/jacs.1c03258. Epub 2021 Jun 17.

Norstictic Acid Is a Selective Allosteric Transcriptional Regulator

Julie M Garlick  1   2 , Steven M Sturlis  1   2 , Paul A Bruno  1   2 , Joel A Yates  3 , Amanda L Peiffer  1   4 , Yejun Liu  1   4 , Laura Goo  3 , LiWei Bao  3 , Samantha N De Salle  1   4 , Giselle Tamayo-Castillo  5 , Charles L Brooks 3rd  2   4   6 , Sofia D Merajver  3 , Anna K Mapp  1   2   4
Affiliations

Norstictic Acid Is a Selective Allosteric Transcriptional Regulator

Julie M Garlick et al. J Am Chem Soc. .

Abstract

Inhibitors of transcriptional protein-protein interactions (PPIs) have high value both as tools and for therapeutic applications. The PPI network mediated by the transcriptional coactivator Med25, for example, regulates stress-response and motility pathways, and dysregulation of the PPI networks contributes to oncogenesis and metastasis. The canonical transcription factor binding sites within Med25 are large (∼900 Å2) and have little topology, and thus, they do not present an array of attractive small-molecule binding sites for inhibitor discovery. Here we demonstrate that the depsidone natural product norstictic acid functions through an alternative binding site to block Med25-transcriptional activator PPIs in vitro and in cell culture. Norstictic acid targets a binding site comprising a highly dynamic loop flanking one canonical binding surface, and in doing so, it both orthosterically and allosterically alters Med25-driven transcription in a patient-derived model of triple-negative breast cancer. These results highlight the potential of Med25 as a therapeutic target as well as the inhibitor discovery opportunities presented by structurally dynamic loops within otherwise challenging proteins.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
(A) The hub protein Med25 is a subunit of the Mediator coactivator complex. The activator interaction domain (AcID) forms a PPI network with transcriptional activators using two binding surfaces (H1 and H2) and in doing so regulates key cellular processes.,– PDB entry 2XNF was used to generate figure. (B) Schematic of the high-throughput screen to identify inhibitors of the Med25 AcID PPI network. See the Supporting Information for full screening details.
Figure 2.
Figure 2.
(A) Chemical structures of the top two hits emerging from the screen of Med25 AcID fl-VP16(465–490) along with (B) their apparent IC50 values, which were determined through titrations of either NA or PA against Med25 AcID fl-VP16(465–490) performed in triplicate with the indicated error (SDOM). Full experimental details are reported in the Supporting Information. (C) Assessment of related structures shows that the orthophenoxyaldehyde moiety is important but not sufficient for inhibitory activity. IC50 values were determined via competition fluorescence polarization against Med25 VP16(465–490). Of these molecules, covalent adducts with Med25 were observed only for baeomycesic acid, indicating that the orthophenoxyl group is integral to stable imine formation. (D) Inhibition of related PPI networks by NA. Apparent IC50 values were measured via fluorescence polarization against a suite of coactivator domains (CBP KIX, p300 TAZ1, CBP IBiD, Med15 KIX) bound to fluorescein-tagged activators. The values are averages of three independent experiments with the indicated error (SDOM). No error bars are shown for the IC50 against IBiD ACTR because the IC50 was greater than the highest concentration of NA tested (250 μM), and thus, we can accurately report the IC50 only as >250 μM. Full details are reported in the Supporting Information.
Figure 3.
Figure 3.
(A) LC–MS analysis of norstictic acid covalent adduct formation with Med25 lysine-to-arginine mutants indicates that K519R leads to the most significant reduction of labeling. No reduction of labeling corresponds to a decrease in abundance of the NA covalent adduct of less than 10%. Minimal reduction in labeling, observed for K520R, corresponds to a 22% reduction in the mass abundance of the NA covalent adduct. Significant reduction in labeling, observed for K519R, corresponds to a 53% reduction in the mass abundance of the +1 covalent adduct. The Supporting Information provides additional quantitative analysis. PDB entry 2XNF was used to generate the figure. (B) Inhibition of the Med25 AcID ETV5 interaction by NA measured using fluorescence polarization. Mutants containing K519R, highlighted in gray, demonstrate the most significant increase in apparent IC50. Values represent averages of three independent experiments with the indicated error (SDOM) (C) (left) Centroid structure of the most populated cluster from molecular dynamics simulations, where NA binds to the H2 face of Med25 and covalently links to K519. (right) The residues that showed the greatest reduction in fluctuations (RMSF) upon activator binding all occur on dynamic substructures on the H1 face.
Figure 4.
Figure 4.
(A) Norstictic acid significantly stabilizes full-length Med25 in HeLa cell extracts. Cellular thermal shift assays were conducted by dosing HeLa cell nuclear extracts with 25 μM NA or equivalent DMSO and subjecting the samples to a range of temperatures. Western blot using a Med25 antibody shows increased band density in NA-dosed samples compared with the control samples, indicating thermal stabilization and target engagement. Quantification and CETSA at additional concentrations are shown in Figure S10. (B) Treatment of HeLa cells with 25 μM NA attenuates the formation of the Med25 ETV5 complex. (left) Representative Western blot showing a reduction in co-immunoprecipitation of ETV5 with Med25. (right) Quantitative assessment of ETV5 co-immunoprecipitation with Med25. The Western blot band density was measured using ImageJ and normalized by comparison to overall Med25 levels. Results are averages of biological triplicates. See Figure S11 for the blot images used. (C) NA shows positive synergy with the on-pathway kinase inhibitor lapatinib in MDA-MB-231 cells. IC50 values of fixed dose ratios of NA and lapatinib were measured after 2 days using a cell viability assay (MTT) and plotted on an isobologram. See the Supporting Information for additional experimental details. (D) Analysis of MMP2 transcript levels by qPCR indicates that NA treatment decreases MMP2 levels to that of a knockout (KO) variant of the cell line. MMP2 transcript levels are normalized to the reference gene RPL19. Results shown are averages of technical triplicate experiments conducted in biological duplicate. (E) Western blot showing that treatment of VARI068 cells with NA attenuates the formation of the Med25 ETV5 complex observed via co-immunoprecipitation.
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