A Small Molecule Causes a Population Shift in the Conformational Landscape of an Intrinsically Disordered Protein

Intrinsically disordered proteins (IDPs) have roles in myriad biological processes and numerous human diseases. However, kinetic and amplitude information regarding their ground-state conformational fluctuations has remained elusive. We demonstrate using nuclear magnetic resonance (NMR)-based relaxation dispersion that the D2 domain of p27^Kip1, a prototypical IDP, samples multiple discrete, rapidly exchanging conformational states. By combining NMR with mutagenesis and small-angle X-ray scattering (SAXS), we show that these states involve aromatic residue clustering through long-range hydrophobic interactions. Theoretical studies have proposed that small molecules bind promiscuously to IDPs, causing expansion of their conformational landscapes. However, on the basis of previous NMR-based screening results, we show here that compound binding only shifts the populations of states that existed within the ground state of apo p27-D2 without changing the barriers between states. Our results provide atomic resolution insight into how a small molecule binds an IDP and emphasize the need to examine motions on the low microsecond time scale when probing these types of interactions

High-throughput screening reveals alsterpaullone, 2-cyanoethyl as a potent p27Kip1 transcriptional inhibitor.

p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a "bioactive" library consisting of 8,904 (4,359 unique) compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies

Four p27^Kip1 inhibitors and calculated IC₅₀ values that passed all primary and secondary screens.

<p>The activity data for individual compounds were fit into sigmoidal dose-response curves (n = 3 per compound, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091173#pone-0091173-g003" target="_blank">Fig. 3C–F</a>) to derive IC₅₀ values.</p

Primary screening for inhibition of p27^Kip1-luciferase.

<p>A. Percentage of luciferase inhibition by each compound screened (12 μM), compared to positive control cycloheximide (green), and negative control DMSO (red). Arbitrary threshold was set at 50% luciferase inhibition (yellow line). Compounds which “passed” (blue) and “failed” (black) are shown. B. Percentage of alamar blue inhibition by each compound screened (12 μM) compared to positive control cyclohexamide (green), and negative control DMSO (red). Arbitrary threshold was set at 30% alamar blue inhibition (yellow line). Compounds which inhibited alamar blue more than 30% “failed” (blue), and less than 30% “passed” (black).</p

Alsterpaullone, 2-Cyanoethyl inhibits <i>p27^Kip1</i> transcription from diverse cellular origins, and reduces p27^Kip1 protein levels.

<p>A. Immunoblot of p27^Kip1 and β-actin with indicated concentration of A2CE in HeLa cells. B. Relative intensity of p27^Kip1 normalized to β-actin by densitometry (n = 3) with indicated concentrations of A2CE. C. Fold changes of endogenous p27^Kip1 mRNA (normalized to 18s) in HEK cells treated with indicated concentrations of A2CE normalized to DMSO (n = 3). D. Alamar blue cell viability assay in HEK cells treated with indicated concentrations of A2CE. E. Fold changes of endogenous p27^Kip1 mRNA (normalized to 18s) in 3T3 cells treated with indicated concentrations of A2CE normalized to DMSO (n = 3). F. Alamar blue cell viability assay in 3T3 cells treated with indicated concentrations of A2CE G. Fold changes of endogenous p27^Kip1 mRNA (normalized to 18s) in cochlear explants treated with indicated concentrations of A2CE normalized to DMSO (n = 3). H. Alamar blue cell viability assay in cochlear explants treated with indicated concentrations of A2CE. Mean ± S.E.M., * p<0.05 (One way ANOVA, followed by bonferroni for means comparison).</p

Secondary screening of primary hits reveals four true <i>p27^Kip1</i> inhibitors.

<p>A. Workflow of the secondary screening procedures. B. Remaining hits after each step of the secondary screen. C–F. Dose-dependent luciferase inhibition (black) and alamar blue inhibition (blue) of each compound (n = 3, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091173#pone-0091173-t001" target="_blank">Table 1</a> lists the calculated IC₅₀’s). Curves were fitted by a sigmoidal dose-response curve. G–J. Luciferase levels (normalized to DMSO) over 3 doses of each compound showing no effect on SV-40 driven luciferase (n = 3). Green line represents no change from vehicle DMSO to A2CE. K–N. Dose-dependent decrease in endogenous p27^Kip1 mRNA of each compound normalized to that in DMSO control (RT-qPCR) in HeLa cells (n = 3). Mean ± S.E.M., * p<0.05 (One way ANOVA, followed by bonferroni for means comparison).</p