A Spiroligomer α-Helix Mimic That Binds HDM2, Penetrates Human Cells and Stabilizes HDM2 in Cell Culture

<div><p>We demonstrate functionalized spiroligomers that mimic the HDM2-bound conformation of the p53 activation domain. Spiroligomers are stereochemically defined, functionalized, spirocyclic monomers coupled through pairs of amide bonds to create spiro-ladder oligomers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045948#pone.0045948-Schafmeister1">[1]</a>. Two series of spiroligomers were synthesized, one of structural analogs and one of stereochemical analogs, from which we identified compound <b>1</b>, that binds HDM2 with a Kd value of 400 nM. The spiroligomer <b>1</b> penetrates human liver cancer cells through passive diffusion and in a dose-dependent and time-dependent manner <em>increases</em> the levels of HDM2 more than 30-fold in Huh7 cells in which the p53/HDM2 negative feed-back loop is inoperative. This is a biological effect that is not seen with the HDM2 ligand nutlin-3a. We propose that compound <b>1</b> modulates the levels of HDM2 by stabilizing it to proteolysis, allowing it to accumulate in the absence of a p53/HDM2 feedback loop.</p> </div

The solid-phase synthesis of spiroligomers 1 and 2 using the HMBA resin.

<p>Building blocks were successively coupled using either esterification conditions for residue <b>13</b> or conditions previously developed to achieve hindered active esters <b>14</b> or <b>15 </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045948#pone.0045948-Brown2" target="_blank">[16]</a>. Further derivatization of the oligomers followed by cyclization of the terminal DKP released spiroligomers <b>1</b> and <b>2</b> from the solid support <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045948#pone.0045948-Brown2" target="_blank">[16]</a>.</p

The structures of spiroligomers, which explored the use of different functional groups as side-chains on a fixed backbone (all centers have “<i>S</i>” stereochemistry).

<p>The structures of spiroligomers, which explored the use of different functional groups as side-chains on a fixed backbone (all centers have “<i>S</i>” stereochemistry).</p

Time dependent Western analysis of spiroligomer 1 (15 µM) and Nutlin-3 (15 µM) added to Huh7 and HepG2 cells.

<p>(A) Relative HDM2 band intensity obtained when compound <b>1</b> was incubated with Huh7 cells. This plot is the average of two gels and shows a greater than 30-fold increase in the levels of HDM2. (B) Representative western gels showing the effect on HDM2 and wt-p53 in HepG2 cells in the presence of compound <b>1</b> and Nutlin-3. (C) Plotted average HDM2 band intensity relative to control (intensity of 1) as a function of time of HepG2 cells incubated with compound <b>1</b> at 15 µM. (D) Plotted average of p53 band intensity relative to control (intensity of 1) as a function of time of HepG2 cells incubated with compound <b>1</b> at 15 µM. In (C) and (D) the results of six gels (see supporting information) are averaged and standard deviations are plotted on the data. Compound <b>1</b> leads to a transient increase in HDM2 levels at 4 hours followed by a decrease. The level of p53 also increases transiently and then decreases again and the p53 changes trail the HDM2 response.</p

The stereoisomers 8–12 and 1 were suggested by CANDO as being able to mimic the presentation of the side-chains of p53 when bound to HDM2.

<p>The stereoisomers 8–12 and 1 were suggested by CANDO as being able to mimic the presentation of the side-chains of p53 when bound to HDM2.</p

(A) Fluorescent imaging of spiroligomer 3 in Huh7 cells (spiroligomer 3 concentration 2 µM, incubated for 24 hours, washed with phosphate-buffered saline (PBS), followed by fixation of the cells.

<p>(B) HepG2 cells that were incubated with spiroligomer <b>1</b> (0.5 mM) for 15 hours, washed with PBS and then imaged with fluorescent microscopy (concentration 0.5 µM), incubated for 15 hours and then imaged.</p