Enhanced Cytotoxicity through Conjugation of a “Clickable” Luminescent Re(I) Complex to a Cell-Penetrating Lipopeptide

Re­(I) tricarbonyl polypyridine-based complexes are particularly attractive metal complexes in the field of inorganic chemical biology due to their luminescent properties, ease of conjugation to targeting biomolecules, and the possibility to prepare their “hot” ^99mTc analogues for radioimaging. In this study, we prepared and characterized a novel, “clickable” complex, [Re­(2,2′-bipyridine)­(3-ethynylpyridine)­(CO)₃]­(BF₄) (<b>[Re­(CO)</b>_<b>3</b><b>(bipy)­(py-alkyne)]­(BF</b>_<b>4</b><b>)</b>), exhibiting the characteristic luminescent properties and moderate cytotoxicity of this general class of compound. Using Cu­(I)-catalyzed “click” chemistry, the complex was efficiently attached to a lipidated peptide known to increase cell permeability, namely, the myristoylated HIV-1 Tat peptide (<b>myr-Tat</b>), to give <b>Re-myr-Tat</b>. Fluorescence microscopy localization in human cervical cancer cells (HeLa) confirmed enhanced cellular uptake of <b>Re-myr-Tat</b> compared with <b>[Re­(CO)</b>_<b>3</b><b>(bipy)­(py-alkyne)]­(BF</b>_<b>4</b><b>)</b>, and cytotoxicity studies showed that this resulted in an increase in potency to a level comparable with cisplatin (13.0 ± 2.0 μM)

Synthetic scheme for N⁷-subtituted 8-MG analogues.

<p>Synthetic scheme for N⁷-subtituted 8-MG analogues.</p

X-ray structure data processing and refinement statistics.

*<p>R_merge = ΣhΣi |<i>I</i>i(h) - &lt;<i>I</i>(h)&gt;|/ΣhΣi<i>I</i>i (h),</p>#<p>R_pim = Σh [1/(N-1)]1/2 Σi |<i>I</i>i(h) - &lt;<i>I</i>(h) &gt;|/ΣhΣi<i>I</i>i (h).</p><p>Values in parentheses refer to the outer resolution shell (1.74–1.65 Å).</p><p>Where <i>I</i> is the observed intensity, &lt;<i>I</i>&gt; is the average intensity of multiple observations from symmetry-related reflections, and N is redundancy.</p><p>R_value = _jjFoj _ jFcjj/_jFoj, where Fo and Fc are the observed and calculated structure factors. For R_free the sum is done on the test set reflections (5% of total reflections), for R_work on the remaining reflections.</p

Thermodynamic parameters for the binding of selected compounds to <i>Sa</i>HPPK as determined by ITC^a.

a<p>Values are the means ± the standard deviation for at least three experiments. All ITC and SPR experiments were performed at 298 K and 293 K, respectively. ^b data from Chhabra <i>et al</i> PlosONE 2012.</p

Structures of <i>C</i>⁸, <i>N</i>⁹ and <i>N</i>⁷-substituted guanine analogues and their binding affinities to <i>Sa</i>HPPK, as determined by SPR.

a<p>in the presence of 10 mM Mg^2+.</p>b<p>in the presence of 10 mM Mg²⁺/1 mM ATP.</p>c<p>nd: not determined.</p

Synthetic scheme for compound 21e.

<p>Synthetic scheme for compound 21e.</p

Synthetic scheme for N⁹-subtituted 8-MG analogues.

<p>Synthetic scheme for N⁹-subtituted 8-MG analogues.</p

Structure of SaHPPK in complex with 8-MG.

<p>A–C) Structure of <i>Sa</i>HPPK (PDB:1QBC) in complex with 8-MG. A–B Intermolecular interactions between 8-MG and <i>Sa</i>HPPK. C) Surface representation of <i>Sa</i>HPPK showing the bound 8-MG (blue) overlayed with the closed loop L3 (green) and the bound AMPCPP as observed in the <i>Ec</i>HPPK/HMDP/AMPCPP (PDB:1Q0N) complex. D) Ribbon representation of the loop structure of several <i>Ec</i>HPPK structures overlayed with <i>Sa</i>HPPK (yellow) in complex with 8-MG (red) to illustrate the range of conformations in loops L2 and L3. The interaction of the Trp89 (brown) and the phenethyl inhibitor (cyan) is highlighted (PDB:1DY3) and the position of the HMDP (pink) and AMPCPP (pink) from <i>Ec</i>HPPK/HMDP/AMPCPP (PDB:1Q0N). Images were produced using the UCSF Chimera package (<a href="http://www.cgl.ucsf.edu/chimera" target="_blank">www.cgl.ucsf.edu/chimera</a>).</p

HPPK function and known inhibitors.

<p>A) HPPK catalysis. B) Known inhibitors of HPPK.</p

Comparing the binding of 21a and 8-MG to apo and cofactor bound SaHPPK as judged by 2D NMR.

<p>A–B) Binding of 8-MG and <b>21a</b> to magnesium bound <i>Sa</i>HPPK are very similar. C–D) Binding of 8-MG and <b>21a</b> to the AMPCPP bound <i>Sa</i>HPPK are very different. Figures A and C are adapted from data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059535#pone-0059535-g006" target="_blank">Figure 6A</a> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059535#pone.0059535-Chhabra1" target="_blank">[8]</a>. The concentration of ¹⁵N-labelled <i>Sa</i>HPPK was ∼100 µM in all cases. The concentration of magnesium, AMPCPP, 8-MG and <b>21a</b> was 10 mM, 1 mM, 0.6 mM and 0.6 mM respectively. The assignment of selected substrate site peaks are shown to highlight the effects of binding of the two compounds on the NMR spectra. The sidechain Hε1–Nε1 peak of Trp89 is labelled as W89sc.</p