New p32/gC1qR Ligands for Targeted Tumor Drug Delivery

Abstract

Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50,000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery.

Keywords: cancer; drug delivery; high-throughput screening; nanoparticles; peptides.

Figure 1. Multistep development of systemic homing compounds using reiterative biopanning and FP-based screening

A) In vivo phage display is used for mapping of systemically accessible diversity of vascular beds. It yields primary homing peptides that can be used for biochemical identification of binding partners, “receptors” (denoted as a red square with cavity). B) In vitro biopanning on purified receptors is used for identification of secondary receptors of improved binding properties. C) Fluorescence polarization assays is used for high-throughput screening of compounds that bind to peptide binding site on target receptors. Top: when the rapidly rotating FAM-labeled peptide binds to its larger partner protein, its rotation approximates the slower rotation of this larger protein, so the polarization of the excitation light is retained in the emitted light (light green emission thunderbolts aligned with light blue excitation thunderbolts). Below: in the presence of competitor compound, an increased concentration of free fast-rotating FAM-labeled peptide is seen as decrease in fluorescence polarization (divergent excitation thunderbolts)

Figure 2. In vitro identification and characterization of novel p32 binding peptides

A) CX7C peptide library was used for in vitro selection on hexahistidine-tagged recombinant p32 protein immobilized on Ni-NTA magnetic beads (Qiagen, Hilden, Germany). Binding of phage particles in each selection round is expressed fold control phage displaying heptaglycine peptide (G7). B) Representative peptide sequences recovered after three rounds of ex vivo selection. TT1 peptide (CKRGARSTC) was present 3 times among 30 sequenced phage clones. Note emergence of RGXRS consensus motif in selected peptide pool. C) Binding of TT1, CKRGNRSMC, CTRGSRSKC and LyP-1 phage to immobilized p32 protein. The data are representative of four independent binding experiments. Binding is expressed as fold over control phage displaying polyglycine heptapeptide (G7). Statistical analysis was performed by one-way ANOVA (F: 15.2; F crit 3.4) Error bars indicate s.e.m.; triple asterisk, p<0.001, n=4..

Figure 3. FP- and phage binding- based validation of p32 binding peptides and hit compounds

A) Fluorescence polarization (FP) measurement of binding affinity of FAM-LyP-1 and FAM-TT1 peptides to p32 protein, and dissociation constants (K_D) based on binding curves fitted on Michaelis-Menten kinetics. Measurements were performed with PheraStar FS plate reader (BMG Labtech, Ortenberg, Germany) on 384-well plate. B) Affinity of the 7 hit compounds in dose-dependent (8.25 μM to 100 μM) inhibition of FAM-TT1 binding to p32 in FP assay. C) Assessment of hit compound binding to a non-target secondary protein, NRP-1 to determine the specificity of the hit compounds in FP assay. A known NRP-1 binding peptide, RPARPAR, was used as a control. D) Effect of the hit compounds on the TT1 phage binding to p32 protein. TT1 phage binding to p32 in the presence of 100 μM hit compounds was determined in ELISA-type of binding assay.Library compounds can be converted into Pubchem Compound identifiers using the utility by Chembridge: http://www.chembridge.com/conversion_tool/index.php. Statistical analysis was performed by one-way ANOVA. Significance of differences between individual data points was determined by paired t-test. *P < 0.05, **P < 0.005 and ***P < 0.001. Each data point presents average ± S.D., n=4.

Figure 4. NMR based evaluation of binding of compounds 4014008 and 7933989 to P32 protein

The 1D ¹H-aliph spectra of 5 μM P32 in the absence (black) and presence of compounds #4014008 (A) and #7933989 (B) (red for 100 μM and blue for 500 μM) were collected. As a control, the spectrum of p32 in the presence of the known peptide binder, TT1 at 70 μM, was also collected (green). In presence of both compounds there is a shift in the peak at around −0.3 ppm (dashed line) and the concomitant appearance of a shoulder peak (indicated by arrows) similar to what observed in presence of the reference peptide TT1.

Figure 5. Nanoparticles functionalized with #4014008 bind to p32 protein and home to p32-positive breast tumors

(A) The compound coupled to fluorescent silver nanoparticles showed specific binding toward plate well bound p32 protein, relative to wells having a non-target control protein N3A. Increased concentration of silver nanoparticles caused a dose-dependent increase in fluorescent signal due to nanoparticle binding to the surface of the well. (B–D) Confocal imaging of tissue sections of MCF10Ca1A breast tumors from mice injected with FAM-#4014008 -NW. Red: p32; green: NW; blue: nuclei. Arrows point to vascular structures that show co-localization of nanoparticle and p32 signals. Representative fields from multiple sections of three independent mice are shown. Scale bars = 100 μm.