Many lead molecules determined in drug discovery campaigns are eliminated from consideration due to poor solubility and low cell permeability. cancer cells in vitro and tumors in vivo without toxic adjuvants. These results suggest SVS-1 can serve as a simple, effective delivery platform for molecules with poor solubility and permeability. design of an anticancer peptide, named SVS-1. This peptide kills cancer cells via a lytic mechanism that involves its cell surface-induced folding . Herein, we report that when cancer ARRY-334543 cells are presented with concentrations of SVS-1 that are below its IC50 for lytic action, the peptide does not kill the cells but rather rapidly translocates across the cell membrane into the cytoplasm, and ultimately the nucleus. We show that cell-surface binding triggers the folding of the peptide into a -hairpin conformation that rapidly partitions into the membrane. Thus, the cell-surface -folding event triggers the internalization activity of SVS-1, a mechanism unique to this peptide. When a drug is attached to SVS-1, this serves as an effective means for its intracellular delivery (Fig. 1). Fig. 1 Design of the SVS-1-Paclitaxel conjugate. SVS-1 peptide (green) is conjugated to paclitaxel (PTX) (red) through a PEG spacer (blue) and self-immolative di-sulfide linker (purple). The PTX-S-S-PEG-SVS-1 conjugate electrostatically engages the negatively … SVS-1 (KVKVKVKVDPPTKVKVKVK-NH2) is an 18 amino acid peptide designed to bind to, and fold at, negatively charged cancer cell surfaces [1, 2]. The peptide contains two strands of alternating lysine and valine residues, which flank a tetrapeptide motif (-VDPPT-) designed to adopt a type II -turn when the peptide is folded. Previous studies have shown that in the absence ARRY-334543 of a cell surface, the SVS-1 peptide adopts an ensemble of random coil, bio-inactive conformations . Electrostatic repulsion between the peptides charged lysine side chains keeps it in the unfolded state. However, when presented with a negatively charged surface, such as that displayed by malignant cells, these side chains electrostatically engage the anionic lipid head groups and glycans at the cells surface. This binding event triggers the folding of the ARRY-334543 -hairpin, where the ARRY-334543 lysine and valine residues are displayed from opposite faces of the folded conformer. In this folding mechanism, the lysine-rich face of the hairpin is engaged in electrostatic interactions with the cell and its valine face is solvated by water. Solvation of the valine-rich hydrophobic face is entropically unfavorable and, as a result, the folded peptide interpolates into the membrane to release the ordered water. We discovered that at peptide concentrations below that needed to induce SVS-1s lytic action, the peptide rapidly enters cells without effecting cell viability. Studies using differentially labeled analogs and enantiomeric peptides show that SVS-1 rapidly and preferentially penetrates cancer cells CDH1 through mechanisms involving physical translocation and to a lesser extent clathrin-dependent endocytosis. Ligating the model hydrophobic drug Paclitaxel (PTX) to SVS-1 improved its aqueous solubility by ~1000-fold and successfully delivered and released PTX to cancer cells in vitro. Although SVS-1 is moderately stable to serum proteolysis (t1/2 ~ 6h), its studies following all animal welfare regulations as detailed by the Animal Care and Use Committee (ACUC). All peptides utilized for experiments were prepared with an amidated C-terminus. 2.2 Synthesis of fluorescently-labeled peptides (2) Synthesis of fluorescently-labeled PEG-GG-SVS-1 conjugates was performed as described in Supplementary Scheme S1. Briefly, resin-bound GG-SVS-1 (GGKVKVKVKVDPPTKVKVKVK-NH2), the mirror image enantiomer peptide GG-on resin was reacted with 3-(tritylthio)propionic acid (3 eq.) in NMP containing DIEA (6 eq.) and HCTU (3 eq.) for 2 h at room temperature while shaking to produce 3. Dried resin-bound trityl-S(CH2)2CO-PEG-GG-SVS-1 (3), and its enantiomer, were cleaved from the resin and simultaneously side-chain deprotected using a trifluoroacetic acid/thioanisole/1,2-ethanedithiol/anisole (90:5:3:2) cocktail for 2 h under argon atmosphere. The crude products were precipitated with cold diethyl ether and then dried. Purification was performed by reverse-phase HPLC following the procedure for compound 2, with fractions collected directly in a round bottom flask on dry-ice to immediately freeze the product. Lyophilization produced (4) and its enantiomer as powders. 2.5 2-(2-(pyridin-2-yldisulfanyl)phenyl)acetic acid ARRY-334543 (5) In a typical reaction, 2-mercaptophenylacetic acid (100 mg, 0.594 mmol) is dissolved in MeOH (0.5.