Linker Hydrophilicity Modulates the Anticancer Activity of RGD-Cryptophycin Conjugates.

Most anticancer agents are hydrophobic and can easily penetrate the tumor cell membrane by passive diffusion. This may impede the development of highly effective and tumor-selective treatment options. A hydrophilic ß-glucuronidase-cleavable linker was used to connect the highly potent antimitotic agent cryptophycin-55 glycinate with the αv ß3 integrin ligand c(RGDfK). Incorporation of the self-immolative linker containing glucuronic acid results in lower cytotoxicity than that of the free payload, suggesting that hydrophilic sugar linkers can preclude passive cellular uptake. In vitro drug-release studies and cytotoxicity assays demonstrated the potential of this small molecule-drug conjugate, providing guidance for the development of therapeutics containing hydrophobic anticancer drugs.

Size-Dependent Cellular Uptake of RGD Peptides.

Monomeric RGD peptides show unspecific fluid-phase uptake in cells, whereas multimeric RGD peptides are thought to be internalized by integrin-mediated endocytosis. However, a potential correlation between uptake mechanism and molecular mass has been neglected so far. A dual derivatization of peptide c(RGDw(7Br)K) was performed to investigate this. A fluorescent probe was installed by chemoselective Suzuki-Miyaura cross-coupling of the 7-bromotryptophan and a poly(ethylene glycol) (PEG) linker was attached to the lysine residue. Flow cytometry and live cell imaging confirmed unspecific uptake of the small, non-PEGylated peptide, whereas the PEG5000 peptide conjugate unveiled a selective internalization by M21 cells overexpressing αv ß3 and no uptake in αv -deficient M21L cells.

Phenothiazine-Biaryl-Containing Fluorescent RGD Peptides.

Cyclic RGD peptides are well-known ligands of integrins. The integrins αV ß3 and α5 ß1 are involved in angiogenesis, and integrin αV ß3 is abundantly present on cancer cells, thus representing a therapeutic target. Hence, synthetic and biophysical studies continuously are being directed towards the understanding of ligand-integrin interaction. In this context, the development of versatile synthetic strategies to obtain fluorescent building blocks that can add molecular diversity and modular spectral characteristics while not compromising binding affinity or selectivity is a relevant task. An on-resin intramolecular Suzuki-Miyaura cross-coupling (SMC) between l- or d-7-bromotryptophan (7BrTrp) and a phenothiazine (Ptz) boronic acid affords fluorescent cyclic RGD pseudopeptides, c(RGD(W/w)Ptz). Ring closure by SMC establishes a phenothiazine-indole moiety with axial chirality. An array of eight novel compounds has been synthesized, among them one fluorescent compound with good affinity to integrin αV ß3 . The fluorescence properties of the analogues can be efficiently tuned depending on the substituents in Ptz moiety even for fluorescence emission in the visible (red) spectral range.

EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids.

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.

Cyclic RGD and isoDGR Integrin Ligands Containing cis-2-amino-1-cyclopentanecarboxylic (cis-ß-ACPC) Scaffolds.

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-ß-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified αvß3 and α5ß1 receptors using biotinylated vitronectin (αvß3) and fibronectin (α5ß1) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for αvß3 over α5ß1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αvß3). The two RGD ligands showed IC50 values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC50 value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the αVß3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.

Synthesis and antimicrobial activity of binaphthyl-based, functionalized oxazole and thiazole peptidomimetics.

Thirty two new binaphthyl-based, functionalized oxazole and thiazole peptidomimetics and over thirty five novel leucine-containing intermediate oxazoles and thiazoles were prepared in this study. This includes the first examples of the direct C-5 arylation of an amino acid dipeptide-derived oxazole. Moderate to excellent antibacterial activity was observed for all new compounds across Gram positive isolates with MICs ranging from 1-16 µg mL(-1). Results for Gram negative E. coli and A. baumannii were more variable, but MICs as low as 4 µg mL(-1) were returned for two examples. Significantly, the in vitro results with a fluoromethyl-oxazole derivative collectively represent the best obtained to date for a member of our binaphthyl peptide antimicrobials.

Tuning the Biological Activity of RGD Peptides with Halotryptophans†.

An array of l- and d-halotryptophans with different substituents at the indole moiety was synthesized employing either enzymatic halogenation by halogenases or incorporation of haloindoles using tryptophan synthase. Introduction of these Trp derivatives into RGD peptides as a benchmark system was performed to investigate their influence on bioactivity. Halotryptophan-containing RGD peptides display increased affinity toward integrin αvß3 and enhanced selectivity over integrin α5ß1. In addition, bromotryptophan was exploited as a platform for late-stage diversification by Suzuki-Miyaura cross-coupling (SMC), resulting in new-to-nature biaryl motifs. These peptides show enhanced affinity toward αvß3, good affinity to αvß8, and remarkable selectivity over α5ß1 and αIIbß3 while featuring fluorogenic properties. Their feasibility as a probe was demonstrated in vitro. Extensive molecular dynamics simulations were undertaken to elucidate NMR and high-performance liquid chromatography (HPLC) data for these late-stage diversified cyclic RGD peptides and to further characterize their conformational preferences.

Cyclization of RGD Peptides by Suzuki-Miyaura Cross-Coupling.

Halogenated l- or d-tryptophan obtained by biocatalytic halogenation was incorporated into RGD peptides together with a variety of alkyl or aryl boronic acids. Suzuki-Miyaura cross-coupling either in solution or on-resin results in side chain-to-tail-cyclized RGD peptides, for example, with biaryl moieties, providing a new dimension of structure-activity relationships. An array of RGD peptides differing in macrocycle size, the presence of d-amino acid, N-methylation, or connectivity between the indole moiety and the boronic acid showed that, in particular, connectivity exhibits a major impact on affinities toward integrins, for example, αVß3. Structure-activity relationship studies yielded peptides with affinities toward αVß3 in the low nanomolar range, good selectivity, and high plasma stability. Structural characteristics of representative molecules have been investigated by molecular dynamics simulations, which allowed understanding the observed activity differences.

Synthesis and Biological Evaluation of RGD⁻Cryptophycin Conjugates for Targeted Drug Delivery.

Cryptophycins are potent tubulin polymerization inhibitors with picomolar antiproliferative potency in vitro and activity against multidrug-resistant (MDR) cancer cells. Because of neurotoxic side effects and limited efficacy in vivo, cryptophycin-52 failed as a clinical candidate in cancer treatment. However, this class of compounds has emerged as attractive payloads for tumor-targeting applications. In this study, cryptophycin was conjugated to the cyclopeptide c(RGDfK), targeting integrin αvß3, across the protease-cleavable Val-Cit linker and two different self-immolative spacers. Plasma metabolic stability studies in vitro showed that our selected payload displays an improved stability compared to the parent compound, while the stability of the conjugates is strongly influenced by the self-immolative moiety. Cathepsin B cleavage assays revealed that modifications in the linker lead to different drug release profiles. Antiproliferative effects of Arg-Gly-Asp (RGD)⁻cryptophycin conjugates were evaluated on M21 and M21-L human melanoma cell lines. The low nanomolar in vitro activity of the novel conjugates was associated with inferior selectivity for cell lines with different integrin αvß3 expression levels. To elucidate the drug delivery process, cryptophycin was replaced by an infrared dye and the obtained conjugates were studied by confocal microscopy.