Fifteen Years of Cell-Penetrating, Guanidinium-Rich Molecular Transporters: Basic Science, Research Tools, and Clinical Applications

Abstract

Conspectus All living systems require biochemical barriers. As a consequence, all drugs, imaging agents, and probes have targets that are either on, in, or inside of these barriers. Fifteen years ago, we initiated research directed at more fully understanding these barriers and at developing tools and strategies for breaching them that could be of use in basic research, imaging, diagnostics and medicine. At the outset of this research and to a lesser extent now, the “rules” for drug design biased the selection of drug candidates to mainly those with an intermediate and narrow log P. At the same time, it was becoming increasingly apparent that Nature had long ago developed clever strategies to circumvent these “rules”. In 1988, for example, independent reports appeared documenting the otherwise uncommon passage of a protein (HIV-Tat) across a membrane. A subsequent study called attention to a highly basic domain in this protein (Tat49–57) being responsible for its cellular entry. This conspicuously contradictory behavior, i.e., a polar, highly charged peptide passing through a non-polar membrane, set the stage for learning how Nature had gotten around the current “rules” of transport. As elaborated in our studies and discussed herein, the key strategy used in Nature rests in part on the ability of a molecule to change its properties as a function of microenvironment, being a polarity chameleon – i.e., being polar in a polar milieu and relatively non-polar in a non-polar environment. Because this research originated in part with the protein Tat and its basic peptide domain, Tat49–57, the field focused heavily on peptides, even limiting its nomenclature to names such as ‘cell-penetrating peptides,’ ‘cell-permeating peptides,’ ‘protein transduction domains,’ and ‘membrane translocating peptides’ to note a few. Starting in 1997, through a systematic reverse engineering approach, we established that the ability of Tat49–57 to enter cells is not a function of its peptide backbone, but rather the number and spatial array of its guanidinium groups. These function-oriented studies allowed one to design more effective peptidic agents and to think beyond the confines of peptidic systems to new and even more effective non-peptidic agents. Because the function of passage across a cell membrane is not limited to or even best achieved with the peptide backbone, we referred to these agents by their shared function, i.e., ‘cell-penetrating molecular transporters’. The scope of this molecular approach to breaching biochemical barriers has expanded remarkably in the past 15 years, enabling or enhancing the delivery of a wide range of cargos into cells and across other biochemical barriers; creating new tools for research, imaging, and diagnostics; and introducing new therapies into clinical trials.

Peptide targeting of fluorescein-based sensors to discrete intracellular locales

“…These results are consistent with an energy-dependent uptake mechanism such as endocytosis. 18 Further support for such a mechanism is the inability of DA-ZP1-R 9 to penetrate the plasma membrane when incubated with live cells at 4 °C (Figure S8). Because the fluorescence turn-on of DA-ZP1-R 9 is pH-insensitive, however, the probe was still able to detect an influx of zinc within vesicles (Figure 6).…”

“…Recent literature, however, reveals that fluorescein is a “non-innocent” reporter that can alter the uptake and localization of a peptide. 18a,21 Achieving significant uptake of fluorescein-labelled peptides often requires high loading concentrations of ≥ 10 μM, co-administration with endosomal disrupting agents, or modification of sidechain residues. 18a,20b,22 Using r(F x r) 3 as a model system, we compared the uptake and localization of peptide derivatives labelled with 2′,7′-dichlorofluorescein (DCF) or 2′,7′-dichlorofluorescein diacetate (DA-DCF), respectively.…”

Insights into solute carriers: physiological functions and implications in disease and pharmacokinetics

“…From the above findings, one can conclude that OATPs can be valid targets for cancer treatment. 59 SLC6A14 is an amino acid transporter that is up-regulated in cancer cells, facilitating the entrance of several amino acids, namely glutamine, arginine, and leucine. These upregulated transporters seem to be relevant targets for selective anticancer drugs.…”

Current Understanding of the Mechanisms by which Membrane-Active Peptides Permeate and Disrupt Model Lipid Membranes

“…carbamates; carbonates; carbohydrates, and dendrimers [44]. Here, we will focus on oligoarginine itself as questions still remain concerning the mechanism and important molecular determinants by which oligoarginine permeates lipid membranes.…”