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.

Multidrug resistance (MDR) is a major cause of chemotherapy failure in the clinic. Drugs that were once effective against naïve disease subsequently prove ineffective against recurrent disease, which often exhibits an MDR phenotype. MDR can be attributed to many factors; often dominating among these is the ability of a cell to suppress or block drug entry through upregulation of membrane-bound drug efflux pumps. Efflux pumps exhibit polyspecificity, recognizing and exporting many different types of drugs, especially those whose lipophilic nature contributes to residence in the membrane. We have developed a general strategy to overcome efflux-based resistance. This strategy involves conjugating a known drug that succumbs to efflux-mediated resistance to a cell-penetrating molecular transporter, specifically, the cell-penetrating peptide (CPP), d-octaarginine. The resultant conjugates are discrete single entities (not particle mixtures) and highly water-soluble. They rapidly enter cells, are not substrates for efflux pumps, and release the free drug only after cellular entry at a rate controlled by linker design and favored by target cell chemistry. This general strategy can be applied to many classes of drugs and allows for an exceptionally rapid advance to clinical testing, especially of drugs that succumb to resistance. The efficacy of this strategy has been successfully demonstrated with Taxol in cellular and animal models of resistant cancer and with ex vivo samples from patients with ovarian cancer. Next generation efforts in this area will involve the extension of this strategy to other chemotherapeutics and other MDR-susceptible diseases.