New method for delivering proteins intracellularly offers a promising opportunity to target the undruggable proteome


It is estimated that 85 percent of the human proteome is ‘undruggable’ which means such proteins could not be targeted pharmacologically and developing effective medications to target these proteins is extremely difficult. While most small molecule medications can easily pass the cell membrane, their pharmacological effects need docking into deep functional or allosteric binding pockets, which are challenging to locate in many significant therapeutic targets. In recent years, interest in cytosolic accessible protein-based therapies has fueled the development of several novel drug delivery systems. Previous studies demonstrated that potent Ras-inhibiting antibodies designed with integrin-binding moieties and an endosomal escape motif improve cellular internalization and cytosolic access. Functionalization using cell-penetrating peptides is another common approach for transporting proteins into cells. Carrier-based protein delivery systems such as virus-like particles, polymers, and inorganic nanoparticles have also been reported with limited success. Despite the fact that cytosolic transport is required, it is a considerable obstacle to the effective delivery of therapeutic peptides, proteins, and nucleic acids, making the entire drug delivery process difficult.

The capacity to distribute small protein scaffolds intracellularly might allow for the targeting and inhibiting numerous therapeutic targets that are now intractable with small-molecule medicines. In a new study published in the scientific journal Molecular Pharmaceutics scientists from the Department of Bioengineering at the University of Pennsylvania, Alexander Chan, Hejia Wang, Rebecca Haley, Cindy Song, David Gonzalez-Martinez, Lukasz Bugaj, Dr. Michael Mitchell and Professor Andrew Tsourkas developed a new effective and adaptable intracellular delivery strategy for a variety of small protein binders. The technique developed by the researchers allows for excellent delivery efficiency (up to 90%), employing cationic lipids and ionizable lipid nanoparticles (LNPs) and functional targeting of two of the most frequent proto-oncogenic pathways. They demonstrated that anti-Ras DARPin (Design Ankyrin Repeat Proteins) reduces canonical MAPK signalling in non-small cell lung cancer (NSCLC) and colorectal cancer cells, allowing them to exhibit intracellular activity. Ras and Myc are two important oncologic targets that are challenging to treat with conventional small molecule medication development. The authors showed the successful delivery of the Myc-inhibiting mini protein Omomyc to A549 NSCLC cells inhibits Myc-responsive transcription.

The research team demonstrated that anionic polypeptide fusions can enable the delivery of a number of small proteins. The splitGFP complementation assay was used to determine the effectiveness of intracellular delivery of several small protein scaffolds. Investigators explored three different forms of protein scaffolds: DARPins, nanobodies, and the mini-protein Omomyc. They were either complexed with off-the-shelf Lipofectamine 2000 reagent or encapsulated in LNPs for facilitating cellular uptake and endosomal-lysosomal escape of these fusion proteins. Flow cytometry was used to confirm intracellular protein delivery in the cytosol. In A549 non-small cell lung cancer (NSCLC) cells, these proteins had enhanced activity when fused with the anionic polypeptide. All ApP-fused constructs exhibited >65% delivery efficiency when complexed with Lipofectamine 2000. Additionally, protein scaffold-ApP fusions showed increased median fluorescence intensity. According to the authors, Lipofectamine 2000 which is commonly used in medical research delivered inadequate cytosolic protein delivery in HCT116 colorectal cancer cells. To address this issue, the researchers designed their ionizable LNPs and they showed successful DARPinK27 distribution coupled with anionic polypeptide over a wide range of concentrations using LNP formulations with low cytotoxicity indicating an excellent safety profile. Furthermore, the LNP of DARPinK27 suppressed the proto-oncogene Ras in HCT116 cells. The authors also showed that Omomyc-ApP fusion proteins complexed with cationic lipids could inhibit Myc activity, another prominent proto-oncogene. Another important application of the new technology. These findings imply that new designed ionizable LNPs are ideal candidates for cytosolic protein delivery.

In a nutshell, several high-profile oncogenic targets that remained undruggable using conventional approaches may be cracked using innovative therapeutic systems. The new study shows that combining minimally designed tiny protein scaffolds with tractable nanocarriers can block intracellular proteins that are currently regarded as “undruggable” using small molecule medicines and biologics. University of Pennsylvania researchers demonstrated the success of employing LNPs to deliver their designed inhibitors, boosting the likelihood of effectively transferring this technique for in vivo applications.

New method for delivering proteins intracellularly offers a promising opportunity to target the undruggable proteome - Medicine Innovates

About the author

Andrew Tsourkas, Ph.D. is a Professor and Undergraduate Chair of Bioengineering at the University of Pennsylvania.  He received his Bachelor’s degree in Mechanical Engineering in 1997 from Cornell University and his Ph.D. in Biomedical Engineering from the Georgia Tech/Emory University joint Ph.D. program in 2002.  He conducted a post-doctoral fellowship in the Department of Radiology at Harvard University, before joining Penn in 2004. Dr. Tsourkas is currently the Co-Director for the Center for Targeted Therapeutics and Translational Nanomedicine as well as the founding Director of the Chemical and Nanoparticle Synthesis Core. He was a recipient of the Coulter Foundation Early Career Award, the National Science Foundation CAREER Award, and was elected fellow of the American Institute for Medical and Biological Engineering. Dr. Tsourkas founded AlphaThera, Inc. in 2016, a biotechnology company that specializes in antibody labeling reagents. Research efforts in the Tsourkas lab are focused on developing improved targeted imaging and therapeutic agents. In particular, his lab (i) develops new nanoformulations for diverse biomedical applications, including image-guided surgery and photodynamic therapy; (ii) investigates new targeting strategies that maximize specificity and sensitivity or improves the accumulation and penetration of nanomaterials within tumors; and (iii) develops new bioconjugation techniques that enable the highly efficient, site-specific labeling of proteins, including antibodies.


Chan A, Wang HH, Haley RM, Song C, Gonzalez-Martinez D, Bugaj L, Mitchell MJ, Tsourkas A. Cytosolic Delivery of Small Protein Scaffolds Enables Efficient Inhibition of Ras and Myc. Molecular Pharmaceutics. 2022 Feb 28;19(4):1104-16.

Go To Molecular Pharmaceutics.