Drug development is a balancing act between ensuring that the drug is suitable for the target and that the drug can penetrate the cell membrane to reach the target. Typically, research into drugs that can cross cell membranes has focused on small, rigid molecules with nonpolar chemical structures. However, new therapeutic strategies break traditional drug design rules by using larger, flexibly linked chemical entities.

Recently, a team of researchers from the University of California, San Francisco (UCSF) published a study in Science, in which they unveiled a new discovery of a cellular uptake pathway critical for macromolecules. These large, complex molecules bind to their targets in unique ways, are efficiently taken up by cells, and have the potential to be used to create new drugs to treat cancer and other diseases. Through a combination of functional genomics and chemical approaches, the scientists discovered an endogenous pathway involving interferon-induced transmembrane (IFITM) proteins that facilitate cellular uptake of different associated chemotypes. These proteins are found in the plasma membrane and normally provide the cell's resistance to viruses.

Most traditional medicines are small molecules that follow simple molecular rules, including limiting the size of the molecule and the number of sticky chemical groups on the surface of the molecule. Many key drug targets, such as kinase enzymes frequently involved in cancer, are difficult to selectively target with conventional drugs.

"There are more than 500 human kinase enzymes that are very similar in the drug-binding region, making it a challenge to selectively target individual members of this family and lead to unwanted drug side effects," said Kevin Lou, lead author of the study. It is increasingly being found that certain linked molecules outside this traditional framework can retain drug-like properties and acquire novel mechanisms of action."

There are many important intracellular drug targets that researchers cannot target with small, compact and rigid molecules. To address this challenge, scientists have begun to link multiple ligands into a single chemical entity (linked chemotype). These associated chemotypes can have enhanced potency, greater selectivity and the ability to induce multiple target associations.

The team designed two new linked drugs that they hypothesized might take advantage of this cellular entry pathway. They generated DasatiLink-1 through the linker conjugation of two known inhibitors of the leukemia protein BCL-ABL1 (dasatinib and asciminib). Since each drug binds a different pocket on the target protein, the researchers reasoned that the tethered version could anchor itself at the two points of contact, like inserting a double-pointed key into two locks, enhancing its specificity and effectiveness.

They also engineered BisRoc-1 to link together two molecules of the chemotherapy drug rocaglamide, allowing it to bridge the drug's two copies of the protein target. Although both drugs violate traditional principles of drug design, the team showed that both enter cells, bind tightly to their intended targets, and are just as effective as unbound versions. The ligated version is uniquely dependent on IFITM protein expression in target cells, supporting a general role for the IFITM pathway in many types of ligated molecules. The researchers showed that DasatiLink-1 has specificity only for the BCL-ABL1 kinase, unlike its two component drugs, which have a more relaxed specificity when unlinked.

Lou writes: "Given the discrepancy between the favorable biological activity of many large, bivalent molecules and the traditional concept of passive permeability, we reasoned that the associated chemotype might hijack cellular processes to facilitate passage through the cell membrane. We chose RapaLink-1, a bitopic inhibitor of mTOR, as an example with a molecular weight well beyond the usual guidelines. Linkage inhibitors that require a multi-pronged binding mechanism are more selective. As long as they can enter cells efficiently, they have huge advantages.”

"Our discovery that the IFITM protein enables entry of bitopic inhibitors into cells may allow us to target previously untargetable proteins in disease," said co-corresponding author Luke Gilbert, Ph.D. "Hopefully, our research can provide new clues for drug design scientists and virologists to study the mechanism of IFITM protein."

Scientists are working to chemically optimize the properties of related BCR-ABL inhibitors to increase their potency and position them as next-generation therapies for BCR-ABL-mutated cancers. "We are also excited to expand the range of intracellular targets for bitopic inhibition," said Gilbert.