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First PROTAC Degradation of Mulit-Pass Transmembrane Protein

Cancer cells exploit proteins involved in energy metabolism to maintain a steady supply of building blocks necessary for increased proliferation. Many transporters play a role in the process of shuttling water, nutrients, ions, and various metabolic products across the cell membrane, including the solute carrier (SLC) group of proteins. The SLC protein group consists of more than 400 proteins organized into more than 50 families. For example, the SLC9 family is involved in intracellular pH homeostasis, with increased SL9 activity resulting in an elevated intracellular pH and cytosolic alkalinization in cancer. Currently there 12 FDA approved drugs targeting SLC proteins for conditions ranging from hypertension to depression, but recent studies suggest SLC proteins may be attractive targets for cancer drug development.


With this end goal, Bensimon et al., aimed to investigate if multi-pass transmembrane proteins like SLCs can be chemically degraded. To first explore this aim, they used the dTAG system to determine that SLCs can undergo phthalimide-mediated degradation using a sample of 13 SLCs. Given that SLCs can localize to differing subcellular locals, they observed various degrees of degradation, but overall all the SLCs tested except those localized to the inner mitochondrial membrane were able to undergo proteasome-mediated degradation. This process was confirmed to require proteolytic machinery using competition experiments including co-treatment of cullin neddylation using a NAE1 inhibitor, addition of excess pomalidomide to saturate CRBN binding, and treatment with proteasome inhibitors. 

Due to limitations with using the dTag approach to genetically degrade SLCs, the group designed a library of SLC9A1 PROTACs based on a previously published SLC9A1 inhibitor to determine if SLC group members can be chemically degraded. SLC9A1 (aka NHE1) exchanges one Na+ ion for one H+ ion contributing to a more alkaline intracellular environment as mentioned above. One of the PROTACs, d9A-2 was able to degrade endogenous SLC9A1 with sub-micromolar concentration in two chronic myelogenous leukemia (CML) cell lines.


The authors also investigated the effect of d9A-2 treatment on SLC9 functionality and cell viability. SLC9 function can be tested by examining the ability of cells to recover from acid load. Compared to the warhead, d9A-2 showed a significant defect in acid recovery with 1 M of PROTAC having the same effect as 25 uM of warhead. The novel SLC PROTAC also showed varied cytotoxicity across cell lines, having the greatest effect on leukemic cancers with an EC50 less than 0.1 uM.  


In conclusion, the Superti-Furga group developed the first SLC PROTAC. This is also the first multi-pass trans-membrane PROTAC, suggesting that in the future even GPCRs can be proteolytically degraded.

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