Originally identified in the platelet protein pleckstrin, pleckstrin homology (PH) domains are evolutionarily conserved across eukaryotic species and represent the 11th most frequently observed three-dimensional protein fold. Although they are primarily recognized for mediating membrane association, PH domains also facilitate proteinprotein interactions and play crucial roles in cellular signaling pathways. In some instances, these domains regulate kinase activity by blocking access to the active site in the absence of stimulation.
Alterations in PH domains of proteins such as Akt1, p210 BCR-ABL, Dbl, Tiam1, and ITK-SYK have been linked to the onset and progression of various cancers. Additionally, PH domain-containing proteins have been associated with metabolic disorders, including insulin resistance and obesity, underscoring their potential as therapeutic targets. Despite their biological importance, PH domains remain underexplored due to challenges such as the absence of catalytic activity, limited assay systems, and a lack of effective chemical probes for functional studies or proteomic applications. Since PH domains often bind lipid molecules like phosphoinositides (PIPs), their study may be hindered by the limited representation of lipid-like compounds in standard chemical libraries.
To overcome these challenges, we developed a novel fragment-based DNA-encoded chemical library (DEL) encompassing 240 000 unique fragment pairings. This approach was designed to incorporate unconventional fragments that typically fall outside the physicochemical parameters of traditional drug-like molecules. As a result, the library includes compounds with greater molecular weight, lower polarity, and lipidlike features such as extended hydrocarbon chains or sterol-based structures.
The primary focus of this work was the PH domain of mSIN1, a unique and essential component of the mTORC2 complex, which regulates cellular growth and proliferation. Our goal was to identify modulators of the mTORC2 pathway without interfering with the well-studied, rapamycin-sensitive mTORC1 complex.
Initial DEL screening identified promising binders, prompting the synthesis of a focused library by covalently linking top candidates. Variations in linker length, orientation, and flexibility were introduced to optimize interactions. This effort led to the identification of a subset of compounds with strong binding affinity for the mSIN1 PH domain. Among them, compound 2DII emerged as a lead candidate, exhibiting partial inhibition of mTORC2 signaling in cancer cell lines.
To better understand compound 2DII’s intracellular targets and specificity, we applied a Dexter energy transfer-based micromapping strategy in living cells. This approach confirmed a direct interaction between compound 2DII and mSIN1, with no detectable binding to other PH domains. Further investigation showed that the compound predominantly localizes to the plasma membrane, where it likely anchors the PH domain of mSIN1, locking mTORC2 in a conformation that is unfavorable for substrate recognition and binding.
This work represents the first identification of small molecules that selectively engage the PH domain of mSIN1. Ongoing and future studies will be essential to fully characterize the mechanism through which these compounds influence mTORC2 signaling.
