Optimization of a Novel DEL Hit That Binds in the Cbl-b SH2 Domain and Blocks Substrate Binding.

We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an ∼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.

Development of Liposome Systems for Enhancing the PK Properties of Bivalent PROTACs.

Proteolysis-Targeting Chimeras (PROTACs) are a promising new technology in drug development. They have rapidly evolved in recent years, with several of them in clinical trials. While most of these advances have been associated with monovalent protein degraders, bivalent PROTACs have also entered clinical trials, although progression to market has been limited. One of the reasons is the complex physicochemical properties of the heterobifunctional PROTACs. A promising strategy to improve pharmacokinetics of highly lipophilic compounds, such as PROTACs, is encapsulation in liposome systems. Here we describe liposome systems for intravenous administration to enhance the PK properties of two bivalent PROTAC molecules, by reducing clearance and increasing systemic coverage. We developed and characterized a PROTAC-in-cyclodextrin liposome system where the drug was retained in the liposome core. In PK studies at 1 mg/kg for GNE-01 the PROTAC-in-cyclodextrin liposome, compared to the solution formulation, showed a 80- and a 380-fold enhancement in AUC for mouse and rat studies, respectively. We further investigated the same PROTAC-in-cyclodextrin liposome system with the second PROTAC (GNE-02), where we monitored both lipid and drug concentrations in vivo. Similarly, in a mouse PK study of GEN-02, the PROTAC-in-cyclodextrin liposome system exhibited enhancement in plasma concentration of a 23× increase over the conventional solution formulation. Importantly, the lipid CL correlated with the drug CL. Additionally, we investigated a conventional liposome approach for GNE-02, where the PROTAC resides in the lipid bilayer. Here, a 5× increase in AUC was observed, compared to the conventional solution formulation, and the drug CL was faster than the lipid CL. These results indicate that the different liposome systems can be tailored to translate across multiple PROTAC systems to modulate and improve plasma concentrations. Optimization of the liposomes could further improve tumor concentration and improve the overall therapeutic index (TI). This delivery technology may be well suited to bring novel protein targeted PROTACs into clinics.