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.

Correlating the Structure and Gene Silencing Activity of Oligonucleotide-Loaded Lipid Nanoparticles Using Small-Angle X-ray Scattering.

With three FDA-approved products, lipid nanoparticles (LNPs) are under intensive development for delivering wide-ranging nucleic acid therapeutics. A significant challenge for LNP development is insufficient understanding of structure-activity relationship (SAR). Small changes in chemical composition and process parameters can affect LNP structure, significantly impacting performance in vitro and in vivo. The choice of polyethylene glycol lipid (PEG-lipid), one of the essential lipids for LNP, has been proven to govern particle size. Here we find that PEG-lipids can further modify the core organization of antisense oligonucleotide (ASO)-loaded LNPs to govern its gene silencing activity. Furthermore, we also have found that the extent of compartmentalization, measured by the ratio of disordered vs ordered inverted hexagonal phases within an ASO-lipid core, is predictive of in vitro gene silencing. In this work, we propose that a lower ratio of disordered/ordered core phases correlates with stronger gene knockdown efficacy. To establish these findings, we developed a seamless high-throughput screening approach that integrated an automated LNP formulation system with structural analysis by small-angle X-ray scattering (SAXS) and in vitro TMEM106b mRNA knockdown assessment. We applied this approach to screen 54 ASO-LNP formulations while varying the type and concentration of PEG-lipids. Representative formulations with diverse SAXS profiles were further visualized using cryogenic electron microscopy (cryo-EM) to help structural elucidation. The proposed SAR was built by combining this structural analysis with in vitro data. Our integrated methods, analysis, and resulting findings on PEG-lipid can be applied to rapidly optimize other LNP formulations in a complex design space.

Impact of non-ionizable lipids and phase mixing methods on structural properties of lipid nanoparticle formulations.

Lipid nanoparticles (LNPs) have been widely investigated for nucleic acid therapeutic delivery, and demonstrated their potential in enabling new mRNA vaccines. LNPs are usually formulated with multi-lipid components and the composition variables may impact their structural properties. Here, we investigated the impact of helper lipids on physicochemical properties of LNPs using a Design of Experiments (DoE) definitive screening design. Phospholipid head group, degree of unsaturation, ratio to cholesterol as well as PEG-lipid content were varied and a series of 14 LNPs were prepared by microfluidic- and solvent-injection mixing. Solvent-injection mixing by a robotic liquid handler yielded 50-225 nm nanoparticles with highly ordered, ∼5 nm inter-lamellar spacing as measured by small angle X-ray scattering (SAXS) and confirmed by cryo-transmission electron microscopy (cryo-EM). In contrast, microfluidic mixing resulted in less ordered, notably smaller (50-75 nm) and more homogenous nanoparticles. Significant impacts of the stealth-lipid DSPE-PEG2000 on nanoparticle size, polydispersity and encapsulation efficiency of an oligonucleotide cargo were observed in LNPs produced by both methods, while varying the phospholipid type and content had only marginal effect on these physicochemical properties. These findings suggest that from a physicochemical perspective, the design space for combinations of helper lipids in LNPs may be considerably larger than anticipated based on the conservative formulation composition of the currently FDA-approved LNPs, thereby opening opportunities for screening and optimization of novel LNP formulations.

Predictive high-throughput screening of PEGylated lipids in oligonucleotide-loaded lipid nanoparticles for neuronal gene silencing.

Lipid nanoparticles (LNPs) are gaining traction in the field of nucleic acid delivery following the success of two mRNA vaccines against COVID-19. As one of the constituent lipids on LNP surfaces, PEGylated lipids (PEG-lipids) play an important role in defining LNP physicochemical properties and biological interactions. Previous studies indicate that LNP performance is modulated by tuning PEG-lipid parameters including PEG size and architecture, carbon tail type and length, as well as the PEG-lipid molar ratio in LNPs. Owing to these numerous degrees of freedom, a high-throughput approach is necessary to fully understand LNP behavioral trends over a broad range of PEG-lipid variables. To this end, we report a low-volume, automated, high-throughput screening (HTS) workflow for the preparation, characterization, and in vitro assessment of LNPs loaded with a therapeutic antisense oligonucleotide (ASO). A library of 54 ASO-LNP formulations with distinct PEG-lipid compositions was prepared using a liquid handling robot and assessed for their physiochemical properties as well as gene silencing efficacy in murine cortical neurons. Our results show that the molar ratio of anionic PEG-lipid in LNPs regulates particle size and PEG-lipid carbon tail length controls ASO-LNP gene silencing activity. ASO-LNPs formulated using PEG-lipids with optimal carbon tail lengths achieved up to 5-fold lower mRNA expression in neurons as compared to naked ASO. Representative ASO-LNP formulations were further characterized using dose-response curves and small-angle X-ray scattering to understand structure-activity relationships. Identified hits were also tested for efficacy in primary murine microglia and were scaled-up using a microfluidic formulation technique, demonstrating a smooth translation of ASO-LNP properties and in vitro efficacy. The reported HTS workflow can be used to screen additional multivariate parameters of LNPs with significant time and material savings, therefore guiding the selection and scale-up of optimal formulations for nucleic acid delivery to a variety of cellular targets.

Fast and versatile analysis of liposome encapsulation efficiency by nanoParticle exclusion chromatography.

Liposomes are an attractive drug delivery platform for a wide variety of pharmaceutical molecules. Encapsulation efficiency, which refers to the amount of drug contained inside liposomes compared with the total amount of drug, is a critical quality attribute of liposome products, as the free drug in a liposomal formulation may cause toxicity or undesired biodistribution. The determination of encapsulation efficiency requires the measurement of at least two of the three drug populations: total drug, encapsulated drug and free drug. However, direct measurement of the encapsulated drug and free drug remains a challenging analytical task. Nanoparticle exclusion chromatography (nPEC), an emerging high-performance liquid chromatography (HPLC) technique, has shown great potential in separating and quantifying the free drug in liposomal formulations. In this study, nPEC was systematically evaluated for two representative liposomal formulations containing either hydrophilic or hydrophobic small molecule drugs. It is reported for the first time that the insoluble free drug suspended in the aqueous formulation can be directly measured by nPEC. This free drug in the suspension sample was quantified with excellent accuracy and precision. On the other hand, the total drug measurement from dissociated liposomes was confirmed by the benchmark methodology of reversed phase liquid chromatography (RPLC). The facile quantitation of free and total drug in the liposome formulation enables the fast and accurate determination of the encapsulation efficiency, which can be used to guide the formulation development and characterize the product quality.