A Multifaceted Hit-Finding Approach Reveals Novel LC3 Family Ligands.

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.

DNA Compatible Multistep Synthesis and Applications to DNA Encoded Libraries.

Complex mixtures of DNA encoded small molecules may be readily interrogated via high-throughput sequencing. These DNA encoded libraries (DELs) are commonly used to discover molecules that interact with pharmaceutically relevant proteins. The chemical diversity displayed by the library is key to successful discovery of potent, novel, and drug-like chemical matter. The small molecule moieties of DELs are generally synthesized though a multistep process, and each chemical step is accomplished while it is simultaneously attached to an encoding DNA oligomer. Hence, library chemical diversity is often limited to DNA compatible synthetic reactions. Herein, protocols for 24 reactions are provided that have been optimized for high-throughput production of DELs. These protocols detail the multistep synthesis of benzimidazoles, imidazolidinones, quinazolinones, isoindolinones, thiazoles, and imidazopyridines. Additionally, protocols are provided for a diverse range of useful chemical reactions including BOC deprotection (under pH neutral conditions), carbamylation, and Sonogashira coupling. Last, step-by-step protocols for synthesizing functionalized DELs from trichloronitropyrimidine and trichloropyrimidine scaffolds are detailed.

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.