Chemical Synthesis and Biological Application of Modified Oligonucleotides.

RNA plays a myriad of roles in the body including the coding, decoding, regulation, and expression of genes. RNA oligonucleotides have garnered significant interest as therapeutics via antisense oligonucleotides or small interfering RNA strategies for the treatment of diseases ranging from hyperlipidemia, HCV, and others. Additionally, the recently developed CRISPR-Cas9 mediated gene editing strategy also relies on Cas9-associated RNA strands. However, RNA presents numerous challenges as both a synthetic target and a potential therapeutic. RNA is inherently unstable, difficult to deliver into cells, and potentially immunogenic by itself or upon modification. Despite these challenges, with the help of chemically modified oligonucleotides, multiple RNA-based drugs have been approved by the FDA. The progress is made possible due to the nature of chemically modified oligonucleotides bearing advantages of nuclease stability, stronger binding affinity, and some other unique properties. This review will focus on the chemical synthesis of RNA and its modified versions. How chemical modifications of the ribose units and of the phosphatediester backbone address the inherent issues with using native RNA for biological applications will be discussed along the way.

Development of Substituted Phenyl Dihydrouracil as the Novel Achiral Cereblon Ligands for Targeted Protein Degradation.

Glutarimides such as thalidomide, pomalidomide, and lenalidomide are the most frequently used ligands to recruit E3 ubiquitin ligase cereblon (CRBN) for the development of proteolysis-targeting chimeras (PROTACs). Due to the rapid and spontaneous racemization of glutarimides, most CRBN-recruiting PROTACs are synthesized as a mixture of racemates or diastereomers. Since the (S)-enantiomer is primarily responsible for binding to CRBN, the existence of the largely inactive (R)-enantiomer complicates the drug development process. Herein, we report that substituted achiral phenyl dihydrouracil (PDHU) can be used as a novel class of CRBN ligands for the development of PROTACs. Although the parent PDHU has a minimal binding affinity to CRBN, we found that some substituted PDHUs had a comparable binding affinity to lenalidomide. Structural modeling provided a further understanding of the molecular interactions between PDHU ligands and CRBN. PDHUs also have greater stability than lenalidomide. Finally, potent BRD4 degraders were developed by employing trisubstituted PDHUs.

Evaluation of the binding affinity of E3 ubiquitin ligase ligands by cellular target engagement and in-cell ELISA assay.

The discovery of potent cell-permeable E3 ubiquitin ligase ligands can significantly facilitate the development of proteolysis targeting chimeras (PROTACs). Here, we present a protocol to determine the binding affinity of ligands toward CRBN E3 ubiquitin ligase, using a cellular target engagement mechanism and in-cell ELISA assay. This protocol is easy to establish, with relatively low cost and rapid time frame. It can also be modified to measure the level of other proteins or determine the ligand affinity toward other E3s. For complete details on the use and execution of this protocol, please refer to Yang et al. (2020).

Two-Stage Strategy for Development of Proteolysis Targeting Chimeras and its Application for Estrogen Receptor Degraders.

Proteolysis targeting chimeras (PROTACs) have emerged as useful chemical probes and potential therapeutics by taking advantage of the ubiquitin-proteasome system to degrade intracellular disease-associated proteins. PROTACs are heterobifunctional molecules composed of a target protein ligand, E3 ubiquitin ligase ligand, and a linker between them. The generation of efficient PROTACs requires screening of many parameters, especially the lengths and types of the linkers. We report our proof-of-concept study using a two-stage strategy to facilitate the development of PROTACs against the estrogen receptor (ER). In stage one, a library of close to 100 PROTACs was synthesized by simply mixing a library of ERα ligands containing a hydrazide functional group at different positions with a preassembled library of E3 ligase ligands bearing different types and lengths of linkers with a terminal aldehyde group in a 1:1 ratio. Cell-based screening occurred without further purification, because the formation of the acylhydrazone linkage is highly efficient and produces water as the only byproduct. Compound A3 was the most potent ER degrader in two ER+ cell lines (DC50= ∼ 10 nM, Dmax= ≥ 95%). Stage two involved transformation to a more stable amide linker to generate a more drug-like molecule. The new compound, AM-A3, showed comparable biological activity (DC50 = 1.1 nM, Dmax = 98%) and induced potent antiproliferation (IC50= 13.2 nM, Imax= 69%) in MCF-7. This proof-of -concept study demonstrates that the two-stage strategy can significantly facilitate the development of PROTACs against ER without the tedious process of making large numbers of PROTACs one by one. It has the potential to be expanded to many other targets.

Transient Compound Treatment Induces a Multigenerational Reduction of Oxysterol-Binding Protein (OSBP) Levels and Prophylactic Antiviral Activity.

Oxysterol-binding protein (OSBP) is a lipid transport and regulatory protein required for the replication of Enterovirus genus viruses, which includes many significant human pathogens. Short-term exposure (i.e., 1-6 h) to a low dose (i.e., 1 nM) of the natural product compound OSW-1 induces a reduction of cellular OSBP levels by ∼90% in multiple different cell lines with no measurable cytotoxicity, defect in cellular proliferation, or global proteome reduction. Interestingly, the reduction of OSBP levels persists multiple days after the low-dose, transient OSW-1 compound treatment is ended and the intracellular OSW-1 compound levels drop to undetectable levels. The reduction in OSBP levels is inherited in multiple generations of cells that are propagated after the OSW-1 compound treatment is stopped. The enduring multiday, multigenerational reduction of OSBP levels triggered by the OSW-1 compound is not due to proteasome degradation of OSBP or due to a reduction in OSBP mRNA levels. OSW-1 compound treatment induces transient autophagy in cells, but blocking autophagy does not rescue OSBP levels. Although the specific cellular mechanism of long-term OSBP repression is not yet identified, these results clearly show the existence of an OSBP specific cellular regulation process that is triggered upon treatment with an OSBP-binding compound. The stable reduction of OSBP levels upon short-term, transient OSW-1 compound treatment will be a powerful tool to understand OSBP regulation and cellular function. Additionally, the persistent reduction in OSBP levels triggered by the transient OSW-1 compound treatment substantially reduces viral replication in treated cells. Therefore, the long-term, compound-induced reduction of OSBP in cells presents a new route to broad spectrum anti- Enterovirus activity, including as a novel route to antiviral prophylactic treatment through small molecule targeting a human host protein.

Differing activities of oxysterol-binding protein (OSBP) targeting anti-viral compounds.

Oxysterol-binding Protein (OSBP) is a human lipid-transport protein required for the cellular replication of many types of viruses, including several human pathogens. The structurally-diverse small molecule compounds OSW-1, itraconazole (ITZ), T-00127-HEV2 (THEV) and TTP-8307 (TTP) inhibit viral replication through interaction with the OSBP protein. The OSW-1 compound reduces intracellular OSBP, and the reduction of OSBP protein levels persists multiple days after the OSW-1-compound treatment is stopped. The OSW-1-induced reduction of OSBP levels inhibited Enterovirus replication prophylactically in cells. In this report, the OSBP-interacting compounds ITZ, THEV, and TTP are shown not to reduce OSBP levels in cells, unlike the OSW-1-compound, and the OSW-1 compound is determined to be the only compound capable of providing prophylactic antiviral activity in cells. Furthermore, OSW-1 and THEV inhibit the binding of 25-hydroxycholesterol (25-OHC) to OSBP indicating that these compounds bind at the conserved sterol ligand binding site. The ITZ and TTP compounds do not inhibit 25-hydroxycholesterol binding to OSBP, and therefore ITZ and TTP interact with OSBP through other, unidentified binding sites. Co-administration of the THEV compound partially blocks the cellular activity of OSW-1, including the reduction of cellular OSBP protein levels; co-administration of the ITZ and TTP compounds have minimal effect on OSW-1 cellular activity further supporting different modes of interaction with these compounds to OSBP. OSW-1, ITZ, THEV, and TTP treatment alter OSBP cellular localization and levels, but in four distinct ways. Co-administration of OSW-1 and ITZ induced OSBP cellular localization patterns with features similar to the effects of ITZ and OSW-1 treatment alone. Based on these results, OSBP is capable of interacting with multiple structural classes of antiviral small molecule compounds at different binding sites, and the different compounds have distinct effects on OSBP cellular activity.