RNA-Targeting Splicing Modifiers: Drug Development and Screening Assays.

RNA splicing is an essential step in producing mature messenger RNA (mRNA) and other RNA species. Harnessing RNA splicing modifiers as a new pharmacological modality is promising for the treatment of diseases caused by aberrant splicing. This drug modality can be used for infectious diseases by disrupting the splicing of essential pathogenic genes. Several antisense oligonucleotide splicing modifiers were approved by the U.S. Food and Drug Administration (FDA) for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD). Recently, a small-molecule splicing modifier, risdiplam, was also approved for the treatment of SMA, highlighting small molecules as important warheads in the arsenal for regulating RNA splicing. The cellular targets of these approved drugs are all mRNA precursors (pre-mRNAs) in human cells. The development of novel RNA-targeting splicing modifiers can not only expand the scope of drug targets to include many previously considered "undruggable" genes but also enrich the chemical-genetic toolbox for basic biomedical research. In this review, we summarized known splicing modifiers, screening methods for novel splicing modifiers, and the chemical space occupied by the small-molecule splicing modifiers.

Synthetic lethality by targeting the RUVBL1/2-TTT complex in mTORC1-hyperactive cancer cells.

Despite considerable efforts, mTOR inhibitors have produced limited success in the clinic. To define the vulnerabilities of mTORC1-addicted cancer cells and to find previously unknown therapeutic targets, we investigated the mechanism of piperlongumine, a small molecule identified in a chemical library screen to specifically target cancer cells with a hyperactive mTORC1 phenotype. Sensitivity to piperlongumine was dependent on its ability to suppress RUVBL1/2-TTT, a complex involved in chromatin remodeling and DNA repair. Cancer cells with high mTORC1 activity are subjected to higher levels of DNA damage stress via c-Myc and displayed an increased dependency on RUVBL1/2 for survival and counteracting genotoxic stress. Examination of clinical cancer tissues also demonstrated that high mTORC1 activity was accompanied by high RUVBL2 expression. Our findings reveal a previously unknown role for RUVBL1/2 in cell survival, where it acts as a functional chaperone to mitigate stress levels induced in the mTORC1-Myc-DNA damage axis.

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Control of Chemo-, Regio-, and Enantioselectivity in Copper Hydride Reductions of Morita-Baylis-Hillman Adducts.

Nonracemically ligated copper hydride can be used to effect tandem SN2'/1,2-reductions of racemic Morita-Baylis-Hillman (MBH) acetates to access enantioenriched chiral allylic alcohols with defined olefin geometry. MBH esters, including those with ß-substitution, can be transformed to stereodefined enoates by taking advantage of a bulky, oligomeric, in situ generated trialkoxysiloxane leaving group. Finally, an atypical conversion of easily arrived at MBH alcohol derivatives to nonracemic allylic alcohols is disclosed.

Efficient Routes to a Diverse Array of Amino Alcohol-Derived Chiral Fragments.

Efficient syntheses of chiral fragments derived from chiral amino alcohols are described. Several unique scaffolds were readily accessed in 1-5 synthetic steps leading to 45 chiral fragments, including oxazolidinones, morpholinones, lactams, and sultams. These fragments have molecular weights ranging from 100 to 255 Da and are soluble in water (0.085 to >15 mM).

Real-Time Biological Annotation of Synthetic Compounds.

Organic chemists are able to synthesize molecules in greater number and chemical complexity than ever before. Yet, a majority of these compounds go untested in biological systems, and those that do are often tested long after the chemist can incorporate the results into synthetic planning. We propose the use of high-dimensional "multiplex" assays, which are capable of measuring thousands of cellular features in one experiment, to annotate rapidly and inexpensively the biological activities of newly synthesized compounds. This readily accessible and inexpensive "real-time" profiling method can be used in a prospective manner to facilitate, for example, the efficient construction of performance-diverse small-molecule libraries that are enriched in bioactives. Here, we demonstrate this concept by synthesizing ten triads of constitutionally isomeric compounds via complexity-generating photochemical and thermal rearrangements and measuring compound-induced changes in cellular morphology via an imaging-based "cell painting" assay. Our results indicate that real-time biological annotation can inform optimization efforts and library syntheses by illuminating trends relating to biological activity that would be difficult to predict if only chemical structure were considered. We anticipate that probe and drug discovery will benefit from the use of optimization efforts and libraries that implement this approach.

Inhibition of Zinc-Dependent Histone Deacetylases with a Chemically Triggered Electrophile.

Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an ortho-quinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound-enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization-mass spectrometry.

Synthesis of piperlogs and analysis of their effects on cells.

Piperlongumine (PL) is a naturally occurring small molecule previously shown to induce cell death preferentially in cancer cells relative to non-cancer cells. An initial effort to synthesize analogs highlighted the reactivities of both of piperlongumine's α,ß-unsaturated imide functionalities as key features determining PL's cellular effects. In this study, a second-generation of analogs was synthesized and evaluated in cells to gain further insight into how the reactivity, number, and orientation of PL's reactive olefins contribute to its ability to alter the physiology of cells.

Discovery of small-molecule enhancers of reactive oxygen species that are nontoxic or cause genotype-selective cell death.

Elevation of reactive oxygen species (ROS) levels has been observed in many cancer cells relative to nontransformed cells, and recent reports have suggested that small-molecule enhancers of ROS may selectively kill cancer cells in various in vitro and in vivo models. We used a high-throughput screening approach to identify several hundred small-molecule enhancers of ROS in a human osteosarcoma cell line. A minority of these compounds diminished the viability of cancer cell lines, indicating that ROS elevation by small molecules is insufficient to induce death of cancer cell lines. Three chemical probes (BRD5459, BRD56491, BRD9092) are highlighted that most strongly elevate markers of oxidative stress without causing cell death and may be of use in a variety of cellular settings. For example, combining nontoxic ROS-enhancing probes with nontoxic doses of L-buthionine sulfoximine, an inhibitor of glutathione synthesis previously studied in cancer patients, led to potent cell death in more than 20 cases, suggesting that even nontoxic ROS-enhancing treatments may warrant exploration in combination strategies. Additionally, a few ROS-enhancing compounds that contain sites of electrophilicity, including piperlongumine, show selective toxicity for transformed cells over nontransformed cells in an engineered cell-line model of tumorigenesis. These studies suggest that cancer cell lines are more resilient to chemically induced increases in ROS levels than previously thought and highlight electrophilicity as a property that may be more closely associated with cancer-selective cell death than ROS elevation.

CuH-catalyzed enantioselective 1,2-reductions of alpha,beta-unsaturated ketones.

The first study on a general technology for arriving at valued nonracemic allylic alcohols using asymmetric ligand-accelerated catalysis by copper hydride is described.

Copper + nickel-in-charcoal (Cu-Ni/C): a bimetallic, heterogeneous catalyst for cross-couplings.

A new heterogeneous catalyst composed of copper and nickel oxide particles supported within charcoal has been developed. It catalyzes cross-couplings that traditionally use palladium, nickel, or copper, including Suzuki-Miyaura reactions, Buchwald-Hartwig aminations, vinylalane alkylations, etherifications of aryl halides, aryl halide reductions, asymmetric conjugate reductions of activated olefins, and azide-alkyne "click" reactions.

(BDP)CuH: a "hot" Stryker's reagent for use in achiral conjugate reductions.

A ligand-modified, economical version of Stryker's reagent (SR) has been developed based on a bidentate, achiral bis-phosphine. Generated in situ, "(BDP)CuH" smoothly effects conjugate reductions of a variety of unsaturated substrates, including those that are normally unreactive toward SR. Substrate-to-ligand ratios typically on the order of 1000-10000:1 can be used leading to products in high yields.