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Although more than 98% of the human genome is noncoding, nearly all drugs on the market target one of about 700 disease-related proteins. However, an increasing number of diseases are now being attributed to noncoding RNA and the ability to target them would vastly expand the chemical space for drug development. We recently devised a screening strategy based upon affinity-selection mass spectrometry and succeeded in identifying bioactive compounds for the noncoding RNA prototype, Xist. One such compound, termed X1, has drug-like properties and binds specifically to the RepA motif of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that X1 changes the conformation of RepA in solution, thereby explaining the displacement of cognate interacting protein factors (PRC2 and SPEN) and inhibition of X-chromosome inactivation. In this Perspective, we discuss lessons learned from these proof-of-concept experiments and suggest that RNA can be systematically targeted by drug-like compounds to disrupt RNA structure and function.

[Image: see text] Determination of target engagement for candidate drug molecules in the native cellular environment is a significant challenge for drug discovery programs. The cellular thermal shift assay (CETSA) has emerged as a powerful tool for determining compound target engagement through measurement of changes to a protein's thermal stability upon ligand binding. Here, we present a HiBiT thermal shift assay (BiTSA) that deploys a quantitative peptide tag for determination of compound target engagement in the native cellular environment using a high throughput, plate-based luminescence readout. We demonstrate that BiTSA can rapidly assess cellular target engagement of small molecule ligands against their cognate targets and highlight two applications of BiTSA for differentiating small molecules targeting mutant KRAS and TP53.

Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with pemetrexed/carbo(cis)platin/pembrolizumab (PCP) chemo-immunotherapy in lung cancer. Despite the strong genetic support and therapeutic potential for a NRF2 transcriptional inhibitor, currently there are no known direct inhibitors of the NRF2 protein or its complexes with MAF and/or DNA. Herein we describe the design of a novel and high-confidence homology model to guide a medicinal chemistry effort that resulted in the discovery of a series of peptides that demonstrate high affinity, selective binding to the Antioxidant Response Element (ARE) DNA and thereby displace NRF2-MAFG from its promoter, which is an inhibitory mechanism that to our knowledge has not been previously described. In addition to their activity in electrophoretic mobility shift (EMSA) and TR-FRET-based assays, we show significant dose-dependent ternary complex disruption of NRF2-MAFG binding to DNA by SPR, as well as cellular target engagement by thermal destabilization of HiBiT-tagged NRF2 in the NCI–H1944 NSCLC cell line upon digitonin permeabilization, and SAR studies leading to improved cellular stability. We report the characterization and unique profile of lead peptide 18, which we believe to be a useful in vitro tool to probe NRF2 biology in cancer cell lines and models, while also serving as an excellent starting point for additional in vivo optimization toward inhibition of NRF2-driven transcription to address a significant unmet medical need in non-small cell lung cancer (NSCLC).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Although more than 98% of the human genome is non-coding

Mass spectrometry-based discovery proteomics is an essential tool for the proximal readout of cellular drug action. Here, we apply a robust proteomic workflow to rapidly profile the proteomes of five lung cancer cell lines in response to more than 50 drugs. Integration of millions of quantitative protein-drug associations substantially improved the mechanism of action (MoA) deconvolution of single compounds. For example, MoA specificity increased after removal of proteins that frequently responded to drugs and the aggregation of proteome changes across cell lines resolved compound effects on proteostasis. We leveraged these findings to demonstrate efficient target identification of chemical protein degraders. Aggregating drug response across cell lines also revealed that one-quarter of compounds modulated the abundance of one of their known protein targets. Finally, the proteomic data led us to discover that inhibition of mitochondrial function is an off-target mechanism of the MAP2K1/2 inhibitor PD184352 and that the ALK inhibitor ceritinib modulates autophagy.

SUMMARYApart from their antimicrobial properties, tetracyclines demonstrate clinically validated effects in the amelioration of pathological inflammation and human cancer. Delineation of the target(s) and mechanism(s) responsible for these effects, however, has remained elusive. Here, employing quantitative mass spectrometry-based proteomics, we identified human 80S ribosomes as targets of the tetracyclines Col-3 and doxycycline. We then developed in cell click selective crosslinking with RNA sequence profiling (icCL-Seq) to map binding sites for these tetracyclines on key human rRNA substructures at nucleotide resolution. Importantly, we found that structurally and phenotypically variant tetracycline analogs could chemically discriminate these rRNA binding sites. We also found that tetracyclines both subtly modify human ribosomal translation and selectively activate the cellular integrated stress response (ISR). Together, the data reveal that targeting of specific rRNA substructures, activation of the ISR, and inhibition of translation are correlated with the anti-proliferative properties of tetracyclines in human cancer cell lines.

A biocatalytic platform that employs the final two monomodular type I polyketide synthases (PKS) of the pikromycin pathway in vitro followed by direct appendage of D-desosamine and final C-H oxidation(s) in vivo was developed and applied toward the synthesis of a suite of 12-and 14-membered ring macrolide natural products. This methodology delivered both compound classes in thirteen steps (longest linear sequence) from commercially available (R)-Roche ester in >10% overall yields.

Natural product biosynthetic pathways have evolved enzymes with myriad activities that represent an expansive array of chemical transformations for constructing secondary metabolites. Recently, harnessing the biosynthetic potential of these enzymes through chemoenzymatic synthesis has provided a powerful tool that often rivals the most sophisticated methodologies in modern synthetic chemistry and provides new opportunities for accessing chemical diversity. Herein, we describe our research efforts with enzymes from a broad collection of biosynthetic systems, highlighting recent progress in this exciting field.

1,10-Phenanthroline reacts with aldehydes and ketones in the presence of samarium diiodide to produce 2-(1-hydroxyalkyl)-1,10-phenanthrolines. The hydroxyalkyl substituent can be functionalized in numerous ways or removed to permit further ligand variation. The carbonyl coupling reaction can also be repeated to provide 2,9-disubstituted phenanthrolines. Taken together, these operations provide ready access to a large number of phenanthroline derivatives to serve as ligand libraries for catalyst exploration.

Natural product biosynthetic pathways have evolved enzymes with myriad activities that represent an expansive array of chemical transformations for constructing secondary metabolites. Recently, harnessing the biosynthetic potential of these enzymes through chemoenzymatic synthesis has provided a powerful tool that often rivals the most sophisticated methodologies in modern synthetic chemistry and provides new opportunities for accessing chemical diversity. Herein, we describe our research efforts with enzymes from a broad collection of biosynthetic systems, highlighting recent progress in this exciting field.

The 6-deoxyerythronolide B synthase (DEBS) and pikromycin (Pik) modular polyketide synthases are unique multifunctional enzyme systems that are responsible for the biosynthesis of the erythromycin and pikromycin 14-membered ring aglycones, respectively. Together, these natural product biosynthetic systems provide excellent platforms to examine the fundamental structural and catalytic elements that govern polyketide assembly, processing and macrocyclization. In these studies, the native pentaketide intermediate for DEBS was synthesized and employed for in vitro chemoenzymatic synthesis of macrolactone products in engineered monomodules Ery5, Ery5-TE and Ery6. A comparative analysis was performed with the corresponding Pik module 5 (PikAIII) and module 6 (PikAIV), dissecting key similarities and differences between these highly related PKSs. The data revealed that individual modules in the DEBS and Pik PKSs possess distinctive molecular selectivity profiles, and suggest that substrate recognition has evolved unique characteristics in each system.

2-Substituted pyridine, quinoline, isoquinoline, bipyridine, and 1,10-phenanthroline analogues of benzylic acetates undergo SmI(2)-promoted coupling with aldehydes and ketones to afford (2-hydroxyalkyl)heteroaromatics. [reaction: see text]