Design and Evaluation of a Cyclobutane Diazirine Alkyne Tag for Photoaffinity Labeling in Cells.

Alkyl diazirines are frequently used in photoaffinity labeling to map small molecule-protein interactions in target identification studies. However, the alkyl diazirines can preferentially label acidic amino acids and acidic protein surfaces in a pH-dependent manner, presumably via a reactive alkyl diazo intermediate. Here, we explore the use of ring strain to alter these reactivity preferences and report the development of a cyclobutane diazirine photoaffinity tag with reduced pH-dependent reactivity, termed PALBOX. We show that PALBOX possesses differential reactivity profiles as compared to other diazirine tags in vitro and is readily incorporated into small molecules to profile their binding interactions in cells. Using a set of small molecule fragments and ligands, we show that photoaffinity probes equipped with PALBOX can label the known protein targets in cells with reduced labeling of known alkyl diazirine off-targets. Finally, we demonstrate that ligands equipped with PALBOX can accurately map small molecule-protein binding sites. Thus, PALBOX is a versatile diazirine-based photoaffinity tag for use in the development of chemical probes for photoaffinity labeling experiments, including the study of small molecule-protein interactions.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

Development of Photolenalidomide for Cellular Target Identification.

The thalidomide analogue lenalidomide (Len) is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photolenalidomide (pLen), a Len probe with a photoaffinity label and enrichment handle, designed for target identification by chemical proteomics. pLen preserves the substrate degradation profile, phenotypic antiproliferative and immunomodulatory properties of Len, and enhances interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using pLen, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by pLen and forms a ternary complex with CRBN in the presence of Len across several epithelial cell lines but is itself not ubiquitylated or degraded. These data point to the existence of a broader array of targets induced by ligands to CRBN that may or may not be degraded, which can be identified by the highly translatable application of pLen to additional biological systems.

Selective Divergent Synthesis of Indanols, Indanones, and Indenes via Acid-Mediated Cyclization of ( Z)- and ( E)-(2-Stilbenyl)methanols and Its Application for the Synthesis of Paucifloral F Derivatives.

Starting from bromo/iodobenzaldehyde derivatives, the corresponding ( Z)- and ( E)-(2-stilbenyl)methanols could be prepared in 2-5 steps via Pd-catalyzed cross-coupling reactions (Sonogashira and Heck reactions) followed by aryllithium/aryl Grignard addition. For the ( E)-stilbenes, subsequent acid-mediated cyclization using p-TsOH immobilized on silica (PTS-Si) at low temperatures furnished the 2,3- trans-1-indanols with complete stereocontrol at the C2-C3. Further oxidization of the alcohol provided the indanones, which are structurally related to the natural product paucifloral F. At higher temperatures, 1,2- and 2,3-disubstituted indenes could be selectively prepared in good to excellent yields. On the other hand, the ( Z)-stilbenes, under similar conditions (PTS-Si), did not give the indanols; only the 1,2-disubstituted indenes could be obtained. To gain further insights into the stereochemistry at C2-C3 for the ( Z)-stilbenes, hydride or azide was employed as a nucleophile; the corresponding indane products were obtained with the cis stereochemistry at the C2-C3. Thus, the ( Z)- or ( E)-olefin geometry of the substrate directed the stereoselective indanyl cyclization to furnish the cis or trans at the C2-C3 ring junction, respectively, while reaction conditions controlled the selectivity of the product types.

Small Molecule Interactome Mapping by Photoaffinity Labeling Reveals Binding Site Hotspots for the NSAIDs.

Many therapeutics elicit cell-type specific polypharmacology that is executed by a network of molecular recognition events between a small molecule and the whole proteome. However, measurement of the structures that underpin the molecular associations between the proteome and even common therapeutics, such as the nonsteroidal anti-inflammatory drugs (NSAIDs), is limited by the inability to map the small molecule interactome. To address this gap, we developed a platform termed small molecule interactome mapping by photoaffinity labeling (SIM-PAL) and applied it to the in cellulo direct characterization of specific NSAID binding sites. SIM-PAL uses (1) photochemical conjugation of NSAID derivatives in the whole proteome and (2) enrichment and isotope-recoding of the conjugated peptides for (3) targeted mass spectrometry-based assignment. Using SIM-PAL, we identified the NSAID interactome consisting of over 1000 significantly enriched proteins and directly characterized nearly 200 conjugated peptides representing direct binding sites of the photo-NSAIDs with proteins from Jurkat and K562 cells. The enriched proteins were often identified as parts of complexes, including known targets of NSAID activity (e.g., NF-κB) and novel interactions (e.g., AP-2, proteasome). The conjugated peptides revealed direct NSAID binding sites from the cell surface to the nucleus and a specific binding site hotspot for the three photo-NSAIDs on histones H2A and H2B. NSAID binding stabilized COX-2 and histone H2A by cellular thermal shift assay. Since small molecule stabilization of protein complexes is a gain of function regulatory mechanism, it is conceivable that NSAIDs affect biological processes through these broader proteomic interactions. SIM-PAL enabled characterization of NSAID binding site hotspots and is amenable to map global binding sites for virtually any molecule of interest.

Transfer of axial chirality through the nickel-catalysed hydrocyanation of chiral allenes.

The transfer of axial chirality of allenes is beginning to be exploited as a powerful method for creating central chirality, particularly through the use of transition metal catalysis. In this communication, the transfer of axial chirality of chiral allenes via nickel-catalysed hydrocyanation is achieved through both regio- and face-selective hydronickelation as well as regioselective reductive elimination. This protocol was applied to 12 substrates and gave chiral carbonitriles with up to 97% ee. Further application to hydrocyanative cyclization using a chiral allene-yne is also presented, along with a discussion of the corresponding mechanism of racemization.

A new protocol for nickel-catalysed regio- and stereoselective hydrocyanation of allenes.

Regio- and stereoselective hydrocyanation under nickel catalysis is described. This report shows that allenyl C-C double bonds are discriminated and converted to the corresponding carbonitriles as a single product. The key functionalities for achieving high regio- and stereocontrol are aryl and cyclopropyl groups in the substrates.

Regioselective hydronickelation of allenes and its application to the hydrocyanative carbocyclization reaction of allene-ynes and bis-allenes.

The carbocyanative cyclization of allene-ynes and bis-allenes under nickel catalysis is described. The key steps are the regioselective hydronickelation of allenes and subsequent cyclization via carbometalation. The former step determines the reaction pathway, and the latter controls the stereochemistry of substituted olefins. The products are useful carbo- and heterocycles that include a cyano group, functionalized double bonds, and quaternary carbons.