Zhenkun Na

“Minimalist” Cyclopropene-Containing Photo-Cross-Linkers Suitable for Live-Cell Imaging and Affinity-Based Protein Labeling

Target identification of bioactive compounds within the native cellular environment is important in biomedical research and drug discovery, but it has traditionally been carried out in vitro. Information about how such molecules interact with their endogenous targets (on and off) is currently highly limited. An ideal strategy would be one that recapitulates protein-small molecule interactions in situ (e.g., in living cells) and at the same time enables enrichment of these complexes for subsequent proteome-wide target identification. Similarly, small molecule-based imaging approaches are becoming increasingly available for in situ monitoring of a variety of proteins including enzymes. Chemical proteomic strategies for simultaneous bioimaging and target identification of noncovalent bioactive compounds in live mammalian cells, however, are currently not available. This is due to a lack of photoaffinity labels that are minimally modified from their parental compounds, yet chemically tractable using copper-free bioorthogonal chemistry. We have herein developed novel minimalist linkers containing both an alkyl diazirine and a cyclopropene. We have shown chemical probes (e.g., BD-2) made from such linkers could be used for simultaneous in situ imaging and covalent labeling of endogenous BRD-4 (an important epigenetic protein) via a rapid, copper-free, tetrazine-cyclopropene ligation reaction (k2 > 5 M(-1) s(-1)). The key features of our cyclopropenes, with their unique C-1 linkage to BRD-4-targeting moiety, are their tunable reactivity and solubility, relative stability, and synthetic accessibility. BD-2, which is a linker-modified analogue of (+)-JQ1 (a recently discovered nanomolar protein-protein-interaction inhibitor of BRD-4), was subsequently used in a cell-based proteome profiling experiment for large-scale identification of potential off-targets of (+)-JQ1. Several newly identified targets were subsequently confirmed by preliminary validation experiments.

Preparation of Small-Molecule Microarrays by trans-Cyclooctene Tetrazine Ligation and Their Application in the High-Throughput Screening of Protein–Protein Interaction Inhibitors of Bromodomains†

The e-N-acetylation of lysine residues (Kac) is one of the most common posttranslational modifications in proteins that are associated with epigenetics, and frequently occurs in large macromolecular complexes that play a role in chromatin remodelling, DNA damage, and cell-cycle control. In histones, acetylated lysines reduce electrostatic interactions with the negatively charged DNA phosphates, thus providing a more relaxed chromatin structure, which is associated with transcriptionally active genes. The cellular histone acetylation levels are strictly controlled by histone acetyltransferases (HATs; epigenetic “writers”) and histone deacetylases (HDACs; epigenetic “erasers”). 3] Furthermore, Kac affects gene transcription through interactions with bromodomain (BRD)-containing proteins (BCPs; epigenetic “readers”). HATs and HDACs have traditionally been the main research focus in medicinal chemistry and drug discovery for epigenetic diseases. As enzymes, they are considered as “druggable”. The development of protein–protein interaction (PPI) inhibitors that target the epigenetic readers, on the other hand, is much more challenging. It is generally believed that small molecules do not bind sufficiently tightly to the much larger protein–protein interface because they contain only a limited number of functional groups. Such simplistic views, however, have recently been challenged by two ground-breaking studies that showed that certain benzodiazepine-containing compounds (e.g., JQ1 and I-BET) are specific nanomolar PPI inhibitors of BET bromodomains (e.g., BRD4). This has spurred interest in compounds that might possess similar biological activities against other BRDs. However, there remains a lack of sensitive assays for rapid and high-throughput screening (HTS) of other BRD-binding compounds. Existing assays, including those based on fluorescence polarization (FP), isothermal titration calorimetry (ITC), protein stability shift, and surface plasmon resonance (SPR), are either applicable to only a few wellknown BRDs (e.g., BRD2/3/ 4), or they suffer from limited throughput because large amounts of proteins are required. Furthermore, for most of the 61 human BRDs, their cognate Kac-binding sequences are not well-understood. Recent large-scale structural studies and histone–peptide membrane arrays failed to identify any sub-micromolar-binding acetylated peptides that could be suitable for HTS assays. It is perhaps not surprising that both JQ1 and I-BET were serendipitously discovered by HTS of random in-house compound libraries using cell-based reporter assays without any prior target information. These assays, though useful in their own right, are not target-oriented, and compounds identified during these assays may not actually act on their intended BRDs. Therefore, a prerequisite for drug-development efforts that focus on the discovery of new BRD inhibitors is the development of an HTS platform that is capable of general, rapid, and systematic large-scale screening of the interactions between BRDs and small molecules. We recently developed a powerful small-molecule microarray (SMM) that is capable of sensitive, quantitative, and rapid identification of cell-permeable small-molecule PPI inhibitors. SMMs are miniaturized assemblies of small molecules that are immobilized across a planar glass slide, on which thousands of protein–ligand interactions (strong and weak) may be measured, with minimal consumption of proteins and ligands (in the nL to mL range). We reasoned that BRDs, which are protein-binding domains that normally bind to cognate acetylated peptides with moderate or weak affinities, would be well-suited for our SMM approach. Herein, in a proof-of-concept study, we have successfully constructed a SMM on which 48 different benzodiazepines that are based on the core structure of JQ 1/I-BET were immobilized, and screened it against 55 fluorescently labeled proteins, twelve of which were human BRDs (Figure 1). Specifically, we have 1) developed the first SMM that is based on bioorthogonal trans-cyclooctene (TCO) tetrazine ligation for site-specific covalent immobilization of TCO-modified benzodiazepines with unprecedented speed; 2) used this SMM for miniaturized HTS of these compounds against [*] C.-J. Zhang, C. Y. J. Tan, Dr. J. Ge, Z. Na, G. Y. J. Chen, Dr. M. Uttamchandani, Prof. Dr. S. Q. Yao Department of Chemistry, National University of Singapore 3 Science Drive 3, Singapore 117543 (Singapore) E-mail: chmyaosq@nus.edu.sg Homepage: http://staff.science.nus.edu.sg/~ syao

A Small‐Molecule Protein–Protein Interaction Inhibitor of PARP1 That Targets Its BRCT Domain

Poly(ADP-ribose)polymerase-1 (PARP1) is a BRCT-containing enzyme (BRCT = BRCA1 C-terminus) mainly involved in DNA repair and damage response and a validated target for cancer treatment. Small-molecule inhibitors that target the PARP1 catalytic domain have been actively pursued as anticancer drugs, but are potentially problematic owing to a lack of selectivity. Compounds that are capable of disrupting protein-protein interactions of PARP1 provide an alternative by inhibiting its activities with improved selectivity profiles. Herein, by establishing a high-throughput microplate-based assay suitable for screening potential PPI inhibitors of the PARP1 BRCT domain, we have discovered that (±)-gossypol, a natural product with a number of known biological activities, possesses novel PARP1 inhibitory activity both in vitro and in cancer cells and presumably acts through disruption of protein-protein interactions. As the first known cell-permeable small-molecule PPI inhibitor of PAPR1, we further established that (-)-gossypol was likely the causative agent of PARP1 inhibition by promoting the formation of a 1:2 compound/PARP1 complex by reversible formation of a covalent imine linkage.

Discovery of Cell-Permeable Inhibitors That Target the BRCT Domain of BRCA1 Protein by Using a Small-Molecule Microarray†

BRCTs are phosphoserine-binding domains found in proteins involved in DNA repair, DNA damage response and cell cycle regulation. BRCA1 is a BRCT domain-containing, tumor-suppressing protein expressed in the cells of breast and other human tissues. Mutations in BRCA1 have been found in ca. 50% of hereditary breast cancers. Cell-permeable, small-molecule BRCA1 inhibitors are promising anticancer agents, but are not available currently. Herein, with the assist of microarray-based platforms, we have discovered the first cell-permeable protein-protein interaction (PPI) inhibitors against BRCA1. By targeting the (BRCT)2 domain, we showed compound 15 a and its prodrug 15 b inhibited BRCA1 activities in tumor cells, sensitized these cells to ionizing radiation-induced apoptosis, and showed synergistic inhibitory effect when used in combination with Olaparib (a small-molecule inhibitor of poly-ADP-ribose polymerase) and Etoposide (a small-molecule inhibitor of topoisomerase II). Unlike previously reported peptide-based PPI inhibitors of BRCA1, our compounds are small-molecule-like and could be directly administered to tumor cells, thus making them useful for future studies of BRCA1/PARP-related pathways in DNA damage and repair response, and in cancer therapy.

Dynamic Monitoring of Newly Synthesized Proteomes: Up‐Regulation of Myristoylated Protein Kinase A During Butyric Acid Induced Apoptosis

Post-translational modification (PTM) is a highly dynamic yet precisely controlled process by which most eukaryotic proteins are chemically diversified. Many critical cellular responses are mediated through PTMs, which lead to modulation of enzyme activity, protein conformation, protein–protein interaction, and cellular localization. Analysis of these modifications at the proteome level could provide invaluable biological insight but remains a technically challenging undertaking. Traditionally, PTMs have been studied by standard molecular biology techniques involving tedious isolation of individual proteins and subsequent direct detection and analysis of amino acids bearing the modification. Recent advances in mass spectrometry, when combined with stable-isotope or metabolic labeling approaches, have enabled several large-scale studies of PTMs and their dynamics. These methods, however, analyze PTM changes of all proteins (old and new) present in the cell at the time of sampling and thus are only able to evaluate PTM dynamics at the ensemble level. With unnatural metabolic building blocks and in vivo compatible conjugation chemistries becoming increasingly available (Scheme 1), we sought to develop a proteomic strategy for the detection and identification of newly synthesized proteomes and their PTMs (Figure 1). We envisioned several advantages of studying the PTM dynamics of newly synthesized proteomes: 1) this method decreases the complexity of the proteome and enables the identification of PTM changes that occur in a predefined protein synthesis window; 2) it gives an accurate estimate of the time scale of different PTM events in transforming newly synthesized, modification-free proteins into mature functional entities; 3) it permits PTM analysis of primary protein synthesis responses to internal and external cues. To isolate a newly synthesized proteome, wemade use of BONCAT (bio-orthogonal noncanonical amino acid tagging), which uses the known methionine surrogates azidohomoalanine (AHA) and homopropargylglycine (HPG) for metabolic incorporation into newly synthesized proteins (Scheme 1, blue) and the corresponding alkyneor azide-modified biotin reporter (Scheme 1, bottom) for subsequent proteome isolation. To monitor dynamic changes of an PTM event, we fed growing cells with an azideor alkynecontaining sugar, fatty acid, or lipid building block (Scheme 1, orange). It should be noted that while our work was in progress, Hang and co-workers reported a tandem labeling and detection method to monitor the dynamic acylation of LCK (a tyrosine kinase) and its turnover. Their work focused on the study of single PTM events (i.e. protein palmitoylation) of a specific protein (i.e., LCK) in a proteome. The work herein, while conceptually similar, greatly expands the scope of this double metabolic incorporation strategy by successfully demonstrating, for the first Scheme 1. Methionine surrogates (blue) and unnatural metabolite PTM probes (orange) form bio-orthogonal pairs for compatible double metabolic incorporation. Those forming pairs are boxed in the same group. Azideand alkyne-containing fluorophore and biotin reporters are also shown (bottom).

Protein–Protein Interaction Inhibitors of BRCA1 Discovered Using Small Molecule Microarrays

Microarray screening technology has transformed the life sciences arena over the last decade. The platform is widely used in the area of mapping interaction networks, to molecular fingerprinting and small molecular inhibitor discovery. The technique has significantly impacted both basic and applied research. The microarray platform can likewise enable high-throughput screening and discovery of protein-protein interaction (PPI) inhibitors. Herein we demonstrate the application of microarray-guided PPI inhibitor discovery, using human BRCA1 as an example. Mutations in BRCA1 have been implicated in ~50 % of hereditary breast cancers. By targeting the (BRCT)2 domain, we showed compound 15a and its prodrug 15b inhibited BRCA1 activities in tumor cells. Unlike previously reported peptide-based PPI inhibitors of BRCA1, the compounds identified could be directly administered to tumor cells, thus making them useful in targeting BRCA1/PARP-related pathways involved in DNA damage and repair response, for cancer therapy.

MSN-on-a-Chip: Cell-Based Screenings Made Possible on a Small-Molecule Microarray of Native Natural Products

Standard small‐molecule microarrays (SMMs) are not well‐suited for cell‐based screening assays. Of the few attempts made thus far to render SMMs cell‐compatible, all encountered major limitations. Here we report the first mesoporous silica nanoparticle (MSN)‐on‐a‐chip platform capable of allowing high‐throughput cell‐based screening to be conducted on SMMs. By making use of a glass surface on which hundreds of MSNs, each encapsulated with a different native natural product, were immobilized in spatially defined manner, followed by on‐chip mammalian cell growth and on‐demand compound release, high‐content screening was successfully carried out with readily available phenotypic detection methods. By combining this new MSN‐on‐a‐chip system with small interfering RNA technology for the first time, we discovered that (+)‐usniacin possesses synergistic inhibitory properties similar to those of olaparib (an FDA‐approved drug) in BRCA1‐knockdown cancer cells.

Screening Mammalian Cells on a Hydrogel: Functionalized Small Molecule Microarray

Mammalian cell-based microarray technology has gained wide attention, for its plethora of promising applications. The platform is able to provide simultaneous information on multiple parameters for a given target, or even multiple target proteins, in a complex biological system. Here we describe the preparation of mammalian cell-based microarrays using selectively captured of human prostate cancer cells (PC-3). This platform was then used in controlled drug release and measuring the associated drug effects on these cancer cells.