Synthesis and Antiproliferative Activities of OSW‐1 Analogues Bearing 2”‐O‐p‐Acylaminobenzoyl Residues†

OSW‐1 is a well‐known natural saponin with potent antitumor activities. We have designed and prepared a small library of 22 OSW‐1 analogues with a variety of p‐acylamino‐benzoyl groups installed at C2” of the xylose residue, wherein a regioselective (1→3)‐glycosylation of arabinoside 3,4‐diol has been achieved by manipulation of the protecting groups on the imidate donors. Bioassays lead to new structure‐activity relationships as well as two applicable fluorescent probes, which are found to localize to lysosomes in HeLa cells and could be used in further antitumor mechanism studies of OSW‐1 in living cells.     

Late-Stage C(sp2)−H Functionalization: A Powerful Toolkit To Arm Natural Products for In Situ Proteome Profiling?

The comprehensive investigation of target interactions from native cellular environments is of paramount importance for natural products and related bioactive compounds in drug discovery and chemical biology. Current chemoproteomic tools, such as in situ proteome profiling can do so effectively, but rely heavily on “tagged” probes that are accessible through traditional organic synthesis at the reactive sites of a compound, which may often be required for target binding. Late-stage functionalization may resolve such limitations by tagging compounds in a single step at biologically inert C−H bonds. Herein, recent advances in late-stage C(sp²)−H functionalization of (hetero)arenes, which are present in many natural products, are summarized, and new toolkits for more widespread use of such strategies to install natural products with next-generation “minimalist” linkers for in situ proteome profiling are suggested.     

Recent advances in target identification by natural product based chemical probes

Natural products have been extensively used to treat diseases throughout human history. These are mainly because natural products normally target biological macromolecules selectively. Target identification could help us to develop new therapeutic agents and discover new biological pathways underlying human diseases. Herein, we highlight some recent examples of using natural products and their derivatives as chemical probes to identify the molecular targets and elucidate mode of action.     

In Vivo Imaging of Mouse Tumors by a Lipidated Cathepsin S Substrate

The synthesis and evaluation of two cathepsin S‐specific probes is described. For long‐term retention of the probe at the target site and a high signal‐to‐noise ratio, we introduced a lipidation approach via the simple attachment of palmitoic acid to the reporter. After cathepsin S‐specific cleavage in cultured cells and in a grafted tumor mouse model, fluorescence increased owing to dequenching and we observed an intracellular accumulation of the fluorescence in the target tissue. The lipidated probe provided a prolonged and strongly fluorescent signal in tumors when compared to the very similar non‐lipidated probe, demonstrating that non‐invasive tumor identification is feasable. The homing principle by probe lipidation might also work for selective administration of cytotoxic compounds to specifically reduce tumor mass.     

Downfalls of Chemical Probes Acting at the Kinase ATP-Site: CK2 as a Case Study

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase’s biology, with wide-reaching implications for drug development.     

Werkzeug für die Chemische Biologie. Semisynthese

Semisynthetic techniques have greatly contributed to the rapid development of Chemical Biology in recent years. In this regard the semisynthesis of complex modified proteins as well as the selective derivatization of natural products has evolved into more than mere proof‐of‐principle concepts but powerful tools to probe protein functions. This technology provides a solid basis for further investigations on proteomics and qualitative and quantitative cell biology. The interdisciplinary charter bridging chemistry and biology is the hallmark of semisynthesis. It can be expected that its scientific impact will further increase in the future.     

Reactivity of functional groups on the protein surface: development of epoxide probes for protein labeling

We present the development of new affinity probes for protein labeling based on an epoxide reactive group. Systematic screening revealed that an epoxide functionality possesses the special combination of stability and reactivity which renders it stable toward proteins in solution but reactive on the protein surface outside the active site (proximity-induced reactivity). Highly efficient and selective labeling of purified HCA II (human carbonic anhydrase II) was achieved. For instance, 2 equiv of epoxide probe 9 was sufficient for nearly quantitative labeling of HCA II (>90% yield, 20 h reaction time). MS analysis of the labeled protein revealed that 1 equiv of the probe was attached and that labeling occurred at a single residue (His 64) outside the active site. Importantly, epoxide probe 9 selectively labeled HCA II both in simple protein mixtures and in cellular extracts. In addition to the chemical insight and its relevance to many epoxide-containing natural products, this study generated a promising lead in the development of new affinity probes for protein labeling.     

High‐Throughput Kinetic Analysis for Target‐Directed Covalent Ligand Discovery

Cysteine‐reactive small molecules are used as chemical probes of biological systems and as medicines. Identifying high‐quality covalent ligands requires comprehensive kinetic analysis to distinguish selective binders from pan‐reactive compounds. Quantitative irreversible tethering (qIT), a general method for screening cysteine‐reactive small molecules based upon the maximization of kinetic selectivity, is described. This method was applied prospectively to discover covalent fragments that target the clinically important cell cycle regulator Cdk2. Crystal structures of the inhibitor complexes validate the approach and guide further optimization. The power of this technique is highlighted by the identification of a Cdk2‐selective allosteric (type IV) kinase inhibitor whose novel mode‐of‐action could be exploited therapeutically.     

Novel Coumarin-based Fluorescent Probe for Selective Detection of Bisulfite Anion in Water

Coumarins and its analogues have been widely used as chromophore in design of fluorescent probe, while less coumarin‐based fluorescent probe was reported for detection of anion in water. In this article, coumarin‐based fluorescent probes with salicylaldehyde functionality as recognition unit have been developed for selective detection of bisulfite anions in water. Four novel fluorescent probes were synthesized from 4‐haloresorcinol in three steps. The chemoprobe exhibited selective response to bisulfite over other anions. Moreover, the detection mechanism was studied. Upon bisulfite added, the fluorescent intensity of the probes was enhanced highly due to the nucleophilic addition reaction of formyl group with bisulfite anion.     

香豆素类荧光探针对硫化氢检测的研究进展

硫化氢(H2S)是目前人们发现的第三类生物内源性“气体信使分子”。其及时检测对人类的健康有着非常大的意义。随着荧光探针技术的发展,有机小分子荧光探针受到广大学者的关注。其中,香豆素因其结构简单,荧光量子产率高以及易于功能化而备受青睐。本文根据探针的识别机理综述近三年来报道的香豆素类H2S荧光探针代表性研究成果,并对其进行了展望,为后续设计开发更具实用价值的H2S荧光探针提供一点有益的参考。     

Imaging of Colorectal Cancers Using Activatable Nanoprobes with Second Near‐Infrared Window Emission

Fluorescent probes in the second near‐infrared window (NIR‐II) allow high‐resolution bioimaging with deep‐tissue penetration. However, existing NIR‐II materials often have poor signal‐to‐background ratios because of the lack of target specificity. Herein, an activatable NIR‐II nanoprobe for visualizing colorectal cancers was devised. This designed probe displays H₂S‐activated ratiometric fluorescence and light‐up NIR‐II emission at 900–1300 nm. By using this activatable and target specific probe for deep‐tissue imaging of H₂S‐rich colon cancer cells, accurate identification of colorectal tumors in animal models were performed. It is anticipated that the development of activatable NIR‐II probes will find widespread applications in biological and clinical systems.     

From Phenotypic Hit to Chemical Probe: Chemical Biology Approaches to Elucidate Small Molecule Action in Complex Biological Systems

Biologically active small molecules have a central role in drug development, and as chemical probes and tool compounds to perturb and elucidate biological processes. Small molecules can be rationally designed for a given target, or a library of molecules can be screened against a target or phenotype of interest. Especially in the case of phenotypic screening approaches, a major challenge is to translate the compound-induced phenotype into a well-defined cellular target and mode of action of the hit compound. There is no “one size fits all” approach, and recent years have seen an increase in available target deconvolution strategies, rooted in organic chemistry, proteomics, and genetics. This review provides an overview of advances in target identification and mechanism of action studies, describes the strengths and weaknesses of the different approaches, and illustrates the need for chemical biologists to integrate and expand the existing tools to increase the probability of evolving screen hits to robust chemical probes.     

Sterically Controlled C−H Olefination of Heteroarenes

The regioselective functionalization of heteroarenes is a highly attractive synthetic target due to the prevalence of multiply substituted heteroarenes in nature and bioactive compounds. Some substitution patterns remain challenging: While highly efficient methods for the C2‐selective olefination of 3‐substituted five‐membered heteroarenes have been reported, analogous methods to access the 5‐olefinated products have remained limited by poor regioselectivities and/or the requirement to use an excess of the valuable heteroarene starting material. Herein we report a sterically controlled C?H olefination using heteroarenes as the limiting reagent. The method enables the highly C5‐selective olefination of a wide range of heteroarenes and is shown to be useful in the context of late‐stage functionalization.     

Diversity-driven chemical probe development for biomolecules: beyond hypothesis-driven approach

Bioprobes are indispensable tools for biological study and clinical diagnosis. A conventional strategy for probe development is hypothesis-driven approach based on known molecular mechanisms of recognition for individual analytes. However, even the most sophisticated rational design does not always guarantee the applicability of probes in complex biological systems, therefore the efficiency and scope of probe development has been intrinsically limited. Diversity-driven approach is a rapidly emerging alternative and has been employed for the development of new probes even in the absence of the knowledge about target recognition mechanism. This tutorial review summarizes the recent advances in probe development along with conceptual advantages and perspectives of the diversity-driven approach.     

Protein Organic Chemistry and Applications for Labeling and Engineering in Live‐Cell Systems

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry‐based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test‐tube settings; 2) the bioorthogonal reactions of proteins with non‐natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag–probe pair for labeling natural amino acids; and 4) ligand‐directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2–4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.     

Apoptolidin, a selective cytotoxic agent, is an inhibitor of F0F1-ATPase

BACKGROUND: Apoptolidin is a macrolide originally identified on the basis of its ability to selectively kill E1A and E1A/E1B19K transformed rat glial cells while not killing untransformed glial cells. The goal of this study was to identify the molecular target of this newly discovered natural product. RESULTS: Our approach to uncovering the mechanism of action of apoptolidin utilized a combination of molecular and cell-based pharmacological assays as well as structural comparisons between apoptolidin and other macrocyclic polyketides with known mechanism of action. Cell killing induced by apoptolidin was independent of p53 status, inhibited by BCL-2, and dependent on the action of caspase-9. PARP was completely cleaved in the presence of 1 microM apoptolidin within 6 h in a mouse lymphoma cell line. Together these results suggested that apoptolidin might target a mitochondrial protein. Structural comparisons between apoptolidin and other macrolides revealed significant similarity between the apoptolidin aglycone and oligomycin, a known inhibitor of mitochondrial F0F1-ATP synthase. The relevance of this similarity was established by demonstrating that apoptolidin is a potent inhibitor of the F0F1-ATPase activity in intact yeast mitochondria as well as Triton X-100-solubilized ATPase preparations. The K(i) for apoptolidin was 4-5 microM. The selectivity of apoptolidin in the NCI-60 cell line panel was found to correlate well with that of several known anti-fungal natural products that inhibit the eukaryotic mitochondrial F0F1-ATP synthase. SIGNIFICANCE: Although the anti-fungal activities of macrolide inhibitors of the mitochondrial F0F1-ATP synthase such as oligomycin, ossamycin and cytovaricin are well-documented, their unusual selectivity toward certain cell types is not widely appreciated. The recent discovery of apoptolidin, followed by the demonstration that it is an inhibitor of the mitochondrial F0F1-ATP synthase, highlights the potential relevance of these natural products as small molecules to modulate apoptotic pathways. The mechanistic basis for selective cytotoxicity of mitochondrial ATP synthase inhibitors is discussed.     

Identification of Tumor Initiating Cells with a Small‐Molecule Fluorescent Probe by Using Vimentin as a Biomarker

Tumor initiating cells (TICs) have been implicated in clinical relapse and metastasis of a variety of epithelial cancers, including lung cancer. While efforts toward the development of specific probes for TIC detection and targeting are ongoing, a universal TIC probe has yet to be developed. We report the first TIC‐specific fluorescent chemical probe, TiY, with identification of the molecular target as vimentin, a marker for epithelial‐to‐mesenchymal transition (EMT). TiY selectively stains TICs over differentiated tumor cells or normal cells, and facilitates the visualization and enrichment of functionally active TICs from patient tumors. At high concentration, TiY also shows anti‐TIC activity with low toxicity to non‐TICs. With the unexplored target vimentin, TiY shows potential as a first universal probe for TIC detection in different cancers.     

Photo-affinity labeling strategies in identifying the protein ligands of bioactive small molecules: examples of targeted synthesis of drug analog photoprobes

The discovery of many new targets by chemical genetics has frequently exploited the fact that their biologically active chemical ligands were reactive and thus could covalently bind to their protein target(s). When experimental compounds or therapeutic agents with unidentified mechanisms of action do not contain reactive groups that can covalently label the putative site of molecular action, it may be possible to create a reactive photo-affinity probe if there is sufficient knowledge of the structure-activity relationship of the chemical series. Two specific examples are presented. These include the use of photo-affinity probes in the identification of the mechanism of action of synthetic oxazolidinones, a class of novel acting antibiotics and in the identification of a novel target for the insulin-sensitizing thiazolidinediones. Developments in photo-affinity labeling and combinatorial library design now imply that the parallel incorporation of photo-probes into screening library design could, at least in principle, greatly facilitate reverse pharmacological and chemical genetics approaches to protein target discovery.     

Switchable Solvatochromic Probes for Live‐Cell Super‐resolution Imaging of Plasma Membrane Organization

Visualization of the nanoscale organization of cell membranes remains challenging because of the lack of appropriate fluorescent probes. Herein, we introduce a new design concept for super‐resolution microscopy probes that combines specific membrane targeting, on/off switching, and environment sensing functions. A functionalization strategy for solvatochromic dye Nile Red that improves its photostability is presented. The dye is grafted to a newly developed membrane‐targeting moiety composed of a sulfonate group and an alkyl chain of varied lengths. While the long‐chain probe with strong membrane binding, NR12A, is suitable for conventional microscopy, the short‐chain probe NR4A, owing to the reversible binding, enables first nanoscale cartography of the lipid order exclusively at the surface of live cells. The latter probe reveals the presence of nanoscopic protrusions and invaginations of lower lipid order in plasma membranes, suggesting a subtle connection between membrane morphology and lipid organization.