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The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down's syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

Combinatorial post-translational modifications (PTMs), which can serve as dynamic “molecular barcodes”, have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways., An erasable single molecule blot (eSiMBlot) assay provides an unprecedentedly simple and versatile platform for analyzing the combinatorial post-translational modifications in biological pathways.

Serendipitously, mono-allyloxylated cucurbit[7]uril (AO1 CB[7]) was discovered to act as an unconventional amphiphile which self-assembles into light-responsive vesicles (AO1 CB[7]VC) in water. Although the mono-allyloxy group, directly tethered on the periphery of CB[7], is much shorter (C4) than the hydrophobic tails of conventional amphiphiles, it played an important role in vesicle formation. Light-activated transformation of the allyloxy group by conjugation with glutathione was exploited as a remote tool to disrupt the vesicle. The vesicle showed on-demand release of cargo upon irradiation by a laser, after they were internalized into cancer cells. This result demonstrated the potential of AO1 CB[7]VC as a new type of light-responsive intracellular delivery vehicle for the release of therapeutic cargo, within cells, on demand.

A supramolecular FRET pair based on the ultrahigh binding affinity between cyanine 3 conjugated cucurbit[7]uril (CB[7]-Cy3) and cyanine 5 conjugated adamantylamine (AdA-Cy5) was exploited as a new synthetic tool for imaging cellular processes in live cells. Confocal laser scanning microscopy revealed that CB[7]-Cy3 and AdA-Cy5 were intracellularly translocated and accumulated in lysosomes and mitochondria, respectively. CB[7]-Cy3 and AdA-Cy5 then formed a host-guest complex, reported by a FRET signal, as a result of the fusion of lysosomes and mitochondria. This observation not only indicated that CB[7] forms a stable complex with AdA in a live cell, but also suggested that this FRET pair can visualize dynamic organelle fusion processes, such as those involved in the degradation of mitochondria through autophagy (mitophagy), by virtue of its small size, chemical stability, and ease of use.

The host-guest complexes between commercially available fluorescence dyes and cucurbit[n]urils (CB[n], n = 6, 7 and 8) were exploited as multiple sensor elements to provide arrays for sensing biogenic amines using principal component analysis (PCA). Since the sensor elements responds differently to each amine, the array generates distinct patterns of fluorescence changes for each amine. We analyzed these results using PCA to allow precise discrimination of individual biogenic amines. This result demonstrated the great potential of these host-guest complexes as useful sensor elements for biogenic molecules, which may be useful to develop a diagnostic tool for diseases including cancers.

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson’s disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LR...

A new non-proteolytic method to harvest a cell-sheet was demonstrated using controllable host-guest interactions which can be dissociated by treating a strong guest on demand. Fibroblast cells (NIH3T3) were grown to confluence on a 1,6-diammoniumhexane conjugated hyaluronic acid (DAH-HA) polymer which was anchored to a cucurbit[7]uril (CB[7]) surface using the host-guest interaction between DAH and CB[7]. Treating with a strong guest allowed the cultured cells to be detached from the surface as a free standing sheet. This approach demonstrated the great potential of controllable host-guest chemistry as a novel tool for non-proteolytic harvesting of cell-sheets useful for regenerative therapy.

Chemical proteomics relies primarily on click-chemistry-based protein labeling and biotin-streptavidin enrichment, but these techniques have inherent limitations. Enrichment of intracellular proteins using a totally synthetic host-guest complex is described, overcoming the problem associated with the classical approach. We achieve this by affinity-based protein labeling with a target-specific probe molecule conjugated to a high-affinity guest (suberanilohydroxamic acid-ammonium-adamantane; SAHA-Ad) and then enriching the labeled species using a cucurbit[7]uril bead. This method shows high specificity for labeled molecules in a MDA-MB-231 breast cancer cell lysate. Moreover, this method shows promise for labeling proteins in live cells.

Cucurbit[6]uril (CB[6])-based polymer nanocapsules (CB[6]PNs) were used as a multimodal platform for cancer-targeted in vivo bioimaging. By taking advantage of the strong and robust host–guest interaction between CB[6] and spermidine (spmd) under in vivo conditions, the surface of the CB[6]PNs was non-covalently modified with various spmd-conjugated functionalities in a modular manner. The CB[6]PNs modified with imaging probes and targeting ligands showed tumor targeted PET and NIR imaging in mice. This proof of concept demonstrates the potential of non-covalently modified nanomaterials in real world in vivo systems.

Tb-based metal-organic framework nanoparticles (Tb-MOF NPs) with good colloidal stability and stable fluorescence properties in an aqueous solution were prepared by a simple mechanical grinding of Tb-MOF with a biocompatible polymer surfactant (F127). The characteristic fluorescence property of Tb-MOF NPs allowed us to use this nanomaterial as a cell imaging probe. Efficient cellular uptake of Tb-MOF NPs apparently via an energy-dependent endocytosis was observed by confocal laser scanning microscopy. By taking advantage of the porous nature of the Tb-MOF NPs an anticancer drug (doxorubicin) was successfully loaded and delivered to kill cancer cells to demonstrate their usage as a drug delivery vehicle. This simple grinding method afforded a nanosized, multifunctional biomaterial that was used for cell imaging and drug delivery, and it can be extended to other MOFs to widen their applications.

In this chapter, the synthesis, mechanism of formation, structural features, and functionalization of cucurbituril family members are introduced. Synthetic methodologies for the preparation of cucurbituril family members ranging from the initial discovery to recent developments are highlighted. A brief summary of the detailed investigation on the mechanism of cucurbituril formation, as well as key structural features of cucurbituril family members are showcased. Two major approaches to incorporate further functional groups on the skeleton of cucurbiturils are also introduced. One involves utilizing the building block approach, where the functional group is installed during the macrocyclization of cucurbiturils. The other applies direct functionalization of cucurbiturils, which installs the desired functional group through chemical and photochemical methods on the skeleton of cucurbituril macrocycles.

In this chapter, ultrastable synthetic binding pairs between cucurbit[7]uril and its guests are introduced as a supramolecular latching system. The unique features of the synthetic binding pairs including their small size with exceptionally high binding affinity, bio-orthogonality in binding, chemical tractability, scalable synthesis, and robust chemical structures are highlighted with a brief summary of their applications. Other interesting unique features of this binding pair system are also introduced such as delicate control of host–guest binding affinities by treatment with stronger guest molecules on-demand and negligible interferences of the binding properties from endogenous biomolecules, enabling accurate and precise analysis of bioimaging and proteomics.

We describe our work to establish structure- and fragment-based drug discovery to identify small molecules that inhibit the anti-apoptotic activity of the proteins Mcl-1 and Bcl-2. This identified hit series of compounds, some of which were subsequently optimized to clinical candidates in trials for treating various cancers. Many protein constructs were designed to identify protein with suitable properties for different biophysical assays and structural methods. Fragment screening using ligand-observed NMR experiments identified several series of compounds for each protein. The series were assessed for their potential for subsequent optimization using 1H and 15N heteronuclear single-quantum correlation NMR, surface plasmon resonance, and isothermal titration calorimetry measurements to characterize and validate binding. Crystal structures could not be determined for the early hits, so NMR methods were developed to provide models of compound binding to guide compound optimization. For Mcl-1, a benzodioxane/benzoxazine series was optimized to a Kd of 40 μM before a thienopyrimidine hit series was identified which subsequently led to the lead series from which the clinical candidate S 64315 (MIK 665) was identified. For Bcl-2, the fragment-derived series were difficult to progress, and a compound derived from a published tetrahydroquinone compound was taken forward as the hit from which the clinical candidate (S 55746) was obtained. For both the proteins, the work to establish a portfolio of assays gave confidence for identification of compounds suitable for optimization.

Here we report a recombinant protein (MS) obtained by genetic fusion of a mussel foot protein (Mfp3) motif into a silk spidroin (MaSp1). The MS not only self-assembled into a supramolecular fibre, as does the parent MaSp1, but also showed enhanced adhesiveness resulting from the DOPA-containing Mfp3 portion. The successful incorporation of the wet adhesiveness of Mfp3 into the well-structured assembly of MaSp1 may provide a new insight for the genetic design of underwater adhesive recombinant proteins by utilizing the structural features of a spidroin protein.

Here we report ultrastable synthetic binding pairs between cucurbit[7]uril (CB[7]) and adamantyl- (AdA) or ferrocenyl-ammonium (FcA) as a supramolecular latching system for protein imaging, overcoming the limitations of protein-based binding pairs. Cyanine 3-conjugated CB[7] (Cy3-CB[7]) can visualize AdA- or FcA-labeled proteins to provide clear fluorescence images for accurate and precise analysis of proteins. Furthermore, controllability of the system is demonstrated by treating with a stronger competitor guest. At low temperature, this allows us to selectively detach Cy3-CB[7] from guest-labeled proteins on the cell surface, while leaving Cy3-CB[7] latched to the cytosolic proteins for spatially conditional visualization of target proteins. This work represents a non-protein-based bioimaging tool which has inherent advantages over the widely used protein-based techniques, thereby demonstrating the great potential of this synthetic system., Although protein-ligand pairs are useful tools for bioimaging, they are susceptible to enzymatic degradation and interference from endogenous species. Here, the authors show that a synthetic and bioorthogonal cucurbit[7]uril-guest binding pair can be used to visualize proteins in cells, overcoming limitations of protein-based platforms.

Single-molecule surface-enhanced Raman spectroscopy (SERS) offers new opportunities for exploring the complex chemical and biological processes that cannot be easily probed using ensemble techniques. However, the ability to place the single molecule of interest reliably within a hot spot, to enable its analysis at the single-molecule level, remains challenging. Here we describe a novel strategy for locating and securing a single target analyte in a SERS hot spot at a plasmonic nanojunction. The "smart" hot spot was generated by employing a thiol-functionalized cucurbit[6]uril (CB[6]) as a molecular spacer linking a silver nanoparticle to a metal substrate. This approach also permits one to study molecules chemically reluctant to enter the hot spot, by conjugating them to a moiety, such as spermine, that has a high affinity for CB[6]. The hot spot can accommodate at most a few, and often only a single, analyte molecule. Bianalyte experiments revealed that one can reproducibly treat the SERS substrate such that 96% of the hot spots contain a single analyte molecule. Furthermore, by utilizing a series of molecules each consisting of spermine bound to perylene bisimide, a bright SERS molecule, with polymethylene linkers of varying lengths, the SERS intensity as a function of distance from the center of the hot spot could be measured. The SERS enhancement was found to decrease as 1 over the square of the distance from the center of the hot spot, and the single-molecule SERS cross sections were found to increase with AgNP diameter.

A permselective membrane with a non-covalently tailorable surface has been fabricated by deposition of a cucurbit[6]uril-based 2D polymer film onto a simple support membrane. Tuneable permselectivity of this membrane has been achieved through non-covalent surface modification using the remarkable host-guest chemistry of the incorporated cucurbit[6]uril units.

In this Commentary, we discuss cucurbit[7]uril-based ultrastable artificial binding pairs as a supramolecular latching system and how we envision this becoming important tools in proteomics. The limitations of current proteomic techniques are described with an emphasis on the lack of tools to answer questions about the complex and dynamic nature of the proteome. Our thoughts as to how artificial ultrastable binding pairs may be able to address these questions are given especially when they are combined with existing methods.

This tutorial review summarizes the continuining exploration of three prominent water-soluble hosts: cucurbiturils, pillar[n]arenes and deep-cavity cavitands. As we describe, these hosts are revealing how orchestrating the Hydrophobic Effect can lead to a broad range of properties and applications, from: nano-reactors, supramolecular polymers, stimuli-responsive biointerfaces, switches, and novel purification devices. We also describe how their study is also revealing more details about the properties of water and aqueous solutions.

A supramolecular hydrogel was formed by a simple mixing of solutions of nor-seco-cucurbit[10]uril (NS-CB[10]) and adamantylamine-terminated 4-armed polyethylene glycol (AdA-4-arm-PEG). In the formation of the hydrogel, NS-CB[10] acted as a noncovalent crosslinker to form a ternary complex with two AdA moieties. The dynamic and selective nature of the host-guest interaction between NS-CB[10] and AdA enabled the supramolecular hydrogel to rapidly recover its physical properties after it was damaged. In addition, the recovered hydrogel retained its physical properties with negligible differences from those of the pristine material, even after multiple self-healing cycles. The NS-CB[10]-based hydrogel with the self-healing property may be useful for various biological applications such as drug delivery, cell therapy and tissue engineering.

Libraries of nonpurified resorcinol amide derivatives were screened by surface plasmon resonance (SPR) to determine the binding dissociation constant (off-rate, kd) for compounds binding to the pyruvate dehydrogenase kinase (PDHK) enzyme. Parallel off-rate measurements against HSP90 and application of structure-based drug design enabled rapid hit to lead progression in a program to identify pan-isoform ATP-competitive inhibitors of PDHK. Lead optimization identified selective sub-100-nM inhibitors of the enzyme which significantly reduced phosphorylation of the E1α subunit in the PC3 cancer cell line in vitro.

Conventional immunoassays rely on antibodies that provide high affinity, specificity, and selectivity against a target analyte. However, the use of antibodies for the detection of small-sized, nonimmunogenic targets, such as pharmaceuticals and environmental contaminants, presents a number of challenges. Recent advances in protein engineering have led to the emergence of antibody mimetics that offer the high affinity and specificity associated with antibodies, but with reduced batch-to-batch variability, high stability, and in vitro selection to ensure rapid discovery of binders against a wide range of targets. In this work we explore the potential of Affimers, a recent example of antibody mimetics, as suitable bioreceptors for the detection of small organic target compounds, here methylene blue. Target immobilization for Affimer characterization was achieved using long-chained alkanethiol linkers coupled with oligoethylene glycol (LCAT–OEG). Using quartz crystal microbalance with dissipation monitoring (QCM-D), we determine the affinity constant, KD, of the methylene blue Affimer to be comparable to that of antibodies. Further, we demonstrate the high selectivity of Affimers for its target in complex matrixes, here a limnetic sample. Finally, we demonstrate an Affimer-based competition assay, illustrating the potential of Affimers as bioreceptors in immunoassays for the detection of small-sized, nonimmunogenic compounds.