Molecular basis for inhibition of adhesin-mediated bacterial-host interactions through a peptide-binding domain.

Infections typically begin with pathogens adhering to host cells. For bacteria, this adhesion can occur through specific ligand-binding domains. We identify a 20-kDa peptide-binding domain (PBD) in a 1.5-MDa RTX adhesin of a Gram-negative marine bacterium that colonizes diatoms. The crystal structure of this Ca2+-dependent PBD suggests that it may bind the C termini of host cell-surface proteins. A systematic peptide library analysis reveals an optimal tripeptide sequence with 30-nM affinity for the PBD, and X-ray crystallography details its peptide-protein interactions. Binding of the PBD to the diatom partner of the bacteria can be inhibited or competed away by the peptide, providing a molecular basis for inhibiting bacterium-host interactions. We further show that this PBD is found in other bacteria, including human pathogens such as Vibrio cholerae and Aeromonas veronii. Here, we produce the PBD ortholog from A. veronii and demonstrate, using the same peptide inhibitor, how pathogens may be prevented from adhering to their hosts.

A Structural Study of the Cytoplasmic Chaperone Effect of 14-3-3 Proteins on Ataxin-1.

Expansion of the polyglutamine tract in the N terminus of Ataxin-1 is the main cause of the neurodegenerative disease, spinocerebellar ataxia type 1 (SCA1). However, the C-terminal part of the protein - including its AXH domain and a phosphorylation on residue serine 776 - also plays a crucial role in disease development. This phosphorylation event is known to be crucial for the interaction of Ataxin-1 with the 14-3-3 adaptor proteins and has been shown to indirectly contribute to Ataxin-1 stability. Here we show that 14-3-3 also has a direct anti-aggregation or "chaperone" effect on Ataxin-1. Furthermore, we provide structural and biophysical information revealing how phosphorylated S776 in the intrinsically disordered C terminus of Ataxin-1 mediates the cytoplasmic interaction with 14-3-3 proteins. Based on these findings, we propose that 14-3-3 exerts the observed chaperone effect by interfering with Ataxin-1 dimerization through its AXH domain, reducing further self-association. The chaperone effect is particularly important in the context of SCA1, as it was previously shown that a soluble form of mutant Ataxin-1 is the major driver of pathology.

Structure-based evolution of a promiscuous inhibitor to a selective stabilizer of protein-protein interactions.

The systematic stabilization of protein-protein interactions (PPI) has great potential as innovative drug discovery strategy to target novel and hard-to-drug protein classes. The current lack of chemical starting points and focused screening opportunities limits the identification of small molecule stabilizers that engage two proteins simultaneously. Starting from our previously described virtual screening strategy to identify inhibitors of 14-3-3 proteins, we report a conceptual molecular docking approach providing concrete entries for discovery and rational optimization of stabilizers for the interaction of 14-3-3 with the carbohydrate-response element-binding protein (ChREBP). X-ray crystallography reveals a distinct difference in the binding modes between weak and general inhibitors of 14-3-3 complexes and a specific, potent stabilizer of the 14-3-3/ChREBP complex. Structure-guided stabilizer optimization results in selective, up to 26-fold enhancement of the 14-3-3/ChREBP interaction. This study demonstrates the potential of rational design approaches for the development of selective PPI stabilizers starting from weak, promiscuous PPI inhibitors.

Ligand-Based Design of Allosteric Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) Inverse Agonists.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor associated with the pathogenesis of autoimmune diseases. Allosteric inhibition of RORγt is conceptually new, unique for this specific nuclear receptor, and offers advantages over traditional orthosteric inhibition. Here, we report a highly efficient in silico-guided approach that led to the discovery of novel allosteric RORγt inverse agonists with a distinct isoxazole chemotype. The the most potent compound, 25 (FM26), displayed submicromolar inhibition in a coactivator recruitment assay and effectively reduced IL-17a mRNA production in EL4 cells, a marker of RORγt activity. The projected allosteric mode of action of 25 was confirmed by biochemical experiments and cocrystallization with the RORγt ligand binding domain. The isoxazole compounds have promising pharmacokinetic properties comparable to other allosteric ligands but with a more diverse chemotype. The efficient ligand-based design approach adopted demonstrates its versatility in generating chemical diversity for allosteric targeting of RORγt.

A study on the effect of synthetic α-to-ß3-amino acid mutations on the binding of phosphopeptides to 14-3-3 proteins.

Here we describe the synthesis of a series of α,ß-phosphopeptides, based on the phosphoepitope site on YAP1 (yes-associated protein 1), and the biochemical, biophysical and structural characterization of their binding to 14-3-3 proteins. The impact of systematic mono- and di-substitution of α → ß3 amino acid residues around the phosphoserine residue are discussed. Our results confirm the important role played by the +2 proline residue in the thermodynamics and structure of the phosphoepitope/14-3-3 interaction.

Cooperativity basis for small-molecule stabilization of protein-protein interactions.

A cooperativity framework to describe and interpret small-molecule stabilization of protein-protein interactions (PPI) is presented. The stabilization of PPIs is a versatile and emerging therapeutic strategy to target specific combinations of protein partners within the protein interactome. Currently, the potency of PPI stabilizers is typically expressed by their apparent affinity or EC50. Here, we propose that the effect of a PPI stabilizer be best described involving the cooperativity factor, α, between the stabilizer and binding partners in addition to the intrinsic affinity, K D II, of the stabilizer for one of the apo-proteins. By way of illustration, we combine fluorescence polarization measurements with thermodynamic modeling to determine the α and K D II for the PPI stabilization of 14-3-3 and TASK3 by fusicoccin-A (FC-A) and validate our approach by studying other PPI-partners of 14-3-3 proteins. Finally, we characterize a library of different stabilizer compounds, and perform structure-activity relationship studies in which molecular changes could be attributed to either changes in cooperativity or intrinsic affinity. Such insights should aid in the development of more effective protein-protein stabilizer drugs.

Tetrazine- trans-Cyclooctene Chemistry Applied to Fabricate Self-Assembled Fluorescent and Radioactive Nanoparticles for in Vivo Dual Mode Imaging.

Multimodal imaging agents combine two or more imaging modalities into one probe. Self-assembling fluorescent nanoparticles are a promising class of modular multimodal imaging probes as they can allow easy blending of imaging and targeting modalities. Our group recently developed a class of self-assembling and intrinsically fluorescent small molecule-based nanoparticles (SMNPs) with excellent optical properties. In this article, we describe the efficient radiolabeling of these SMNPs via a two-step bioconjugation strategy involving the inverse-electron-demand Diels-Alder ligation between a tetrazine (Tz)-tagged radiolabel and a trans-cyclooctene (TCO)-tagged fluorescent small molecule building block of the SMNPs. Studies in mice revealed that the SMNPs are well tolerated and could be monitored by both radioactivity and fluorescence, thereby demonstrating the potential of SMNPs in optical and dual-mode imaging in vivo. The work also testifies to the utility of the Tz-TCO conjugation chemistry for the labeling of self-assembled nanoparticles.

Mutually Exclusive Cellular Uptake of Combinatorial Supramolecular Copolymers.

The cellular uptake of self-assembled biological and synthetic matter results from their multicomponent properties. However, the interplay of the building block composition of self-assembled materials and uptake mechanisms urgently requires addressing. It is shown here that supramolecular polymers that self-assemble in aqueous media, are a modular and controllable platform to modulate cellular delivery by the introduction of small ligands or cationic moieties, with concomitantly different cellular uptake kinetics and valence dependence. A library of supramolecular copolymers revealed stringent mutually exclusive uptake behavior in which either of the uptake pathways dominated, with sharp compositional transition. Supramolecular biomaterial engineering thus provides for adaptive platforms with great potential for efficient tuning of multivalent and multicomponent systems interfacing with biological matter.

Inhibition of 14-3-3/Tau by Hybrid Small-Molecule Peptides Operating via Two Different Binding Modes.

Current molecular hypotheses have not yet delivered marketable treatments for Alzheimer's disease (AD), arguably due to a lack of understanding of AD biology and an overreliance on conventional drug modalities. Protein-protein interactions (PPIs) are emerging drug targets, which show promise for the treatment of, e.g., cancer, but are still underexploited for treating neurodegenerative diseases. 14-3-3 binding to phosphorylated Tau is a promising PPI drug target based on its reported destabilizing effect on microtubules, leading to enhanced neurofibrillary tangle formation as a potential cause of AD-related neurodegeneration. Inhibition of 14-3-3/Tau may therefore be neuroprotective. Previously, we reported the structure-guided development of modified peptide inhibitors of 14-3-3/Tau. Here, we report further efforts to optimize the binding mode and activity of our modified Tau peptides through a combination of chemical synthesis, biochemical assays, and X-ray crystallography. Most notably, we were able to characterize two different high-affinity binding modes, both of which inhibited 14-3-3-binding to full-length PKA-phosphorylated Tau protein in vitro as measured by NMR spectroscopy. Our findings, besides producing useful tool inhibitor compounds for studying 14-3-3/Tau, have enhanced our understanding of the molecular parameters for inhibiting 14-3-3/Tau, which are important milestones toward the establishment of our 14-3-3 PPI hypothesis.

A multi-gram-scale stereoselective synthesis of Z-endoxifen.

Z-Endoxifen is widely regarded as the most active metabolite of tamoxifen, and has recently demonstrated a 26.3% clinical benefit in a phase I clinical trial to treat metastatic breast cancer after the failure of standard endocrine therapy. Future pharmacological and pre-clinical studies of Z-endoxifen would benefit from reliable and efficient synthetic access to the drug. Here, we describe a short and efficient, stereoselective synthesis of Z-endoxifen capable of delivering multi-gram (37 g) quantities of the drug in >97% purity with a Z/E ratio >99% after trituration.

Synthesis and Self-Assembly of Bay-Substituted Perylene Diimide Gemini-Type Surfactants as Off-On Fluorescent Probes for Lipid Bilayers.

Interest in bay-substituted perylene-3,4:9,10-tetracarboxylic diimides (PDIs) for solution-based applications is growing due to their improved solubility and altered optical and electronic properties compared to unsubstituted PDIs. Synthetic routes to 1,12-bay-substituted PDIs have been very demanding due to issues with steric hindrance and poor regioselectivity. Here we report a simple one-step regioselective and high yielding synthesis of a 1,12-dihydroxylated PDI derivative that can subsequently be alkylated in a straightforward fashion to produce nonplanar 1,12-dialkoxy PDIs. These PDIs show a large Stokes shift, which is specifically useful for bioimaging applications. A particular cationic PDI gemini-type surfactant has been developed that forms nonfluorescent self-assembled particles in water ("off state"), which exerts a high fluorescence upon incorporation into lipophilic bilayers ("on state"). Therefore, this probe is appealing as a highly sensitive fluorescent labelling marker with a low background signal for imaging artificial and cellular membranes.

Characterization of Coding/Noncoding Variants for SHROOM3 in Patients with CKD.

Background Interpreting genetic variants is one of the greatest challenges impeding analysis of rapidly increasing volumes of genomic data from patients. For example, SHROOM3 is an associated risk gene for CKD, yet causative mechanism(s) of SHROOM3 allele(s) are unknown.Methods We used our analytic pipeline that integrates genetic, computational, biochemical, CRISPR/Cas9 editing, molecular, and physiologic data to characterize coding and noncoding variants to study the human SHROOM3 risk locus for CKD.Results We identified a novel SHROOM3 transcriptional start site, which results in a shorter isoform lacking the PDZ domain and is regulated by a common noncoding sequence variant associated with CKD (rs17319721, allele frequency: 0.35). This variant disrupted allele binding to the transcription factor TCF7L2 in podocyte cell nuclear extracts and altered transcription levels of SHROOM3 in cultured cells, potentially through the loss of repressive looping between rs17319721 and the novel start site. Although common variant mechanisms are of high utility, sequencing is beginning to identify rare variants involved in disease; therefore, we used our biophysical tools to analyze an average of 112,849 individual human genome sequences for rare SHROOM3 missense variants, revealing 35 high-effect variants. The high-effect alleles include a coding variant (P1244L) previously associated with CKD (P=0.01, odds ratio=7.95; 95% CI, 1.53 to 41.46) that we find to be present in East Asian individuals at an allele frequency of 0.0027. We determined that P1244L attenuates the interaction of SHROOM3 with 14-3-3, suggesting alterations to the Hippo pathway, a known mediator of CKD.Conclusions These data demonstrate multiple new SHROOM3-dependent genetic/molecular mechanisms that likely affect CKD.

Dual-Input Regulation and Positional Control in Hybrid Oligonucleotide/Discotic Supramolecular Wires.

The combination of oligonucleotides and synthetic supramolecular systems allows for novel and long-needed modes of regulation of the self-assembly of both molecular elements. Discotic molecules were conjugated with short oligonucleotides and their assembly into responsive supramolecular wires studied. The self-assembly of the discotic molecules provides additional stability for DNA-duplex formation owing to a cooperative effect. The appended oligonucleotides allow for positional control of the discotic elements within the supramolecular wire. The programmed assembly of these hybrid architectures can be modulated through the DNA, for example, by changing the number of base pairs or salt concentration, and through the discotic platform by the addition of discotic elements without oligonucleotide handles. These hybrid supramolecular-DNA structures allow for advanced levels of control over 1D dynamic platforms with responsive regulatory elements at the interface with biological systems.

Fragmentation of organic ions bearing fixed multiple charges observed in MALDI MS.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) was used to analyze a series of synthetic organic ions bearing fixed multiple charges. Despite the multiple intrinsic charges, only singly charged ions were recorded in each case. In addition to the pseudo-molecular ions formed by counterion adduction, deprotonation and electron capture, a number of fragment ions were also observed. Charge splitting by fragmentation was found to be a viable route for charge reduction leading to the formation of the observed singly charged fragment ions. Unlike multivalent metal ions, organic ions can rearrange and/or fragment during charge reduction. This fragmentation process will evidently complicate the interpretation of the MALDI MS spectrum. Because MALDI MS is usually considered as a soft ionization technique, the fragment ion peaks can easily be erroneously interpreted as impurities. Therefore, the awareness and understanding of the underlying MALDI-induced fragmentation pathways is essential for a proper interpretation of the corresponding mass spectra. Due to the fragment ions generated during charge reduction, special care should be taken in the MALDI MS analysis of multiply charged ions. In this work, the possible mechanisms by which the organic ions bearing fixed multiple charges fragment are investigated. With an improved understanding of the fragmentation mechanisms, MALDI TOF MS should still be a useful technique for the characterization of organic ions with fixed multiple charges.

Semisynthetic Bioluminescent Sensor Proteins for Direct Detection of Antibodies and Small Molecules in Solution.

Single-step immunoassays that can be performed directly in solution are ideally suited for point-of-care diagnostics. Our group recently developed a new platform of bioluminescent sensor proteins (LUMABS; LUMinescent AntiBody Sensor) that allow antibody detection in blood plasma. Thus far, LUMABS has been limited to the detection of antibodies recognizing natural peptide epitopes. Here, we report the development of semisynthetic LUMABS sensors that recognize nonpeptide epitopes. The non-natural amino acid para-azidophenylalanine was introduced at the position of the original antibody-recognition sites as a chemical handle to enable site-specific conjugation of synthetic epitope molecules coupled to a dibenzocylcooctyne moiety via strain-promoted click chemistry. The approach was successfully demonstrated by developing semisynthetic LUMABS sensors for antibodies targeting the small molecules dinitrophenol and creatinine (DNP-LUMABS and CR-LUMABS) with affinities of 5.8 pM and 1.3 nM, respectively. An important application of these semisynthetic LUMABS is the detection of small molecules using a competitive assay format, which is demonstrated here for the detection of creatinine. Using a preassembled complex of CR-LUMABS and an anti-creatinine antibody, the detection of high micromolar concentrations of creatinine was possible both in buffer and in 1:1 diluted blood plasma. The use of semisynthetic LUMABS sensors significantly expands the range of antibody targets and enables the application of LUMABS sensors for the ratiometric bioluminescent detection of small molecules using a competitive immunoassay format.

Supramolecular Chemistry Targeting Proteins.

The specific recognition of protein surface elements is a fundamental challenge in the life sciences. New developments in this field will form the basis of advanced therapeutic approaches and lead to applications such as sensors, affinity tags, immobilization techniques, and protein-based materials. Synthetic supramolecular molecules and materials are creating new opportunities for protein recognition that are orthogonal to classical small molecule and protein-based approaches. As outlined here, their unique molecular features enable the recognition of amino acids, peptides, and even whole protein surfaces, which can be applied to the modulation and assembly of proteins. We believe that structural insights into these processes are of great value for the further development of this field and have therefore focused this Perspective on contributions that provide such structural data.

Stabilization of protein-protein interactions in drug discovery.

INTRODUCTION: PPIs are involved in every disease and specific modulation of these PPIs with small molecules would significantly improve our prospects of developing therapeutic agents. Both industry and academia have engaged in the identification and use of PPI inhibitors. However in comparison, the opposite strategy of employing small-molecule stabilizers of PPIs is underrepresented in drug discovery. Areas covered: PPI stabilization has not been exploited in a systematic manner. Rather, this concept validated by a number of therapeutically used natural products like rapamycin and paclitaxel has been shown retrospectively to be the basis of the activity of synthetic molecules originating from drug discovery projects among them lenalidomide and tafamidis. Here, the authors cover the growing number of synthetic small-molecule PPI stabilizers to advocate for a stronger consideration of this as a drug discovery approach. Expert opinion: Both the natural products and the growing number of synthetic molecules show that PPI stabilization is a viable strategy for drug discovery. There is certainly a significant challenge to adapt compound libraries, screening techniques and downstream methodologies to identify, characterize and optimize PPI stabilizers, but the examples of molecules reviewed here in our opinion justify these efforts.

Ligand Dependent Switch from RXR Homo- to RXR-NURR1 Heterodimerization.

Retinoid X receptors (RXRs) play key roles in many physiological processes in both the periphery and central nervous system. In addition, RXRs form heterodimers with other nuclear receptors to exert their physiological effects. The nuclear receptor related 1 protein (NURR1) is particularly interesting because of its role in promoting differentiation and survival of dopamine neurons. However, only a small number of RXR-heterodimer selective modulators are available, with limited chemical diversity. This work describes the synthesis, biochemical evaluation, and structural elucidation of a novel series of RXR ligands with strongly biased interactions with RXRα-NURR1 heterodimers. Targeted modifications to the small molecule biaryl scaffold caused local RXRα side-chain disturbances and displacement of secondary structural elements upon ligand binding. This resulted in the repositioning of protein helices in the heterodimer interface of RXRα, alterations in homo- versus heterodimer formation, and modulation of activation function 2 (AF2). The data provide a rationale for the design of RXR ligands consisting of a highly conserved hydrophilic region, strongly contributing to the ligand affinity, and a variable hydrophobic region, which efficiently probes the effects of structural changes at the level of the ligand on co-regulator recruitment or the RXRα-NURR1 dimerization interface.

A Binary Bivalent Supramolecular Assembly Platform Based on Cucurbit[8]uril and Dimeric Adapter Protein 14-3-3.

Interactions between proteins frequently involve recognition sequences based on multivalent binding events. Dimeric 14-3-3 adapter proteins are a prominent example and typically bind partner proteins in a phosphorylation-dependent mono- or bivalent manner. Herein we describe the development of a cucurbit[8]uril (Q8)-based supramolecular system, which in conjunction with the 14-3-3 protein dimer acts as a binary and bivalent protein assembly platform. We fused the phenylalanine-glycine-glycine (FGG) tripeptide motif to the N-terminus of the 14-3-3-binding epitope of the estrogen receptor α (ERα) for selective binding to Q8. Q8-induced dimerization of the ERα epitope augmented its affinity towards 14-3-3 through a binary bivalent binding mode. The crystal structure of the Q8-induced ternary complex revealed molecular insight into the multiple supramolecular interactions between the protein, the peptide, and Q8.

Relationship between Side-Chain Polarity and the Self-Assembly Characteristics of Perylene Diimide Derivatives in Aqueous Solution.

Perylene-3,4,9,10-tetracarboxylic acid diimides (PDIs) have recently gained considerable interest for water-based biosensing applications. PDIs have been studied intensively in the bulk state, but their physical properties in aqueous solution in interplay with side-chain polarity are, however, poorly understood. Therefore, three perylene diimide based derivatives were synthesized to study the relationship between side-chain polarity and their self-assembly characteristics in water. The polarity of the side chains was found to dictate the size and morphology of the formed aggregates. Side-chain polarity rendered the self-assembly and photophysical properties of the PDIs-both important for imminent water-based applications-and these were revealed to be especially responsive to changes in solvent composition.