Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Discovery of new imidazotetrazinones with potential to overcome tumor resistance.

We describe the design, organic synthesis, and characterization, including X-ray crystallography, of a series of novel analogues of the clinically used antitumor agent temozolomide, together with their in vitro biological evaluation. The work has resulted in the discovery of a new series of anticancer imidazotetrazines that offer the potential to overcome the resistance mounted by tumors against temozolomide. The rationally designed compounds that incorporate a propargyl alkylating moiety and a thiazole ring as isosteric replacement for a carboxamide, are readily synthesized (gram-scale), exhibit defined solid-state structures, and enhanced growth-inhibitory activity against human tumor cell lines, including MGMT-expressing and MMR-deficient lines, molecular features that confer tumor resistance. The cell proliferation data were confirmed by clonogenic cell survival assays, and DNA flow cytometry analysis was undertaken to determine the effects of new analogues on cell cycle progression. Detailed 1H NMR spectroscopic studies showed that the new agents are stable in solution, and confirmed their mechanism of action. The propargyl and thiazole substituents significantly improve potency and physicochemical, drug metabolism and permeability properties, suggesting that the thiazole 13 should be prioritized for further preclinical evaluation.

Understanding the Molecular Conformation and Viscoelasticity of Low Sol-Gel Transition Temperature Gelatin Methacryloyl Suspensions.

For biomedical applications, gelatin is usually modified with methacryloyl groups to obtain gelatin methacryloyl (GelMA), which can be crosslinked by a radical reaction induced by low wavelength light to form mechanically stable hydrogels. The potential of GelMA hydrogels for tissue engineering has been well established, however, one of the main disadvantages of mammalian-origin gelatins is that their sol-gel transitions are close to room temperature, resulting in significant variations in viscosity that can be a problem for biofabrication applications. For these applications, cold-water fish-derived gelatins, such as salmon gelatin, are a good alternative due to their lower viscosity, viscoelastic and mechanical properties, as well as lower sol-gel transition temperatures, when compared with mammalian gelatins. However, information regarding GelMA (with special focus on salmon GelMA as a model for cold-water species) molecular conformation and the effect of pH prior to crosslinking, which is key for fabrication purposes since it will determine final hydrogel's structure, remains scarce. The aim of this work is to characterize salmon gelatin (SGel) and salmon methacryloyl gelatin (SGelMA) molecular configuration at two different acidic pHs (3.6 and 4.8) and to compare them to commercial porcine gelatin (PGel) and methacryloyl porcine gelatin (PGelMA), usually used for biomedical applications. Specifically, we evaluated gelatin and GelMA samples' molecular weight, isoelectric point (IEP), their molecular configuration by circular dichroism (CD), and determined their rheological and thermophysical properties. Results showed that functionalization affected gelatin molecular weight and IEP. Additionally, functionalization and pH affected gelatin molecular structure and rheological and thermal properties. Interestingly, the SGel and SGelMA molecular structure was more sensitive to pH changes, showing differences in gelation temperatures and triple helix formation than PGelMA. This work suggests that SGelMA presents high tunability as a biomaterial for biofabrication, highlighting the importance of a proper GelMA molecular configuration characterization prior to hydrogel fabrication.

Relative Abundance of Lipid Metabolites in Spermatozoa across Three Compartments.

Male fertility, as manifest by the quantity and progressive motility of spermatozoa, is negatively impacted by obesity, dyslipidaemia and metabolic disease. However, the relative distribution of lipids in spermatozoa and the two compartments which supply lipids for spermatogenesis (seminal fluid and blood serum) has not been studied. We hypothesised that altered availability of lipids in blood serum and seminal fluid may affect the lipid composition and progressive motility of sperm. 60 men of age 35 years (median (range 20-45) and BMI 30.4 kg/m2 (24-36.5) under preliminary investigation for subfertility were recruited at an NHS clinic. Men provided samples of serum and semen, subject to strict acceptance criteria, for analysis of spermatozoa count and motility. Blood serum (n = 60), spermatozoa (n = 26) and seminal fluid (n = 60) were frozen for batch lipidomics analysis. Spermatozoa and seminal fluid had comparable lipid composition but showed marked differences with the serum lipidome. Spermatozoa demonstrated high abundance of ceramides, very-long-chain fatty acids (C20-22), and certain phospholipids (sphingomyelins, plasmalogens, phosphatidylethanolamines) with low abundance of phosphatidylcholines, cholesterol and triglycerides. Men with spermatozoa of low progressive motility had evidence of fewer concentration gradients for many lipid species between blood serum and spermatozoa compartments. Spermatozoa are abundant in multiple lipid species which are likely to contribute to key cellular functions. Lipid metabolism shows reduced regulation between compartments in men with spermatozoa with reduced progressive motility.

Paternal nutritional programming of lipid metabolism is propagated through sperm and seminal plasma.

BACKGROUND: The paternal diet affects lipid metabolism in offspring for at least two generations through nutritional programming. However, we do not know how this is propagated to the offspring. OBJECTIVES: We tested the hypothesis that the changes in lipid metabolism that are driven by paternal diet are propagated through spermatozoa and not seminal plasma. METHODS: We applied an updated, purpose-built computational network analysis tool to characterise control of lipid metabolism systemically (Lipid Traffic Analysis v2.3) on a known mouse model of paternal nutritional programming. RESULTS: The analysis showed that the two possible routes for programming effects, the sperm (genes) and seminal plasma (influence on the uterine environment), both have a distinct effect on the offspring's lipid metabolism. Further, the programming effects in offspring suggest that changes in lipid distribution are more important than alterations in lipid biosynthesis. CONCLUSIONS: These results show how the uterine environment and genes both affect lipid metabolism in offspring, enhancing our understanding of the link between parental diet and metabolism in offspring.

Site-Selective Installation of Nϵ -Modified Sidechains into Peptide and Protein Scaffolds via Visible-Light-Mediated Desulfurative C-C Bond Formation.

Post-translational modifications (PTMs) enhance the repertoire of protein function and mediate or influence the activity of many cellular processes. The preparation of site-specifically and homogeneously modified proteins, to apply as tools to understand the biological role of PTMs, is a challenging task. Herein, we describe a visible-light-mediated desulfurative C(sp3 )-C(sp3 ) bond forming reaction that enables the site-selective installation of Nϵ -modified sidechains into peptides and proteins of interest. Rapid, operationally simple, and tolerant to ambient atmosphere, we demonstrate the installation of a range of lysine (Lys) PTMs into model peptide systems and showcase the potential of this technology by site-selectively installing an Nϵ Ac sidechain into recombinantly expressed ubiquitin (Ub).

An ALS-associated variant of the autophagy receptor SQSTM1/p62 reprograms binding selectivity toward the autophagy-related hATG8 proteins.

Recognition of human autophagy-related 8 (hATG8) proteins by autophagy receptors represents a critical step within this cellular quality control system. Autophagy impairment is known to be a pathogenic mechanism in the motor neuron disorder amyotrophic lateral sclerosis (ALS). Overlapping but specific roles of hATG8 proteins belonging to the LC3 and GABARAP subfamilies are incompletely understood, and binding selectivity is typically overlooked. We previously showed that an ALS-associated variant of the SQSTM1/p62 (p62) autophagy receptor bearing an L341V mutation within its ATG8-interacting motif (AIM) impairs recognition of LC3B in vitro, yielding an autophagy-deficient phenotype. Improvements in understanding of hATG8 recognition by AIMs now distinguish LC3-interaction and GABARAP-interaction motifs and predict the effects of L341V substitution may extend beyond loss of function to biasing AIM binding preference. Through biophysical analyses, we confirm impaired binding of the L341V-AIM mutant to LC3A, LC3B, GABARAP, and GABARAPL1. In contrast, p62 AIM interactions with LC3C and GABARAPL2 are unaffected by this mutation. Isothermal titration calorimetry and NMR investigations provided insights into the entropy-driven GABARAPL2/p62 interaction and how the L341V mutation may be tolerated. Competition binding demonstrated reduced association of the L341V-AIM with one hATG8 manifests as a relative increase in association with alternate hATG8s, indicating effective reprogramming of hATG8 selectivity. These data highlight how a single AIM peptide might compete for binding with different hATG8s and suggest that the L341V-AIM mutation may be neomorphic, representative of a disease mechanism that likely extends into other human disorders.

A pipeline for making 31P NMR accessible for small- and large-scale lipidomics studies.

Detailed molecular analysis is of increasing importance in research into the regulation of biochemical pathways, organismal growth and disease. Lipidomics in particular is increasingly sought after as it provides insight into molecular species involved in energy storage, signalling and fundamental cellular structures. This has led to the use of a range of tools and techniques to acquire lipidomics data. 31P NMR for lipidomics offers well-resolved head group/lipid class analysis, structural data that can be used to inform and strengthen interpretation of mass spectrometry data and part of a priori structural determination. In the present study, we codify the use of 31P NMR for lipidomics studies to make the technique more accessible to new users and more useful for a wider range of questions. The technique can be used in isolation (phospholipidomics) or as a part of determining lipid composition (lipidomics). We describe the process from sample extraction to data processing and analysis. This pipeline is important because it allows greater thoroughness in lipidomics studies and increases scope for answering scientific questions about lipid-containing systems.

Lipid Traffic Analysis reveals the impact of high paternal carbohydrate intake on offsprings' lipid metabolism.

In this paper we present an investigation of parental-diet-driven metabolic programming in offspring using a novel computational network analysis tool. The impact of high paternal carbohydrate intake on offsprings' phospholipid and triglyceride metabolism in F1 and F2 generations is described. Detailed lipid profiles were acquired from F1 neonate (3 weeks), F1 adult (16 weeks) and F2 neonate offspring in serum, liver, brain, heart and abdominal adipose tissues by MS and NMR. Using a purpose-built computational tool for analysing both phospholipid and fat metabolism as a network, we characterised the number, type and abundance of lipid variables in and between tissues (Lipid Traffic Analysis), finding a variety of reprogrammings associated with paternal diet. These results are important because they describe the long-term metabolic result of dietary intake by fathers. This analytical approach is important because it offers unparalleled insight into possible mechanisms for alterations in lipid metabolism throughout organisms.

Structural and electronic studies of substituted m-terphenyl lithium complexes.

The effect of para-substitution upon the structural and electronic properties of a series of m-terphenyl lithium complexes [R-Ar#-Li]2 (R = t-Bu 1, SiMe32, H 3, Cl 4, CF35; where R-Ar# = 2,6-{2,6-Xyl}2-4-R-C6H2 and 2,6-Xyl = 2,6-Me2C6H3) has been investigated. X-ray crystallography reveals the complexes to be structurally similar, with little variation in C-M-C bond lengths and angles across the series. However, in-depth NMR spectroscopic studies reveal notable electronic differences, showing a linear correlation between the 7Li{1H} NMR chemical shifts of the para-substituted complexes and their Hammett constants. The flanking methyl protons exhibit a similar electronic shift in the 1H NMR spectra, which has been rationalised by the presence of through-space LiH interactions, as evidenced by two-dimensional 7Li-1H heteronuclear Overhauser spectroscopy (HOESY). In both cases, electron-withdrawing substituents are found to cause an upfield peak shift. A computational analysis is employed to account for these trends.

Structural, Functional, and Metabolic Alterations in Human Cerebrovascular Endothelial Cells during Toxoplasma gondii Infection and Amelioration by Verapamil In Vitro.

Toxoplasma gondii (T. gondii), the causative agent of toxoplasmosis, is a frequent cause of brain infection. Despite its known ability to invade the brain, there is still a dire need to better understand the mechanisms by which this parasite interacts with and crosses the blood-brain barrier (BBB). The present study revealed structural and functional changes associated with infection and replication of T. gondii within human brain microvascular endothelial cells (BMECs) in vitro. T. gondii proliferated within the BMECs and disrupted the integrity of the cerebrovascular barrier through diminishing the cellular viability, disruption of the intercellular junctions and increasing permeability of the BMEC monolayer, as well as altering lipid homeostasis. Proton nuclear magnetic resonance (1H NMR)-based metabolomics combined with multivariate data analysis revealed profiles that can be attributed to infection and variations in the amounts of certain metabolites (e.g., amino acids, fatty acids) in the extracts of infected compared to control cells. Notably, treatment with the Ca2+ channel blocker verapamil rescued BMEC barrier integrity and restricted intracellular replication of the tachyzoites regardless of the time of treatment application (i.e., prior to infection, early- and late-infection). This study provides new insights into the structural and functional changes that accompany T. gondii infection of the BMECs, and sheds light upon the ability of verapamil to inhibit the parasite proliferation and to ameliorate the adverse effects caused by T. gondii infection.

Site-Selective Modification of Peptides and Proteins via Interception of Free-Radical-Mediated Dechalcogenation.

The development of site-selective chemistry targeting the canonical amino acids enables the controlled installation of desired functionalities into native peptides and proteins. Such techniques facilitate the development of polypeptide conjugates to advance therapeutics, diagnostics, and fundamental science. We report a versatile and selective method to functionalize peptides and proteins through free-radical-mediated dechalcogenation. By exploiting phosphine-induced homolysis of the C-Se and C-S bonds of selenocysteine and cysteine, respectively, we demonstrate the site-selective installation of groups appended to a persistent radical trap. The reaction is rapid, operationally simple, and chemoselective. The resulting aminooxy linker is stable under a variety of conditions and selectively cleavable in the presence of a low-oxidation-state transition metal. We have explored the full scope of this reaction using complex peptide systems and a recombinantly expressed protein.

G-quadruplex ligands mediate downregulation of DUX4 expression.

Abnormal DUX4 expression in skeletal muscles plays a key role in facioscapulohumeral muscular dystrophy (FSHD) pathogenesis, although the molecular mechanisms regulating DUX4 expression are not fully defined. Using bioinformatic analysis of the genomic DUX4 locus, we have identified a number of putative G-quadruplexes (GQs) forming sequences. Their presence was confirmed in synthetic oligonucleotiode sequences derived from the enhancer, promoter and transcript of DUX4 through circular dichroism and nuclear magnetic resonance analysis. We further examined the binding affinity of a naturally occurring GQ stabilizing compound, berberine, to these non-canonical genetic structures using UV-Vis and fluorescence spectroscopy. Subsequent in vitro study in FSHD patient myoblasts indicated that berberine treatment reduced DUX4 expression and also expression of genes normally switched on by DUX4. Further investigation in a mouse model overexpressing exogenous DUX4 confirmed the therapeutic effects of berberine in downregulating DUX4 protein expression, inhibiting muscle fibrosis, and consequently rescuing muscle function. Our data demonstrate for the first time that GQs are present in the DUX4 locus and that the GQ interactive ligand reduces DUX4 expression suggesting potential role of GQs in FSHD pathogenesis. Our work provides the basis of a novel therapeutic strategy for the treatment of FSHD.

A high-throughput platform for detailed lipidomic analysis of a range of mouse and human tissues.

Lipidomics is of increasing interest in studies of biological systems. However, high-throughput data collection and processing remains non-trivial, making assessment of phenotypes difficult. We describe a platform for surveying the lipid fraction for a range of tissues. These techniques are demonstrated on a set of seven different tissues (serum, brain, heart, kidney, adipose, liver, and vastus lateralis muscle) from post-weaning mouse dams that were either obese (> 12 g fat mass) or lean (<5 g fat mass). This showed that the lipid metabolism in some tissues is affected more by obesity than others. Analysis of human serum (healthy non-pregnant women and pregnant women at 28 weeks' gestation) showed that the abundance of several phospholipids differed between groups. Human placenta from mothers with high and low BMI showed that lean placentae contain less polyunsaturated lipid. This platform offers a way to map lipid metabolism with immediate application in metabolic research and elsewhere. Graphical abstract.

Synthesis of folic acid functionalized gold nanoclusters for targeting folate receptor-positive cells.

We report on the synthesis of water-soluble gold nanoclusters capped with polyethylene glycol (PEG)-based ligands and further functionalized with folic acid for specific cellular uptake. The dihydrolipoic acid-PEG-based ligands terminated with -OMe, -NH2 and -COOH functional groups are produced and used for surface passivation of Au nanoclusters (NCs) with diameters <2 nm. The produced sub 2 nm Au NCs possess long-shelf life and are stable in physiologically relevant environments (temperature and pH), are paramagnetic and biocompatible. The paramagnetism of Au NCs in solution is also reported. The functional groups on the capping ligands are used for direct conjugation of targeting molecules onto Au NCs without the need for post synthesis modification. Folic acid (FA) is attached via an amide group and effectively target cells expressing the folate receptor. The combination of targeting ability, biocompatibility and paramagnetism in FA-functionalized Au NCs is of relevance for their exploitation in nanomedicine for targeted imaging.

Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib.

Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMß2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.

Accelerated 19F·MRI Detection of Matrix Metalloproteinase-2/-9 through Responsive Deactivation of Paramagnetic Relaxation Enhancement.

Paramagnetic gadolinium ions (GdIII), complexed within DOTA-based chelates, have become useful tools to increase the magnetic resonance imaging (MRI) contrast in tissues of interest. Recently, "on/off" probes serving as 19F·MRI biosensors for target enzymes have emerged that utilize the increase in transverse (T 2 ∗ or T 2) relaxation times upon cleavage of the paramagnetic GdIII centre. Molecular 19F·MRI has the advantage of high specificity due to the lack of background signal but suffers from low signal intensity that leads to low spatial resolution and long recording times. In this work, an "on/off" probe concept is introduced that utilizes responsive deactivation of paramagnetic relaxation enhancement (PRE) to generate 19F longitudinal (T 1) relaxation contrast for accelerated molecular MRI. The probe concept is applied to matrix metalloproteinases (MMPs), a class of enzymes linked with many inflammatory diseases and cancer that modify bioactive extracellular substrates. The presence of these biomarkers in extracellular space makes MMPs an accessible target for responsive PRE deactivation probes. Responsive PRE deactivation in a 19F biosensor probe, selective for MMP-2 and MMP-9, is shown to enable molecular MRI contrast at significantly reduced experimental times compared to previous methods. PRE deactivation was caused by MMP through cleavage of a protease substrate that served as a linker between the fluorine-containing moiety and a paramagnetic GdIII-bound DOTA complex. Ultrashort echo time (UTE) MRI and, alternatively, short echo times in standard gradient echo (GE) MRI were employed to cope with the fast 19F transverse relaxation of the PRE active probe in its "on-state." Upon responsive PRE deactivation, the 19F·MRI signal from the "off-state" probe diminished, thereby indicating the presence of the target enzyme through the associated negative MRI contrast. Null point 1H·MRI, obtainable within a short time course, was employed to identify false-positive 19F·MRI responses caused by dilution of the contrast agent.

A Highly Active Bidentate Magnesium Catalyst for Amine-Borane Dehydrocoupling: Kinetic and Mechanistic Studies.

A magnesium complex (1) featuring a bidentate aminopyridinato ligand is a remarkably selective catalyst for the dehydrocoupling of amine-boranes. This reaction proceeds to completion with low catalyst loadings (1 mol %) under mild conditions (60 °C), exceeding previously reported s-block systems in terms of selectivity, rate, and turnover number (TON). Mechanistic studies by in situ NMR analysis reveals the reaction to be first order in both catalyst and substrate. A reaction mechanism is proposed to account for these findings, with the high TON of the catalyst attributed to the bidentate nature of the ligand, which allows for reversible deprotonation of the substrate and regeneration of 1 as a stable resting state.

DNA replication initiation in Bacillus subtilis: structural and functional characterization of the essential DnaA-DnaD interaction.

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA-DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.