Investigating Interaction Dynamics of an Enantioselective Peptide-Catalyzed Acylation Reaction.

Modern nuclear magnetic resonance (NMR) methods like carbon relaxation dispersion in the rotating frame (C-R) and proton chemical exchange saturation transfer (H-CEST) are key methods to investigate molecular recognition in biomacromolecules and to detect molecular motions on the μs to s timescale, revealing transient conformational states. Changes in kinetics can be linked to binding, folding, or catalytic events. Here, we investigated whether these methods allow detection of changes in the dynamics of a small, highly selective peptide catalyst during recognition of its enantiomeric substrates.

Evaluation of Potency and Specificity of Cryptophycin-Loaded Antibody-Drug Conjugates.

An enhanced variant of the antimitotic toxin cryptophycin was conjugated to the anti-Her2 monoclonal antibody (mAb) Trastuzumab upon Michael addition. Either antibodies with freed hinge-region cysteines or THIOMAB formats with engineered cysteines in the mAbs light chain were added to a maleimide derivative of cryptophycin. These Antibody-Drug Conjugates (ADCs) showed retained binding to Her2 positive tumor cells and highly efficient cell killing in double-digit pM range on high Her2-expressing SK-BR-3 cells.

Exciting Novel Polyaspartates: Design, Synthesis, and Photo-Responsive Behavior in Solution and Lyotropic Liquid Crystalline Phase Upon Irradiation with Visible Light.

Many polypeptides form stable, helical secondary structures enabling the formation of lyotropic liquid crystalline (LLC) phases. Contrary to the well-studied polyglutamate, their counterparts based on polyaspartates exhibit a much lower helix inversion barrier. Therefore, the helix sense is not solely dictated by the chirality of the amino acid used, but additionally by the nature and conformation of the polymer sidechain.

2,5-[C+C] Ringtransformation of Pyrylium Salts with α-Sulfinylacetaldehydes.

A rapid synthesis of chiral sulfoxide-functionalized -terphenyl derivatives by a 2,5-[C+C] ring transformation reaction of pyrylium salts with in situ generated enantiomerically pure α-sulfinylacetaldehydes is described in this paper. This synthetic method demonstrates, for the first time, the use of α-sulfinylacetaldehydes in a reaction sequence initiated by the nucleophilic attack of pyrylium salts by α-sulfinylcarbanions to generate chiral aromatic systems. The method presented shows a broad applicability starting with various methyl sulfoxides and a number of functionalized pyrylium salts, furnishing -terphenyls with complex substitution patterns from readily accessible starting compounds.

Proteasome inhibition potentiates Kv1.3 potassium channel expression as therapeutic target in drug-sensitive and -resistant human melanoma cells.

Primary and acquired therapy resistance is a major problem in patients with BRAF-mutant melanomas being treated with BRAF and MEK inhibitors (BRAFI, MEKi). Therefore, development of alternative therapy regimes is still required. In this regard, new drug combinations targeting different pathways to induce apoptosis could offer promising alternative approaches.

Parahydrogen-induced polarization enables the single-scan NMR detection of a 236 kDa biopolymer at nanomolar concentrations.

Nuclear magnetic resonance (NMR) experiments utilizing parahydrogen-induced polarization (PHIP) were performed to elucidate the PHIP activity of the synthetic 236 kDa biopolymer poly-γ-(4-propargyloxy)-benzyl-L-glutamate) (PPOBLG). The homopolypeptide was successfully hyperpolarized and the enhanced signals were detected in 11.7 T solution NMR as a function of the PPOBLG concentration.

Novel amino-Li resin for water-based solid-phase peptide synthesis.

We report the first application of a novel amino-Li resin to water-based solid-phase peptide synthesis (SPPS) applying the Smoc-protecting group approach. We demonstrated that it is a suitable support for the sustainable water-based alternative to a classical SPPS approach. The resin possesses good swelling properties in aqueous milieu, provides significant coupling sites, and may be applicable to the synthesis of difficult sequences and aggregation-prone peptides.

Parahydrogen-induced polarization allows 2000-fold signal enhancement in biologically active derivatives of the peptide-based drug octreotide.

Octreotide, a somatostatin analogue, has shown its efficacy for the diagnostics and treatment of various types of cancer, i.e., in octreotide scan, as radio-marker after labelling with a radiopharmaceutical.

Effect of macrocyclization and tetramethylrhodamine labeling on chemokine binding peptides.

Receptor-derived peptides have played an important role in elucidating chemokine-receptor interactions. For the inflammatory chemokine CXC-class chemokine ligand 8 (CXCL8), a site II-mimetic peptide has been derived from parts of extracellular loops 2 and 3 and adjacent transmembrane helices of its receptor CXC-class chemokine receptor 1 (Helmer et al., RSC Adv.

Detection and Verification of a Key Intermediate in an Enantioselective Peptide Catalyzed Acylation Reaction.

Until now, the intermediate responsible for the acyl transfer of a highly enantioselective tetrapeptide organocatalyst for the kinetic resolution of trans-cycloalkane-1,2-diols has never been directly observed. It was proposed computationally that a π-methylhistidine moiety is acylated as an intermediate step in the catalytic cycle. In this study we set out to investigate whether we can detect and characterize this key intermediate using NMR-spectroscopy and mass spectrometry.

Triplet States Reveal Slow Local Dynamics in the Solvation Shell of Biomolecules.

Protein hydration shell dynamics plays a pivotal role in biochemical processes such as protein folding, enzyme function, molecular recognition and interaction with biological membranes. Thus, it is crucial to understand the mobility of the solvation shell at the surface of biomolecules. Triplet state solvation dynamics can reveal the slow dynamics of the solvation shell.

Atropisomerism in a polyglutamate-based thermoresponsive alignment medium.

Structure elucidation residual dipolar couplings (RDCs) relies on alignment media. We report on lyotropic liquid crystals (LLCs) of poly-γ--biphenyl(2'-methoxy-2-methyl)methyl-L-glutamate (PBPMLG). Temperature dependent atropisomerism within the biphenyl group enables the acquisition of multiple RDC-datasets within one sample.

Concentration gradients in evaporating binary droplets probed by spatially resolved Raman and NMR spectroscopy.

Understanding the evaporation process of binary sessile droplets is essential for optimizing various technical processes, such as inkjet printing or heat transfer. Liquid mixtures whose evaporation and wetting properties may differ significantly from those of pure liquids are particularly interesting. Concentration gradients may occur in these binary droplets.

A supramolecular and liquid crystalline water-based alignment medium based on azobenzene-substituted 1,3,5-benzenetricarboxamides.

A supramolecular, lyotropic liquid crystalline alignment medium based on an azobenzene-containing 1,3,5-benzenetricarboxamide (BTA) building block is described and investigated. As we demonstrate, this water-based system is suitable for the investigation of various water-soluble analytes and allows for a scaling of alignment strength through variation of temperature. Additionally, alignment is shown to reversibly collapse above a certain temperature, yielding an isotropic solution.

Structural and Biochemical Characterization of a Dye-Decolorizing Peroxidase from .

A novel cytoplasmic dye-decolorizing peroxidase from was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in DyPA. In solution, DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers.

Model-Free Approach for the Configurational Analysis of Marine Natural Products.

The NMR-based configurational analysis of complex marine natural products is still not a routine task. Different NMR parameters are used for the assignment of the relative configuration: NOE/ROE, homo- and heteronuclear couplings as well as anisotropic parameters. The combined distance geometry (DG) and distance bounds driven dynamics (DDD) method allows a model-free approach for the determination of the relative configuration that is invariant to the choice of an initial starting structure and does not rely on comparisons with (DFT) calculated structures.

Cell-based actin polymerization assay to analyze chemokine inhibitors.

Chemokines play an important role in various diseases as signaling molecules for immune cells. Therefore, the inhibition of the chemokine-receptor interaction and the characterization of potential inhibitors are important steps in the development of new therapies. Here, we present a new cell-based assay for chemokine-receptor interaction, using chemokine-dependent actin polymerization as a readout.

Treating anisotropic artefacts in circular dichroism spectroscopy enables investigation of lyotropic liquid crystalline polyaspartate solutions.

Circular dichroism (CD) spectroscopy is commonly used for investigation of the secondary structure of biomolecular compounds as well as polymers in isotropic solution. In anisotropic solution, the usage of the apparent CD is prone to misinterpretations due to artefacts from contributions of e.g.

Sex differences in the effect of the FKBP5 inhibitor SAFit2 on anxiety and stress-induced reinstatement following cocaine self-administration.

Cocaine use and withdrawal prompt stress system responses. Stress and the negative affective state produced by cocaine withdrawal are major triggers for relapse. FKBP5 is a co-chaperone of the glucocorticoid receptor and regulates HPA axis negative feedback.

Valine derived poly (acetylenes) as versatile chiral lyotropic liquid crystalline alignment media for RDC-based structure elucidations.

Anisotropic samples of lyotropic liquid crystalline (LLC) phases of valine derived polyaryl acetylenes were employed as chiral alignment media for the measurement of residual dipolar couplings (RDCs) of 12 small, chiral, organic molecules. The quadrupolar splitting of the deuterium signal of CDCl can be adjusted by temperature and concentration changes from 0 to 350 Hz. The LLC phases showed excellent orienting properties for all analytes bearing various functional groups.

Structural and Dynamical Basis of G Protein Inhibition by YM-254890 and FR900359: An Inhibitor in Action.

Specific inhibition of G proteins holds a great pharmacological promise to, e.g., target oncogenic G proteins and can be achieved by the two natural products FR900359 (FR) and YM-254890 (YM).

The selective FKBP51 inhibitor SAFit2 reduces alcohol consumption and reinstatement of conditioned alcohol effects in mice.

There is still no widely effective pharmacotherapy for alcohol addiction available in the clinic. FK506-binding protein 51 (FKBP51) is a negative regulator of the glucocorticoid receptor signaling pathway that regulates the stress-induced glucocorticoid feedback circuit. Here we asked whether selective inhibitors of FKBP51, exemplified by SAFit2, may serve as a new pharmacological strategy to reduce alcohol consumption and conditioned alcohol effects in a mouse model.

SpyLigase-Catalyzed Modification of Antibodies.

Immunoconjugates are essential tools in diagnostics for the detection and quantification of proteins and in cell biology for the characterization of different cell populations as well as for tracking intracellular pathways. In recent years, antibody-drug conjugates (ADCs) have emerged as promising therapeutics to treat cancer and have moved into the focus of interest of the pharmaceutical industry. Here we describe a conjugation method for the generation of antibody conjugates that relies on the formation of a spontaneous isopeptide bond between two peptide tags referred to as SpyTag and KTag.

Tailoring Activity and Selectivity of Microbial Transglutaminase.

Microbial transglutaminase (mTG), a protein-glutamine γ-glutamyltransferase from Streptomyces mobaraensis, is an enzyme capable of forming isopeptide bonds between the nearly inert (from the chemical point of view) γ-carboxamides present in the side chain of glutamine residues and primary amines. Its high substrate tolerance, compared to other bond-forming enzymes, makes it a versatile tool for numerous applications including food manufacturing, material science, and biotechnology. Although an mTG-mediated bioconjugation is a well-established technique, some major drawbacks of this approach need to be bypassed, with the poor substrate specificity being among the most essential ones.

Site-Specific Antibody-Drug Conjugation Using Microbial Transglutaminase.

Antibody-drug conjugates (ADCs) are a relatively young class of cancer therapeutics that combine the superior selectivity of monoclonal antibodies (mAbs) with the high potency of cytotoxic agents. In the first generation of ADCs, the toxic payload is attached to the mAb via chemical conjugation to endogenous lysine or cysteine residues providing only limited control over site specificity and drug-to-antibody ratio (DAR). The resulting product is a heterogeneous population of different ADC species, each with individual characteristics concerning pharmacokinetics, toxicology, and efficacy.