Expanding the toolbox for predictive parameters describing antibody stability considering thermodynamic and kinetic determinants.

Purpose: Introduction of the activation energy (E) as a kinetic parameter to describe and discriminate monoclonal antibody (mAb) stability.

Methods: E is derived from intrinsic fluorescence (IF) unfolding thermograms. An apparent irreversible three-state fit model based on the Arrhenius integral is developed to determine E of respective unfolding transitions.

Quantifying the Interaction of Phosphite with ABC Transporters: MicroScale Thermophoresis and a Novel His-Tag Labeling Approach.

The combination of MicroScale Thermophoresis (MST) and near-native site-specific His-tag labeling enables simple, robust, and reliable determination of the binding affinity between proteins and ligands. To demonstrate its applicability for periplasmic proteins, we provide a detailed protocol for determination of the binding affinity of phosphite to three ABC transporter periplasmic-binding proteins from environmental microorganisms. ABC transporters are central to many important biomedical phenomena, including resistance of cancers and pathogenic microbes to drugs.

Development of the First Potential Nonpeptidic Positron Emission Tomography Tracer for the Imaging of CCR2 Receptors.

Herein we report the design and synthesis of a series of highly selective CCR2 antagonists as F-labeled PET tracers. The derivatives were evaluated extensively for their off-target profile at 48 different targets. The most potent and selective candidate was applied in vivo in a biodistribution study, demonstrating a promising profile for further preclinical development.

Development of a fast screening method for selecting excipients in formulations using MD simulations, NMR and microscale thermophoresis.

Development of peptide therapeutics generally involves screening of excipients that inhibit peptide-peptide interactions, hence aggregation, and improve peptide stability. We used the therapeutic peptide plectasin to develop a fast screening method that combines microscale thermophoresis titration assays and molecular dynamics simulations to relatively rank the excipients with respect to binding affinity and to study key peptide-excipient interaction hotspots on a molecular level, respectively. Additionally, H-C-HSQC NMR titration experiments were performed to validate the fast screening approach.

The Chemokine Receptor CXCR3 Isoform B Drives Breast Cancer Stem Cells.

We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The chemokine receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this receptor to promote tumor cell migration and invasion.

Insight into structural requirements for selective and/or dual CXCR3 and CXCR4 allosteric modulators.

Based on the previously published pyrazolopyridine-based hit compound for which negative allosteric modulation of both CXCR3 and CXCR4 receptors was disclosed, we designed, synthesized and biologically evaluated a set of novel, not only negative, but also positive allosteric modulators with preserved pyrazolopyridine core. Compound 9e is a dual negative modulator, inhibiting G protein activity of both receptors. For CXCR4 receptor para-substituted aromatic group of compounds distinguishes between negative and positive modulation.

Near-native, site-specific and purification-free protein labeling for quantitative protein interaction analysis by MicroScale Thermophoresis.

MicroScale Thermophoresis (MST) is a frequently used method for the quantitative characterization of intermolecular interactions with several advantages over other technologies. One of these is its capability to determine equilibrium constants in solution including complex biological matrices such as cell lysates. MST requires one binding partner to be fluorescent, which is typically achieved by labeling target proteins with a suitable fluorophore.

Molecular Mechanisms of Biased and Probe-Dependent Signaling at CXC-Motif Chemokine Receptor CXCR3 Induced by Negative Allosteric Modulators.

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 (-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)--[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[(-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([H]-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]--[(1-methylpiperidin-4-yl)methyl]acetamide), we identified that F131 and Y308 contribute specifically to the binding pocket of BD064, whereas D186 solely participates in the stabilization of binding conformation of BD103.

Synthesis and biological evaluation of chemokine receptor ligands with 2-benzazepine scaffold.

Targeting CCR2 and CCR5 receptors is considered as promising concept for the development of novel antiinflammatory drugs. Herein, we present the development of the first probe-dependent positive allosteric modulator (PAM) of CCR5 receptors with a 2-benzazepine scaffold. Compound 14 (2-isobutyl-N-({[N-methyl-N-(tetrahydro-2H-pyran-4-yl)amino]methyl}phenyl)-1-oxo-2,3-dihydro-1H-2-benzazepine-4-carboxamide) activates the CCR5 receptor in a CCL4-dependent manner, but does not compete with [H]TAK-779 binding at the CCR5.

Attenuation of chemokine receptor function and surface expression as an immunomodulatory strategy employed by human cytomegalovirus is linked to vGPCR US28.

Background: Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency.

Identification of Two Distinct Sites for Antagonist and Biased Agonist Binding to the Human Chemokine Receptor CXCR3.

The chemokine receptor CXCR3 is a G protein-coupled receptor that conveys extracellular signals into cells by changing its conformation upon ligand binding. We previously hypothesized that small-molecule allosteric CXCR3-agonists do not bind to the same allosteric binding pocket as 8-azaquinazolinone-based negative allosteric modulators. We have now performed molecular-dynamics (MD) simulations with metadynamics enhanced sampling on the CXCR3 system to refine structures and binding modes and to predict the CXCR3-binding affinities of the biased allosteric agonist FAUC1036 and the negative allosteric modulator RAMX3.

Real time monitoring of membrane GPCR reconstitution by plasmon waveguide resonance: on the role of lipids.

G-protein coupled receptors (GPCRs) are important therapeutic targets since more than 40% of the drugs on the market exert their action through these proteins. To decipher the molecular mechanisms of activation and signaling, GPCRs often need to be isolated and reconstituted from a detergent-solubilized state into a well-defined and controllable lipid model system. Several methods exist to reconstitute membrane proteins in lipid systems but usually the reconstitution success is tested at the end of the experiment and often by an additional and indirect method.

Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level.

G protein-coupled receptors (GPCRs), including dopamine receptors, represent a group of important pharmacological targets. An increased formation of dopamine receptor D2 homodimers has been suggested to be associated with the pathophysiology of schizophrenia. Selective labeling and ligand-induced modulation of dimerization may therefore allow the investigation of the pathophysiological role of these dimers.

Allosteric Modulators of the Class A G Protein Coupled Receptors.

Allosteric modulation is the regulation of a protein by binding of an effector molecule at the proteins allosteric site (a site other than that of the endogenous ligand). Allosteric modulators, by virtue of the fact that they may stabilize different global conformations of a receptor, have the potential to disrupt protein-protein interactions of very large proteins and elicit diverse functional responses. The existence of ligands that allosterically modulate the G protein receptor (GPCR) functions provides both challenges and opportunities for drug development campaigns.

Development of Photoactivatable Allosteric Modulators for the Chemokine Receptor CXCR3.

The CXCR3 receptor, a class A G protein-coupled receptor (GPCR), is involved in the regulation and trafficking of various immune cells. CXCR3 antagonists have been proposed to be beneficial for the treatment of a wide range of disorders including but not limited to inflammatory and autoimmune diseases. The structure-based design of CXCR3 ligands remains, however, hampered by a lack of structural information describing in detail the interactions between an allosteric ligand and the receptor.

Discovery and Characterization of Biased Allosteric Agonists of the Chemokine Receptor CXCR3.

In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment.

Ligand selectivity of a synthetic CXCR4 mimetic peptide.

The chemokine receptor CXCR4 belongs to the family of seven-transmembrane G-protein coupled receptors (GPCRs). It is activated by its natural ligand SDF-1α. In addition, CXCR4, along with CCR5, serve as coreceptors during HIV-1 entry into its target cell.

Ligand-biased and probe-dependent modulation of chemokine receptor CXCR3 signaling by negative allosteric modulators.

Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target G protein-coupled receptors (GPCRs). The CXC chemokine receptor 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer.

Identifying modulators of CXC receptors 3 and 4 with tailored selectivity using multi-target docking.

The G protein-coupled receptors of the C-X-C subfamily form a group among the chemokine receptors whose endogenous ligands are peptides with a common Cys-X-Cys motif. The CXC chemokine receptors 3 and 4 (CXCR3, CXCR4), which are investigated in this study, are linked to severe diseases such as cancer, multiple sclerosis, and HIV infections. Of particular interest, this receptor pair potentially forms a target for a polypharmacological drug treatment.

Boronic acids as probes for investigation of allosteric modulation of the chemokine receptor CXCR3.

The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking.

Allosteric modulation of the G protein-coupled US28 receptor of human cytomegalovirus: are the small-weight inverse agonist of US28 'camouflaged' agonists?

The highly constitutively active G protein-coupled receptor US28 of human cytomegalovirus (HCMV) is thought to camouflage agonism by mediating constitutive endocytosis. With the use of the US28Δ300 mutant, which is largely devoid of constitutive internalization, I have demonstrated that the coupling of the receptor to its downstream signaling partners is responsible for the inverse agonism to agonism efficacy switch in some small-weight ligands of US28.

Active-state model of a dopamine D2 receptor-Gαi complex stabilized by aripiprazole-type partial agonists.

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gαi protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better.

Radiosynthesis and validation of ¹⁸F-FP-CMT, a phenyltropane with superior properties for imaging the dopamine transporter in living brain.

To date there is no validated, (18)F-labeled dopamine transporter (DAT) radiotracer with a rapid kinetic profile suitable for preclinical small-animal positron emission tomography (PET) studies in rodent models of human basal ganglia disease. Herein we report radiosynthesis and validation of the phenyltropane (18)F-FP-CMT. Dynamic PET recordings were obtained for (18)F-FP-CMT in six untreated rats, and six rats pretreated with the high-affinity DAT ligand GBR 12909; mean parametric maps of binding potential (BPND) relative to the cerebellum reference region, and maps of total distribution volume (VT) relative to the metabolite-corrected arterial input were produced.

Fast and efficient (18) F-labeling by [(18) f]fluorophenylazocarboxylic esters.

Introduction of [(18) F]fluoride ion into the aromatic core of phenylazocarboxylic esters was achieved in only 30 seconds, with radiochemical yields of up to 95 % (85(±10) %). For labeling purposes, the resulting (18) F-substituted azoester can be further converted in radical-arylation reactions to give biaryls, or in substitutions at its carbonyl unit to produce azocarboxamides.

Synthesis and biological evaluation of biphenyl amides that modulate the US28 receptor.

To prepare and biologically evaluate 38 new potential US28 allosteric modulators, we employed a straightforward synthetic route involving radical arylation. The study was based on a former lead structure but with the dihydroisoquinolinone moiety replaced by substituted biphenyls. The investigation of structure-activity relationships among the new biphenyl-derived ligands led to a preliminary pharmacophore model and the discovery of four promising candidates with full inverse agonist properties.