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

PURPOSE: Introduction of the activation energy (Ea) as a kinetic parameter to describe and discriminate monoclonal antibody (mAb) stability. METHODS: Ea is derived from intrinsic fluorescence (IF) unfolding thermograms. An apparent irreversible three-state fit model based on the Arrhenius integral is developed to determine Ea of respective unfolding transitions. These activation energies are compared to the thermodynamic parameter of van´t Hoff enthalpies (∆Hvh). Using a set of 34 mAbs formulated in four different formulations, both the apparent thermodynamic and kinetic parameters together with apparent melting temperatures are correlated collectively with each other to storage stabilities to evaluate its predictive power with respect to long-term effects potentially reflected in shelf-life. RESULTS: Ea allows for the discrimination of (i) different parent mAbs, (ii) different variants that originate from parent mAbs, and (iii) different formulations. Interestingly, we observed that the Ea of the CH2 unfolding transition shows strongest correlations with monomer and aggregate content after storage at accelerated and stress conditions when collectively compared to ∆Hvh and Tm of the CH2 transition. Moreover, the predictive parameters determined for the CH2 domain show generally stronger correlations with monomer and aggregate content than those derived for the Fab. Qualitative assessment by ranking Ea of the Fab domain showed good agreement with monomer content in storage stabilities of individual mAb sub-sets. CONCLUSION: Ea from IF unfolding transitions can be used in addition to other commonly used thermodynamic predictive parameters to discriminate and characterize thermal stability of different mAbs in different formulations. Hence, it shows great potential for antibody engineering and formulation scientists.

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, 1H-13C-HSQC NMR titration experiments were performed to validate the fast screening approach. The NMR results are in qualitative agreement with results from the fast screening method demonstrating that this approach can be reliably applied to other peptides and proteins as a fast screening method to relatively rank excipients and predict possible excipient binding sites.

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 18 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. This compound represents the first potential nonpeptidic PET tracer for the imaging of CCR2 receptors.

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. The protocol described here can be used to quantify protein-ligand and protein-protein interactions for other soluble, membrane-associated and integral membrane proteins.

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. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44+CD24- phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target.

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. Para-methoxy substitution leads to functional antagonism, while para-chloro triggers agonism. Additionally, we discovered that chemotaxis is not completely correlated with G protein pathways. This is the first work in which we have on a series of compounds successfully demonstrated that it is possible to produce selective as well as dual-acting modulators of chemokine receptors, which is very promising for future research in the field of discovery of selective or dual modulators of chemokine receptors.

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. Here, we present a near-native, site-specific in situ labeling strategy for MST experiments that enables reliable measurements in cell lysates and that has distinct advantages over routine covalent labeling techniques. To this end, we exploited the high-affinity interaction of tris-NTA with oligohistidine-tags, which are popular for purification, immobilization or detection of recombinant proteins. We used various DYE-tris-NTA conjugates to successfully label His-tagged proteins that were either purified or a component of cell lysate. The RED-tris-NTA was identified as the optimal dye conjugate with a high affinity towards oligohistidine-tags, a high fluorescence signal and an optimal signal-to-noise ratio in MST binding experiments. Owing to its emission in the red region of the spectrum, it also enables reliable measurements in complex biological matrices such as cell lysates allowing a more physiologically realistic assessment and eliminating the need for protein purification.

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 (N-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[(N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]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 ([3H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide), we identified that F1313.32 and Y3087.43 contribute specifically to the binding pocket of BD064, whereas D1864.60 solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and ß-arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F1313.32, S3047.39, and Y3087.43 emerged as key residues for the compounds to modulate the chemokine response. Notably, D1864.60, W2686.48, and S3047.39 turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but ß-arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties.

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 [3H]TAK-779 binding at the CCR5. Furthermore, introduction of a p-tolyl moiety at 7-position of the 2-benzazepine scaffold turns the CCR5 PAM 14 into the selective CCR2 receptor antagonist 26b. The structure affinity and activity relationships presented here offer new insights into ligand recognition by CCR2 and CCR5 receptors.

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. RESULTS: We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection. CONCLUSIONS: We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.

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. Irrespective of the method used, the reconstitution process is often an intractable and time-consuming trial-and-error procedure. Herein, we present a method that allows directly monitoring the reconstitution of GPCRs in model planar lipid membranes. Plasmon waveguide resonance (PWR) allows following GPCR lipid reconstitution process without any labeling and with high sensitivity. Additionally, the method is ideal to probe the lipid effect on receptor ligand binding as demonstrated by antagonist binding to the chemokine CCR5 receptor.

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. We have identified two distinct binding sites; a "shallow" and a second "deeper" pocket to which the biased allosteric agonist FAUC1036 and negative allosteric modulator RAMX3 bind, respectively.

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. Using TIRF microscopy at the single molecule level, transient formation of homodimers of dopamine receptors in the membrane of stably transfected CHO cells has been observed. The equilibrium between dimers and monomers was modulated by the binding of ligands; whereas antagonists showed a ratio that was identical to that of unliganded receptors, agonist-bound D2 receptor-ligand complexes resulted in an increase in dimerization. Addition of bivalent D2 receptor ligands also resulted in a large increase in D2 receptor dimers. A physical interaction between the protomers was confirmed using high resolution cryogenic localization microscopy, with ca. 9 nm between the centers of mass.

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. A number of therapeutic advantages of allosteric modulators over classic orthosteric ligands were proposed, involving nature of response, improved selectivity and ligand-directed signaling. In this review I discuss various aspects of allosteric modulation of GPCRs, which arise from the interactions of receptors with synthetic or endogenous small molecules, ions, lipids and diverse proteins. Detection and quantification of allosteric modulation will be also addressed. In the conclusion I will present future opportunities and challenges in the development of allosteric modulators as therapeutics.

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. We designed and synthesized photoactivatable probes for the structural and functional characterization, using photoaffinity labeling followed by mass spectrometry, of the CXCR3 allosteric binding pocket of AMG 487 and RAMX3, two potent and selective CXCR3 negative allosteric modulators. Photoaffinity labeling is a common approach to elucidate binding modes of small-molecule ligands of GPCRs through the aid of photoactivatable probes that convert to extremely reactive intermediates upon photolysis. The photolabile probe N-[({1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-{1-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl}piperidin-4-yl)methyl]acetamide (10) showed significant labeling of the CXCR3 receptor (80%) in a [(3) H]RAMX3 radioligand displacement assay. Compound 10 will serve as an important tool compound for the detailed investigation of the binding pocket of CXCR3 by mass spectrometry.

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 ß-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 ß-arrestin 2 recruitment. Concomitant structure-activity relationship studies indicated very steep structure-activity relationships, which steer the ligand bias between the ß-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further development and rational design of strongly biased allosteric agonists of CXCR3.

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. Recently, we introduced a CXCR4 mimetic peptide, termed CX4-M1, which presents the three extracellular loops (ECLs) of the receptor. CX4-M1 was shown to selectively bind to gp120 of X4-tropic, that is, CXCR4 using, HIV-1, as well as to peptides that present the V3-loops of these gp120 proteins. Furthermore, CX4-M1 selectively inhibits infection of cells with X4-tropic HIV-1. We have now adapted the sequence of the ECLs presented by CX4-M1 to the recently published crystal structure of CXCR4. The binding behavior, as well as the effect on HIV-1 infection, of the resulting peptide (CX4-Mc) was very similar to CX4-M1, validating retrospectively the original design of CX4-M1. A peptide presenting the ECLs of CCR5 (CR5-M), on the other hand, did neither bind to gp120 from X4-tropic HIV-1, nor did it inhibit infection of cells with X4-tropic HIV-1. Furthermore, we could show that CX4-M1, as well as CX4-Mc, but not CR5-M, are selectively recognized by anti-CXCR4 antibodies, bind to SDF-1α, and also inhibit SDF-1α signaling, extending the scope of selective functional CXCR4 mimicry through CX4-M1.

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. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC chemokine 11 (CXCL11)-dependent G protein activation over ß-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated G protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.

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. Considering known ligands from public databases, such dual binders have not been identified yet. We therefore applied large-scale docking to the structure of CXCR4 and a homology model of CXCR3 with the goal to predict such dual binders, as well as compounds selective for either one of the receptors. Using signaling and biochemical assays, we showed that more than 50% of these predictions were correct in each category, yielding ligands with excellent binding efficiencies. These results highlight that docking is a suitable tool for the identification of ligands with tailored binding profiles to GPCRs, even when using homology models. More importantly, we present novel CXCR3-CXCR4 dual modulators that might pave the road to understanding the mechanisms of polypharmacological inhibition of these receptors.

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. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.