Molecular determinants of antagonist interactions with chemokine receptors CCR2 and CCR5.

By driving monocyte chemotaxis, the chemokine receptor CCR2 shapes inflammatory responses and the formation of tumor microenvironments. This makes it a promising target in inflammation and immuno-oncology; however, despite extensive efforts, there are no FDA-approved CCR2-targeting therapeutics. Cited challenges include the redundancy of the chemokine system, suboptimal properties of compound candidates, and species differences that confound the translation of results from animals to humans. Structure-based drug design can rationalize and accelerate the discovery and optimization of CCR2 antagonists to address these challenges. The prerequisites for such efforts include an atomic-level understanding of the molecular determinants of action of existing antagonists. In this study, using molecular docking and artificial-intelligence-powered compound library screening, we uncover the structural principles of small molecule antagonism and selectivity towards CCR2 and its sister receptor CCR5. CCR2 orthosteric inhibitors are shown to universally occupy an inactive-state-specific tunnel between receptor helices 1 and 7; we also discover an unexpected role for an extra-helical groove accessible through this tunnel, suggesting its potential as a new targetable interface for CCR2 and CCR5 modulation. By contrast, only shape complementarity and limited helix 8 hydrogen bonding govern the binding of various chemotypes of allosteric antagonists. CCR2 residues S1012.63 and V2446.36 are implicated as determinants of CCR2/CCR5 and human/mouse orthosteric and allosteric antagonist selectivity, respectively, and the role of S1012.63 is corroborated through experimental gain-of-function mutagenesis. We establish a critical role of induced fit in antagonist recognition, reveal strong chemotype selectivity of existing structures, and demonstrate the high predictive potential of a new deep-learning-based compound scoring function. Finally, this study expands the available CCR2 structural landscape with computationally generated chemotype-specific models well-suited for structure-based antagonist design.

Ligand-Dependent Mechanisms of C-C Chemokine Receptor 5 (CCR5) Trafficking Revealed by APEX2 Proximity Labeling Proteomics.

CC chemokine receptor 5 (CCR5) contributes to inflammatory responses by driving cell migration and scavenging chemokine to shape directional chemokine gradients. A drug against CCR5 has been approved for blocking HIV entry into cells. However, targeting CCR5 for the treatment of inflammatory diseases and cancer has had limited success because of the complex biology and pharmacology of this receptor. CCR5 is activated by many natural and engineered chemokines that elicit distinct receptor signaling and trafficking responses, including some that sequester the receptor inside the cell. The sequestration phenomenon may be therapeutically exploitable, but the mechanisms by which different ligands traffic CCR5 to different cellular locations are poorly understood. Here we employed live cell ascorbic acid peroxidase proximity labeling and quantitative mass spectrometry proteomics for unbiased discovery of temporally resolved protein neighborhoods of CCR5 following stimulation with its endogenous agonist, CCL5, and two CCL5 variants that promote intracellular retention of the receptor. Along with targeted pharmacological assays, the data reveals distinct ligand-dependent CCR5 trafficking patterns with temporal resolution. All three chemokines internalize CCR5 via ß-arrestin- dependent, clathrin-mediated endocytosis but to different extents, with different kinetics and with varying dependencies on GPCR kinase subtypes. The agonists differ in their ability to target the receptor to lysosomes for degradation, as well as to the Golgi compartment and the trans-Golgi network, and these trafficking patterns translate into distinct levels of ligand scavenging. The results provide insight into the molecular mechanisms behind CCR5 intracellular sequestration and suggest actionable patterns for the development of chemokine-based CCR5 targeting molecules. Significance Statement: CCR5 plays a crucial role in the immune system and is important in numerous physiological and pathological processes such as inflammation, cancer and HIV transmission. Along with its functional diversity, different CCR5 ligands can induce distinct receptor signaling responses and trafficking behaviors; the latter includes intracellular receptor sequestration which offers a potential therapeutic strategy for inhibiting CCR5 function. Using time-resolved proximity labeling proteomics and targeted pharmacological experiments, this study reveals the molecular basis for receptor sequestration including information that can be exploited for the development of CCR5 targeting molecules that promote retention of the receptor inside the cell.

Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods.

BACKGROUND: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. RESULTS: Here we generated highly soluble and stable recombinant protein constructs GST-CB1414-472 and GST-CB1414-442 containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. CONCLUSIONS: Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.

Combined docking and machine learning identify key molecular determinants of ligand pharmacological activity on ß2 adrenoceptor.

G protein-coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands that bind them. Ligands exert their action via the interactions in the ligand binding pocket. We hypothesized that there is a common set of receptor interactions made by ligands of diverse structures that mediate their action and that among a large dataset of different ligands, the functionally important interactions will be over-represented. We computationally docked ~2700 known ß2AR ligands to multiple ß2AR structures, generating ca 75 000 docking poses and predicted all atomic interactions between the receptor and the ligand. We used machine learning (ML) techniques to identify specific interactions that correlate with the agonist or antagonist activity of these ligands. We demonstrate with the application of ML methods that it is possible to identify the key interactions associated with agonism or antagonism of ligands. The most representative interactions for agonist ligands involve K972.68×67 , F194ECL2 , S2035.42×43 , S2045.43×44 , S2075.46×641 , H2966.58×58 , and K3057.32×31 . Meanwhile, the antagonist ligands made interactions with W2866.48×48 and Y3167.43×42 , both residues considered to be important in GPCR activation. The interpretation of ML analysis in human understandable form allowed us to construct an exquisitely detailed structure-activity relationship that identifies small changes to the ligands that invert their pharmacological activity and thus helps to guide the drug discovery process. This approach can be readily applied to any drug target.

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

The Absence of the Transient Receptor Potential Vanilloid 1 Directly Impacts on the Expression and Localization of the Endocannabinoid System in the Mouse Hippocampus.

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective ligand-gated cation channel involved in synaptic transmission, plasticity, and brain pathology. In the hippocampal dentate gyrus, TRPV1 localizes to dendritic spines and dendrites postsynaptic to excitatory synapses in the molecular layer (ML). At these same synapses, the cannabinoid CB1 receptor (CB1R) activated by exogenous and endogenous cannabinoids localizes to the presynaptic terminals. Hence, as both receptors are activated by endogenous anandamide, co-localize, and mediate long-term depression of the excitatory synaptic transmission at the medial perforant path (MPP) excitatory synapses though by different mechanisms, it is plausible that they might be exerting a reciprocal influence from their opposite synaptic sites. In this anatomical scenario, we tested whether the absence of TRPV1 affects the endocannabinoid system. The results obtained using biochemical techniques and immunoelectron microscopy in a mouse with the genetic deletion of TRPV1 show that the expression and localization of components of the endocannabinoid system, included CB1R, change upon the constitutive absence of TRPV1. Thus, the expression of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) drastically increased in TRPV1-/- whole homogenates. Furthermore, CB1R and MAGL decreased and the cannabinoid receptor interacting protein 1a (CRIP1a) increased in TRPV1-/- synaptosomes. Also, CB1R positive excitatory terminals increased, the number of excitatory terminals decreased, and CB1R particles dropped significantly in inhibitory terminals in the dentate ML of TRPV1-/- mice. In the outer 2/3 ML of the TRPV1-/- mutants, the proportion of CB1R particles decreased in dendrites, and increased in excitatory terminals and astrocytes. In the inner 1/3 ML, the proportion of labeling increased in excitatory terminals, neuronal mitochondria, and dendrites. Altogether, these observations indicate the existence of compensatory changes in the endocannabinoid system upon TRPV1 removal, and endorse the importance of the potential functional adaptations derived from the lack of TRPV1 in the mouse brain.