Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91.

SUCNR1 is an auto- and paracrine sensor of the metabolic stress signal succinate. Using unsupervised molecular dynamics (MD) simulations (170.400 ns) and mutagenesis across human, mouse, and rat SUCNR1, we characterize how a five-arginine motif around the extracellular pole of TM-VI determines the initial capture of succinate in the extracellular vestibule (ECV) to either stay or move down to the orthosteric site. Metadynamics demonstrate low-energy succinate binding in both sites, with an energy barrier corresponding to an intermediate stage during which succinate, with an associated water cluster, unlocks the hydrogen-bond-stabilized conformationally constrained extracellular loop (ECL)-2b. Importantly, simultaneous binding of two succinate molecules through either a "sequential" or "bypassing" mode is a frequent endpoint. The mono-carboxylate NF-56-EJ40 antagonist enters SUCNR1 between TM-I and -II and does not unlock ECL-2b. It is proposed that occupancy of both high-affinity sites is required for selective activation of SUCNR1 by high local succinate concentrations.

Biosynthesis of natural and halogenated plant monoterpene indole alkaloids in yeast.

Monoterpenoid indole alkaloids (MIAs) represent a large class of plant natural products with marketed pharmaceutical activities against a wide range of indications, including cancer, malaria and hypertension. Halogenated MIAs have shown improved pharmaceutical properties; however, synthesis of new-to-nature halogenated MIAs remains a challenge. Here we demonstrate a platform for de novo biosynthesis of two MIAs, serpentine and alstonine, in baker's yeast Saccharomyces cerevisiae and deploy it to systematically explore the biocatalytic potential of refactored MIA pathways for the production of halogenated MIAs. From this, we demonstrate conversion of individual haloindole derivatives to a total of 19 different new-to-nature haloserpentine and haloalstonine analogs. Furthermore, by process optimization and heterologous expression of a modified halogenase in the microbial MIA platform, we document de novo halogenation and biosynthesis of chloroalstonine. Together, this study highlights a microbial platform for enzymatic exploration and production of complex natural and new-to-nature MIAs with therapeutic potential.

Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Molecular dynamics-guided discovery of an ago-allosteric modulator for GPR40/FFAR1.

The long-chain fatty acid receptor FFAR1/GPR40 binds agonists in both an interhelical site between the extracellular segments of transmembrane helix (TM)-III and TM-IV and a lipid-exposed groove between the intracellular segments of these helices. Molecular dynamics simulations of FFAR1 with agonist removed demonstrated a major rearrangement of the polar and charged anchor point residues for the carboxylic acid moiety of the agonist in the interhelical site, which was associated with closure of a neighboring, solvent-exposed pocket between the extracellular poles of TM-I, TM-II, and TM-VII. A synthetic compound designed to bind in this pocket, and thereby prevent its closure, was identified through structure-based virtual screening and shown to function both as an agonist and as an allosteric modulator of receptor activation. This discovery of an allosteric agonist for a previously unexploited, dynamic pocket in FFAR1 demonstrates both the power of including molecular dynamics in the drug discovery process and that this specific, clinically proven, but difficult, antidiabetes target can be addressed by chemotypes different from existing ligands.

The Zinc-Sensing Receptor GPR39 in Physiology and as a Pharmacological Target.

The G-protein coupled receptor GPR39 is abundantly expressed in various tissues and can be activated by changes in extracellular Zn2+ in physiological concentrations. Previously, genetically modified rodent models have been able to shed some light on the physiological functions of GPR39, and more recently the utilization of novel synthetic agonists has led to the unraveling of several new functions in the variety of tissues GPR39 is expressed. Indeed, GPR39 seems to be involved in many important metabolic and endocrine functions, but also to play a part in inflammation, cardiovascular diseases, saliva secretion, bone formation, male fertility, addictive and depression disorders and cancer. These new discoveries offer opportunities for the development of novel therapeutic approaches against many diseases where efficient therapeutics are still lacking. This review focuses on Zn2+ as an endogenous ligand as well as on the novel synthetic agonists of GPR39, placing special emphasis on the recently discovered physiological functions and discusses their pharmacological potential.

Preassociation between the 5-HT7 serotonin receptor and G protein Gs: molecular determinants and association with low potency activation of adenylyl cyclase.

According to early models of GPCR signaling, G proteins only interact with activated receptors. However, some GPCRs were shown to assemble with G proteins before receptor activation, in accordance with more recent models. Previously, we found that the 5-HT7 receptor, as opposed to the 5-HT4 receptor, was preassociated with Gs, but the molecular determinants for this interaction are still elusive. In a series of chimeric 5-HT7 receptors with intracellular segments from 5-HT4, we determined the receptor-G protein interaction by performing antibody-immobilized fluorescence recovery after photobleaching and fluorescence resonance energy transfer. We identified the intracellular loop 3 and C-tail of the 5-HT7 receptor to be responsible for the preassociation with Gs, and we further delineated the TM5 extension in the intracellular loop 3 and helix 8 in the C-tail as the molecular determinants. These chimeric exchanges converted the 5-HT7 receptor into a collision-coupled receptor that recruited G proteins only upon agonist activation, whereas reciprocal exchanges converted 5-HT4 to a preassociated receptor. The 5-HT7 receptor displayed 2-component agonist-induced Gs signaling with high and low potency. In addition, the same segments were involved in low-potency signaling and preassociation. The correspondence between Gs preassociation and low-potency Gs signaling is a novel aspect of GPCR pharmacology.-Ulsund, A. H., Dahl, M., Frimurer, T. M., Manfra, O., Schwartz, T. W., Levy, F. O., Andressen, K. W. Preassociation between the 5-HT7 serotonin receptor and G protein Gs: molecular determinants and association with low potency activation of adenylyl cyclase.

Allostery in Coagulation Factor VIIa Revealed by Ensemble Refinement of Crystallographic Structures.

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.

Inhibiting RHOA Signaling in Mice Increases Glucose Tolerance and Numbers of Enteroendocrine and Other Secretory Cells in the Intestine.

BACKGROUND & AIMS: Glucagon-like peptide 1 (GLP1) is produced by L cells in the intestine, and agonists of the GLP1 receptor are effective in the treatment of diabetes. Levels of GLP1 increase with numbers of L cells. Therefore, agents that increase numbers of L cell might be developed for treatment of diabetes. Ras homologue family member A (RhoA) signaling through Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) controls cell differentiation, but it is not clear whether this pathway regulates enteroendocrine differentiation in the intestinal epithelium. We investigated the effects of Y-27632, an inhibitor of ROCK1 and ROCK2, on L-cell differentiation. METHODS: We collected intestinal tissues from GLU-Venus, GPR41-RFP, and Neurog3-RFP mice, in which the endocrine lineage is fluorescently labeled, for in vitro culture and histologic analysis. Small intestine organoids derived from these mice were cultured with Y-27632 and we measured percentages of L cells, expression of intestinal cell-specific markers, and secretion of GLP1 in medium. Mice were fed a normal chow or a high-fat diet and given Y-27632 or saline (control) and blood samples were collected for measurement of GLP1, insulin, and glucose. RESULTS: Incubation of intestinal organoids with Y-27632 increased numbers of L cells and secretion of GLP1. These increases were associated with upregulated expression of genes encoding intestinal hormones, neurogenin 3, neurogenic differentiation factor 1, forkhead box A1 and A2, and additional markers of secretory cells. Mice fed the normal chow diet and given Y-27632 had increased numbers of L cells in intestinal tissues, increased plasma levels of GLP1 and insulin, and lower blood levels of glucose compared with mice fed the normal chow diet and given saline. In mice with insulin resistance induced by the high-fat diet, administration of Y-27632 increased secretion of GLP1 and glucose tolerance compared with administration of saline. CONCLUSIONS: In mouse intestinal organoids, an inhibitor of RhoA signaling increased the differentiation of the secretory lineage and the development of enteroendocrine cells. Inhibitors of RhoA signaling or other strategies to increase numbers of L cells might be developed for treatment of patients with type 2 diabetes or for increasing glucose tolerance.

Structural changes at the myrtenol backbone reverse its positive allosteric potential into inhibitory GABAA receptor modulation.

GABAA receptors are ligand-gated anion channels that form pentameric arrangements of various subunits. Positive allosteric modulators of GABAA receptors have been reported as being isolated either from plants or synthesized analogs of known GABAA receptor targeting drugs. Recently, we identified monoterpenes, e.g. myrtenol as a positive allosteric modulator at α1ß2 GABAA receptors. Here, along with pharmacophore-based virtual screening studies, we demonstrate that scaffold modifications of myrtenol resulted in the loss of modulatory activity. Two independent approaches, fluorescence-based compound analysis and electrophysiological recordings in whole-cell configurations were used for analysis of transfected cells. C-atoms 1 and 2 of the myrtenol backbone were identified as crucial to preserve positive allosteric potential. A modification at C-atom 2 and lack of the hydroxyl group at C-atom 1 exhibited significantly reduced GABAergic currents at α1ß2, α1ß2γ, α2ß3, α2ß3γ and α4ß3δ receptors. This effect was independent of the γ2 subunit. A sub-screen with side chain length and volume differences at the C-atom 1 identified two compounds that inhibited GABAergic responses but without receptor subtype specificity. Our combined approach of pharmacophore-based virtual screening and functional readouts reveals that side chain modifications of the bridged six-membered ring structure of myrtenol are crucial for its modulatory potential at GABAA receptors.

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

Biased Gs versus Gq proteins and ß-arrestin signaling in the NK1 receptor determined by interactions in the water hydrogen bond network.

X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and ß-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na(+) ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gq only; 2) ß-arrestin only; and 3) Gq and ß-arrestin but not through Gs. Interestingly, increased constitutive Gs but not Gq signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans-membrane receptor conformations activating different signal transduction pathways.

Optimisation of in silico derived 2-aminobenzimidazole hits as unprecedented selective kappa opioid receptor agonists.

Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treatment of various pains. Pharmacophore based mining of databases led to the identification of 2-aminobenzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR and MOR. A short SAR exploration with the objective of identifying more polar and hence less brain penetrant agonists is described herewith. Modeling studies of the recently published structures of KOR, DOR and MOR are used to explain the receptor selectivity. The synthesis, biological evaluation and SAR of novel benzimidazole derivatives as KOR agonists are described. The in vivo proof of principle for anti-nociceptive effect with a lead compound from this series is exemplified.

Biased and constitutive signaling in the CC-chemokine receptor CCR5 by manipulating the interface between transmembrane helices 6 and 7.

The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biased signaling. Thus, ß-arrestin recruitment was eliminated, whereas constitutive activity was observed in Gαi-mediated signaling. Furthermore, the CCR5 antagonist aplaviroc was converted to a full agonist (a so-called efficacy switch). Computational modeling revealed that the position of the 7TM receptor-conserved Trp in TM6 (Trp-248 in position VI:13/6.48, part of the CWXP motif) was influenced by the G286F mutation, causing Trp-248 to change orientation away from TM7. The essential role of Trp-248 in CCR5 activation was supported by complete inactivity of W248A-CCR5 despite maintaining chemokine binding. Furthermore, replacing Trp-248 with a smaller aromatic amino acid (Tyr/Phe) impaired the ß-arrestin recruitment, yet with maintained G protein activity (biased signaling); also, here aplaviroc switched to a full agonist. Thus, the altered positioning of Trp-248, induced by G286F, led to a constraint of G protein active, but ß-arrestin inactive and thus biased, CCR5 conformation. These results provide important information on the molecular interplay and impact of TM6 and TM7 for CCR5 activity, which may be extrapolated to other chemokine receptors and possibly to other 7TM receptors.

Multiple recent HCAR2 structures demonstrate a highly dynamic ligand binding and G protein activation mode.

A surprisingly clear picture of the allosteric mechanism connecting G protein-coupled receptor agonists with G protein binding-and back - is revealed by a puzzle of thirty novel 3D structures of the hydroxycarboxylic acid receptor 2 (HCAR2) in complex with eight different orthosteric and a single allosteric agonist. HCAR2 is a sensor of ß-hydroxybutyrate, niacin and certain anti-inflammatory drugs. Surprisingly, agonists with and without on-target side effects bound very similarly and in a completely occluded orthosteric binding site. Thus, despite the many structures we are still left with a pertinent need to understand the molecular dynamics of this and similar systems.

PheVI:09 (Phe6.44) as a sliding microswitch in seven-transmembrane (7TM) G protein-coupled receptor activation.

In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the ß(2)-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the ß(2)-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.

Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop.

Based on a rare, natural Glu for Ala-204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2b (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional analysis. Introduction of Glu in different positions demonstrated that although the constitutive signaling was partly reduced when introduced in position 205(C+7) it was only totally eliminated in position 204(C+6). No charge-charge interaction partner could be identified for the Glu(C+6) variant despite mutational analysis of a number of potential partners in the extracellular loops and outer parts of the transmembrane segments. Systematic probing of position 204(C+6) with amino acid residues of different physicochemical properties indicated that a positively charged Lys surprisingly provided phenotypes similar to those of the negatively charged Glu residue. Computational chemistry analysis indicated that the propensity for the C-terminal segment of extracellular loop 2b to form an extended α-helix was increased from 15% in the wild type to 89 and 82% by introduction in position 204(C+6) of a Glu or a Lys residue, respectively. Moreover, the constitutive activity of the receptor was inhibited by Zn(2+) binding in an engineered metal ion site, stabilizing an α-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment of extracellular loop 2 and that mutations or ligand binding that constrains this segment and thereby conceivably the movements of transmembrane domain V relative to transmembrane domain III inhibits the high constitutive signaling.

The arginine of the DRY motif in transmembrane segment III functions as a balancing micro-switch in the activation of the ß2-adrenergic receptor.

Recent high resolution x-ray structures of the ß2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected "ionic lock" interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased G(s) signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of G(s) signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on G(s) signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.

Structure-activity relationships and identification of optmized CC-chemokine receptor CCR1, 5, and 8 metal-ion chelators.

Chemokine receptors are involved in trafficking of leukocytes and represent targets for autoimmune conditions, inflammatory diseases, viral infections, and cancer. We recently published CCR1, CCR8, and CCR5 agonists and positive modulators based on a three metal-ion chelator series: 2,2'-bipyridine, 1,10-phenanthroline, and 2,2';6',2″-terpyridine. Here, we have performed an in-depth structure-activity relationship study and tested eight new optimized analogs. Using density functional theory calculations we demonstrate that the chelator zinc affinities depend on how electron-donating and -withdrawing substituents modulate the partial charges of chelating nitrogens. The zinc affinity was found to constitute the major factor for receptor potency, although the activity of some chelators deviate suggesting favorable or unfavorable interactions. Hydrophobic and halogen substituents are generally better accommodated in the receptors than polar groups. The new analog brominated terpyridine (29) resulted in the highest chelator potencies observed so far CCR1 (EC50: 0.49 µM) and CCR8 (EC50: 0.28 µM). Furthermore, we identified the first selective CCR5 agonist chelator, meta dithiomethylated bipyridine (23). The structure-activity relationships contribute to small-molecule drug development, and the novel chelators constitute valuable tools for studies of structural mechanisms for chemokine receptor activation.

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach.

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.