Negative Cooperativity Research Articles

Transthyretin Binding Heterogeneity and Anti-amyloidogenic Activity of Natural Polyphenols and Their Metabolites

Transthyretin (TTR) is an amyloidogenic protein, the amyloidogenic potential of which is enhanced by a number of specific point mutations. The ability to inhibit TTR fibrillogenesis is known for several classes of compounds, including natural polyphenols, which protect the native state of TTR by specifically interacting with its thyroxine binding sites. Comparative analyses of the interaction and of the ability to protect the TTR native state for polyphenols, both stilbenoids and flavonoids, and some of their main metabolites have been carried out. A main finding of this investigation was the highly preferential binding of resveratrol and thyroxine, both characterized by negative binding cooperativity, to distinct sites in TTR, consistent with the data of x-ray analysis of TTR in complex with both ligands. Although revealing the ability of the two thyroxine binding sites of TTR to discriminate between different ligands, this feature has allowed us to evaluate the interactions of polyphenols with both resveratrol and thyroxine preferential binding sites, by using resveratrol and radiolabeled T4 as probes. Among flavonoids, genistein and apigenin were able to effectively displace resveratrol from its preferential binding site, whereas genistein also showed the ability to interact, albeit weakly, with the preferential thyroxine binding site. Several glucuronidated polyphenol metabolites did not exhibit significant competition for resveratrol and thyroxine preferential binding sites and lacked the ability to stabilize TTR. However, resveratrol-3-O-sulfate was able to significantly protect the protein native state. A rationale for the in vitro properties found for polyphenol metabolites was provided by x-ray analysis of their complexes with TTR.

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins.

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones.

Structural basis for cpSRP43 chromodomain selectivity and dynamics in Alb3 insertase interaction.

Canonical membrane protein biogenesis requires co-translational delivery of ribosome-associated proteins to the Sec translocase and depends on the signal recognition particle (SRP) and its receptor (SR). In contrast, high-throughput delivery of abundant light-harvesting chlorophyll a,b-binding proteins (LHCPs) in chloroplasts to the Alb3 insertase occurs post-translationally via a soluble transit complex including the cpSRP43/cpSRP54 heterodimer (cpSRP). Here we describe the molecular mechanisms of tethering cpSRP to the Alb3 insertase by specific interaction of cpSRP43 chromodomain 3 with a linear motif in the Alb3 C-terminal tail. Combining NMR spectroscopy, X-ray crystallography and biochemical analyses, we dissect the structural basis for selectivity of chromodomains 2 and 3 for their respective ligands cpSRP54 and Alb3, respectively. Negative cooperativity in ligand binding can be explained by dynamics in the chromodomain interface. Our study provides a model for membrane recruitment of the transit complex and may serve as a prototype for a functional gain by the tandem arrangement of chromodomains.

Bacterial Thymidylate Synthase Binds Two Molecules of Substrate and Cofactor without Cooperativity.

Thymidylate synthase (TSase) is a clinically important enzyme because it catalyzes synthesis of the sole de novo source of deoxy-thymidylate. Without this enzyme, cells die a "thymineless death" since they are starved of a crucial DNA synthesis precursor. As a drug target, TSase is well studied in terms of its structure and reaction mechanism. An interesting mechanistic feature of dimeric TSase is that it is "half-the-sites reactive", which is a form of negative cooperativity. Yet, the basis for this is not well-understood. Some experiments point to cooperativity at the binding steps of the reaction cycle as being responsible for the phenomenon, but the literature contains conflicting reports. Here we use ITC and NMR to resolve these inconsistencies. This first detailed thermodynamic dissection of multisite binding of dUMP to E. coli TSase shows the nucleotide binds to the free and singly bound forms of the enzyme with nearly equal affinity over a broad range of temperatures and in multiple buffers. While small but significant differences in ΔC°P for the two binding events show that the active sites are not formally equivalent, there is little-to-no allostery at the level of ΔG°bind. In addition NMR titration data reveal that there is minor intersubunit cooperativity in formation of a ternary complex with the mechanism based inhibitor, 5F-dUMP, and cofactor. Taken together, the data show that functional communication between subunits is minimal for both binding steps of the reaction coordinate.

VU0477573: Partial Negative Allosteric Modulator of the Subtype 5 Metabotropic Glutamate Receptor with In Vivo Efficacy.

Negative allosteric modulators (NAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) have potential applications in the treatment of fragile X syndrome, levodopa-induced dyskinesia in Parkinson disease, Alzheimer disease, addiction, and anxiety; however, clinical and preclinical studies raise concerns that complete blockade of mGlu5 and inverse agonist activity of current mGlu5 NAMs contribute to adverse effects that limit the therapeutic use of these compounds. We report the discovery and characterization of a novel mGlu5 NAM, N,N-diethyl-5-((3-fluorophenyl)ethynyl)picolinamide (VU0477573) that binds to the same allosteric site as the prototypical mGlu5 NAM MPEP but displays weak negative cooperativity. Because of this weak cooperativity, VU0477573 acts as a "partial NAM" so that full occupancy of the MPEP site does not completely inhibit maximal effects of mGlu5 agonists on intracellular calcium mobilization, inositol phosphate (IP) accumulation, or inhibition of synaptic transmission at the hippocampal Schaffer collateral-CA1 synapse. Unlike previous mGlu5 NAMs, VU0477573 displays no inverse agonist activity assessed using measures of effects on basal [(3)H]inositol phosphate (IP) accumulation. VU0477573 acts as a full NAM when measuring effects on mGlu5-mediated extracellular signal-related kinases 1/2 phosphorylation, which may indicate functional bias. VU0477573 exhibits an excellent pharmacokinetic profile and good brain penetration in rodents and provides dose-dependent full mGlu5 occupancy in the central nervous system (CNS) with systemic administration. Interestingly, VU0477573 shows robust efficacy, comparable to the mGlu5 NAM MTEP, in models of anxiolytic activity at doses that provide full CNS occupancy of mGlu5 and demonstrate an excellent CNS occupancy-efficacy relationship. VU0477573 provides an exciting new tool to investigate the efficacy of partial NAMs in animal models.

2-Oxoglutarate levels control adenosine nucleotide binding by Herbaspirillum seropedicae PII proteins.

Nitrogen metabolism in Proteobacteria is controlled by the Ntr system, in which PII proteins play a pivotal role, controlling the activity of target proteins in response to the metabolic state of the cell. Characterization of the binding of molecular effectors to these proteins can provide information about their regulation. Here, the binding of ATP, ADP and 2-oxoglutarate (2-OG) to the Herbaspirillum seropedicae PII proteins, GlnB and GlnK, was characterized using isothermal titration calorimetry. Results show that these proteins can bind three molecules of ATP, ADP and 2-OG with homotropic negative cooperativity, and 2-OG binding stabilizes the binding of ATP. Results also show that the affinity of uridylylated forms of GlnB and GlnK for nucleotides is significantly lower than that of the nonuridylylated proteins. Furthermore, fluctuations in the intracellular concentration of 2-OG in response to nitrogen availability are shown. Results suggest that under nitrogen-limiting conditions, PII proteins tend to bind ATP and 2-OG. By contrast, after an ammonium shock, a decrease in the 2-OG concentration is observed causing a decrease in the affinity of PII proteins for ATP. This phenomenon may facilitate the exchange of ATP for ADP on the ligand-binding pocket of PII proteins, thus it is likely that under low ammonium, low 2-OG levels would favor the ADP-bound state.

The observation of ion-pairing effect based on substituent effect of ferrocene derivatives

Abstract The ion-pairing effect was investigated based on the substituent effect of ferrocene (Fc) derivatives using cyclic voltammetry. It was shown that the presence of ion-pairing strongly affected the electrochemical redox behavior in the organic solvent. The formal redox potential ( E 0′ , the average of anodic and cathodic peak potential) shifted negatively with the increasing ion-pairing effect. That was because the formation of ion pair (Fc + ·ClO 4 − ) was beneficial to equilibrium shift from Fc to Fc + in thermodynamics. In this work, electron-donating and electron-withdrawing substituents of ferrocene derivatives were employed for a deep study of ion-pairing effect, respectively. It is confirmed that both ion-pairing effect and electron-donating substituent effect facilitated the negative shift of E 0′ for ferrocene derivatives, showing the positive cooperativity. While the electron-withdrawing substituent effect resulted in the positive shift of E 0′ for ferrocene derivatives and was unfavorable for the oxidation of Fc derivatives, reflecting the negative cooperativity with ion-pairing effect. In addition, the reversal phenomenon of weak electron-withdrawing substituent was revealed when the ion-pairing effect was stronger than the electron-withdrawing substituent effect, indicating that the ion-pairing function has a significant effect on electrochemical behavior of ferrocene derivatives.

Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6′ in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1′ (with a ~32–93% preference for leucine depending on the MMP), and basic and small residues in P2′ and P3′, respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1′ leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1′-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1′. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.

Measuring Multivalent Binding Interactions by Isothermal Titration Calorimetry.

Multivalent glycoconjugate-protein interactions are central to many important biological processes. Isothermal titration calorimetry (ITC) can potentially reveal the molecular and thermodynamic basis of such interactions. However, calorimetric investigation of multivalency is challenging. Binding of multivalent glycoconjugates to proteins (lectins) often leads to a stoichiometry-dependent precipitation process due to noncovalent cross-linking between the reactants. Precipitation during ITC titration severely affects the quality of the baseline as well as the signals. Hence, the resulting thermodynamic data are not dependable. We have made some modifications to address this problem and successfully studied multivalent glycoconjugate binding to lectins. We have also modified the Hill plot equation to analyze high quality ITC raw data obtained from multivalent binding. As described in this chapter, ITC-driven thermodynamic parameters and Hill plot analysis of ITC raw data can provide valuable information about the molecular mechanism of multivalent lectin-glycoconjugate interactions. The methods described herein revealed (i) the importance of functional valence of multivalent glycoconjugates, (ii) that favorable entropic effects contribute to the enhanced affinities associated with multivalent binding, (iii) that with the progression of lectin binding, the microscopic affinities of the glycan epitopes of a multivalent glycoconjugate decrease (negative cooperativity), (iv) that lectin binding to multivalent glycoconjugates, especially to mucins, involves internal diffusion jumps, (bind and jump) and (v) that scaffolds of glycoconjugates influence their entropy of binding.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.

Cooperative binding: a multiple personality.

Cooperative binding has been described in many publications and has been related to or defined by several different properties of the binding behavior of the ligand to the target molecule. In addition to the commonly used Hill coefficient, other characteristics such as a sigmoidal shape of the overall titration curve in a linear plot, a change of ligand affinity of the other binding sites when a site of the target molecule becomes occupied, or complex roots of the binding polynomial have been used to define or to quantify cooperative binding. In this work, we analyze how the different properties are related in the most general model for binding curves based on the grand canonical partition function and present several examples which highlight differences between the cooperativity characterizing properties which are discussed. Our results mainly show that among the presented definitions there are not two which fully coincide. Moreover, this work poses the question whether it can make sense to distinguish between positive and negative cooperativity based on the macroscopic binding isotherm only. This article shall emphasize that scientists who investigate cooperative effects in biological systems could help avoiding misunderstandings by stating clearly which kind of cooperativity they discuss.

Discovery of a Novel Class of Negative Allosteric Modulator of the Dopamine D2 Receptor Through Fragmentation of a Bitopic Ligand.

Recently, we have demonstrated that N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide (SB269652) (1) adopts a bitopic pose at one protomer of a dopamine D2 receptor (D2R) dimer to negatively modulate the binding of dopamine at the other protomer. The 1H-indole-2-carboxamide moiety of 1 extends into a secondary pocket between the extracellular ends of TM2 and TM7 within the D2R protomer. To target this putative allosteric site, we generated and characterized fragments that include and extend from the 1H-indole-2-carboxamide moiety of 1. N-Isopropyl-1H-indole-2-carboxamide (3) displayed allosteric pharmacology and sensitivity to mutations of the same residues at the top of TM2 as was observed for 1. Using 3 as an "allosteric lead", we designed and synthesized an extensive fragment library to generate novel SAR and identify N-butyl-1H-indole-2-carboxamide (11d), which displayed both increased negative cooperativity and affinity for the D2R. These data illustrate that fragmentation of extended compounds can expose fragments with purely allosteric pharmacology.

A Review of Tafamidis for the Treatment of Transthyretin-Related Amyloidosis.

Transthyretin (TTR)-related amyloidosis (ATTR) is a devastating disease which affects a combination of organs including the heart and the peripheral nerves, and which has a fatal outcome if not treated within a average of 10 years. Tafamidis, or 2-(3,5-dichloro-phenyl)-benzoxazole-6-carboxylic acid, selectively binds to TTR with negative cooperativity and kinetically stabilizes wild-type native TTR and mutant TTR; tafamidis therefore has the potential to halt the amyloidogenic cascade initiated by TTR tetramer dissociation, monomer misfolding, and aggregation. The first tafamidis trial, Fx-005, evaluated the effect of 18 months of tafamidis treatment (20 mg once daily) on disease progression, as well as assessing its safety in TTR-FAP Val30Met patients. The secondary objective of this trial was to study the pharmacodynamic stabilization of mutated TTR. Tafamidis proved effective in reducing the progress of neuropathy, and in maintaining the nutritional status and quality of life of stage 1 (able to walk without support) Val3OMet TTR-FAP patients. Furthermore, TTR stabilization was achieved in more than 90% of patients. An extension study, Fx-006, was conducted to determine the long-term safety and tolerability of tafamidis and to assess the efficacy of the drug on slowing disease progression. No significant safety or tolerability issues were noticed. Taken together, the results from both trials indicated that the beneficial effects of tafamidis were sustained over a 30-month period and that starting treatment early is desirable. Results are expected from an extended open-label study but data that have already been presented show that long-term use of tafamidis in Val30Met patients is associated with reduced progression in polyneuropathy. Tafamidis was initially approved for commercial use in Europe in 2011 and has since been approved for use in Japan, Mexico, and Argentina where it is used as a first-line treatment option for patients with early-stage TTR-FAP. Patients should be carefully followed at referral centers to ascertain the individual response to treatment. In cases of discontinuation, liver transplantation and enrollment in clinical trials of novel drugs aimed mostly toward suppression of TTR production are options.Electronic supplementary materialThe online version of this article (doi:10.1007/s40120-015-0031-3) contains supplementary material, which is available to authorized users.

Selectivity and Cooperativity in the Binding of Multiple Guests to a Pillar[5]arene-Crown Ether Fused Tricyclic Host.

A novel tricylic host molecule 1 that consists of two pillar[5]arene units and a crown ether ring was found to selectively bind two kinds of guest molecules with different shapes, sizes, and electronic constitutions, namely 1,4-dicyanobutane G1 and paraquat G2, with its two macrocyclic subunits, to form a four-component complex 2G1⊂1⊃G2. An (1)H NMR study of stepwise bindings of G1 and G2 to host 1 in CDCl3/DMSO-d6 revealed that the strength of the association between complex 2G1⊂1 and guest G2 was only one-fourth of that between free 1 and G2, demonstrating a negative heterotropic cooperativity of G1 in the binding of G2 to host 1.

YbiB from Escherichia coli, the Defining Member of the Novel TrpD2 Family of Prokaryotic DNA-binding Proteins

We present the crystal structure and biochemical characterization of Escherichia coli YbiB, a member of the hitherto uncharacterized TrpD2 protein family. Our results demonstrate that the functional diversity of proteins with a common fold can be far greater than predictable by computational annotation. The TrpD2 proteins show high structural homology to anthranilate phosphoribosyltransferase (TrpD) and nucleoside phosphorylase class II enzymes but bind with high affinity (KD = 10-100 nM) to nucleic acids without detectable sequence specificity. The difference in affinity between single- and double-stranded DNA is minor. Results suggest that multiple YbiB molecules bind to one longer DNA molecule in a cooperative manner. The YbiB protein is a homodimer that, therefore, has two electropositive DNA binding grooves. But due to negative cooperativity within the dimer, only one groove binds DNA in in vitro experiments. A monomerized variant remains able to bind DNA with similar affinity, but the negative cooperative effect is eliminated. The ybiB gene forms an operon with the DNA helicase gene dinG and is under LexA control, being induced by DNA-damaging agents. Thus, speculatively, the TrpD2 proteins may be part of the LexA-controlled SOS response in bacteria.

The nonadditivity of stacking interactions in adenine–thymine and guanine–cytosine stacked structures: Study by MP2 and SCS-MP2 calculations

The nonadditivity of stacking interactions in stacked structures of adenine–thymine and guanine–cytosine base pairs is investigated by MP2 and SCS-MP2 calculations with 6-311++G** and aug-cc-pvdz basis sets. The calculation results indicate that the intermolecular distances in the multi-stacked structures do not become shorter obviously as the stacked structure added. The middle stacking interaction energies in the multi-stacked structures also become weaker than that of dimer structures. It is found that the total stacking interaction energies of the trimer and tetramer stacked structures do not increase proportionally. Based on the results, we suggest that there is negative cooperativity of the stacking interactions in the adenine–thymine and guanine–cytosine stacked structures.

Cooperativity in CYP2E1 metabolism of acetaminophen and styrene mixtures

Risk assessment for exposure to mixtures of drugs and pollutants relies heavily on in vitro characterization of their bioactivation and/or metabolism individually and extrapolation to mixtures assuming no interaction. Herein, we demonstrated that in vitro CYP2E1 metabolic activation of acetaminophen and styrene mixtures could not be explained through the Michaelis–Menten mechanism or any models relying on that premise. As a baseline for mixture studies with styrene, steady-state analysis of acetaminophen oxidation revealed a biphasic kinetic profile that was best described by negative cooperativity (Hill coefficient=0.72). The best-fit mechanism for this relationship involved two binding sites with differing affinities (Ks=830μM and Kss=32mM). Introduction of styrene inhibited that reaction less than predicted by simple competition and thus provided evidence for a cooperative mechanism within the mixture. Likewise, acetaminophen acted through a mixed-type inhibition mechanism to impact styrene epoxidation. In this case, acetaminophen competed with styrene for CYP2E1 (Ki=830μM and Ksi=180μM for catalytic and effector sites, respectively) and resulted in cooperative impacts on binding and catalysis. Based on modeling of in vivo clearance, cooperative interactions between acetaminophen and styrene resulted in profoundly increased styrene activation at low styrene exposure levels and therapeutic acetaminophen levels. Current Michaelis–Menten based toxicological models for mixtures such as styrene and acetaminophen would fail to detect this concentration-dependent relationship. Hence, future studies must assess the role of alternate CYP2E1 mechanisms in bioactivation of compounds to improve the accuracy of interpretations and predictions of toxicity.

Structure-activity study of N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide (SB269652), a bitopic ligand that acts as a negative allosteric modulator of the dopamine D2 receptor.

We recently demonstrated that SB269652 (1) engages one protomer of a dopamine D2 receptor (D2R) dimer in a bitopic mode to allosterically inhibit the binding of dopamine at the other protomer. Herein, we investigate structural determinants for allostery, focusing on modifications to three moieties within 1. We find that orthosteric "head" groups with small 7-substituents were important to maintain the limited negative cooperativity of analogues of 1, and replacement of the tetrahydroisoquinoline head group with other D2R "privileged structures" generated orthosteric antagonists. Additionally, replacement of the cyclohexylene linker with polymethylene chains conferred linker length dependency in allosteric pharmacology. We validated the importance of the indolic NH as a hydrogen bond donor moiety for maintaining allostery. Replacement of the indole ring with azaindole conferred a 30-fold increase in affinity while maintaining negative cooperativity. Combined, these results provide novel SAR insight for bitopic ligands that act as negative allosteric modulators of the D2R.

Investigation of GPCR allosterism and dimerization in single living cells using fluorescent ligands.

Previous work, using fluorescent adenosine receptor agonists and antagonists, has provided novel insights into the allosteric regulation of adenosine A3 (A3AR) and A1 (A1AR) receptors by allosteric ligands and receptor dimerization in single living cells [1, 2]. We have also used a fluorescent analogue of {type:entrez-protein,attrs:{text:CGP12177,term_id:877152897,term_text:CGP12177}}CGP12177 to investigate ligand binding to the human β1-adrenoceptor. This work has demonstrated that there is negative cooperativity between the two different ligand-binding conformations of the β1-adrenoceptor activated by catecholamines and {type:entrez-protein,attrs:{text:CGP12177,term_id:877152897,term_text:CGP12177}}CGP12177 respectively [3]. Finally, we have used fluorescence correlation spectroscopy (FCS) to investigate ligand binding to A1AR and A3AR in small 0.2 µm2 microdomains of single living cells [4]. FCS studies with a fluorescent A3-agonist have enabled high affinity labeling of the active conformation (R*) of the receptor [4]. We have also used a fluorescent adenosine A3-antagonist ({type:entrez-nucleotide,attrs:{text:CA200645,term_id:35234116,term_text:CA200645}}CA200645) to study the binding characteristics of antagonist-occupied receptor conformations (R) in membrane microdomains of single cells [5]. Investigation of the dissociation kinetics of {type:entrez-nucleotide,attrs:{text:CA200645,term_id:35234116,term_text:CA200645}}CA200645 provided further support for allosteric regulation of this receptor by homodimerization [5].

Biased Agonism and Biased Allosteric Modulation at the CB1 Cannabinoid Receptor.

CB1 cannabinoid receptors (CB1Rs) are attractive therapeutic targets for numerous central nervous system disorders. However, clinical application of cannabinoid ligands has been hampered owing to their adverse on-target effects. Ligand-biased signaling from, and allosteric modulation of, CB1Rs offer pharmacological approaches that may enable the development of improved CB1R drugs, through modulation of only therapeutically desirable CB1R signaling pathways. There is growing evidence that CB1Rs are subject to ligand-biased signaling and allosterism. Therefore, in the present study, we quantified ligand-biased signaling and allosteric modulation at CB1Rs. Cannabinoid agonists displayed distinct biased signaling profiles at CB1Rs. For instance, whereas 2-arachidonylglycerol and WIN55,212-2 [(R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone] showed little preference for inhibition of cAMP and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2), N-arachidonoylethanolamine (anandamide), methanandamide, CP55940 [2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol], and HU-210 [11-hydroxy-Δ(8)-THC-dimethylheptyl] were biased toward cAMP inhibition. The small-molecule allosteric modulator Org27569 [5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide] displayed biased allosteric effects by blocking cAMP inhibition mediated by all cannabinoid ligands tested, at the same time having little or no effect on ERK1/2 phosphorylation mediated by a subset of these ligands. Org27569 also displayed negative binding cooperativity with [(3)H]SR141716A [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide]; however, it had minimal effects on binding of cannabinoid agonists. Furthermore, we highlight the need to validate the reported allosteric effects of the endogenous ligands lipoxin A4 and pregnenolone at CB1Rs. Pregnenolone but not lipoxin A4 displaced [(3)H]SR141716A, but there was no functional interaction between either of these ligands and cannabinoid agonists. This study demonstrates an approach to validating and quantifying ligand-biased signaling and allosteric modulation at CB1Rs, revealing ligand-biased "fingerprints" that may ultimately allow the development of improved CB1R-targeted therapies.