Design, Synthesis, and Application of Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the CXC Chemokine Receptor 2.

The inhibition of CXC chemokine receptor 2 (CXCR2), a key inflammatory mediator, is a potential strategy in the treatment of several pulmonary diseases and cancers. The complexity of endogenous chemokine interaction with the orthosteric binding site has led to the development of CXCR2 negative allosteric modulators (NAMs) targeting an intracellular pocket near the G protein binding site. Our understanding of NAM binding and mode of action has been limited by the availability of suitable tracer ligands for competition studies, allowing direct ligand binding measurements. Here, we report the rational design, synthesis, and pharmacological evaluation of a series of fluorescent NAMs, based on navarixin (2), which display high affinity and preferential binding for CXCR2 over CXCR1. We demonstrate their application in fluorescence imaging and NanoBRET binding assays, in whole cells or membranes, capable of kinetic and equilibrium analysis of NAM binding, providing a platform to screen for alternative chemophores targeting these receptors.

Assessment of the potential of novel and classical opioids to induce respiratory depression in mice.

BACKGROUND AND PURPOSE: Opioid-induced respiratory depression limits the use of µ-opioid receptor agonists in clinical settings and is the main cause of opioid overdose fatalities. The relative potential of different opioid agonists to induce respiratory depression at doses exceeding those producing analgesia is understudied despite its relevance to assessments of opioid safety. Here we evaluated the respiratory depressant and anti-nociceptive effects of three novel opioids and relate these measurements to their in vitro efficacy. EXPERIMENTAL APPROACH: Respiration was measured in awake, freely moving male CD-1 mice using whole body plethysmography. Anti-nociception was measured using the hot plate test. Morphine, oliceridine and tianeptine were administered intraperitoneally, whereas methadone, oxycodone and SR-17018 were administered orally. Receptor activation and arrestin-3 recruitment were measured in HEK293 cells using BRET assays. KEY RESULTS: Across the dose ranges examined, all opioids studied depressed respiration in a dose-dependent manner, with similar effects at the highest doses, and with tianeptine and oliceridine showing reduced duration of effect, when compared with morphine, oxycodone, methadone and SR-17018. When administered at doses that induced similar respiratory depression, all opioids induced similar anti-nociception, with tianeptine and oliceridine again showing reduced duration of effect. These data were consistent with the in vitro agonist activity of the tested compounds. CONCLUSION AND IMPLICATIONS: In addition to providing effective anti-nociception, the novel opioids, oliceridine, tianeptine and SR-17018 depress respiration in male mice. However, the different potencies and kinetics of effect between these novel opioids may be relevant to their therapeutic application in different clinical settings.

Synthesis, in vitro cytotoxic activity and molecular docking study of androstene and estrone derivatives.

The development and discovery of steroidal drugs to cure cervical cancer is of the most important. The Claisen condensation of androstene and estrone with aromatic aldehydes was catalyzed by potassium tert. butoxide in tert. butanol to give the corresponding 2-arylidene and 16-arylidene estrone. Subsequently, the 16-arylidene estrone reacted with acid chloride in presence of quaternary amine in halogenated solvent resulting in the steroidal arylidene derivatives. Synthesis, Characterization and in vitro cytotoxic activity of arylidenes are rationalized. Fifteen compounds are synthesized and six of them were evaluated for cytotoxic activity against cervical cancer cell line. HT-3 cell line examination revealed a considerable growth inhibition. Compounds 4a, 4b, 6b, 8c, and 8d, which are estrone-based arylidenes, are the most potent of the series, with IC50 value of 7.15, 10.76, 6.37, 3.56, and 1.55 µM/ml against HT-3 cell line. In addition, molecular docking studies were performed for the steroidal arylidenes to elucidate the binding interactions. Compound 4a, 4b, 6b, 8c and 8d showed excellent binding energy. Docking studies agreed well with in vitro studies. The end result offers an alternative approach to develop steroidal arylidenes that are more effective and are based on estrone, leading to the development of novel anticancer agents.

M1 muscarinic receptor activation reduces the molecular pathology and slows the progression of prion-mediated neurodegenerative disease.

Many dementias are propagated through the spread of "prion-like" misfolded proteins. This includes prion diseases themselves (such as Creutzfeldt-Jakob disease) and Alzheimer's disease (AD), for which no treatments are available to slow or stop progression. The M1 acetylcholine muscarinic receptor (M1 receptor) is abundant in the brain, and its activity promotes cognitive function in preclinical models and in patients with AD. Here, we investigated whether activation of the M1 receptor might slow the progression of neurodegeneration associated with prion-like misfolded protein in a mouse model of prion disease. Proteomic and transcriptomic analysis of the hippocampus revealed that this model had a molecular profile that was similar to that of human neurodegenerative diseases, including AD. Chronic enhancement of the activity of the M1 receptor with the positive allosteric modulator (PAM) VU0486846 reduced the abundance of prion-induced molecular markers of neuroinflammation and mitochondrial dysregulation in the hippocampus and normalized the abundance of those associated with neurotransmission, including synaptic and postsynaptic signaling components. PAM treatment of prion-infected mice prolonged survival and maintained cognitive function. Thus, allosteric activation of M1 receptors may reduce the severity of neurodegenerative diseases caused by the prion-like propagation of misfolded protein.

Development of fluorescent peptide G protein-coupled receptor activation biosensors for NanoBRET characterization of intracellular allosteric modulators.

G protein-coupled receptors (GPCRs) are widely therapeutically targeted, and recent advances in allosteric modulator development at these receptors offer further potential for exploitation. Intracellular allosteric modulators (IAM) represent a class of ligands that bind to the receptor-effector interface (e.g., G protein) and inhibit agonist responses noncompetitively. This potentially offers greater selectivity between receptor subtypes compared to classical orthosteric ligands. However, while examples of IAM ligands are well described, a more general methodology for assessing compound interactions at the IAM site is lacking. Here, fluorescent labeled peptides based on the Gα peptide C terminus are developed as novel binding and activation biosensors for the GPCR-IAM site. In TR-FRET binding studies, unlabeled peptides derived from the Gαs subunit were first characterized for their ability to positively modulate agonist affinity at the ß2 -adrenoceptor. On this basis, a tetramethylrhodamine (TMR) labeled tracer was synthesized based on the 19 amino acid Gαs peptide (TMR-Gαs19cha18, where cha = cyclohexylalanine). Using NanoBRET technology to detect binding, TMR-Gαs19cha18 was recruited to Gs coupled ß2 -adrenoceptor and EP2 receptors in an agonist-dependent manner, but not the Gi-coupled CXCR2 receptor. Moreover, NanoBRET competition binding assays using TMR-Gαs19cha18 enabled direct assessment of the affinity of unlabeled ligands for ß2 -adrenoceptor IAM site. Thus, the NanoBRET platform using fluorescent-labeled G protein peptide mimetics offers novel potential for medium-throughput screens to identify IAMs, applicable across GPCRs coupled to a G protein class. Using the same platform, Gs peptide biosensors also represent useful tools to probe orthosteric agonist efficacy and the dynamics of receptor activation.

Optimization of Peptide Linker-Based Fluorescent Ligands for the Histamine H1 Receptor.

The histamine H1 receptor (H1R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H1R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H1R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H1R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H1R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a, both potent and subtype-selective, enabled H1R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H1R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

Hit Identification of New Potent PqsR Antagonists as Inhibitors of Quorum Sensing in Planktonic and Biofilm Grown Pseudomonas aeruginosa.

Current treatments for Pseudomonas aeruginosa infections are becoming less effective because of the increasing rates of multi-antibiotic resistance. Pharmacological targeting of virulence through inhibition of quorum sensing (QS) dependent virulence gene regulation has considerable therapeutic potential. In P. aeruginosa, the pqs QS system regulates the production of multiple virulence factors as well as biofilm maturation and is a promising approach for developing antimicrobial adjuvants for combatting drug resistance. In this work, we report the hit optimisation for a series of potent novel inhibitors of PqsR, a key regulator of the pqs system, bearing a 2-((5-methyl-5H-[1,2,4]triazino[5,6-b]indol-3-yl)thio) acetamide scaffold. The initial hit compound 7 (PAO1-L IC50 0.98 ± 0.02 µM, PA14 inactive at 10 µM) was obtained through a virtual screening campaign performed on the PqsR ligand binding domain using the University of Nottingham Managed Chemical Compound Collection. Hit optimisation gave compounds with enhanced potency against strains PAO1-L and PA14, evaluated using P. aeruginosa pqs-based QS bioreporter assays. Compound 40 (PAO1-L IC50 0.25 ± 0.12 µM, PA14 IC50 0.34 ± 0.03 µM) is one of the most potent PqsR antagonists reported showing significant inhibition of P. aeruginosa pyocyanin production and pqs system signaling in both planktonic cultures and biofilms. The co-crystal structure of 40 with the PqsR ligand binding domain revealed the specific binding interactions occurring between inhibitor and this key regulatory protein.

27th Annual GP2A Medicinal Chemistry Conference.

The 27th annual GP2A (Groupement des Pharmacochimistes de l'Arc Atlantique/Group of Medicinal Chemists in the Atlantic Arc) conference took place from 21 to 23 August 2019, at the East Midlands Conference Centre (University Park, Nottingham, United Kingdom) and was hosted by the Division of Biomolecular Science and Medicinal Chemistry (BSMC), within the School of Pharmacy at the University of Nottingham. The event brought together an international delegation of researchers with interests in medicinal chemistry and interfacing disciplines. In addition, a pre-conference workshop provided an opportunity for younger researchers to develop their theoretical knowledge in quantitative pharmacology. Abstracts of presentations by the 14 invited speakers and 6 young researchers, in addition to 41 posters, are included in this report.

Structure-Kinetic Profiling of Haloperidol Analogues at the Human Dopamine D2 Receptor.

Haloperidol is a typical antipsychotic drug (APD) associated with an increased risk of extrapyramidal side effects (EPSs) and hyperprolactinemia relative to atypical APDs such as clozapine. Both drugs are dopamine D2 receptor (D2R) antagonists, with contrasting kinetic profiles. Haloperidol displays fast association/slow dissociation at the D2R, whereas clozapine exhibits relatively slow association/fast dissociation. Recently, we have provided evidence that slow dissociation from the D2R predicts hyperprolactinemia, whereas fast association predicts EPS. Unfortunately, clozapine can cause severe side effects independent of its D2R action. Our results suggest an optimal kinetic profile for D2R antagonist APDs that avoids EPS. To begin exploring this hypothesis, we conducted a structure-kinetic relationship study of haloperidol and revealed that subtle structural modifications dramatically change binding kinetic rate constants, affording compounds with a clozapine-like kinetic profile. Thus, optimization of these kinetic parameters may allow development of novel APDs based on the haloperidol scaffold with improved side-effect profiles.

Subtle modifications to a thieno[2,3-d]pyrimidine scaffold yield negative allosteric modulators and agonists of the dopamine D2 receptor.

We recently described a structurally novel series of negative allosteric modulators (NAMs) of the dopamine D2 receptor (D2R) based on thieno[2,3-d]pyrimidine 1, showing it can be structurally simplified to reveal low molecular weight, fragment-like NAMs that retain robust negative cooperativity, such as 3. Herein, we report the synthesis and functional profiling of analogues of 3, placing specific emphasis on examining secondary and tertiary amino substituents at the 4-position, combined with a range of substituents at the 5/6-positions (e.g. aromatic/aliphatic carbocycles). We identify analogues with diverse pharmacology at the D2R including NAMs with sub-µM affinity (9h) and, surprisingly, low efficacy partial agonists (9d and 9i).

A Thieno[2,3- d]pyrimidine Scaffold Is a Novel Negative Allosteric Modulator of the Dopamine D2 Receptor.

Recently, a novel negative allosteric modulator (NAM) of the D2-like dopamine receptors 1 was identified through virtual ligand screening. This ligand comprises a thieno[2,3- d]pyrimidine scaffold that does not feature in known dopaminergic ligands. Herein, we provide pharmacological validation of an allosteric mode of action for 1, revealing that it is a NAM of dopamine efficacy and identify the structural determinants of this allostery. We find that key structural moieties are important for functional affinity and negative cooperativity, while functionalization of the thienopyrimidine at the 5- and 6-positions results in analogues with divergent cooperativity profiles. Successive compound iterations have yielded analogues exhibiting a 10-fold improvement in functional affinity, as well as enhanced negative cooperativity with dopamine affinity and efficacy. Furthermore, our study reveals a fragment-like core that maintains low µM affinity and robust negative cooperativity with markedly improved ligand efficiency.

Assessment of the Molecular Mechanisms of Action of Novel 4-Phenylpyridine-2-One and 6-Phenylpyrimidin-4-One Allosteric Modulators at the M1 Muscarinic Acetylcholine Receptors.

Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism but whose molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, benzyl quinolone carboxylic acid (BQCA), in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signal-regulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and ß-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated.

Novel selective ß1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease.

ß-Blockers reduce mortality and improve symptoms in people with heart disease; however, current clinically available ß-blockers have poor selectivity for the cardiac ß1-adrenoceptor (AR) over the lung ß2-AR. Unwanted ß2-blockade risks causing life-threatening bronchospasm and reduced efficacy of ß2-agonist emergency rescue therapy. Thus, current life-prolonging ß-blockers are contraindicated in patients with both heart disease and asthma. Here, we describe NDD-713 and -825, novel highly ß1-selective neutral antagonists with good pharmaceutical properties that can potentially overcome this limitation. Radioligand binding studies and functional assays that use human receptors expressed in Chinese hamster ovary cells demonstrate that NDD-713 and -825 have nanomolar ß1-AR affinity >500-fold ß1-AR vs ß2-AR selectivity and no agonism. Studies in conscious rats demonstrate that these antagonists are orally bioavailable and cause pronounced ß1-mediated reduction of heart rate while showing no effect on ß2-mediated hindquarters vasodilatation. These compounds also have good disposition properties and show no adverse toxicologic effects. They potentially offer a truly cardioselective ß-blocker therapy for the large number of patients with heart and respiratory or peripheral vascular comorbidities.-Baker, J. G., Gardiner, S. M., Woolard, J., Fromont, C., Jadhav, G. P., Mistry, S. N., Thompson, K. S. J., Kellam, B., Hill, S. J., Fischer, P. M. Novel selective ß1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease.

GPCRs through the keyhole: the role of protein flexibility in ligand binding to ß-adrenoceptors.

G protein-coupled receptors (GPCRs) are proteins of pharmaceutical importance, with over 30% of all drugs in clinical use targeting them. Increasing numbers of X-ray crystal (XRC) structures of GPCRs offer a wealth of data relating to ligand binding. For the ß-adrenoceptors (ß-ARs), XRC structures are available for human ß2- and turkey ß1-subtypes, in complexes with a range of ligands. While these structures provide insight into the origins of ligand structure-activity relationships (SARs), questions remain. The ligands in all published complexed XRC structures lack extensive substitution, with no obvious way the ligand-binding site can accommodate ß1-AR-selective antagonists with extended side-chains para- to the common aryloxypropanolamine pharmacophore. Using standard computational docking tools with such ligands generally returns poses that fail to explain known SARs. Application of our Active Site Pressurisation modelling method to ß-AR XRC structures and homology models, however, reveals a dynamic area in the ligand-binding pocket that, through minor changes in amino acid side chain orientations, opens a fissure between transmembrane helices H4 and H5, exposing intra-membrane space. This fissure, which we term the "keyhole", is ideally located to accommodate extended moieties present in many high-affinity ß1-AR-selective ligands, allowing the rest of the ligand structure to adopt a canonical pose in the orthosteric binding site. We propose the keyhole may be a feature of both ß1- and ß2-ARs, but that subtle structural differences exist between the two, contributing to subtype-selectivity. This has consequences for the rational design of future generations of subtype-selective ligands for these therapeutically important targets.

Positive Allosteric Modulation of the Muscarinic M1 Receptor Improves Efficacy of Antipsychotics in Mouse Glutamatergic Deficit Models of Behavior.

Current antipsychotics are effective in treating the positive symptoms associated with schizophrenia, but they remain suboptimal in targeting cognitive dysfunction. Recent studies have suggested that positive allosteric modulation of the M1 muscarinic acetylcholine receptor (mAChR) may provide a novel means of improving cognition. However, very little is known about the potential of combination therapies in extending coverage across schizophrenic symptom domains. This study investigated the effect of the M1 mAChR positive allosteric modulator BQCA [1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid], alone or in combination with haloperidol (a first-generation antipsychotic), clozapine (a second-generation atypical antipsychotic), or aripiprazole (a third-generation atypical antipsychotic), in reversing deficits in sensorimotor gating and spatial memory induced by the N-methyl-d-aspartate receptor antagonist, MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]. Sensorimotor gating and spatial memory induction are two models that represent aspects of schizophrenia modeled in rodents. In prepulse inhibition (an operational measure of sensorimotor gating), BQCA alone had minimal effects but exhibited different levels of efficacy in reversing MK-801-induced prepulse inhibition disruptions when combined with a subeffective dose of each of the three (currently prescribed) antipsychotics. Furthermore, the combined effect of BQCA and clozapine was absent in M1-/- mice. Interestingly, although BQCA alone had no effect in reversing MK-801-induced memory impairments in a Y-maze spatial test, we observed a reversal upon the combination of BQCA with atypical antipsychotics, but not with haloperidol. These findings provide proof of concept that a judicious combination of existing antipsychotics with a selective M1 mAChR positive allosteric modulator can extend antipsychotic efficacy in glutamatergic deficit models of behavior.

Novel Fused Arylpyrimidinone Based Allosteric Modulators of the M1 Muscarinic Acetylcholine Receptor.

Benzoquinazolinone 1 is a positive allosteric modulator (PAM) of the M1 muscarinic acetylcholine receptor (mAChR), which is significantly more potent than the prototypical PAM, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA). In this study, we explored the structural determinants that underlie the activity of 1 as a PAM of the M1 mAChR. We paid particular attention to the importance of the tricyclic scaffold of compound 1, for the activity of the molecule. Complete deletion of the peripheral fused benzene ring caused a significant decrease in affinity and binding cooperativity with acetylcholine (ACh). This loss of affinity was rescued with the addition of either one or two methyl groups in the 7- and/or 8-position of the quinazolin-4(3H)-one core. These results demonstrate that the tricyclic benzo[h]quinazolin-4(3H)-one core could be replaced with a quinazolin-4(3H)-one core and maintain functional affinity. As such, the quinazolin-4(3H)-one core represents a novel scaffold to further explore M1 mAChR PAMs with improved physicochemical properties.

Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions.

Malaria remains a global health problem, and though international efforts for treatment and eradication have made some headway, the emergence of drug-resistant parasites threatens this progress. Antimalarial therapeutics acting via novel mechanisms are urgently required. Plasmodium falciparum M1 and M17 are neutral aminopeptidases which are essential for parasite growth and development. Previous work in our group has identified inhibitors capable of dual inhibition of PfA-M1 and PfA-M17, and revealed further regions within the protease S1 pockets that could be exploited in the development of ligands with improved inhibitory activity. Herein, we report the structure-based design and synthesis of novel hydroxamic acid analogues that are capable of potent inhibition of both PfA-M1 and PfA-M17. Furthermore, the developed compounds potently inhibit Pf growth in culture, including the multi-drug resistant strain Dd2. The ongoing development of dual PfA-M1/PfA-M17 inhibitors continues to be an attractive strategy for the design of novel antimalarial therapeutics.

4-Phenylpyridin-2-one Derivatives: A Novel Class of Positive Allosteric Modulator of the M1 Muscarinic Acetylcholine Receptor.

Positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (M1 mAChR) are a promising strategy for the treatment of the cognitive deficits associated with diseases including Alzheimer's and schizophrenia. Herein, we report the design, synthesis, and characterization of a novel family of M1 mAChR PAMs. The most active compounds of the 4-phenylpyridin-2-one series exhibited comparable binding affinity to the reference compound, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA) (1), but markedly improved positive cooperativity with acetylcholine, and retained exquisite selectivity for the M1 mAChR. Furthermore, our pharmacological characterization revealed ligands with a diverse range of activities, including modulators that displayed both high intrinsic efficacy and PAM activity, those that showed no detectable agonism but robust PAM activity and ligands that displayed robust allosteric agonism but little modulatory activity. Thus, the 4-phenylpyridin-2-one scaffold offers an attractive starting point for further lead optimization.

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.

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.