Structure of the Adenosine A1 Receptor Reveals the Basis for Subtype Selectivity.

The adenosine A1 receptor (A1-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We determined a 3.2 Å crystal structure of the A1-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A2A receptor (A2A-AR) structure. Mutational and computational analysis of A1-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a molecular basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.

Positive allosteric mechanisms of adenosine A1 receptor-mediated analgesia.

The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.

Biocompatible Cationic Lipoamino Acids as Counterions for Oral Administration of API-Ionic Liquids.

PURPOSE: The use of ionic liquids (ILs) in drug delivery has focused attention on non-toxic IL counterions. Cationic lipids can be used to form ILs with weakly acidic drugs to enhance drug loading in lipid-based formulations (LBFs). However, cationic lipids are typically toxic. Here we explore the use of lipoaminoacids (LAAs) as cationic IL counterions that degrade or digest in vivo to non-toxic components. METHODS: LAAs were synthesised via esterification of amino acids with fatty alcohols to produce potentially digestible cationic LAAs. The LAAs were employed to form ILs with tolfenamic acid (Tol) and the Tol ILs loaded into LBF and examined in vitro and in vivo. RESULTS: Cationic LAAs complexed with Tol to generate lipophilic Tol ILs with high drug loading in LBFs. Assessment of the LAA under simulated digestion conditions revealed that they were susceptible to enzymatic degradation under intestinal conditions, forming biocompatible FAs and amino acids. In vitro dispersion and digestion studies of Tol ILs revealed that formulations containing digestible Tol ILs were able to maintain drug dispersion and solubilisation whilst the LAA were breaking down under digesting conditions. Finally, in vivo oral bioavailability studies demonstrated that oral delivery of a LBF containing a Tol IL comprising a digestible cationic lipid counterion was able to successfully support effective oral delivery of Tol. CONCLUSIONS: Digestible LAA cationic lipids are potential IL counterions for weakly acidic drug molecules and digest in situ to form non-toxic breakdown products.

Lipophilic Salts and Lipid-Based Formulations: Enhancing the Oral Delivery of Octreotide.

PURPOSE: Successful oral peptide delivery faces two major hurdles: low enzymatic stability in the gastro-intestinal lumen and poor intestinal membrane permeability. While lipid-based formulations (LBF) have the potential to overcome these barriers, effective formulation of peptides remains challenging. Lipophilic salt (LS) technology can increase the apparent lipophilicity of peptides, making them more suitable for LBF. METHODS: As a model therapeutic peptide, octreotide (OCT) was converted to the docusate LS (OCT.DoS2), and compared to the commercial acetate salt (OCT.OAc2) in oral absorption studies and related in vitro studies, including parallel artificial membrane permeability assay (PAMPA), Caco-2, in situ intestine perfusion, and simulated digestion in vitro models. The in vivo oral absorption of OCT.DoS2 and OCT.OAc2 formulated in self-emulsifying drug delivery systems (SEDDS) was studied in rats. RESULTS: LS formulation improved the solubility and loading of OCT in LBF excipients and OCT.DoS2 in combination with SEDDS showed higher OCT absorption than the acetate comparator in the in vivo studies in rats. The Caco-2 and in situ intestine perfusion models indicated no increases in permeability for OCT.DoS2. However, the in vitro digestion studies showed reduced enzymatic degradation of OCT.DoS2 when formulated in the SEDDS formulations. Further in vitro dissociation and release studies suggest that the enhanced bioavailability of OCT from SEDDS-incorporating OCT.DoS2 is likely a result of higher partitioning into and prolonged retention within lipid colloid structures. CONCLUSION: The combination of LS and LBF enhanced the in vivo oral absorption of OCT primarily via the protective effect of LBF sheltering the peptide from gastrointestinal degradation.

Enantioenriched Positive Allosteric Modulators Display Distinct Pharmacology at the Dopamine D1 Receptor.

(1) Background: Two first-in-class racemic dopamine D1 receptor (D1R) positive allosteric modulator (PAM) chemotypes (1 and 2) were identified from a high-throughput screen. In particular, due to its selectivity for the D1R and reported lack of intrinsic activity, compound 2 shows promise as a starting point toward the development of small molecule allosteric modulators to ameliorate the cognitive deficits associated with some neuropsychiatric disease states; (2) Methods: Herein, we describe the enantioenrichment of optical isomers of 2 using chiral auxiliaries derived from (R)- and (S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one (d- and l-pantolactone, respectively); (3) Results: We confirm both the racemate and enantiomers of 2 are active and selective for the D1R, but that the respective stereoisomers show a significant difference in their affinity and magnitude of positive allosteric cooperativity with dopamine; (4) Conclusions: These data warrant further investigation of asymmetric syntheses of optically pure analogues of 2 for the development of D1R PAMs with superior allosteric properties.

Driving antimalarial design through understanding of target mechanism.

Malaria continues to be a global health threat, affecting approximately 219 million people in 2018 alone. The recurrent development of resistance to existing antimalarials means that the design of new drug candidates must be carefully considered. Understanding of drug target mechanism can dramatically accelerate early-stage target-based development of novel antimalarials and allows for structural modifications even during late-stage preclinical development. Here, we have provided an overview of three promising antimalarial molecular targets, PfDHFR, PfDHODH and PfA-M1, and their associated inhibitors which demonstrate how mechanism can inform drug design and be effectively utilised to generate compounds with potent inhibitory activity.

Overcoming P-Glycoprotein-Mediated Drug Resistance with Noscapine Derivatives.

The antitussive agent noscapine has been shown to inhibit the proliferation of cancer cells by disruption of tubulin dynamic. However, the efficacy of several anticancer drugs that inhibit tublin dynamics (vinca alkaloids and taxanes) is reduced by the multidrug resistance phenotype. These compounds are substrates for P-glycoprotein (P-gp)-mediated extrusion from cells. Consequently, the antiproliferative activity of noscapine and a series of derivatives was measured in drug-sensitive and drug-resistant cells that overexpress P-gp. None of the noscapine derivatives displayed lower potency in cells overexpressing P-gp, thereby suggesting a lack of interaction with this pump. However, the cellular efflux of a fluorescent substrate by P-gp was potently inhibited by noscapine and most derivatives. Further investigation with purified, reconstituted P-gp demonstrated that inhibition of P-gp function was due to direct interaction of noscapine derivatives with the transporter. Moreover, coadministration of vinblastine with two of the noscapine derivatives displayed synergistic inhibition of proliferation, even in P-gp-expressing resistant cell lines. Therefore, noscapine derivatives offer a dual benefit of overcoming the significant impact of P-gp in conferring multidrug resistance and synergy with tubulin-disrupting anticancer drugs.

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.

Enhancing the Oral Absorption of Kinase Inhibitors Using Lipophilic Salts and Lipid-Based Formulations.

The absolute bioavailability of many small molecule kinase inhibitors (smKIs) is low. The reasons for low bioavailability are multifaceted and include constraints due to first pass metabolism and poor absorption. For smKIs where absorption limits oral bioavailability, low aqueous solubility and high lipophilicity, often in combination with high-dose requirements have been implicated in low and variable absorption, food-effects, and absorption-related drug-drug interactions. The current study has evaluated whether preparation of smKIs as lipophilic salts/ionic liquids in combination with coadministration with lipid-based formulations is able to enhance absorption for examples of this compound class. Lipophilic (docusate) salt forms of erlotinib, gefitinib, ceritinib, and cabozantinib (as example smKIs demonstrating low aqueous solubility and high lipophilicity) were prepared and isolated as workable powder solids. In each case, the lipophilic salt exhibited high and significantly enhanced solubility in lipidic excipients (>100 mg/g) when compared to the free base or commercial salt form. Isolation as the lipophilic salt facilitated smKI loading in model lipid-based formulations at high concentration, increased in vitro solubilization at gastric and intestinal pH and in some cases increased oral absorption (∼2-fold for cabozantinib formulations in rats). Application of a lipophilic salt approach can therefore facilitate the use of lipid-based formulations for examples of the smKI compound class where low solubility limits absorption and is a risk factor for increased variability due to food-effects.

Ionic Liquid Forms of Weakly Acidic Drugs in Oral Lipid Formulations: Preparation, Characterization, in Vitro Digestion, and in Vivo Absorption Studies.

This study aimed to transform weakly acidic poorly water-soluble drugs (PWSD) into ionic liquids (ILs) to promote solubility in, and the utility of, lipid-based formulations. Ionic liquids (ILs) were formed directly from tolfenamic acid (Tolf), meclofenamic acid, diclofenac, and ibuprofen by pairing with lipophilic counterions. The drug-ILs were obtained as liquids or low melting solids and were significantly more soluble (either completely miscible or highly soluble) in lipid based, self-emulsifying drug delivery systems (SEDDS) when compared to the equivalent free acid. In vivo assessment of a SEDDS lipid solution formulation of Tolf didecyldimethylammonium salt and the same formulation of Tolf free acid at low dose (18 mg/kg, where the free acid was soluble in the SEDDS), resulted in similar absorption profiles and overall exposure. At high dose (100 mg/kg), solution SEDDS formulations of the Tolf ILs (didecyldimethylammonium, butyldodecyldimethylammonium or didecylmethylammonium salts) were possible, but the lower lipid solubility of Tolf free acid dictated that administration of the free acid was only possible as a suspension in the SEDDS formulation or as an aqueous suspension. Under these conditions, total drug plasma exposure was similar for the IL formulations and the free acid, but the plasma profiles were markedly different, resulting in flatter, more prolonged exposure profiles and reduced Cmax for the IL formulations. Isolation of a weakly acidic drug as an IL may therefore provide advantage as it allows formulation as a solution SEDDS rather than a lipid suspension, and in some cases may provide a means of slowing or sustaining absorption. The current studies compliment previous studies with weakly basic PWSD and demonstrate that transformation into highly lipophilic ILs is also possible for weakly acidic compounds.

Structure and substrate fingerprint of aminopeptidase P from Plasmodium falciparum.

Malaria is one of the world's most prevalent parasitic diseases, with over 200 million cases annually. Alarmingly, the spread of drug-resistant parasites threatens the effectiveness of current antimalarials and has made the development of novel therapeutic strategies a global health priority. Malaria parasites have a complicated lifecycle, involving an asymptomatic 'liver stage' and a symptomatic 'blood stage'. During the blood stage, the parasites utilise a proteolytic cascade to digest host hemoglobin, which produces free amino acids absolutely necessary for parasite growth and reproduction. The enzymes required for hemoglobin digestion are therefore attractive therapeutic targets. The final step of the cascade is catalyzed by several metalloaminopeptidases, including aminopeptidase P (APP). We developed a novel platform to examine the substrate fingerprint of APP from Plasmodium falciparum (PfAPP) and to show that it can catalyze the removal of any residue immediately prior to a proline. Further, we have determined the crystal structure of PfAPP and present the first examination of the 3D structure of this essential malarial enzyme. Together, these analyses provide insights into potential mechanisms of inhibition that could be used to develop novel antimalarial therapeutics.

Separation of on-target efficacy from adverse effects through rational design of a bitopic adenosine receptor agonist.

The concepts of allosteric modulation and biased agonism are revolutionizing modern approaches to drug discovery, particularly in the field of G protein-coupled receptors (GPCRs). Both phenomena exploit topographically distinct binding sites to promote unique GPCR conformations that can lead to different patterns of cellular responsiveness. The adenosine A1 GPCR (A1AR) is a major therapeutic target for cardioprotection, but current agents acting on the receptor are clinically limited for this indication because of on-target bradycardia as a serious adverse effect. In the current study, we have rationally designed a novel A1AR ligand (VCP746)--a hybrid molecule comprising adenosine linked to a positive allosteric modulator--specifically to engender biased signaling at the A1AR. We validate that the interaction of VCP746 with the A1AR is consistent with a bitopic mode of receptor engagement (i.e., concomitant association with orthosteric and allosteric sites) and that the compound displays biased agonism relative to prototypical A1AR ligands. Importantly, we also show that the unique pharmacology of VCP746 is (patho)physiologically relevant, because the compound protects against ischemic insult in native A1AR-expressing cardiomyoblasts and cardiomyocytes but does not affect rat atrial heart rate. Thus, this study provides proof of concept that bitopic ligands can be designed as biased agonists to promote on-target efficacy without on-target side effects.

Role of the Second Extracellular Loop of the Adenosine A1 Receptor on Allosteric Modulator Binding, Signaling, and Cooperativity.

Allosteric modulation of adenosine A1 receptors (A1ARs) offers a novel therapeutic approach for the treatment of numerous central and peripheral disorders; however, despite decades of research, there is a relative paucity of structural information regarding the A1AR allosteric site and mechanisms governing cooperativity with orthosteric ligands. We combined alanine-scanning mutagenesis of the A1AR second extracellular loop (ECL2) with radioligand binding and functional interaction assays to quantify effects on allosteric ligand affinity, cooperativity, and efficacy. Docking and molecular dynamics (MD) simulations were performed using an A1AR homology model based on an agonist-bound A2AAR structure. Substitution of E172ECL2 for alanine reduced the affinity of the allosteric modulators PD81723 and VCP171 for the unoccupied A1AR. Residues involved in cooperativity with the orthosteric agonist NECA were different in PD81723 and VCP171; positive cooperativity between PD81723 and NECA was reduced on alanine substitution of a number of ECL2 residues, including E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and W156ECL2 decreased VCP171 cooperativity with NECA. Molecular modeling localized a likely allosteric pocket for both modulators to an extracellular vestibule that overlaps with a region used by orthosteric ligands as they transit into the canonical A1AR orthosteric site. MD simulations confirmed a key interaction between E172ECL2 and both modulators. Bound PD81723 is flanked by another residue, E170ECL2, which forms hydrogen bonds with adjacent K168ECL2 and K173ECL2. Collectively, our data suggest E172ECL2 is a key allosteric ligand-binding determinant, whereas hydrogen-bonding networks within the extracellular vestibule may facilitate the transmission of cooperativity between orthosteric and allosteric sites.

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.

Solution NMR characterization of apical membrane antigen 1 and small molecule interactions as a basis for designing new antimalarials.

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) plays an important role in the invasion by merozoites of human red blood cells during a malaria infection. A key region of PfAMA1 is a conserved hydrophobic cleft formed by 12 hydrophobic residues. As anti-apical membrane antigen 1 antibodies and other inhibitory molecules that target this hydrophobic cleft are able to block the invasion process, PfAMA1 is an attractive target for the development of strain-transcending antimalarial agents. As solution nuclear magnetic resonance spectroscopy is a valuable technique for the rapid characterization of protein-ligand interactions, we have determined the sequence-specific backbone assignments for PfAMA1 from two P. falciparum strains, FVO and 3D7. Both selective labelling and unlabelling strategies were used to complement triple-resonance experiments in order to facilitate the assignment process. We have then used these assignments for mapping the binding sites for small molecules, including benzimidazoles, pyrazoles and 2-aminothiazoles, which were selected on the basis of their affinities measured from surface plasmon resonance binding experiments. Among the compounds tested, benzimidazoles showed binding to a similar region on both FVO and 3D7 PfAMA1, suggesting that these compounds are promising scaffolds for the development of novel PfAMA1 inhibitors. Copyright © 2016 John Wiley & Sons, Ltd.

A new mechanism of allostery in a G protein-coupled receptor dimer.

SB269652 is to our knowledge the first drug-like allosteric modulator of the dopamine D2 receptor (D2R), but it contains structural features associated with orthosteric D2R antagonists. Using a functional complementation system to control the identity of individual protomers within a dimeric D2R complex, we converted the pharmacology of the interaction between SB269652 and dopamine from allosteric to competitive by impairing ligand binding to one of the protomers, indicating that the allostery requires D2R dimers. Additional experiments identified a 'bitopic' pose for SB269652 extending from the orthosteric site into a secondary pocket at the extracellular end of the transmembrane (TM) domain, involving TM2 and TM7. Engagement of this secondary pocket was a requirement for the allosteric pharmacology of SB269652. This suggests a new mechanism whereby a bitopic ligand binds in an extended pose on one G protein-coupled receptor protomer to allosterically modulate the binding of a ligand to the orthosteric site of a second protomer.

VCP746, a novel A1 adenosine receptor biased agonist, reduces hypertrophy in a rat neonatal cardiac myocyte model.

VCP746 is a novel A1 adenosine receptor (A1 AR) biased agonist previously shown to be cytoprotective with no effect on heart rate. The aim of this study was to investigate the potential anti-hypertrophic effect of VCP746 in neonatal rat cardiac myocytes (NCM). NCM hypertrophy was stimulated with interleukin (IL)-1ß (10 ng/mL), tumour necrosis factor (TNF)-α (10 ng/mL) or Ang II (100 nmol/L) and was assessed by (3) H-leucine incorporation assay. VCP746 significantly inhibited IL-1ß-, TNF-α- and Ang II-stimulated NCM hypertrophy as determined by (3) H-leucine incorporation. The anti-hypertrophic effect of VCP746 was also more potent than that of the prototypical A1 AR agonist, N(6) -cyclopentyladenosine (CPA). Further investigation with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay showed that neither CPA nor VCP746 had any effect on cell viability, confirming that the reduction in (3) H-leucine incorporation mediated by CPA and VCP746 was not due to a reduction in cell viability. IL-1ß, TNF-α and Ang II were also shown to increase the mRNA expression of hypertrophy biomarkers, ANP, ß-MHC and α-SKA in NCM. Treatment with VCP746 at concentrations as low as 1 nmol/L suppressed mRNA expression of ANP, ß-MHC and α-SKA stimulated by IL-1ß, TNF-α or Ang II, demonstrating the broad mechanistic basis of the potent anti-hypertrophic effect of VCP746. This study has shown that the novel A1 AR agonist, VCP746, is able to attenuate cardiac myocyte hypertrophy. As such, VCP746 is potentially useful as a pharmacological agent in attenuating cardiac remodelling, especially in the post-myocardial infarction setting, given its previously established cytoprotective properties.

Mechanistic insights into allosteric structure-function relationships at the M1 muscarinic acetylcholine receptor.

Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400(7.35) in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs.

Molecular mechanisms of bitopic ligand engagement with the M1 muscarinic acetylcholine receptor.

TBPB and 77-LH-28-1 are selective agonists of the M1 muscarinic acetylcholine receptor (mAChR) that may gain their selectivity through a bitopic mechanism, interacting concomitantly with the orthosteric site and part of an allosteric site. The current study combined site-directed mutagenesis, analytical pharmacology,and molecular modeling to gain further insights into the structural basis underlying binding and signaling by these agonists. Mutations within the orthosteric binding site caused similar reductions in affinity and signaling efficacy for both selective and prototypical orthosteric ligands. In contrast, the mutation of residues within transmembrane helix (TM) 2 and the second extracellular loop (ECL2) discriminated between the different classes of ligand. In particular, ECL2 appears to be involved in the selective binding of bitopic ligands and in coordinating biased agonism between intracellular calcium mobilization and ERK1/2 phosphorylation. Molecular modeling of the interaction between TBPB and the M1 mAChR revealed a binding pose predicted to extend from the orthosteric site up toward a putative allosteric site bordered by TM2, TM3, and TM7, thus consistent with a bitopic mode of binding. Overall, these findings provide valuable structural and mechanistic insights into bitopic ligand actions and receptor activation and support a role for ECL2 in dictating the active states that can be adopted by a G protein-coupled receptor. This may enable greater selective ligand design and development for mAChRs and facilitate improved identification of bitopic ligands.

Transformation of poorly water-soluble drugs into lipophilic ionic liquids enhances oral drug exposure from lipid based formulations.

Absorption after oral administration is a requirement for almost all drug products but is a challenge for drugs with intrinsically low water solubility. Here, the weakly basic, poorly water-soluble drugs (PWSDs) itraconazole, cinnarizine, and halofantrine were converted into lipophilic ionic liquids to facilitate incorporation into lipid-based formulations and integration into lipid absorption pathways. Ionic liquids were formed via metathesis reactions of the hydrochloride salt of the PWSDs with a range of lipophilic counterions. The resultant active pharmaceutical ingredient-ionic liquids (API-ILs) were liquids or low melting point solids and either completely miscible or highly soluble in lipid based, self-emulsifying drug delivery systems (SEDDS) comprising mixtures of long or medium chain glycerides, surfactants such as Kolliphor-EL and cosolvents such as ethanol. They also readily incorporated into the colloids formed in intestinal fluids during lipid digestion. Itraconazole docusate or cinnarizine decylsulfate API-ILs were subsequently dissolved in long chain lipid SEDDS at high concentration, administered to rats and in vivo exposure assessed. The data were compared to control formulations based on the same SEDDS formulations containing the same concentrations of drug as the free base, but in this case as a suspension (since the solubility of the free base in the SEDDS was much lower than the API-ILs). For itraconazole, comparison was also made to a physical mixture of itraconazole free base and sodium docusate in the same SEDDS formulation. For both drugs plasma exposure was significantly higher for the API-IL containing formulations (2-fold for cinnarizine and 20-fold for itraconazole), when compared to the suspension formulations (or the physical mixture in the case of itraconazole) at the same dose. The liquid SEDDS formulations, made possible by the use of the API-ILs, also provide advantages in dose uniformity, capsule filling, and stability compared to similar suspension formulations. The data suggest that the formation of lipophilic ionic liquids provides a means of increasing dissolved-drug loading in lipid based formulations and thereby promoting the exposure of poorly water-soluble drugs after oral administration.