Publications by authors named "Peter J Scammells"

128 Publications

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A Receptor in Living Cells.

J Med Chem 2021 Mar 16. Epub 2021 Mar 16.

Medicinal Chemistry, Monash University, Parkville, Victoria 3052, Australia.

The adenosine A receptor (AAR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of AAR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying AAR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of AAR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel AAR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of AAR molecular pharmacology and signaling in living cells.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02067DOI Listing
March 2021

Stabilising disproportionation of lipophilic ionic liquid salts in lipid-based formulations.

Int J Pharm 2021 Mar 11;597:120292. Epub 2021 Feb 11.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052 Australia; ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash University, 381 Royal Parade, Parkville, Victoria 3052 Australia. Electronic address:

Lipid based formulations (LBFs) can enhance oral bioavailability, however, their utility may be restricted by low drug loading in the formulation. Converting drugs to drug-ionic liquids (drug-ILs) or lipophilic salts can significantly increase lipid solubility but this approach is complicated in some cases by salt disproportionation, leading to a reduction in solubility and physical instability. Here we explore the physical stability of the weakly basic model drug, cinnarizine (CIN), when paired with a decanoate counterion (Dec) to form the drug-IL, cinnarizine decanoate (CIN.Dec). Consistent with published studies of salt disproportionation in aqueous solution, weakly acidic counterions such as Dec lead to the generation of drug-IL lipid solutions with pHs below pH. This leads to CIN deprotonation to the less soluble free base and precipitation. Subsequent studies however, show that these effects can be reversed by acidification of the formulation (either with excess decanoic acid or other lipid soluble acids), stimulating a pH shift to the salt plateau of CIN.Dec and the formation of stable lipid solutions of CIN.Dec. Altering formulation pH to more acidic conditions, therefore stabilises drug-ILs formed using weakly acidic lipophilic counterions, and is a viable method to promote formulation stability via inhibition of disproportionation of some drug-ILs.
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http://dx.doi.org/10.1016/j.ijpharm.2021.120292DOI Listing
March 2021

Driving antimalarial design through understanding of target mechanism.

Biochem Soc Trans 2020 10;48(5):2067-2078

Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.

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.
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http://dx.doi.org/10.1042/BST20200224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609028PMC
October 2020

Development of Novel 4-Arylpyridin-2-one and 6-Arylpyrimidin-4-one Positive Allosteric Modulators of the M Muscarinic Acetylcholine Receptor.

ChemMedChem 2021 Jan 25;16(1):216-233. Epub 2020 Sep 25.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052, Victoria, Australia.

This study investigated the structure-activity relationships of 4-phenylpyridin-2-one and 6-phenylpyrimidin-4-one M muscarinic acetylcholine receptor (M mAChRs) positive allosteric modulators (PAMs). The presented series focuses on modifications to the core and top motif of the reported leads, MIPS1650 (1) and MIPS1780 (2). Profiling of our novel analogues showed that these modifications result in more nuanced effects on the allosteric properties compared to our previous compounds with alterations to the biaryl pendant. Further pharmacological characterisation of the selected compounds in radioligand binding, IP accumulation and β-arrestin 2 recruitment assays demonstrated that, despite primarily acting as affinity modulators, the PAMs displayed different pharmacological properties across the two cellular assays. The novel PAM 7 f is a potential lead candidate for further development of peripherally restricted M PAMs, due to its lower blood-brain-barrier (BBB) permeability and improved exposure in the periphery compared to lead 2.
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http://dx.doi.org/10.1002/cmdc.202000540DOI Listing
January 2021

Rapid Elaboration of Fragments into Leads by X-ray Crystallographic Screening of Parallel Chemical Libraries (REFiL).

J Med Chem 2020 07 24;63(13):6863-6875. Epub 2020 Jun 24.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.

A bottleneck in fragment-based lead development is the lack of systematic approaches to elaborate the initial fragment hits, which usually bind with low affinity to their target. Herein, we describe an analysis using X-ray crystallography of a diverse library of compounds prepared using microscale parallel synthesis. This approach yielded an 8-fold increase in affinity and detailed structural information for the resulting complex, providing an efficient and broadly applicable approach to early fragment development.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00111DOI Listing
July 2020

Subtype-Selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A Receptor.

J Med Chem 2020 03 9;63(5):2656-2672. Epub 2020 Jan 9.

Division of Biomolecular Sciences and Medicinal Chemistry, School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, U.K.

Among class A G protein-coupled receptors (GPCR), the human adenosine A receptor (hAAR) remains an attractive drug target. However, translation of AAR ligands into the clinic has proved challenging and an improved understanding of AAR pharmacology could promote development of more efficacious therapies. Subtype-selective fluorescent probes would allow detailed real-time pharmacological investigations both in vitro and in vivo. In the present study, two families of fluorescent probes were designed around the known hAAR selective antagonist preladenant (SCH 420814). Both families of fluorescent antagonists retained affinity at the hAAR, selectivity over all other adenosine receptor subtypes and allowed clear visualization of specific receptor localization through confocal imaging. Furthermore, the Alexa Fluor 647-labeled conjugate allowed measurement of ligand binding affinities of unlabeled hAAR antagonists using a bioluminescence resonance energy transfer (NanoBRET) assay. The fluorescent ligands developed here can therefore be applied to a range of fluorescence-based techniques to further interrogate hAAR pharmacology and signaling.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01856DOI Listing
March 2020

Ionic Liquid Forms of the Antimalarial Lumefantrine in Combination with LFCS Type IIIB Lipid-Based Formulations Preferentially Increase Lipid Solubility, In Vitro Solubilization Behavior and In Vivo Exposure.

Pharmaceutics 2019 Dec 22;12(1). Epub 2019 Dec 22.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.

Lipid based formulations (LBFs) are commonly employed to enhance the absorption of highly lipophilic, poorly water-soluble drugs. However, the utility of LBFs can be limited by low drug solubility in the formulation. Isolation of ionizable drugs as low melting, lipophilic salts or ionic liquids (ILs) provides one means to enhance drug solubility in LBFs. However, whether different ILs benefit from formulation in different LBFs is largely unknown. In the current studies, lumefantrine was isolated as a number of different lipophilic salt/ionic liquid forms and performance was assessed after formulation in a range of LBFs. The solubility of lumefantrine in LBF was enhanced 2- to 80-fold by isolation as the lumefantrine docusate IL when compared to lumefantrine free base. The increase in drug loading subsequently enhanced concentrations in the aqueous phase of model intestinal fluids during in vitro dispersion and digestion testing of the LBF. To assess in vivo performance, the systemic exposure of lumefantrine docusate after administration in Type II-MCF, IIIB-MCF, IIIB-LCF, and IV formulations was evaluated after oral administration to rats. In vivo exposure was compared to control lipid and aqueous suspension formulations of lumefantrine free base. Lumefantrine docusate in the Type IIIB-LCF showed significantly higher plasma exposure compared to all other formulations (up to 35-fold higher). The data suggest that isolation of a lipid-soluble IL, coupled with an appropriate formulation, is a viable means to increase drug dose in an oral formulation and to enhance exposure of lumefantrine .
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http://dx.doi.org/10.3390/pharmaceutics12010017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7023222PMC
December 2019

A Novel Class of N-Sulfonyl and N-Sulfamoyl Noscapine Derivatives that Promote Mitotic Arrest in Cancer Cells.

ChemMedChem 2019 12 12;14(23):1968-1981. Epub 2019 Nov 12.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia.

Noscapine displays weak anticancer efficacy and numerous research efforts have attempted to generate more potent noscapine analogues. These modifications included the replacement of the N-methyl group in the 6'-position with a range of substituents, where N-ethylcarbamoyl substitution was observed to possess enhanced anticancer activity. Herein, we describe advances in this area, namely the synthesis and pharmacological evaluation of a series of N-sulfonyl and N-sulfamoyl noscapine derivatives. A number of these sulfonyl-containing noscapinoids demonstrated improved activities compared to noscapine. ((R)-5-((S)-4,5-Dimethoxy-1,3-dihydroisobenzofuran-1-yl)-4-methoxy-6-((1-methyl-1H-imidazol-4-yl)sulfonyl)-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline) (14 q) displayed sub-micromolar activities of 560, 980, 271 and 443 nM against MCF-7, PANC-1, MDA-MB-435 and SK-MEL-5 cells, respectively. This antiproliferative effect was also maintained against drug-resistant NCI/Adr cells despite high expression of the multidrug efflux pump, P-glycoprotein.
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http://dx.doi.org/10.1002/cmdc.201900477DOI Listing
December 2019

The effect of two selective A -receptor agonists and the bitopic ligand VCP746 on heart rate and regional vascular conductance in conscious rats.

Br J Pharmacol 2020 01 1;177(2):346-359. Epub 2020 Jan 1.

Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK.

Background And Purpose: Adenosine is a local mediator that regulates physiological and pathological processes via activation of four GPCRs (A , A , A , and A ). We have investigated the effect of two A -receptor-selective agonists and the novel A -receptor bitopic ligand VCP746 on the rat cardiovascular system.

Experimental Approach: The regional haemodynamic responses of these agonist was investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal, mesenteric arteries and the descending abdominal aorta and the jugular vein and caudal artery catheterized. Cardiovascular responses were measured following intravenous infusion (3 min each dose) of CCPA (120, 400, and 1,200 ng·kg ·min ), capadenoson or adenosine (30, 100, and 300 μg·kg ·min ), or VCP746 (6, 20, and 60 μg·kg ·min ) following pre-dosing with DPCPX (0.1 mg·kg , i.v.) or vehicle.

Key Results: CCPA produced a significant A -receptor-mediated decrease in heart rate that was accompanied by vasoconstrictions in the renal and mesenteric vascular beds but an increase in hindquarters vascular conductance. The partial agonist capadenoson also produced an A -receptor-mediated bradycardia. In contrast, VCP746 produced increases in heart rate and renal and mesenteric vascular conductance that were not mediated by A -receptors. In vitro studies confirmed that VCP746 had potent agonist activity at both A - and A -receptors.

Conclusions And Implications: These results suggest VCP746 mediates its cardiovascular effects via activation of A rather than A adenosine receptors. This has implications for the design of future bitopic ligands that incorporate A allosteric ligand moieties.
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http://dx.doi.org/10.1111/bph.14870DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989947PMC
January 2020

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

J Med Chem 2019 11 23;62(21):9488-9520. Epub 2019 Oct 23.

School of Pharmacy, Centre for Biomolecular Sciences , University of Nottingham , Nottingham NG7 2RD , U.K.

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 D receptor (DR) antagonists, with contrasting kinetic profiles. Haloperidol displays fast association/slow dissociation at the DR, whereas clozapine exhibits relatively slow association/fast dissociation. Recently, we have provided evidence that slow dissociation from the DR predicts hyperprolactinemia, whereas fast association predicts EPS. Unfortunately, clozapine can cause severe side effects independent of its DR action. Our results suggest an optimal kinetic profile for DR 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.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00864DOI Listing
November 2019

Molecular Determinants of the Intrinsic Efficacy of the Antipsychotic Aripiprazole.

ACS Chem Biol 2019 08 5;14(8):1780-1792. Epub 2019 Aug 5.

Division of Pharmacology, Physiology and Neuroscience, School of Life Sciences, Queen's Medical Centre , University of Nottingham , Nottingham NG7 2UH , U.K.

Partial agonists of the dopamine D receptor (DR) have been developed to treat the symptoms of schizophrenia without causing the side effects elicited by antagonists. The receptor-ligand interactions that determine the intrinsic efficacy of such drugs, however, are poorly understood. Aripiprazole has an extended structure comprising a phenylpiperazine primary pharmacophore and a 1,2,3,4-tetrahydroquinolin-2-one secondary pharmacophore. We combined site-directed mutagenesis, analytical pharmacology, ligand fragments, and molecular dynamics simulations to identify the DR-aripiprazole interactions that contribute to affinity and efficacy. We reveal that an interaction between the secondary pharmacophore of aripiprazole and a secondary binding pocket defined by residues at the extracellular portions of transmembrane segments 1, 2, and 7 determines the intrinsic efficacy of aripiprazole. Our findings reveal a hitherto unappreciated mechanism for fine-tuning the intrinsic efficacy of DR agonists.
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http://dx.doi.org/10.1021/acschembio.9b00342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365685PMC
August 2019

Cryptic pocket formation underlies allosteric modulator selectivity at muscarinic GPCRs.

Nat Commun 2019 07 23;10(1):3289. Epub 2019 Jul 23.

Departments of Computer Science, Molecular and Cellular Physiology, and Structural Biology, and Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA, 94305, USA.

Allosteric modulators are highly desirable as drugs, particularly for G-protein-coupled receptor (GPCR) targets, because allosteric drugs can achieve selectivity between closely related receptors. The mechanisms by which allosteric modulators achieve selectivity remain elusive, however, particularly given recent structures that reveal similar allosteric binding sites across receptors. Here we show that positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (mAChR) achieve exquisite selectivity by occupying a dynamic pocket absent in existing crystal structures. This cryptic pocket forms far more frequently in molecular dynamics simulations of the M1 mAChR than in those of other mAChRs. These observations reconcile mutagenesis data that previously appeared contradictory. Further mutagenesis experiments validate our prediction that preventing cryptic pocket opening decreases the affinity of M1-selective PAMs. Our findings suggest opportunities for the design of subtype-specific drugs exploiting cryptic pockets that open in certain receptors but not in other receptors with nearly identical static structures.
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http://dx.doi.org/10.1038/s41467-019-11062-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650467PMC
July 2019

Novel Human Aminopeptidase N Inhibitors: Discovery and Optimization of Subsite Binding Interactions.

J Med Chem 2019 08 18;62(15):7185-7209. Epub 2019 Jul 18.

Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology , Monash University , Clayton Campus , Clayton , VIC 3800 , Australia.

Aminopeptidase N (APN/CD13) is a zinc-dependent M1 aminopeptidase that contributes to cancer progression by promoting angiogenesis, metastasis, and tumor invasion. We have previously identified hydroxamic acid-containing analogues that are potent inhibitors of the APN homologue from the malarial parasite M1 aminopeptidase (A-M1). Herein, we describe the rationale that underpins the repurposing of A-M1 inhibitors as novel APN inhibitors. A series of novel hydroxamic acid analogues were developed using a structure-based design approach and evaluated their inhibition activities against APN. -(2-(Hydroxyamino)-2-oxo-1-(3',4',5'-trifluoro-[1,1'-biphenyl]-4-yl)ethyl)-4-(methylsulfonamido)benzamide () proved to be an extremely potent inhibitor of APN activity in vitro, selective against other zinc-dependent enzymes such as matrix metalloproteases, and possessed limited cytotoxicity against Ad293 cells and favorable physicochemical and metabolic stability properties. The combined results indicate that compound may be a useful lead for the development of anticancer agents.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00757DOI Listing
August 2019

Unlocking the full potential of lipid-based formulations using lipophilic salt/ionic liquid forms.

Adv Drug Deliv Rev 2019 03 29;142:75-90. Epub 2019 May 29.

Oral Drug Delivery Innovation, Global Research & Development, Lonza, Strasbourg, France. Electronic address:

Lipid-based formulations (LBF) are widely used by industry and accepted by the regulatory authorities for oral drug delivery in the pharmaceutical and consumer healthcare market. Innovation in the LBF field is however needed in order to meet the demands of modern drugs, their more challenging problem statements and growing needs for achieving optimal pharmacokinetics (i.e., no food-effects, low variability) on approval. This review describes a new lipophilic salt / ionic liquid approach in combination with LBF, and how this salt strategy can be used to better tailor the properties of a drug to LBFs. The potential advantages of lipophilic salts are discussed in the context of dose escalation studies during toxicological evaluation, reducing the pill burden, increasing drug absorption of new drugs and in life-cycle management. Commentary on lipophilic salt synthesis, scale-up, LBF design and the regulatory aspects are also provided. These topics are discussed in the broad context of bringing the widely recognized advantages of LBFs to a broader spectrum of drugs.
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http://dx.doi.org/10.1016/j.addr.2019.05.008DOI Listing
March 2019

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

Eur J Med Chem 2019 Apr 11;168:474-490. Epub 2019 Feb 11.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia. Electronic address:

We recently described a structurally novel series of negative allosteric modulators (NAMs) of the dopamine D receptor (DR) 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 DR including NAMs with sub-μM affinity (9h) and, surprisingly, low efficacy partial agonists (9d and 9i).
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http://dx.doi.org/10.1016/j.ejmech.2019.01.061DOI Listing
April 2019

Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe.

ChemMedChem 2019 03 13;14(5):603-612. Epub 2019 Feb 13.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.
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http://dx.doi.org/10.1002/cmdc.201800802DOI Listing
March 2019

Probe dependence of allosteric enhancers on the binding affinity of adenosine A -receptor agonists at rat and human A -receptors measured using NanoBRET.

Br J Pharmacol 2019 04 6;176(7):864-878. Epub 2019 Mar 6.

Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, UK.

Background And Purpose: Adenosine is a local mediator that regulates a number of physiological and pathological processes via activation of adenosine A -receptors. The activity of adenosine can be regulated at the level of its target receptor via drugs that bind to an allosteric site on the A -receptor. Here, we have investigated the species and probe dependence of two allosteric modulators on the binding characteristics of fluorescent and nonfluorescent A -receptor agonists.

Experimental Approach: A Nano-luciferase (Nluc) BRET (NanoBRET) methodology was used. This used N-terminal Nluc-tagged A -receptors expressed in HEK293T cells in conjunction with both fluorescent A -receptor agonists (adenosine and NECA analogues) and a fluorescent antagonist CA200645.

Key Results: PD 81,723 and VCP171 elicited positive allosteric effects on the binding affinity of orthosteric agonists at both the rat and human A -receptors that showed clear probe dependence. Thus, the allosteric effect on the highly selective partial agonist capadenoson was much less marked than for the full agonists NECA, adenosine, and CCPA in both species. VCP171 and, to a lesser extent, PD 81,723, also increased the specific binding of three fluorescent A -receptor agonists in a species-dependent manner that involved increases in B and pK .

Conclusions And Implications: These results demonstrate the power of the NanoBRET ligand-binding approach to study the effect of allosteric ligands on the binding of fluorescent agonists to the adenosine A -receptor in intact living cells. Furthermore, our studies suggest that VCP171 and PD 81,723 may switch a proportion of A -receptors to an active agonist conformation (R*).
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http://dx.doi.org/10.1111/bph.14575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433648PMC
April 2019

6-Phenylpyrimidin-4-ones as Positive Allosteric Modulators at the M mAChR: The Determinants of Allosteric Activity.

ACS Chem Neurosci 2019 03 28;10(3):1099-1114. Epub 2018 Dec 28.

Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences , University of Glasgow , Glasgow G12 8QQ , United Kingdom.

Targeting allosteric sites of the M muscarinic acetylcholine receptor (mAChR) is an enticing approach to overcome the lack of receptor subtype selectivity observed with orthosteric ligands. This is a promising strategy for obtaining novel therapeutics to treat cognitive deficits observed in Alzheimer's disease and schizophrenia, while reducing the peripheral side effects such as seen in the current treatment regimes, which are non-subtype selective. We previously described compound 2, the first positive allosteric modulator (PAM) of the M mAChR based on a 6-phenylpyrimidin-4-one scaffold, which has been further developed in this study. Herein, we present the synthesis, characterization, and pharmacological evaluation of a series of 6-phenylpyrimidin-4-ones with modifications to the 4-(1-methylpyrazol-4-yl)benzyl pendant. Selected compounds, BQCA, 1, 2, 9i, 13, 14b, 15c, and 15d, were further profiled in terms of their allosteric affinity, cooperativity with acetylcholine (ACh), and intrinsic efficacy. Additionally, 2 and 9i were tested in mouse primary cortical neurons, displaying various degrees of intrinsic agonism and potentiation of the acetylcholine response. Overall, the results suggest that the pendant moiety is important for allosteric binding affinity and the direct agonistic efficacy of the 6-phenylpyrimidin-4-one based M mAChR PAMs.
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http://dx.doi.org/10.1021/acschemneuro.8b00613DOI Listing
March 2019

Hydroxamic Acid Inhibitors Provide Cross-Species Inhibition of Plasmodium M1 and M17 Aminopeptidases.

J Med Chem 2019 01 4;62(2):622-640. Epub 2019 Jan 4.

Department of Microbiology, Biomedicine Discovery Institute , Monash University, Clayton , Melbourne , VIC 3800 , Australia.

There is an urgent clinical need for antimalarial compounds that target malaria caused by both Plasmodium falciparum and Plasmodium vivax. The M1 and M17 metalloexopeptidases play key roles in Plasmodium hemoglobin digestion and are validated drug targets. We used a multitarget strategy to rationally design inhibitors capable of potent inhibition of the M1 and M17 aminopeptidases from both P. falciparum ( Pf-M1 and Pf-M17) and P. vivax ( Pv-M1 and Pv-M17). The novel chemical series contains a hydroxamic acid zinc binding group to coordinate catalytic zinc ion/s, and a variety of hydrophobic groups to probe the S1' pockets of the four target enzymes. Structural characterization by cocrystallization showed that selected compounds utilize new and unexpected binding modes; most notably, compounds substituted with bulky hydrophobic substituents displace the Pf-M17 catalytic zinc ion. Excitingly, key compounds of the series potently inhibit all four molecular targets and show antimalarial activity comparable to current clinical candidates.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01310DOI Listing
January 2019

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

Drug Metab Dispos 2019 02 26;47(2):164-172. Epub 2018 Nov 26.

Division of Biomedical Science and Biochemistry, Research School of Biology and Medical School, Australian National University, Canberra, Australian Capital Territory (D.M., G.K.H., R.C.), and Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria (A.J.D., B.C., P.J.S.), Australia

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.
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http://dx.doi.org/10.1124/dmd.118.083188DOI Listing
February 2019

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

Mol Pharm 2018 12 13;15(12):5678-5696. Epub 2018 Nov 13.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology , Monash University , 381 Royal Parade , Parkville , Victoria 3052 , Australia.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00858DOI Listing
December 2018

Synthesis and Pharmacological Evaluation of Noscapine-Inspired 5-Substituted Tetrahydroisoquinolines as Cytotoxic Agents.

J Med Chem 2018 09 14;61(18):8444-8456. Epub 2018 Sep 14.

Research School of Biology , Australian National University , Canberra , ACT 0200 , Australia.

A series of 5-substituted tetrahydroisoquinolines was synthesized via a 10-step linear synthesis to assess whether replacement of noscapine's southern isobenzofuranone with other moieties resulted in retained cytotoxic activity. One such molecule, 18g, bearing a para-methoxybenzyl functionality with N-ethylcarbamoyl substitution, produced cell-cycle arrest at the G2/M phase with an EC of 2.7 μM in the MCF-7 breast-cancer cell line, a 7-fold increase compared with that of noscapine (5). This molecule had similar activity (EC of 2.5 μM) against the resistant NCI/Adr cell line, demonstrating its potential to overcome or avert known resistance mechanisms, unlike current cytotoxic agents. Compound 18g was found to modify the drug-efflux activity of P-gp and, in combination studies, potentiate the antiproliferative activity of vinblastine. These results provide insights into structural modifications to noscapine that will guide future development toward more potent cytotoxic agents that are active against resistant cancer cells.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00986DOI Listing
September 2018

Subtle Modifications to the Indole-2-carboxamide Motif of the Negative Allosteric Modulator N-(( trans)-4-(2-(7-Cyano-3,4-dihydroisoquinolin-2(1 H)-yl)ethyl)cyclohexyl)-1 H-indole-2-carboxamide (SB269652) Yield Dramatic Changes in Pharmacological Activity at the Dopamine D Receptor.

J Med Chem 2019 01 2;62(1):371-377. Epub 2018 Jul 2.

SB269652 (1) is a negative allosteric modulator of the dopamine D receptor. Herein, we present the design, synthesis, and pharmacological evaluation of "second generation" analogues of 1 whereby subtle modifications to the indole-2-carboxamide motif confer dramatic changes in functional affinity (5000-fold increase), cooperativity (100-fold increase), and a novel action to modulate dopamine efficacy. Thus, structural changes to this region of 1 allows the generation of a novel set of analogues with distinct pharmacological properties.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00192DOI Listing
January 2019

Probing the binding site of novel selective positive allosteric modulators at the M muscarinic acetylcholine receptor.

Biochem Pharmacol 2018 08 17;154:243-254. Epub 2018 May 17.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

Subtype-selective allosteric modulation of the M muscarinic acetylcholine (ACh) receptor (M mAChR) is an attractive approach for the treatment of numerous disorders, including cognitive deficits. The discovery of benzyl quinolone carboxylic acid, BQCA, a selective M mAChR positive allosteric modulator (PAM), spurred the subsequent development of newer generation M PAMs representing diverse chemical scaffolds, different pharmacodynamic properties and, in some instances, improved pharmacokinetics. Key exemplar molecules from such efforts include PF-06767832 (N-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-4-(4-(thiazol-4-yl)benzyl)pyridine-2-carboxamide), VU6004256 (4,6-difluoro-N-(1S,2S)-2-hydroxycyclohexyl-1-((6-(1-methyl-1H-pyrazol-4-yl)pyridine-3-yl)methyl)-1H-indole-3-carboxamide) and MIPS1780 (3-(2-hydroxycyclohexyl)-6-(2-((4-(1-methyl-1H-pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3H)-one). Given these diverse scaffolds and pharmacodynamics, the current study combined pharmacological analysis and site-directed mutagenesis to explore the potential binding site and function of newer M mAChR PAMs relative to BQCA. Interestingly, the mechanism of action of the novel PAMs was consistent with a common model of allostery, as previously described for BQCA. Key residues involved in the activity of BQCA, including Y179 in the second extracellular loop (ECL) and W400 in transmembrane domain (TM) 7, were critical for the activity of all PAMs tested. Overall, our data indicate that structurally distinct PAMs share a similar binding site with BQCA, specifically, an extracellular allosteric site defined by residues in TM2, TM7 and ECL2. These findings provide valuable insights into the structural basis underlying modulator binding, cooperativity and signaling at the M mAChR, which is essential for the rational design of PAMs with tailored pharmacological properties.
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http://dx.doi.org/10.1016/j.bcp.2018.05.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6066355PMC
August 2018

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

Mol Pharmacol 2018 07 24;94(1):770-783. Epub 2018 Apr 24.

Drug Discovery Biology (E.T.W., A.S., E.K., P.M.S., C.V., A.C.) and Medicinal Chemistry (M.J., S.N.M., B.C., P.J.S.), Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia; and Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom (A.B.T.)

Positive allosteric modulators (PAMs) that target the M muscarinic acetylcholine (ACh) receptor (M 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 M 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 M 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 (IP) accumulation, and -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 M 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 M mAChR PAM effects than previously appreciated.
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http://dx.doi.org/10.1124/mol.118.111633DOI Listing
July 2018

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

J Med Chem 2019 01 15;62(1):174-206. Epub 2018 May 15.

Department of Biological Sciences, Bridge Institute , University of Southern California , Los Angeles , California 90089 , United States.

Recently, a novel negative allosteric modulator (NAM) of the D-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.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01565DOI Listing
January 2019

Synthesis and Pharmacological Evaluation of Heterocyclic Carboxamides: Positive Allosteric Modulators of the M Muscarinic Acetylcholine Receptor with Weak Agonist Activity and Diverse Modulatory Profiles.

J Med Chem 2018 04 23;61(7):2875-2894. Epub 2018 Mar 23.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences , Monash University , 381 Royal Parade , Parkville , Victoria 3052 , Australia.

Targeting allosteric sites at M muscarinic acetylcholine receptors is a promising strategy for the treatment of Alzheimer's disease. Positive allosteric modulators not only may potentiate binding and/or signaling of the endogenous agonist acetylcholine (ACh) but also may possess direct agonist activity (thus referred to as PAM-agonists). Recent studies suggest that PAM-agonists with robust intrinsic efficacy are more likely to produce adverse effects in vivo. Herein we present the synthesis and pharmacological evaluation of a series of pyrrole-3-carboxamides with a diverse range of allosteric profiles. We proposed structural modifications at top, core, or pendant moieties of a prototypical molecule. Although generally there was a correlation between the degree of agonist activity and the modulatory potency of the PAMs, some derivatives displayed weak intrinsic efficacy yet maintained strong allosteric modulation. We also identified molecules with the ability to potentiate mainly the affinity or both affinity and efficacy of ACh.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01812DOI Listing
April 2018

A Structure-Activity Relationship Study of Bitopic N-Substituted Adenosine Derivatives as Biased Adenosine A Receptor Agonists.

J Med Chem 2018 03 23;61(5):2087-2103. Epub 2018 Feb 23.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences , Monash University , Parkville , Victoria 3052 , Australia.

The adenosine A receptor (AAR) is a potential novel therapeutic target for myocardial ischemia-reperfusion injury. However, to date, clinical translation of prototypical AAR agonists has been hindered due to dose limiting adverse effects. Recently, we demonstrated that the biased bitopic agonist 1, consisting of an adenosine pharmacophore linked to an allosteric moiety, could stimulate cardioprotective AAR signaling in the absence of unwanted bradycardia. Therefore, this study aimed to investigate the structure-activity relationship of compound 1 biased agonism. A series of novel derivatives of 1 were synthesized and pharmacologically profiled. Modifications were made to the orthosteric adenosine pharmacophore, linker, and allosteric 2-amino-3-benzoylthiophene pharmacophore to probe the structure-activity relationships, particularly in terms of biased signaling, as well as AAR activity and subtype selectivity. Collectively, our findings demonstrate that the allosteric moiety, particularly the 4-(trifluoromethyl)phenyl substituent of the thiophene scaffold, is important in conferring bitopic ligand bias at the AAR.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00047DOI Listing
March 2018

The action of a negative allosteric modulator at the dopamine D receptor is dependent upon sodium ions.

Sci Rep 2018 01 19;8(1):1208. Epub 2018 Jan 19.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 399 Royal Parade, Parkville, VIC 3052, Australia.

Sodium ions (Na) allosterically modulate the binding of orthosteric agonists and antagonists to many class A G protein-coupled receptors, including the dopamine D receptor (DR). Experimental and computational evidences have revealed that this effect is mediated by the binding of Na to a conserved site located beneath the orthosteric binding site (OBS). SB269652 acts as a negative allosteric modulator (NAM) of the DR that adopts an extended bitopic pose, in which the tetrahydroisoquinoline moiety interacts with the OBS and the indole-2-carboxamide moiety occupies a secondary binding pocket (SBP). In this study, we find that the presence of a Na within the conserved Na-binding pocket is required for the action of SB269652. Using fragments of SB269652 and novel full-length analogues, we show that Na is required for the high affinity binding of the tetrahydroisoquinoline moiety within the OBS, and that the interaction of the indole-2-carboxamide moiety with the SBP determines the degree of Na-sensitivity. Thus, we extend our understanding of the mode of action of this novel class of NAM by showing it acts synergistically with Na to modulate the binding of orthosteric ligands at the DR, providing opportunities for fine-tuning of modulatory effects in future allosteric drug design efforts.
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http://dx.doi.org/10.1038/s41598-018-19642-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775417PMC
January 2018

The structural determinants of the bitopic binding mode of a negative allosteric modulator of the dopamine D receptor.

Biochem Pharmacol 2018 02 9;148:315-328. Epub 2018 Jan 9.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville campus), 399 Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

SB269652 is a negative allosteric modulator of the dopamine D receptor (DR) yet possesses structural similarity to ligands with a competitive mode of interaction. In this study, we aimed to understand the ligand-receptor interactions that confer its allosteric action. We combined site-directed mutagenesis with molecular dynamics simulations using both SB269652 and derivatives from our previous structure activity studies. We identify residues within the conserved orthosteric binding site (OBS) and a secondary binding pocket (SBP) that determine affinity and cooperativity. Our results indicate that interaction with the SBP is a requirement for allosteric pharmacology, but that both competitive and allosteric derivatives of SB269652 can display sensitivity to the mutation of a glutamate residue (E95) within the SBP. Our findings provide the molecular basis for the differences in affinity between SB269652 derivatives, and reveal how changes to interactions made by the primary pharmacophore of SB269652 in the orthosteric pocket can confer changes in the interactions made by the secondary pharmacophore in the SBP. Our insights provide a structure-activity framework towards rational optimization of bitopic ligands for DR with tailored competitive versus allosteric properties.
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http://dx.doi.org/10.1016/j.bcp.2018.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800995PMC
February 2018