Synthesis of a group of novel Xanomeline/77-LH-28-1 hybrid ligands and their FRET investigation at muscarinic acetylcholine receptor subtypes

In connection with our interest in investigating novel rationally designed bitopic (i.e., orthosteric/allosteric) derivatives targeting muscarinic acetylcholine receptor (mAChR) subtypes (1,2,3), in this study we designed and synthesized a new set of ligands that integrate in the same molecular skeleton the pharmacophoric moieties of Xanomeline and of 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone). Xanomeline is a well-known M1/M4-preferring orthosteric agonist, which ameliorated cognitive impairments in Alzheimer\u2019s disease patients and showed activity in various models of schizophrenia, thus being potentially beneficial for treatment of positive, negative and cognitive symptoms (4). On the other hand, 77-LH-28-1 was characterized as an M1-selective, positive allosteric modulator, thus representing an interesting pharmacological tool with cognition enhancing properties (5). As illustrated below, we planned the novel bipharmacophoric derivatives as merged structures, with the tetrahydropyridine nucleus of Xanomeline as the central core. In the last years, different receptor sensors, based on the fluorescence resonance energy transfer (FRET), were generated for various G protein-coupled receptors, and represented a valuable tool to investigate real time receptor activation as well as ligand-receptor interactions. Recently, this analysis was performed also on a set of bitopic ligands designed for a selective interaction with M1 mAChRs (6). Our preliminary results on the group of Xanomeline/77-LH-28-1 hybrid compounds indicate, for the M1 sensor, a reproducible activation response, which depends on the linker length. Conversely, no FRET-related effect could be detected at the M2 sensor. Thus, a critical spacer length of the hybrid compounds induces conformational changes with a degree of selectively for the M1 muscarinic receptor. The synthesis and the results of pharmacological investigation will be presented and discussed. References: 1. J. Antony, K. Kellershohn, M. Mohr-Andr\ue4, A. Kebig, S. Prilla, M. Muth, E. Heller, T. Disingrini, C. Dallanoce et al., FASEB J 2009, 23, 442-450. 2. A. Bock, B. Chirinda, F. Krebs, R. Messerer, J. B\ue4tz, M. Muth, C. Dallanoce et al., Nat. Chem. Biol. 2014, 10, 18-20. 3. A. Bock, M. Bermudez, F. Krebs, C. Matera, B. Chirinda, D. Sydow, C. Dallanoce et al., J. Biol. Chem. 2016, 291, 16375-16389. 4. S. Barak, I. Weiner, Int. J. Neuropsychoph. 2011, 14, 1233-1246. 5. C. J. Langmead, N. E. Austin, C. L. Branch, J. T. Brown, K. A. Buchanan, C. H. Davies, I. T. Forbes et al., Br. J. Pharmacol. 2008, 154, 1104-1115. 6. R. Messerer, M. Kauk, D. Volpato, M. C. Alonso Canizal, J. Kl\uf6ckner, U. Zabel, S. Nuber, C. Hoffmann, U. Holzgrabe, ACS Chem. Biol. 2017, 12, 833-843

Population pharmacokinetics at two dose levels and pharmacodynamic profiling of flucloxacillin

Flucloxacillin is often used for the treatment of serious infections due to sensitive staphylococci. The pharmacokinetic (PK)-pharmacodynamic (PD) breakpoint of flucloxacillin has not been determined by the use of population PK. Targets based on the duration of non-protein-bound concentrations above the MIC (fT(> MIC)) best correlate with clinical cure rates for beta-lactams. We compared the breakpoints for flucloxacillin between several dosage regimens. In a randomized, two-way crossover study, 10 healthy volunteers received 500 mg and 1,000 mg flucloxacillin as 5-min intravenous infusions. Drug concentrations were determined by high-pressure liquid chromatography. We used the programs WinNonlin for noncompartmental analysis and statistics and NONMEM for population PK and Monte Carlo simulation. We compared the probability of target attainment (PTA) for intermittent- and continuous-dosage regimens based on the targets of fT(> MIS)s of >= 50% and >= 30% of the dosing interval. The clearance and the volume of distribution were very similar after the administration of 500 mg and 1,000 mg flucloxacillin. We estimated renal and nonrenal clearances of 5.37 liters/h (coefficient of variation, 19%) and 2.73 liters/h (33%). For near maximal killing (target, fT(> MIC) of >= 50%) flucloxacillin showed a robust (>= 90%) PTA up to MICs of 0.75 to 1 mg/liter (PTA of 860/v at 1 mg/liter) for a continuous or a prolonged infusion of 6 g/day. Short-term infusions of 6 g/day had a lower breakpoint of 0.25 to 0.375 mg/liter. The flucloxacillin PK was linear for doses of 500 mg and 1,000 mg. Prolonged and continuous infusion at a 66% lower daily dose achieved the same PK-PD breakpoints as short-term infusions. Prolonged infusion and continuous infusion are appealing options for the treatment of serious infections caused by sensitive staphylococci

A New Bioactive Compound From the Marine Sponge-Derived Streptomyces sp. SBT348 Inhibits Staphylococcal Growth and Biofilm Formation

Staphylococcus epidermidis, the common inhabitant of human skin and mucosal surfaces has emerged as an important pathogen in patients carrying surgical implants and medical devices. Entering the body via surgical sites and colonizing the medical devices through formation of multi-layered biofilms leads to refractory and persistent device-related infections (DRIs). Staphylococci organized in biofilms are more tolerant to antibiotics and immune responses, and thus are difficult-to-treat. The consequent morbidity and mortality, and economic losses in health care systems has strongly necessitated the need for development of new anti-bacterial and anti-biofilm-based therapeutics. In this study, we describe the biological activity of a marine sponge-derived Streptomyces sp. SBT348 extract in restraining staphylococcal growth and biofilm formation on polystyrene, glass, medically relevant titan metal, and silicone surfaces. A bioassay-guided fractionation was performed to isolate the active compound (SKC3) from the crude SBT348 extract. Our results demonstrated that SKC3 effectively inhibits the growth (MIC: 31.25 μg/ml) and biofilm formation (sub-MIC range: 1.95–<31.25 μg/ml) of S. epidermidis RP62A in vitro. Chemical characterization of SKC3 by heat and enzyme treatments, and mass spectrometry (HRMS) revealed its heat-stable and non-proteinaceous nature, and high molecular weight (1258.3 Da). Cytotoxicity profiling of SKC3 in vitro on mouse fibroblast (NIH/3T3) and macrophage (J774.1) cell lines, and in vivo on the greater wax moth larvae Galleria mellonella revealed its non-toxic nature at the effective dose. Transcriptome analysis of SKC3 treated S. epidermidis RP62A has further unmasked its negative effect on central metabolism such as carbon flux as well as, amino acid, lipid, and energy metabolism. Taken together, these findings suggest a potential of SKC3 as a putative drug to prevent staphylococcal DRIs

The ameliorative effects of a phenolic derivative of Moringa oleifera leave against vanadium-induced neurotoxicity in mice

Vanadium, a transition series metal released during some industrial activities, induces oxidative stress and lipid peroxidation. Ameliorative effect of a pure compound from the methanolic extract of Moringa oleifera leaves, code-named MIMO2, in 14-day old mice administered with vanadium (as sodium metavanadate 3 mg/kg) for 2 weeks was assessed. Results from body weight monitoring, muscular strength, and open field showed slight reduction in body weight and locomotion deficit in vanadium-exposed mice, ameliorated with MIMO2 co-administration. Degeneration of the Purkinje cell layer and neuronal death in the hippocampal CA1 region were observed in vanadium-exposed mice and both appeared significantly reduced with MIMO2 co-administration. Demyelination involving the midline of the corpus callosum, somatosensory and retrosplenial cortices was also reduced with MIMO2. Microglia activation and astrogliosis observed through immunohistochemistry were also alleviated. Immunohistochemistry for myelin, axons and oligodendrocyte lineage cells were also carried out and showed that in vanadium-treated mice brains, oligodendrocyte progenitor cells increased NG2 immunolabelling with hypertrophy and bushy, ramified appearance of their processes. MIMO2 displayed ameliorative and antioxidative effects in vanadium-induced neurotoxicity in experimental murine species. This is likely the first time MIMO2 is being used in vivo in an animal model

Muscarinic dualsteric ligands behave as partial or protean agonists depending on the affinity of their orthosteric moiety

Muscarinic acetylcholine receptors have been extensively studied with the purpose of finding selective ligands for their modulation. In the last years, a new strategy was developed towards this aim, i.e. the synthesis of so-called dualsteric ligands that bind simultaneously to both the orthosteric and the allosteric site of the M2 receptor.Two dualsteric compounds, Iper-6-naph and Isox- 6-naph, and their orthosteric fragments were studied in GTP\u3b3S assays and [3H]NMS binding studies, performed with membranes of CHO cells expressing the human M2 receptor. The experiments were carried out in Tris buffer supplemented with either 2 mM or 200 mM NaCl, in order to have the receptor in its spontaneously active or inactive conformations, respectively. In both buffers, Iper-6-naph was a partial agonist and it almost completely abolished [3H]NMS binding. In contrast, Isox-6-naph revealed a protean nature, behaving either as inverse or partial agonist, respectively, in 2 mM and 200 mM NaCl, and it promoted [3H] NMS binding. Since the two hybrid compounds share the same allosteric moiety, these discrepancies have to be attributed to pharmacological differences of the orthosteric moieties, whose potencies and affinities were indeed reported to be significantly divergent. In conclusion, this study suggests that slight structural modifications in the orthosteric building block of dualsteric ligands may cause a reversal of ligand efficacy and, most importantly, it provides a deeper insight into the molecular determinants of protean agonism at the M2 receptor

Novel pharmacological tools which activate mAChRs: a question of &quot;dualsterism&quot;

Muscarinic acetylcholine receptors (mAChRs) represent an excellent model system to study orthosteric and allosteric interactions. The high sequence homology shown by orthosteric sites of mAChRs has hampered the development of subtype selective agonists. On the other hand, allosteric recognition sites are less conserved among the various mAChR subtypes. We synthesized a series of hybrid ligands designed to simultaneously interact with both orthosteric and allosteric sites (\u201cdualsteric\u201d compounds) by fusing orthosteric activators with M2-selective allosteric fragments (W84 and Naphmethonium). In particular, among the oxotremorine-like orthosteric agents, iperoxo emerged as a potent agonist with supraphysiological efficacy but devoid of subtype selectivity.1 To explore the whole chemical space of the binding region, we modified the structure of the three component parts (orthosteric and allosteric moieties and spacer) of dualsteric ligands.2 These ligands permitted to prove for the first time that GPCR\u2019s allosteric vestibule is able to control the extent of receptor movement to govern a hierarchical order of G-protein coupling.3 In addition, they were found to dynamically switch between two distinct binding orientations, engendering both active and inactive populations of receptors bound by a given ligand.4 More recently, some of these ligands (notably N-8-IPER) revealed interesting antinociceptive properties and good tolerability.5 The synthetic approaches together with relevant results and implications of the biological investigation will be presented. References 1. Schrage R et al. Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor. Br J Pharmacol 2013;169(2):357-70. 2. Disingrini T et al. Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors. J Med Chem 2006;49(1):366-72; Antony J et al. Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity. FASEB J 2009;23(2):442-50. 3. Bock A et al. The allosteric vestibule of a seven transmembrane helical receptor controls G-protein coupling. Nat Commun 2012;3:1044. 4. Bock A et al. Dynamic ligand binding dictates partial agonism at a G protein-coupled receptor. Nat Chem Biol 2014;10(1):18-20. 5. Matera C et al. Bis(ammonio)alkane-type agonists of muscarinic acetylcholine receptors: Synthesis, in vitro functional characterization, and in vivo evaluation of their analgesic activity. Eur J Med Chem 2014;75:222-232

Dualsteric compounds modulate the signaling pattern of muscarinic M1 acetylcholine receptors

G protein-coupled receptors (GPCRs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric G proteins [1]. GPCR-induced cell signaling can be rather complex as several GPCRs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other \u201callosteric\u201d binding sites [2]. In the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (mAChRs). To this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). Binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [2]. Therefore, application of different linker length may control the signaling outcome. Here, we applied the human M1 mAChR which preferentially activates G proteins of the Gq/11 type but can also promiscuously stimulate Gs proteins. Gq/11- and Gs- dependent signaling pathways were analyzed by application of CHO cells stably transfected with the human M1 mAChR in IP1 and cAMP accumulation assays, respectively. In comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both Gq-mediated and Gs-mediated signaling. Our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. Particularly, the compound iper-6-naph completely lost intrinsic activity for both Gq/11 and Gs activation at the M1 mAChR. Moreover, Gs-mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than Gq-signaling. Interestingly, longer linker length led to improved signaling for both pathways (Gq and Gs) in both hybrid series. Iper-7-phth seems to be an exception as it had a higher intrinsic efficacy for Gs-dependent signaling than the other phth hybrids with longer linker chains. Strikingly, only iper-fri-phth, which corresponds to iper-8-phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on M1 mAChR for both signaling pathways under investigation. Taken together, these data strongly suggest that, in comparison to Gq/11-mediated signaling, activation of the Gs protein in M1 mAChR is more sensitive to spatial restriction in the allosteric vestibule. Thus, it appears to be possible to control signaling of the M1 mAChR by allosteric constraint of the receptor\u2019s conformational flexibility. [1] Magalhaes, A. et al.: Br. J. Pharmacol. 2011, 165(6): 1717-36 [2] Bock, A. et al.: Nat. Commun. 2012, 3:1044 doi: 10.1038/ncomms202

Fluorescent probe for the identification of potent inhibitors of the macrophage infectivity potentiator (Mip) protein of Burkholderia pseudomallei.

This is the final version. Available from Elsevier via the DOI in this record. The macrophage infectivity potentiator (Mip) protein belongs to the immunophilin superfamily. This class of enzymes catalyzes the interconversion between the cis and trans configuration of proline-containing peptide bonds. Mip has been shown to be important for the virulence of a wide range of pathogenic microorganisms, including the Gram-negative bacterium Burkholderia pseudomallei. Small molecules derived from the natural product rapamycin, lacking its immunosuppression-inducing moiety, inhibit Mip's peptidyl-prolyl cis-trans isomerase (PPIase) activity and lead to a reduction in pathogen load in vitro. Here, a fluorescence polarization assay (FPA) to enable the screening and effective development of BpMip inhibitors was established. A fluorescent probe was prepared, derived from previous pipecolic scaffold Mip inhibitors labeled with fluorescein. This probe showed moderate affinity for BpMip and enabled a highly robust FPA suitable for screening large compound libraries with medium- to high-throughput (Z factor ∼ 0.89) to identify potent new inhibitors. The FPA results are consistent with data from the protease-coupled PPIase assay. Analysis of the temperature dependence of the probe's binding highlighted that BpMip's ligand binding is driven by enthalpic rather than entropic effects. This has considerable consequences for the use of low-temperature kinetic assays.North Atlantic Treaty OrganizationGerman Research Foundation (DFG, Deutsche Forschungsgemeinschaft)Federal Ministry of Education and Research (Germany)Biotechnology and Biological Sciences Research CouncilDMTC Limited (Australia)UK Research and Innovatio

Ligand Binding Ensembles Determine Graded Agonist Efficacies at a G Protein-Coupled Receptor

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to GPCRs induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist-receptor complexes on a molecular level. Employing all-atom molecular dynamics (MD) simulations, dynophores (i.e. a combination of static 3D-pharmacophores and MD-based conformational sampling), ligand design and receptor mutagenesis, we show that inactive agonist-receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) vs. dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist-receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists, where binding modes will be only subtly different and confined to only one binding site

Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major

Background The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. Objectives In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. Methods Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. Results Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. Conclusions These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications