Discovery of Procognitive Antipsychotics by Combining Muscarinic M1 Receptor Structure-Activity Relationship with Systems Response Profiles in Zebrafish Larvae.

Current antipsychotic drugs are notably ineffective at addressing the cognitive deficits associated with schizophrenia. N-Desmethylclozapine (NDMC), the major metabolite of clozapine, displays muscarinic M1 receptor (M1) agonism, an activity associated with improvement in cognitive functioning. Preclinical and clinical data support that M1 agonism may be a desired activity in antipsychotic drugs. However, NDMC failed clinical phase II studies in acute psychotic patients. NDMC analogues were synthesized to establish a structure-activity relationship (SAR) at the M1 receptor as an indication of potential procognitive properties. In vitro evaluation revealed a narrow SAR in which M1 agonist activity was established by functionalization in the 4- and 8-positions in the tricyclic core. In vivo behavioral response profiles were used to evaluate antipsychotic efficacy and exposure in zebrafish larvae and peripheral side effect related M1 activity in adult zebrafish. The NDMC analogue 13f demonstrated antipsychotic activity similar to clozapine including M1 agonist activity. Cotreatment with trospium chloride, an M1 peripheral acting antagonist, counteracted peripheral side effects. Thus, the NDMC analogue 13f, in combination with a peripherally acting anticholinergic compound, could be suitable for further development as an antipsychotic compound with potential procognitive activity.

Discovery of potential antipsychotic agents possessing pro-cognitive properties.

Current antipsychotic drug therapies for schizophrenia have limited efficacy and are notably ineffective at addressing the cognitive deficits associated with this disorder. The present study was designed to develop effective antipsychotic agents that would also ameliorate the cognitive deficits associated with this disease. In vitro studies comprised of binding and functional assays were utilized to identify compounds with the receptor profile that could provide both antipsychotic and pro-cognitive features. Antipsychotic and cognitive models assessing in vivo activity of these compounds included locomotor activity assays and novel object recognition assays. We developed a series of potential antipsychotic agents with a novel receptor activity profile comprised of muscarinic M(1) receptor agonism in addition to dopamine D(2) antagonism and serotonin 5-HT(2A) inverse agonism. Like other antipsychotic agents, these compounds reverse both amphetamine and dizocilpine-induced hyperactivity in animals. In addition, unlike other antipsychotic drugs, these compounds demonstrate pro-cognitive actions in the novel object recognition assay. The dual attributes of antipsychotic and pro-cognitive actions distinguish these compounds from other antipsychotic drugs and suggest that these compounds are prototype molecules in the development of novel pro-cognitive antipsychotic agents.

Measurements of the binding force between the Helicobacter pylori adhesin BabA and the Lewis b blood group antigen using optical tweezers.

Helicobacter pylori is a world-wide spread bacterium that causes persistent infections and chronic inflammations that can develop into gastritis and peptic ulcer disease. It expresses several adhesin proteins on its surface that bind to specific receptors in the gastric epithelium. The most well-known adhesin is BabA, which has previously been shown to bind specifically to the fucosylated blood group antigen Lewis b (Leb). The adhesion forces between BabA and the Leb antigen are investigated in this work and assessed by means of optical tweezers. A model system for in situ measurements of the interaction forces between individual bacteria and beads coated with Leb is developed. It is found that the de-adhesion force in this model system, measured with a loading rate of approximately 100 pNs, ranges from 20 to 200 pN. The de-adhesion force appears predominantly as multiples of an elementary force, which is determined to 25+/-1.5 pN and identified as the unbinding force of an individual BabA-Leb binding. It is concluded that adhesion in general is mediated by a small number of bindings (most often 1 to 4) despite that the contact surface between the bacterium and the bead encompassed significantly more binding sites.

Structure-activity relationships of galabioside derivatives as inhibitors of E. coli and S. suis adhesins: nanomolar inhibitors of S. suis adhesins.

Four collections of Gal alpha1-4Gal derivatives were synthesised and evaluated as inhibitors of the PapG class II adhesin of uropathogenic Escherichia coli and of the P(N) and P(O) adhesins of Streptococcus suis strains. Galabiosides carrying aromatic structures at C1, methoxyphenyl O-galabiosides in particular, were identified as potent inhibitors of the PapG adhesin. Phenylurea derivatisation at C3' and methoxymethylation at O2' of galabiose provided inhibitors of the S. suis strains type P(N) adhesin with remarkably high affinities (30 and 50 nM, respectively). In addition, quantitative structure-activity relationship models for E. coli PapG adhesin and S. suis adhesin type P(O) were developed using multivariate data analysis. The inhibitory lead structures constitute an advancement towards high-affinity inhibitors as potential anti-adhesion therapeutic agents targeting bacterial infections.

Physical properties of Escherichia coli P pili measured by optical tweezers.

The mechanical behavior of individual P pili of uropathogenic Escherichia coli has been investigated using optical tweezers. P pili, whose main part constitutes the PapA rod, composed of approximately 10(3) PapA subunits in a helical arrangement, are distributed over the bacterial surface and mediate adhesion to host cells. They are particularly important in the pathogenesis of E. coli colonizing the upper urinary tract and kidneys. A biological model system has been established for in situ measurements of the forces that occur during mechanical stretching of pili. A mathematical model of the force-versus-elongation behavior of an individual pilus has been developed. Three elongation regions of pili were identified. In region I, P pili stretch elastically, up to a relative elongation of 16 +/- 3%. The product of elasticity modulus and area of a P pilus, EA, was assessed to 154 +/- 20 pN (n=6). In region II, the quaternary structure of the PapA rod unfolds under a constant force of 27 +/- 2 pN (n approximately 100) by a sequential breaking of the interactions between adjacent layers of PapA subunits. This unfolding can elongate the pilus up to 7 +/- 2 times. In region III, pili elongate in a nonlinear manner as a result of stretching until the bond ruptures.

Quantitative studies of the binding of the class II PapG adhesin from uropathogenic Escherichia coli to oligosaccharides.

Binding of the class II PapG adhesin, found at the tip of filamentous pili on Escherichia coli, to the carbohydrate moiety of globoseries glycolipids in the human kidney is a key step in development of pyelonephritis, a severe form of urinary tract infection. An assay based on surface plasmon resonance for quantification of the binding of the class II PapG adhesin to oligosaccharides has been developed. Using this assay dissociation constants ranging from 80 to 540 microM were determined for binding of the PapG adhesin to di-pentasaccharide fragments from the globoseries of glycolipids. A series of galabiose derivatives, modified at the anomeric position, O-2' or O-3', was also investigated. The anomeric position appeared to be the most promising for development of improved inhibitors of PapG-mediated adhesion of E. coli. p-Methoxyphenyl galabioside was found to be most potent (K(d)=140 microM), and binds to PapG almost as well as the Forssman pentasaccharide.

Conformational studies on phenyl thioglycosides: a remote effect on disaccharide linkage by phenyl aglycons attenuates recognition of galabiosides by a bacterial adhesin.

Phenyl S-galabiosides display altered conformational properties, as compared to phenyl O-galabiosides, characterised by a remote effect on the galabiose intersaccharidic glycoside bond by the phenyl aglycon, resulting in significantly lowered affinity for the PapG class II adhesin of uropathogenic E. coli.

Discovery of potent inhibitors of PapG adhesins from uropathogenic Escherichia coli through synthesis and evaluation of galabiose derivatives.

The synthesis of two galabioside (Galalpha1-4Gal) collections based on diversification at the O-1 and O-3' atoms is reported. The galabiosides were evaluated as inhibitors of hemagglutination of human erythrocytes by two strains of Escherichia coli that expressed the class I and class II PapG adhesins, respectively. The class I adhesin was found to prefer aromatic substituents both at the O-1 and the O-3' position of the galabiose disaccharide. One galabioside, p-methoxyphenyl [3-O-(m-nitrobenzyl)-alpha-D-galactopyranosyl]-(1-4)-beta-D-galactopyranoside], had an IC(50) value of 4.1 microM, which is the best inhibition of the class I adhesin to date. In the case of the class II adhesin, one inhibitor, 2-[(S)-2-methoxycarbonyl-2-acetamido-ethylthio]ethyl (3-O-3-[2-(methoxycarbonylphenylthio)propyl]-alpha-D-galactopyranosyl)-(1-4)-alpha-D-galactopyranoside, was found to have an IC(50) value of 68 microM, which is the best artificial inhibition of the class II adhesin reported so far with an affinity for the adhesin comparable to that of the natural tetrasaccharide ligand globotetraose.