Safety and target engagement of an oral small-molecule sequestrant in adolescents with autism spectrum disorder: an open-label phase 1b/2a trial.

Autism spectrum disorder (ASD) is defined by hallmark behaviors involving reduced communication and social interaction as well as repetitive activities and restricted interests. ASD represents a broad spectrum, from minimally affected individuals to those requiring intense support, with additional manifestations often including anxiety, irritability/aggression and altered sensory processing. Gastrointestinal (GI) issues are also common in ASD, and studies have identified changes in the gut microbiome of individuals with ASD compared to control populations, complementing recent findings of differences in gut-derived metabolites in feces and circulation. However, a role for the GI tract or microbiome in ASD remains controversial. Here we report that an oral GI-restricted adsorbent (AB-2004) that has affinity for small aromatic or phenolic molecules relieves anxiety-like behaviors that are driven by a gut microbial metabolite in mice. Accordingly, a pilot human study was designed and completed to evaluate the safety of AB-2004 in an open-label, single-cohort, multiple-ascending-dose clinical trial that enrolled 30 adolescents with ASD and GI symptoms in New Zealand and Australia. AB-2004 was shown to have good safety and tolerability across all dose levels, and no drug-related serious adverse events were identified. Significant reductions in specific urinary and plasma levels of gut bacterial metabolites were observed between baseline and end of AB-2004 treatment, demonstrating likely target engagement. Furthermore, we observed improvements in multiple exploratory behavioral endpoints, most significantly in post hoc analysis of anxiety and irritability, as well as GI health, after 8 weeks of treatment. These results from an open-label study (trial registration no. ACTRN12618001956291) suggest that targeting gut-derived metabolites with an oral adsorbent is a safe and well-tolerated approach to improving symptoms associated with ASD, thereby emboldening larger placebo-controlled trials.

Colesevelam ameliorates non-alcoholic steatohepatitis and obesity in mice.

BACKGROUND: Obesity, non-alcoholic fatty liver disease (NAFLD) and its more advanced form non-alcoholic steatohepatitis (NASH) are important causes of morbidity and mortality worldwide. Bile acid dysregulation is a pivotal part in their pathogenesis. The aim of this study was to evaluate the bile acid sequestrant colesevelam in a microbiome-humanized mouse model of diet-induced obesity and steatohepatitis. METHODS: Germ-free C57BL/6 mice were associated with stool from patients with NASH and subjected to 20 weeks of Western diet feeding with and without colesevelam. RESULTS: Colesevelam reduced Western diet-induced body and liver weight gain in microbiome-humanized mice compared with controls. It ameliorated Western diet-induced hepatic inflammation, steatosis, fibrosis and insulin resistance. Colesevelam increased de novo bile acid synthesis and decreased hepatic cholesterol content in microbiome-humanized mice fed a Western diet. It further induced the gene expression of the antimicrobials Reg3g and Reg3b in the distal small intestine and decreased plasma levels of LPS. CONCLUSIONS: Colesevelam ameliorates Western diet-induced steatohepatitis and obesity in microbiome-humanized mice.

Colesevelam Reduces Ethanol-Induced Liver Steatosis in Humanized Gnotobiotic Mice.

Alcohol-related liver disease is associated with intestinal dysbiosis. Functional changes in the microbiota affect bile acid metabolism and result in elevated serum bile acids in patients with alcohol-related liver disease. The aim of this study was to identify the potential role of the bile acid sequestrant colesevelam in a humanized mouse model of ethanol-induced liver disease. We colonized germ-free (GF) C57BL/6 mice with feces from patients with alcoholic hepatitis and subjected humanized mice to the chronic-binge ethanol feeding model. Ethanol-fed gnotobiotic mice treated with colesevelam showed reduced hepatic levels of triglycerides and cholesterol, but liver injury and inflammation were not decreased as compared with non-treated mice. Colesevelam reduced hepatic cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1) protein expression, although serum bile acids were not lowered. In conclusion, our findings indicate that colesevelam treatment mitigates ethanol-induced liver steatosis in mice.

Plasma and Fecal Metabolite Profiles in Autism Spectrum Disorder.

BACKGROUND: Autism spectrum disorder (ASD) is a neurodevelopmental condition with hallmark behavioral manifestations including impaired social communication and restricted repetitive behavior. In addition, many affected individuals display metabolic imbalances, immune dysregulation, gastrointestinal dysfunction, and altered gut microbiome compositions. METHODS: We sought to better understand nonbehavioral features of ASD by determining molecular signatures in peripheral tissues through mass spectrometry methods (ultrahigh performance liquid chromatography-tandem mass spectrometry) with broad panels of identified metabolites. Herein, we compared the global metabolome of 231 plasma and 97 fecal samples from a large cohort of children with ASD and typically developing control children. RESULTS: Differences in amino acid, lipid, and xenobiotic metabolism distinguished ASD and typically developing samples. Our results implicated oxidative stress and mitochondrial dysfunction, hormone level elevations, lipid profile changes, and altered levels of phenolic microbial metabolites. We also revealed correlations between specific metabolite profiles and clinical behavior scores. Furthermore, a summary of metabolites modestly associated with gastrointestinal dysfunction in ASD is provided, and a pilot study of metabolites that can be transferred via fecal microbial transplant into mice is identified. CONCLUSIONS: These findings support a connection between metabolism, gastrointestinal physiology, and complex behavioral traits and may advance discovery and development of molecular biomarkers for ASD.

Structure-based discovery of LpxC inhibitors.

The emergence and spread of multidrug-resistant (MDR) Gram negative bacteria presents a serious threat for public health. Novel antimicrobials that could overcome the resistance problems are urgently needed. UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase (LpxC) is a cytosolic zinc-based deacetylase that catalyzes the first committed step in the biosynthesis of lipid A, which is essential for the survival of Gram-negative bacteria. Our efforts toward the discovery of novel LpxC inhibitors are presented herein.

Targeting Bacterial Cell Wall Peptidoglycan Synthesis by Inhibition of Glycosyltransferase Activity.

Synthesis of bacterial cell wall peptidoglycan requires glycosyltransferase enzymes that transfer the disaccharide-peptide from lipid II onto the growing glycan chain. The polymerization of the glycan chain precedes cross-linking by penicillin-binding proteins and is essential for growth for key bacterial pathogens. As such, bacterial cell wall glycosyltransferases are an attractive target for antibiotic drug discovery. However, significant challenges to the development of inhibitors for these targets include the development of suitable assays and chemical matter that is suited to the nature of the binding site. We developed glycosyltransferase enzymatic activity and binding assays using the natural products moenomycin and vancomycin as model inhibitors. In addition, we designed a library of disaccharide compounds based on the minimum moenomycin fragment with peptidoglycan glycosyltransferase inhibitory activity and based on a more drug-like and synthetically versatile disaccharide building block. A subset of these disaccharide compounds bound and inhibited the glycosyltransferase enzymes, and these compounds could serve as chemical entry points for antibiotic development.

LC-MS/MS characterization of phospholipid content in daptomycin-susceptible and -resistant isolates of Staphylococcus aureus with mutations in mprF.

Daptomycin (DAP) is a cyclic lipopeptide antibiotic used for the treatment of certain Staphylococcus aureus infections. Although rare, strains have been isolated that are DAP resistant. These strains usually have mutations in mprF, a gene encoding a membrane protein with both lysylphosphatidylglycerol (LPG) synthase and flippase activities. Because ΔmprF strains have increased DAP susceptibility, the mechanism of resistance is not likely due to a loss of mprF function. In this study, we developed an LC-MS assay to examine the effect of different mprF mutations on the ratio of phosphatidylglycerol (PG) to LPG in the membrane. Our assay demonstrated that some, but not all, mutations in the flippase and synthase domains result in small but reproducible increases in the proportion of LPG relative to PG. Techniques described herein represent a higher throughput and more sensitive method for measuring relative phospholipids levels. These results offer guidance in the understanding of how mprF confers DAP resistance; namely, mprF-mediated resistance may be through more than one mechanism, including increased overall LPG synthesis and increased LPG present on the outer leaflet of the cytoplasmic membrane.

Regulation of mprF by antisense RNA restores daptomycin susceptibility to daptomycin-resistant isolates of Staphylococcus aureus.

Mutations in mprF have been shown to result in reduced susceptibility to daptomycin and other cationic antibacterials. An mprF antisense-inducible plasmid was constructed and used to demonstrate that depletion of mprF can reestablish susceptibility to daptomycin. Inducing antisense to mprF also resulted in increased susceptibility to vancomycin and gentamicin but, paradoxically, decreased susceptibility to oxacillin. These results suggest that mprF mutations that reduce susceptibility to cationic antibacterials result in a gain-of-function phenotype.

Mutagenesis and functional characterization of the four domains of GlnD, a bifunctional nitrogen sensor protein.

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme (UTase/UR) and is believed to be the primary sensor of nitrogen status in the cell by sensing the level of glutamine in enteric bacteria. It plays an important role in nitrogen assimilation and metabolism by reversibly regulating the modification of P(II) protein; P(II) in turn regulates a variety of other proteins. GlnD appears to have four distinct domains: an N-terminal nucleotidyltransferase (NT) domain; a central HD domain, named after conserved histidine and aspartate residues; and two C-terminal ACT domains, named after three of the allosterically regulated enzymes in which this domain is found. Here we report the functional analysis of these domains of GlnD from Escherichia coli and Rhodospirillum rubrum. We confirm the assignment of UTase activity to the NT domain and show that the UR activity is a property specifically of the HD domain: substitutions in this domain eliminated UR activity, and a truncated protein lacking the NT domain displayed UR activity. The deletion of C-terminal ACT domains had little effect on UR activity itself but eliminated the ability of glutamine to stimulate that activity, suggesting a role for glutamine sensing by these domains. The deletion of C-terminal ACT domains also dramatically decreased UTase activity under all conditions tested, but some of these effects are due to the competition of UTase activity with unregulated UR activity in these variants.

Roles of the heme and heme ligands in the activation of CooA, the CO-sensing transcriptional activator.

CooA of Rhodospirillum rubrum is a CO-sensing, heme-containing transcriptional activator that regulates the expression of the genes responsible for CO oxidation. We randomized the codons for residues 75-77 of CooA which include two proximal heme ligands, screened for both CO-dependent and CO-independent variants, and characterized in vivo and in vitro properties of selected CooA variants. The analysis showed that small residues at position 75 are critical and that, as previously suspected, His77 is absolutely necessary for CO responsiveness of CooA. Many hemeless variants altered at those residues were found to be constitutively active. We propose that proximal heme pocket residues of wild-type CooA have important role in stabilizing both active and inactive heme positions for its CO-sensing function.

The poor growth of Rhodospirillum rubrum mutants lacking PII proteins is due to an excess of glutamine synthetase activity.

The P(II) family of proteins is found in all three domains of life and serves as a central regulator of the function of proteins involved in nitrogen metabolism, reflecting the nitrogen and carbon balance in the cell. The genetic elimination of the genes encoding these proteins typically leads to severe growth problems, but the basis of this effect has been unknown except with Escherichia coli. We have analysed a number of the suppressor mutations that correct such growth problems in Rhodospirillum rubrum mutants lacking P(II) proteins. These suppressors map to nifR3, ntrB, ntrC, amtB(1) and the glnA region and all have the common property of decreasing total activity of glutamine synthetase (GS). We also show that GS activity is very high in the poorly growing parental strains lacking P(II) proteins. Consistent with this, overexpression of GS in glnE mutants (lacking adenylyltransferase activity) also causes poor growth. All of these results strongly imply that elevated GS activity is the causative basis for the poor growth seen in R. rubrum mutants lacking P(II) and presumably in mutants of some other organisms with similar genotypes. The result underscores the importance of proper regulation of GS activity for cell growth.

Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum.

The AmtB protein transports uncharged NH(3) into the cell, but it also interacts with the nitrogen regulatory protein P(II), which in turn regulates a variety of proteins involved in nitrogen fixation and utilization. Three P(II) homologues, GlnB, GlnK and GlnJ, have been identified in the photosynthetic bacterium Rhodospirillum rubrum, and they have roles in at least four overlapping and distinct functions, one of which is the post-translational regulation of nitrogenase activity. In R. rubrum, nitrogenase activity is tightly regulated in response to addition or energy depletion (shift to darkness), and this regulation is catalysed by the post-translational regulatory system encoded by draTG. Two amtB homologues, amtB(1) and amtB(2), have been identified in R. rubrum, and they are linked with glnJ and glnK, respectively. Mutants lacking AmtB(1) are defective in their response to both addition and darkness, while mutants lacking AmtB(2) show little effect on the regulation of nitrogenase activity. These responses to darkness and appear to involve different signal transduction pathways, and the poor response to darkness does not seem to be an indirect result of perturbation of internal pools of nitrogen. It is also shown that AmtB(1) is necessary to sequester detectable amounts GlnJ to the cell membrane. These results suggest that some element of the AmtB(1)-P(II) regulatory system senses energy deprivation and a consistent model for the integration of nitrogen, carbon and energy signals by P(II) is proposed. Other results demonstrate a degree of specificity in interaction of AmtB(1) with the different P(II) homologues in R. rubrum. Such interaction specificity might be important in explaining the way in which P(II) proteins regulate processes involved in nitrogen acquisition and utilization.

The role of the DNA-binding domains in CooA activation.

Carbon monoxide oxidation activator protein (CooA) is a dimeric carbon monoxide (CO) binding transcription factor that in the presence of CO promotes the transcription of genes involved in CO oxidation in Rhodospirillum rubrum. The off state (inactive) of Fe(II) CooA has His and Pro as the two axial heme ligands. In contrast, in the on state, which is active in DNA binding, the Pro ligand bond has been replaced by CO. This occurs by the transient loss of the Pro ligand, thus generating a pentacoordinate heme that can bind CO. The active on state of CooA has two major structural differences from the off state, in addition to the displacement of the Pro ligand by CO. There is a repositioning of long C helices at the dimer interface and a concomitant reorientation of the hinge region between the DNA- and effector-binding domains within each monomer [Roberts et al. (2005) J. Inorg. Biochem. 99, 280-292]. To better understand the relationship of these conformational changes, we have removed the DNA-binding domains and compared CO binding to the truncated and full-length protein. The crystal structure of truncated Fe(II) CooA is the same as that of the effector-binding domain of full-length Fe(II) CooA, including the relative orientation of the homodimer and the ligation environment of the heme. Thus, removing the DNA-binding domains has little obvious effect on the structure of CooA in the inactive off state. However, CO binds about 2-fold more tightly and about 10 times more rapidly to truncated CooA. The rate of CO binding is known to be limited by the dissociation of the Pro heme ligand [Puranik et al. (2004) J. Biol. Chem. 279, 21096-21108]. Therefore, the absence of the DNA-binding domain makes it easier for the Pro ligand to dissociate from the heme iron, which also makes it easier for truncated CooA to adopt the on-state structure.

Identification of Rhodospirillum rubrum GlnB variants that are altered in their ability to interact with different targets in response to nitrogen status signals.

In Rhodospirillum rubrum, NifA, the transcriptional activator for the nif genes, is posttranslationally activated only by the uridylylated form of GlnB, one of three P(II) homologs in the organism. We have used the yeast two-hybrid system to detect variants of GlnB that interact better with NifA than does wild-type GlnB. When examined for physiological effects in R. rubrum, these GlnB* variants activated NifA in the presence of NH(4)(+), which normally blocks NifA activation completely, and in the absence of GlnD, whose uridylylation of GlnB is also normally essential for NifA activation. When these variants were tested in the two-hybrid system for their interaction with NtrB, a receptor that should interact with the nonuridylylated form of GlnB, they were uniformly weaker than wild-type GlnB in that interaction. When expressed in R. rubrum either as single-copy integrants or on multiple-copy plasmids, these variants were also dramatically altered in terms of their ability to regulate several other receptors involved in nitrogen metabolism, including GlnE, NtrB/NtrC, and DRAT (dinitrogenase reductase ADP-ribosyl transferase)-DRAG (dinitrogenase reductase-activating glycohydrolase). The consistent pattern throughout is that these GlnB variants partially mimic the uridylylated form of wild-type GlnB, even under nitrogen-excess conditions and in strains lacking GlnD. The results suggest that the role of uridylylation of GlnB is primarily to shift the equilibrium of GlnB from a "nitrogen-sufficient" form to a "nitrogen-deficient" form, each of which interacts with different but overlapping receptor proteins in the cell. These GlnB variants apparently shift that equilibrium through direct structural changes.

Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein.

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.

Dual roles of an E-helix residue, Glu167, in the transcriptional activator function of CooA.

CooA is a transcriptional activator that mediates CO-dependent expression of the genes responsible for CO oxidation in Rhodospirillum rubrum. In this study, we suggest in vitro and in vivo models explaining an unusual requirement of CooA for millimolar levels of divalent cations for high-affinity DNA binding. Several lines of evidence indicate that an E-helix residue, Glu167, plays a central role in this requirement by inhibiting sequence-specific DNA binding via charge repulsion in the absence of any divalent cation and that divalent cations relieve such repulsion in the process of DNA binding by CooA. Unexpectedly, the Glu167 residue is the optimal residue for in vivo transcriptional activity of CooA. We present a model in which the Glu167 from the downstream subunit of CooA helps the protein to interact with RNA polymerase, probably through an interaction between activating region 3 and sigma subunit. The study was further extended to a homologous protein, cyclic AMP receptor protein (CRP), which revealed similar, but not identical, roles of the residue in this protein as well. The results show a unique mechanism of CooA modulating its DNA binding and transcriptional activation in response to divalent cations among the CRP/FNR (fumarate and nitrate reductase activator protein) superfamily of regulators.

CooA, a paradigm for gas sensing regulatory proteins.

The heme-containing transcriptional factor CooA regulates the expression of genes involved in the oxidation of carbon monoxide (CO) in the bacterium Rhodospirillum rubrum. CooA is both a redox sensor and a specific CO sensor, a combination of properties that is unique among heme proteins. Extensive biochemical and genetic analyses, interpreted in the context of a crystal structure of one form of the protein, have allowed the creation of hypotheses concerning the mechanism of CooA activation by CO as well as the basis for its CO specificity. The article details the data in support of these hypotheses and indicates future lines of research.

Functionally critical elements of CooA-related CO sensors.

CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. We have identified a number of CooA homologs through database searches, expressed these heterologously in Escherichia coli, and monitored their ability to respond to CO in vivo. Further in vitro analysis of two CooA homologs from Azotobacter vinelandii and Carboxydothermus hydrogenoformans corroborated the in vivo data by revealing the ability of CO to bind to these hemoproteins and stimulate their binding at specific DNA sequences. These data, as well as the patterns of conserved residues in the homologs, are compared to what is already known about functionally important residues in the CooA protein of R. rubrum. The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins.