Allen K. Bourdon

Exogenous Fatty Acids Protect Enterococcus faecalis from Daptomycin-Induced Membrane Stress Independently of the Response Regulator LiaR

ABSTRACT Enterococcus faecalis is a commensal bacterium of the gastrointestinal tract that can cause nosocomial infections in immunocompromised humans. The hallmarks of this organism are its ability to survive in a variety of stressful habitats and, in particular, its ability to withstand membrane damage. One strategy used by E. faecalis to protect itself from membrane-damaging agents, including the antibiotic daptomycin, involves incorporation of exogenous fatty acids from bile or serum into the cell membrane. Additionally, the response regulator LiaR (a member of the LiaFSR [lipid II-interacting antibiotic response regulator and sensor] system associated with cell envelope stress responses) is required for the basal level of resistance E. faecalis has to daptomycin-induced membrane damage. This study aimed to determine if membrane fatty acid changes could provide protection against membrane stressors in a LiaR-deficient strain of E. faecalis. We noted that despite the loss of LiaR, the organism readily incorporated exogenous fatty acids into its membrane, and indeed growth in the presence of exogenous fatty acids increased the survival of LiaR-deficient cells when challenged with a variety of membrane stressors, including daptomycin. Combined, our results suggest that E. faecalis can utilize both LiaR-dependent and -independent mechanisms to protect itself from membrane damage. IMPORTANCE Enterococcus faecalis is responsible for a significant number of nosocomial infections. Worse, many of the antibiotics used to treat E. faecalis infection are no longer effective, as this organism has developed resistance to them. The drug daptomycin has been successfully used to treat some of these resistant strains; however, daptomycin-resistant isolates have been identified in hospitals. Many daptomycin-resistant isolates are found to harbor mutations in the genetic locus liaFSR, which is involved in membrane stress responses. Another mechanism shown to increase tolerance to daptomycin involves the incorporation of exogenous fatty acids from host fluids like serum or bile. This improved tolerance was found to be independent of liaFSR and suggests that there are additional ways to impact sensitivity to daptomycin. Thus, further studies are needed to understand how host fatty acid sources can influence antibiotic susceptibility.

Purinyl-cobamide is a native prosthetic group of reductive dehalogenases

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules with essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon–halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base, and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

Metabolomics reveals distinct neurochemical profiles associated with stress resilience

Acute social defeat represents a naturalistic form of conditioned fear and is an excellent model in which to investigate the biological basis of stress resilience. While there is growing interest in identifying biomarkers of stress resilience, until recently, it has not been feasible to associate levels of large numbers of neurochemicals and metabolites to stress-related phenotypes. The objective of the present study was to use an untargeted metabolomics approach to identify known and unknown neurochemicals in select brain regions that distinguish susceptible and resistant individuals in two rodent models of acute social defeat. In the first experiment, male mice were first phenotyped as resistant or susceptible. Then, mice were subjected to acute social defeat, and tissues were immediately collected from the ventromedial prefrontal cortex (vmPFC), basolateral/central amygdala (BLA/CeA), nucleus accumbens (NAc), and dorsal hippocampus (dHPC). Ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS) was used for the detection of water-soluble neurochemicals. In the second experiment, male Syrian hamsters were paired in daily agonistic encounters for 2 weeks, during which they formed stable dominant-subordinate relationships. Then, 24 h after the last dominance encounter, animals were exposed to acute social defeat stress. Immediately after social defeat, tissue was collected from the vmPFC, BLA/CeA, NAc, and dHPC for analysis using UPLC-HRMS. Although no single biomarker characterized stress-related phenotypes in both species, commonalities were found. For instance, in both model systems, animals resistant to social defeat stress also show increased concentration of molecules to protect against oxidative stress in the NAc and vmPFC. Additionally, in both mice and hamsters, unidentified spectral features were preliminarily annotated as potential targets for future experiments. Overall, these findings suggest that a metabolomics approach can identify functional groups of neurochemicals that may serve as novel targets for the diagnosis, treatment, or prevention of stress-related mental illness.

MSDC-0160 and MSDC-0602 Binding with Human Mitochondrial Pyruvate Carrier (MPC) 1 and 2 Heterodimer: PPARγ Activating and Sparing TZDs as Therapeutics

Themitochondrialpyruvatecarrier(MPC)isanoveltargetfortherapeuticdrugstotreatAlzheimer’s andParkinson’sdisease, diabetesmellitus, andnon-alcoholic steatohepatitis (NASH).Metabolic SolutionsDevelopmentCompany(MSDC)hastwothiazolidinediones,MSDC-0160andMSDC-0602, inthepipeline.ThisreportdescribesresultsforaMPC1/2heterodimerhomologymodel.TheFASTA sequencesforMPC1andMPC2wereaccessedfromUniProtandsubmittedtoRaptorX,resultingin bestcandidatemonomeric“proteindatabase”filesforeach.OnemutantformofMPC1,L36I,was alsoprocessed.TheseweresubmittedtoPyDocktogeneratebestcandidateMPC1/2heterodimer modelsthatwereusedforliganddockinganalyseswithAutoDockVinaand“RosettaOnlineServer thatIncludesEveryone”(ROSIE).Multiplebindingsitesforpyruvateandbothdrugswerefoundon bothMPC1andMPC2subunitswithdrugshavingnearlydoubletheaffinityineachcaseexceptthe intermediateandopen-instatesfortheL36Imutanttransporter. KeywoRDS Protein Homology Modeling, Inhibitor Docking, L36I Mutant, Michael Addition, MPC1, MPC2, Protein Protein Docking, Pyruvate, Single Nucleotide Polymorphism, Thiohemiacetal, UK-5099, Wild Type

Modeling non-clinical and clinical drug tests in Gaucher disease

There is need for modeling biological systems to accelerate drug pipelines for treating metabolic diseases. The eliglustat treatment for Gaucher disease is approved by the FDA with a companion genomic test. The Transcriptome-To-Metabolome™ biosimulation technology was used to model, in silico, a standard non-clinical eliglustat test with an in vitro canine kidney cell system over-expressing a human gene; and a clinical test using human fibroblasts from control and Gaucher disease subjects. Protein homology modeling and docking studies were included to gather affinity parameters for the kinetic metabolic model. Pharmacodynamics and metabolomics analyses of the results replicated published findings and demonstrated that processing and transport of lysosomal proteins alone cannot explain the metabolic disorder. This technology shows promise for application to other diseases.

Novobiocin and Peptide Analogs of α-factor are Positive Allosteric Modulators of the Yeast G Protein-Coupled Receptor Ste2p

G protein-coupled receptors (GPCRs) are the target of many drugs prescribed for human medicine and are therefore the subject of intense study. It has been recognized that compounds called allosteric modulators can regulate GPCR activity by binding to the receptor at sites distinct from, or overlapping with, that occupied by the orthosteric ligand. The purpose of this study was to investigate the nature of the interaction between putative allosteric modulators and Ste2p, a model GPCR expressed in the yeast Saccharomyces cerevisiae that binds the tridecapeptide mating pheromone α-factor. Biological assays demonstrated that an eleven amino acid α-factor analog and the antibiotic novobiocin were positive allosteric modulators of Ste2p. Both compounds enhanced the biological activity of α-factor, but did not compete with α-factor binding to Ste2p. To determine if novobiocin and the 11-mer shared a common allosteric binding site, a biologically-active analog of the 11-mer peptide ([Bio-DOPA]11-mer) was chemically cross-linked to Ste2p in the presence and absence of novobiocin. Immunoblots probing for the Ste2p-[Bio-DOPA]11-mer complex revealed that novobiocin markedly decreased cross-linking of the [Bio-DOPA]11-mer to the receptor, but cross-linking of the α-factor analog [Bio-DOPA]13-mer, which interacts with the orthosteric binding site of the receptor, was minimally altered. This finding suggests that both novobiocin and [Bio-DOPA]11-mer compete for an allosteric binding site on the receptor. These results indicate that Ste2p may provide an excellent model system for studying allostery in a GPCR.

Metabolomic analysis of mouse prefrontal cortex reveals upregulated analytes during wakefulness compared to sleep

By identifying endogenous molecules in brain extracellular fluid metabolomics can provide insight into the regulatory mechanisms and functions of sleep. Here we studied how the cortical metabolome changes during sleep, sleep deprivation and spontaneous wakefulness. Mice were implanted with electrodes for chronic sleep/wake recording and with microdialysis probes targeting prefrontal and primary motor cortex. Metabolites were measured using ultra performance liquid chromatography-high resolution mass spectrometry. Sleep/wake changes in metabolites were evaluated using partial least squares discriminant analysis, linear mixed effects model analysis of variance, and machine-learning algorithms. More than 30 known metabolites were reliably detected in most samples. When used by a logistic regression classifier, the profile of these metabolites across sleep, spontaneous wake, and enforced wake was sufficient to assign mice to their correct experimental group (pair-wise) in 80–100% of cases. Eleven of these metabolites showed significantly higher levels in awake than in sleeping mice. Some changes extend previous findings (glutamate, homovanillic acid, lactate, pyruvate, tryptophan, uridine), while others are novel (D-gluconate, N-acetyl-beta-alanine, N-acetylglutamine, orotate, succinate/methylmalonate). The upregulation of the de novo pyrimidine pathway, gluconate shunt and aerobic glycolysis may reflect a wake-dependent need to promote the synthesis of many essential components, from nucleic acids to synaptic membranes.

Gibbs free-energy gradient along the path of glucose transport through human glucose transporter 3

Fourteen glucose transporters (GLUTs) play essential roles in human physiology by facilitating glucose diffusion across the cell membrane. Due to its central role in the energy metabolism of the central nervous system, GLUT3 has been thoroughly investigated. However, the Gibbs free-energy gradient (what drives the facilitated diffusion of glucose) has not been mapped out along the transport path. Some fundamental questions remain. Here we present a molecular dynamics study of GLUT3 embedded in a lipid bilayer to quantify the free-energy profile along the entire transport path of attracting a β-d-glucose from the interstitium to the inside of GLUT3 and, from there, releasing it to the cytoplasm by Arrhenius thermal activation. From the free-energy profile, we elucidate the unique Michaelis–Menten characteristics of GLUT3, low KM and high VMAX, specifically suitable for neurons’ high and constant demand of energy from their low-glucose environments. We compute GLUT3’s binding free energy for β-d-glucose to be −4.6 kcal/mol in agreement with the experimental value of −4.4 kcal/mol (KM = 1.4 mM). We also compute the hydration energy of β-d-glucose, −18.0 kcal/mol vs the experimental data, −17.8 kcal/mol. In this, we establish a dynamics-based connection from GLUT3’s crystal structure to its cellular thermodynamics with quantitative accuracy. We predict equal Arrhenius barriers for glucose uptake and efflux through GLUT3 to be tested in future experiments.

Alzheimer's and Parkinson's Disease Novel Therapeutic Target: The Mitochondrial Pyruvate Carrier - Ligand Docking to Screen Natural Compounds Related to Classic Inhibitors

Thiazolidinedione (TZD) drugs (Takeda Pharmaceuticals and Metabolic Solutions Development Company) targeting inhibition of the mitochondrial pyruvate carrier (MPC) are currently being tested in clinical trials to prevent progression into mild cognitive impairment of Alzheimer’s disease (AD) or in the pipeline to prevent neurodegeneration in Parkinson’s disease (PD). These have Ki values in the μM range. This study was focused on identifying candidate drug precursors of the natural cinnamic acid products that might have good bioavailability in the nM ranges forming covalent thiol bonds with targets. In silico protein homology modeling and ligand docking has demonstrated that binding cysteine residues within the transport channel is a key part of the inhibitory mechanism. These are covalent thiohemiacetal bonds with the alpha-carbon, carboxylate group, off a phenol ring. Like the classic MPC inhibitors, these natural derivatives of hydroxycinnamic acid have a conjugated pi-system used to form thiol bonds with the cysteine residue via Michael addition. Alzheimer’s and Parkinson’s Disease Novel Therapeutic Target: The Mitochondrial Pyruvate Carrier Ligand Docking to Screen Natural Compounds Related to Classic Inhibitors

In Silico Biosimulation of Isoflurane Effects on Brain Using Transcriptome-To-MetabolomeTM Technology: Anesthesia Effects on Rat Amygdala & Cortex Metabolism

Anesthetics are a widely used class of drugs with a fast onset and comparatively slow offset, which consequently equates to a low therapeutic index. Unfortunately, the mechanism of action for this class of drugs is considered unknown. For that reason, there is great medical need to study effects of anesthetics on the brain. In this study transcriptomes, generated 6 hours after a 15 minute exposure to isoflurane, from the rat cortex and basolateral amygdala were used to determine parameters for a deterministic biosimulation model of metabolic pathways. Metabolomic results indicated involvement of lipid pathways known for anesthetic effects on membrane function and alternate energy sources due to reduced glucose utilization. Key insights are revealed for potential mechanisms by which anesthetics block memory of the medical procedures.