New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis

Background

In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target.

Methodology/Principal Findings

To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the ‘hits’ for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells.

Conclusions/Significance

This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.     

Molecular Basis of Vancomycin Dependence in VanA-Type Staphylococcus aureus VRSA-9

The vancomycin-resistant Staphylococcus aureus VRSA-9 clinical isolate was partially dependent on glycopeptide for growth. The responsible vanA operon had the same organization as that of Tn1546 and was located on a plasmid. The chromosomal d-Ala:d-Ala ligase (ddl) gene had two point mutations that led to Q260K and A283E substitutions, resulting in a 200-fold decrease in enzymatic activity compared to that of the wild-type strain VRSA-6. To gain insight into the mechanism of enzyme impairment, we determined the crystal structure of VRSA-9 Ddl and showed that the A283E mutation induces new ion pair/hydrogen bond interactions, leading to an asymmetric rearrangement of side chains in the dimer interface. The Q260K substitution is located in an exposed external loop and did not induce any significant conformational change. The VRSA-9 strain was susceptible to oxacillin due to synthesis of pentadepsipeptide precursors ending in d-alanyl-d-lactate which are not substrates for the β-lactam-resistant penicillin binding protein PBP2′. Comparison with the partially vancomycin-dependent VRSA-7, whose Ddl is 5-fold less efficient than that of VRSA-9, indicated that the levels of vancomycin dependence and susceptibility to β-lactams correlate with the degree of Ddl impairment. Ddl drug targeting could therefore be an effective strategy against vancomycin-resistant S. aureus.Methicillin-resistant Staphylococcus aureus (MRSA) bacteria that have acquired the vancomycin resistance vanA operon from glycopeptide-resistant enterococci are designated vancomycin-resistant S. aureus (VRSA) (29). Vancomycin acts by binding to the C-terminal acyl-d-Ala-d-Ala of the undecaprenol-diphosphate MurNAc-pentapeptide intermediate and inhibits transglycosylation and transpeptidation reactions in cell wall peptidoglycan polymerization and cross-linking (30). d-Ala-d-Ala is synthesized by the ATP-dependent d-Ala:d-Ala ligase (Ddl) (EC 6.3.2.4) before its incorporation in peptidoglycan precursors (26, 35). VanA-type vancomycin resistance results from the incorporation into peptidoglycan intermediates of a d-alanyl-d-lactate (d-Ala-d-Lac) depsipeptide, synthesized by a d-Ala:d-Lac ligase, which is responsible for diminished binding affinity of glycopeptides for their target. Kinetic analyses of Ddls have established two subsites in the active site for d-Ala binding (24, 27). The reaction mechanism culminates in the transfer of the γ-phosphoryl of ATP to the carboxyl group of d-Ala₁ to produce an acylphosphate and ADP. The acyl carbon atom of the acylphosphate then reacts with the amino group of d-Ala₂ to yield a tetrahedral intermediate. Finally, the intermediate releases phosphate to yield d-Ala-d-Ala.Mutants of Enterococcus faecium (8, 14), Enterococcus faecalis (34), and S. aureus (23) with an impaired Ddl are able to grow because they use the vancomycin resistance pathway for cell wall synthesis. Since resistance is inducible by the drug, these bacteria require the presence of vancomycin in the culture medium for growth. Ddls from vancomycin-dependent enterococci (14) have mutations affecting amino acids highly conserved in the d-Ala:d-Ala ligase superfamily (10). Molecular modeling based on the X-ray structure of Escherichia coli DdlB (11) revealed that all the mutated residues interact directly with one of the substrates of the enzymatic reaction or stabilize the position of critical residues in the active site. However, the degree of enzyme impairment was not evaluated biochemically. Recently, we reported the mechanism of vancomycin dependence in VanA-type S. aureus VRSA-7 and showed that the chromosomal Ddl had the single mutation N308K, which probably affects the binding of the transition-state intermediate, leading to a 1,000-fold decrease in activity relative to that of the wild-type enzyme (23). Glycopeptide-dependent mutants could therefore be considered useful tools to explore structure-activity relationships of the Ddl, which represents an attractive target for designing new drugs. Here we describe the partially vancomycin-dependent VanA-type S. aureus strain VRSA-9 and report the biochemical and structural characterization of its mutated Ddl.     

Efficient Production of (R)-2-Hydroxy-4-Phenylbutyric Acid by Using a Coupled Reconstructed d-Lactate Dehydrogenase and Formate Dehydrogenase System

Background

(R)-2-Hydroxy-4-phenylbutyric acid [(R)-HPBA] is a key precursor for the production of angiotensin-converting enzyme inhibitors. However, the product yield and concentration of reported (R)-HPBA synthetic processes remain unsatisfactory.

Methodology/Principal Findings

The Y52L/F299Y mutant of NAD-dependent d-lactate dehydrogenase (d-nLDH) in Lactobacillus bulgaricus ATCC 11842 was found to have high bio-reduction activity toward 2-oxo-4-phenylbutyric acid (OPBA). The mutant d-nLDH^Y52L/F299Y was then coexpressed with formate dehydrogenase in Escherichia coli BL21 (DE3) to construct a novel biocatalyst E. coli DF. Thus, a novel bio-reduction process utilizing whole cells of E. coli DF as the biocatalyst and formate as the co-substrate for cofactor regeneration was developed for the production of (R)-HPBA from OPBA. The biocatalysis conditions were then optimized.

Conclusions/Significance

Under the optimum conditions, 73.4 mM OPBA was reduced to 71.8 mM (R)-HPBA in 90 min. Given its high product enantiomeric excess (>99%) and productivity (47.9 mM h⁻¹), the constructed coupling biocatalysis system is a promising alternative for (R)-HPBA production.     

Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth

Background

cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/Principal Finding

The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/Significance

Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.     

A Novel Whole-Cell Biocatalyst with NAD+ Regeneration for Production of Chiral Chemicals

Background

The high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)⁺ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.

Methodology/Principal Findings

Simultaneous overexpression of an NAD⁺ dependent enzyme and an NAD⁺ regenerating enzyme (H₂O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD⁺-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.

Conclusions/Significance

A recombinant strain, in which an NAD⁺ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)⁺ for catalysis.     

Halothiobacillus neapolitanus carboxysomes sequester heterologous and chimeric RubisCO species

Background

The carboxysome is a bacterial microcompartment that consists of a polyhedral protein shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme that catalyzes the first step of CO₂ fixation via the Calvin-Benson-Bassham cycle.

Methodology/Principal Findings

To analyze the role of RubisCO in carboxysome biogenesis in vivo we have created a series of Halothiobacillus neapolitanus RubisCO mutants. We identified the large subunit of the enzyme as an important determinant for its sequestration into α-carboxysomes and found that the carboxysomes of H. neapolitanus readily incorporate chimeric and heterologous RubisCO species. Intriguingly, a mutant lacking carboxysomal RubisCO assembles empty carboxysome shells of apparently normal shape and composition.

Conclusions/Significance

These results indicate that carboxysome shell architecture is not determined by the enzyme they normally sequester. Our study provides, for the first time, clear evidence that carboxysome contents can be manipulated and suggests future nanotechnological applications that are based upon engineered protein microcompartments.     

Microbiology of Urinary Tract Infections in Gaborone,Botswana

Objective

The microbiology and epidemiology of UTI pathogens are largely unknown in Botswana, a high prevalence HIV setting. Using laboratory data from the largest referral hospital and a private hospital, we describe the major pathogens causing UTI and their antimicrobial resistance patterns.

Methods

This retrospective study examined antimicrobial susceptibility data for urine samples collected at Princess Marina Hospital (PMH), Bokamoso Private Hospital (BPH), or one of their affiliated outpatient clinics. A urine sample was included in our dataset if it demonstrated pure growth of a single organism and accompanying antimicrobial susceptibility and subject demographic data were available.

Results

A total of 744 samples were included. Greater than 10% resistance was observed for amoxicillin, co-trimoxazole, amoxicillin-clavulanate, and ciprofloxacin. Resistance of E. coli isolates to ampicillin and co-trimoxazole was greater than 60% in all settings. HIV status did not significantly impact the microbiology of UTIs, but did impact antimicrobial resistance to co-trimoxazole.

Conclusions

Data suggests that antimicrobial resistance has already emerged to most oral antibiotics, making empiric management of outpatient UTIs challenging. Ampicillin, co-trimoxazole, and ciprofloxacin should not be used as empiric treatment for UTI in this context. Nitrofurantoin could be used for simple cystitis; aminoglycosides for uncomplicated UTI in inpatients.     

Microbial-resistant Salmonella enteritidis isolated from poultry samples

Background:

Multidrug resistance in Salmonella enteritidis isolates is a public health problem worldwide; the present study, therefore, was designed for antimicrobial-resistance determination in this strain.

Methods:

Salmonella strains isolated from poultry samples by biochemical positive and negative tests were subjected to PCR and identified as Salmonella enteritidis. For detection and identification of Salmonella enteritidis isolates, sdfI gene-specific primers were used.

Results:

We found that 100% of isolates were resistant to ampicillin, 90% were resistant to cephalothin and streptomycin, 70% were resistant to cefotaxime, and 60% were resistant to kanamycin and gentamicin.

Conclusion:

Salmonella enteritidis isolates had antimicrobial resistance to mentioned antibiotics. Key Words: Antibiotic Resistance, PCR, Poultry, Salmonella enteritidis     

Crystallographic and molecular dynamics analysis of loop motions unmasking the peptidoglycan-binding site in stator protein MotB of flagellar motor

Background

The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues.

Methodology/Principal Findings

We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan.

Conclusion/Significance

Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall.     

Self-guided psychological treatment for depressive symptoms: a meta-analysis

Background

A number of trials have examined the effects of self-guided psychological intervention, without any contact between the participants and a therapist or coach. The results and sizes of these trials have been mixed. This is the first quantitative meta-analysis, aimed at organizing and evaluating the literature, and estimating effect size.

Method

We conducted systematic literature searches in PubMed, PsycINFO and Embase up to January 2010, and identified additional studies through earlier meta-analyses, and the references of included studies. We identified seven randomized controlled trials that met our inclusion criteria, with a total of 1,362 respondents. The overall quality of the studies was high. A post-hoc power calculation showed that the studies had sufficient statistical power to detect an effect size of d = 0.19.

Results

The overall mean effect size indicating the difference between self-guided psychological treatment and control groups at post-test was d = 0.28 (p<0.001), which corresponds to a NNT of 6.41. At 4 to 12 months follow-up the effect size was d = 0.23. There was no indication for significant publication bias.

Conclusions

We found evidence that self-guided psychological treatment has a small but significant effect on participants with increased levels of depressive symptomatology.     

Nanoparticle-Encapsulated Chlorhexidine against Oral Bacterial Biofilms

Background

Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms.

Methodology/Principal Findings

The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50–200 µg/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h.

Conclusions/Significance

This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.     

The Influence of the Patient-Clinician Relationship on Healthcare Outcomes: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Objective

To determine whether the patient-clinician relationship has a beneficial effect on either objective or validated subjective healthcare outcomes.

Design

Systematic review and meta-analysis.

Data Sources

Electronic databases EMBASE and MEDLINE and the reference sections of previous reviews.

Eligibility Criteria for Selecting Studies

Included studies were randomized controlled trials (RCTs) in adult patients in which the patient-clinician relationship was systematically manipulated and healthcare outcomes were either objective (e.g., blood pressure) or validated subjective measures (e.g., pain scores). Studies were excluded if the encounter was a routine physical, or a mental health or substance abuse visit; if the outcome was an intermediate outcome such as patient satisfaction or adherence to treatment; if the patient-clinician relationship was manipulated solely by intervening with patients; or if the duration of the clinical encounter was unequal across conditions.

Results

Thirteen RCTs met eligibility criteria. Observed effect sizes for the individual studies ranged from d = −.23 to .66. Using a random-effects model, the estimate of the overall effect size was small (d = .11), but statistically significant (p = .02).

Conclusions

This systematic review and meta-analysis of RCTs suggests that the patient-clinician relationship has a small, but statistically significant effect on healthcare outcomes. Given that relatively few RCTs met our eligibility criteria, and that the majority of these trials were not specifically designed to test the effect of the patient-clinician relationship on healthcare outcomes, we conclude with a call for more research on this important topic.     

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Methods

We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.

Results

In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC₅₀ concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.

Conclusion

Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.     

The Effectiveness of Internet Cognitive Behavioural Therapy (iCBT) for Depression in Primary Care: A Quality Assurance Study

Background

Depression is a common, recurrent, and debilitating problem and Internet delivered cognitive behaviour therapy (iCBT) could offer one solution. There are at least 25 controlled trials that demonstrate the efficacy of iCBT. The aim of the current paper was to evaluate the effectiveness of an iCBT Program in primary care that had been demonstrated to be efficacious in two randomized controlled trials (RCTs).

Method

Quality assurance data from 359 patients prescribed the Sadness Program in Australia from October 2010 to November 2011 were included.

Results

Intent-to-treat marginal model analyses demonstrated significant reductions in depressive symptoms (PHQ9), distress (K10), and impairment (WHODAS-II) with medium-large effect sizes (Cohen''s d = .51–1.13.), even in severe and/or suicidal patients (Cohen''s d = .50–1.49.) Secondary analyses on patients who completed all 6 lessons showed levels of clinically significant change as indexed by established criteria for remission, recovery, and reliable change.

Conclusions

The Sadness Program is effective when prescribed by primary care practitioners and is consistent with a cost-effective stepped-care framework.     

The effectiveness of online cognitive behavioral treatment in routine clinical practice

Context

Randomized controlled trails have identified online cognitive behavioral therapy as an efficacious intervention in the management of common mental health disorders.

Objective

To assess the effectiveness of online CBT for different mental disorders in routine clinical practice.

Design

An uncontrolled before-after study, with measurements at baseline, posttest, 6-week follow-up, and 1-year follow-up.

Participants & Setting

1500 adult patients (female: 67%; mean age: 40 years) with a GP referral for psychotherapy were treated at a Dutch online mental health clinic for symptoms of depression (n = 413), panic disorder (n = 139), posttraumatic stress (n = 478), or burnout (n = 470).

Interventions

Manualized, web-based, therapist-assisted CBT, of which the efficacy was previously demonstrated in a series of controlled trials. Standardized duration of treatment varied from 5 weeks (online CBT for Posttraumatic stress) to 16 weeks (online CBT for Depression).

Main Outcome Measures

Validated self-report questionnaires of specific and general psychopathology, including the Beck Depression Inventory, the Impact of Event Scale, the Panic Disorder Severity Scale-Self Report, the Oldenburg Burnout Inventory, and the Depression Anxiety Stress Scales.

Results

Treatment adherence was 71% (n = 1071). Study attrition was 21% at posttest, 33% at 6-week FU and 65% at 1-year FU. Mixed-model repeated measures regression identified large short-term reductions in all measures of primary symptoms (d = 1.9±0.2 to d = 1.2±0.2; P<.001), which sustained up to one year after treatment. At posttest, rates of reliable improvement and recovery were 71% and 52% in the completer sample (full sample: 55%/40%). Patient satisfaction was high.

Conclusions

Results suggest that online therapist-assisted CBT may be as effective in routine practice as it is in clinical trials. Although pre-treatment withdrawal and long-term outcomes require further study, results warrant continued implementation of online CBT.     

Lysozyme resistance in Streptococcus suis is highly variable and multifactorial

Background

Streptococcus suis is an important infectious agent for pigs and occasionally for humans. The host innate immune system plays a key role in preventing and eliminating S. suis infections. One important constituent of the innate immune system is the protein lysozyme, which is present in a variety of body fluids and immune cells. Lysozyme acts as a peptidoglycan degrading enzyme causing bacterial lysis. Several pathogens have developed mechanisms to evade lysozyme-mediated killing. In the present study we compared the lysozyme sensitivity of various S. suis isolates and investigated the molecular basis of lysozyme resistance for this pathogen.

Results

The lysozyme minimal inhibitory concentrations of a wide panel of S. suis isolates varied between 0.3 to 10 mg/ml. By inactivating the oatA gene in a serotype 2 and a serotype 9 strain, we showed that OatA-mediated peptidoglycan modification partly contributes to lysozyme resistance. Furthermore, inactivation of the murMN operon provided evidence that additional peptidoglycan crosslinking is not involved in lysozyme resistance in S. suis. Besides a targeted approach, we also used an unbiased approach for identifying factors involved in lysozyme resistance. Based on whole genome comparisons of a lysozyme sensitive strain and selected lysozyme resistant derivatives, we detected several single nucleotide polymorphisms (SNPs) that were correlated with the lysozyme resistance trait. Two SNPs caused defects in protein expression of an autolysin and a capsule sugar transferase. Analysis of specific isogenic mutants, confirmed the involvement of autolysin activity and capsule structures in lysozyme resistance of S. suis.

Conclusions

This study shows that lysozyme resistance levels are highly variable among S. suis isolates and serotypes. Furthermore, the results show that lysozyme resistance in S. suis can involve different mechanisms including OatA-mediated peptidolycan modification, autolysin activity and capsule production.     

Trypanosoma brucei glycogen synthase kinase-3, a target for anti-trypanosomal drug development: a public-private partnership to identify novel leads

Background

Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3β (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT.

Methodology/Principal Findings

A subset of over 16,000 inhibitors of HsGSK-3 β from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed.

Conclusions/Significance

We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.     

Intestine-Targeted DGAT1 Inhibition Improves Obesity and Insulin Resistance without Skin Aberrations in Mice

Objective

Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final committed step in triglyceride biosynthesis. DGAT1 null mice are known to be resistant to diet-induced obesity, and more insulin sensitive relative to the wild-type; however, the mice exhibit abnormalities in the skin. This work determined whether the intestine-targeted DGAT1 inhibitor could improve obesity and insulin resistance without skin aberrations in mice.

Design and Methods

We synthesized 2 DGAT1 inhibitors: Compound A, described in the patent application from the Japan Tobacco, and Compound B (A-922500), reported by Abbott Laboratories. Both compounds were evaluated for inhibitory activities against DGAT1 enzymes and effects on the skin in mice in vivo. Compound B was further investigated for effects on obesity and insulin resistance in diet-induced-obese (DIO) mice.

Results

The 2 compounds comparably inhibited the DGAT1 enzyme activity and the cellular triglyceride synthesis in vitro, while they showed different distribution patterns in mice in vivo. Compound A, which distributed systemically, caused skin aberrations, while Compound B, which preferentially distributed to the intestine, improved obesity and insulin resistance without skin aberrations in DIO mice.

Conclusions

Our results suggest that the intestine is the key tissue in which DGAT1 plays a role in promoting obesity and insulin resistance.