Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Structure-Permeability Relationship of Semipeptidic Macrocycles-Understanding and Optimizing Passive Permeability and Efflux Ratio.

We herein report the first thorough analysis of the structure-permeability relationship of semipeptidic macrocycles. In total, 47 macrocycles were synthesized using a hybrid solid-phase/solution strategy, and then their passive and cellular permeability was assessed using the parallel artificial membrane permeability assay (PAMPA) and Caco-2 assay, respectively. The results indicate that semipeptidic macrocycles generally possess high passive permeability based on the PAMPA, yet their cellular permeability is governed by efflux, as reported in the Caco-2 assay. Structural variations led to tractable structure-permeability and structure-efflux relationships, wherein the linker length, stereoinversion, N-methylation, and peptoids site-specifically impact the permeability and efflux. Extensive nuclear magnetic resonance, molecular dynamics, and ensemble-based three-dimensional polar surface area (3D-PSA) studies showed that ensemble-based 3D-PSA is a good predictor of passive permeability.

Fundamental aspects of DMPK optimization of targeted protein degraders.

Targeted protein degraders are an emerging modality. Their properties fall outside the traditional small-molecule property space and are in the 'beyond rule of 5' space. Consequently, drug discovery programs focused on developing orally bioavailable degraders are expected to face complex drug metabolism and pharmacokinetics (DMPK) challenges compared with traditional small molecules. Nevertheless, little information is available on the DMPK optimization of oral degraders. Therefore, in this review, we discuss our experience of these DMPK optimization challenges and present methodologies and strategies to overcome the hurdles dealing with this new small-molecule modality, specifically in developing oral degraders to treat cancer.

The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri.

BACKGROUND: Members of the genus Naegleria are free-living eukaryotes with the capability to transform from the amoeboid form into resting cysts or moving flagellates in response to environmental conditions. More than 40 species have been characterized, but only Naegleria fowleri (N. fowleri) is known as a human pathogen causing primary amoebic meningoencephalitis (PAM), a fast progressing and mostly fatal disease of the central nervous system. Several studies report an involvement of phospholipases and other molecular factors, but the mechanisms involved in pathogenesis are still poorly understood. To gain a better understanding of the relationships within the genus of Naegleria and to investigate pathogenicity factors of N. fowleri, we characterized the genome of its closest non-pathogenic relative N. lovaniensis. RESULTS: To gain insights into the taxonomy of Naegleria, we sequenced the genome of N. lovaniensis using long read sequencing technology. The assembly of the data resulted in a 30 Mb genome including the circular mitochondrial sequence. Unravelling the phylogenetic relationship using OrthoMCL protein clustering and maximum likelihood methods confirms the close relationship of N. lovaniensis and N. fowleri. To achieve an overview of the diversity of Naegleria proteins and to assess characteristics of the human pathogen N. fowleri, OrthoMCL protein clustering including data of N. fowleri, N. lovaniensis and N. gruberi was performed. GO enrichment analysis shows an association of N. fowleri specific proteins to the GO terms "Membrane" and "Protein Secretion." CONCLUSION: In this study, we characterize the hitherto unknown genome of N. lovaniensis. With the description of the 30 Mb genome, a further piece is added to reveal the complex taxonomic relationship of Naegleria. Further, the whole genome sequencing data confirms the hypothesis of the close relationship between N. fowleri and N. lovaniensis. Therefore, the genome of N. lovaniensis provides the basis for further comparative approaches on the molecular and genomic level to unravel pathogenicity factors of its closest human pathogenic relative N. fowleri and possible treatment options for the rare but mostly fatal primary meningoencephalitis.

Transporters in Drug Development: 2018 ITC Recommendations for Transporters of Emerging Clinical Importance.

This white paper provides updated International Transporter Consortium (ITC) recommendations on transporters that are important in drug development following the 3rd ITC workshop. New additions include prospective evaluation of organic cation transporter 1 (OCT1) and retrospective evaluation of organic anion transporting polypeptide (OATP)2B1 because of their important roles in drug absorption, disposition, and effects. For the first time, the ITC underscores the importance of transporters involved in drug-induced vitamin deficiency (THTR2) and those involved in the disposition of biomarkers of organ function (OAT2 and bile acid transporters).

ITC Commentary on Metformin Clinical Drug-Drug Interaction Study Design That Enables an Efficacy- and Safety-Based Dose Adjustment Decision.

Metformin drug-drug interaction (DDI) studies are conducted during development of drugs that inhibit organic cation transporters and/or multidrug and toxin extrusion proteins (OCTs/MATEs). Monitoring solely changes in systemic exposure, the typical DDI study endpoint appears inadequate for metformin, which is metabolically stable, has poor passive membrane permeability, and undergoes transporter-mediated tissue distribution and clearance. Evaluation of renal clearance, antihyperglycemic effects, and potentially lactate as an exploratory safety marker, can support rational metformin dose adjustment. The proposed DDI study design aims to adequately inform metformin dosing during comedication.

Computational Discovery and Experimental Validation of Inhibitors of the Human Intestinal Transporter OATP2B1.

Human organic anion transporters (OATPs) are vital for the uptake and efflux of drugs and endogenous compounds. Current identification of inhibitors of these transporters is based on experimental screening. Virtual screening remains a challenge due to a lack of experimental three-dimensional protein structures. Here, we describe a workflow to identify inhibitors of the OATP2B1 transporter in the DrugBank library of over 5,000 drugs and druglike molecules. OATP member 2B1 transporter is highly expressed in the intestine, where it participates in oral absorption of drugs. Predictions from a Random forest classifier, prioritized by docking against multiple comparative protein structure models of OATP2B1, indicated that 33 of the 5,000 compounds were putative inhibitors of OATP2B1. Ten predicted inhibitors that are prescription drugs were tested experimentally in cells overexpressing the OATP2B1 transporter. Three of these ten were validated as potent inhibitors of estrone-3-sulfate uptake (defined as more than 50% inhibition at 20 µM) and tested in multiple concentrations to determine exact IC50. The IC50 values of bicalutamide, ticagrelor, and meloxicam suggest that they might inhibit intestinal OATP2B1 at clinically relevant concentrations and therefore modulate the absorption of other concomitantly administered drugs.

Chemical shift assignment of the intrinsically disordered N-terminus and the rubredoxin domain in the folded metal bound and unfolded oxidized state of mycobacterial protein kinase G.

Mycobacterium tuberculosis protein kinase G (PknG) is a 82 kDa multidomain eukaryotic-like serine/threonine kinase mediating the survival of pathogenic mycobacteria within host macrophages. The N-terminal sequence preceding the catalytic kinase domain contains an approximately 75 residues long tail, which was predicted to show no regulatory secondary structure (1-75 = NORS) but harbors the major in vivo phosphorylation site (T63), and a rubredoxin-like metal binding motif (74-147 = RD). In the reduced rubredoxin motif, four conserved cysteine residues that are present as two C-X-X-C-G motifs coordinate a metal ion. The cysteines are further involved in sensing the redox environment to regulate PknG catalytic activity. Here, we report the (1)H, (13)C, and (15)N resonance assignments of the highly dynamic unstructured N-terminal region NORS and the RD in the reduced, metal bound, presumably folded and the oxidized, presumably unfolded state. Chemical shifts have been deposited at the BioMagResBank under the BMRB accession numbers 26,028 for the His-PknG1-147 with the RD in reduced, metal bound state, 26,027 for His-PknG1-75, and 26,030 and 26,029 for PknG74-147 either in the reduced, metal bound or oxidized state, respectively. The presented chemical shift assignments pave the route for the structural characterization of the regulation of PknG by redox changes and posttranslational modifications (phosphorylation).

A pharmacogenetic candidate gene study of tenofovir-associated Fanconi syndrome.

BACKGROUND: Tenofovir disoproxil fumarate (TDF) is a widely used antiretroviral agent with favorable efficacy, safety, and tolerability profiles. However, renal adverse events, including the rare Fanconi syndrome (FS), may occur in a small subset of patients treated for HIV infections. OBJECTIVES: The aim of this study was to identify genetic variants that may be associated with TDF-associated FS (TDF-FS). METHODS: DNA samples collected from 19 cases with TDF-FS and 36 matched controls were sequenced, and genetic association studies were conducted on eight candidate genes: ATP-binding cassette (ABC) transporters ABCC2 (MRP2) and ABCC4 (MRP4), solute carrier family members SLC22A6 (OAT1) and SLC22A8 (OAT3), adenylate kinases 2 (AK2) and 4 (AK4), chloride transporter CIC-5 CLCN5, and Lowe syndrome protein OCRL. The functional effects of a single nucleotide polymorphism (SNP) predicted to alter the transport of tenofovir were then investigated in cells expressing an identified variant of ABCC4. RESULTS: The case group showed a trend toward a higher proportion of rare alleles. Six SNPs in ABCC2 (three SNPs), ABCC4 (one SNP), and OCRL (two SNPs) were associated with TDF-FS case status; however, this association did not remain significant after correction for multiple testing. Six SNPs, present in OCRL (four SNPs) and ABCC2 (two SNPs), were significantly associated with increased serum creatinine levels in the cases, and this association remained significant after multiple test correction (P < 2 × 10). One synonymous SNP in ABCC2 (rs8187707, P = 2.10 × 10, ß = -73.3 ml/min/1.73 m(2)) was also significantly associated with the decreased estimated glomerular filtration rate of creatinine among cases. However, these results were driven by rare SNPs present in a small number of severely affected cases. Finally, a previously uncharacterized, nonsynonymous SNP, rs11568694, that was predicted to alter MRP4 function had no significant effect on tenofovir cellular accumulation in vitro. CONCLUSION: Although no single predictive genetic marker for the development of TDF-FS was identified, the findings from our study suggest that rare variants in multiple genes involved in the renal handling of tenofovir, and/or renal cell homeostasis, may be associated with increased susceptibility to TDF-FS.

Chemical development of intracellular protein heterodimerizers.

Cell activation initiated by receptor ligands or oncogenes triggers complex and convoluted intracellular signaling. Techniques initiating signals at defined starting points and cellular locations are attractive to elucidate the output of selected pathways. Here, we present the development and validation of a protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags. Chemical dimerizers of HaloTag and SNAP-tag (HaXS) show excellent selectivity and have been optimized for intracellular reactivity. HaXS force protein-protein interactions and can translocate proteins to various cellular compartments. Due to the covalent nature of the HaloTag-HaXS-SNAP-tag complex, intracellular dimerization can be easily monitored. First applications include protein targeting to cytoskeleton, to the plasma membrane, to lysosomes, the initiation of the PI3K/mTOR pathway, and multiplexed protein complex formation in combination with the rapamycin dimerization system.

Comparison of rapid methods for detection of Giardia spp. and Cryptosporidium spp. (oo)cysts using transportable instrumentation in a field deployment.

Reliable, sensitive, quantitative, and mobile rapid screening methods for pathogenic organisms are not yet readily available, but would provide a great benefit to humanitarian intervention units in disaster situations. We compared three different methods (immunofluorescent microscopy, IFM; flow cytometry, FCM; polymerase chain reaction, PCR) for the rapid and quantitative detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in a field campaign. For this we deployed our mobile instrumentation and sampled canal water and vegetables during a 2 week field study in Thailand. For purification and concentrations of (oo)cysts, we used filtration and immunomagnetic separation. We were able to detect considerably high oo(cysts) concentrations (ranges: 15-855 and 0-240 oo(cysts)/liter for Giardia and Cryptosporidium, respectively) in 85 to 300 min, with FCM being fastest, followed by PCR, and IFM being slowest due to the long analysis time per sample. FCM and IFM performed consistently well, whereas PCR reactions often failed. The recovery, established by FCM, was around 30% for Giardia and 13% for Cryptosporidium (oo)cysts. It was possible to track (oo)cysts from the wastewater further downstream to irrigation waters and confirm contamination of salads and water vegetables. We believe that rapid detection, in particular FCM-based methods, can substantially help in disaster management and outbreak prevention.

Hepatic gene expression profile in mice perorally infected with Echinococcus multilocularis eggs.

BACKGROUND: Alveolar echinococcosis (AE) is a severe chronic hepatic parasitic disease currently emerging in central and eastern Europe. Untreated AE presents a high mortality (>90%) due to a severe hepatic destruction as a result of parasitic metacestode proliferation which behaves like a malignant tumor. Despite this severe course and outcome of disease, the genetic program that regulates the host response leading to organ damage as a consequence of hepatic alveolar echinococcosis is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We used a mouse model of AE to assess gene expression profiles in the liver after establishment of a chronic disease status as a result of a primary peroral infection with eggs of the fox tapeworm Echinococcus multilocularis. Among 38 genes differentially regulated (false discovery rate adjusted p, while 3 associated with the functional group . Upregulated genes associated with could be clustered into functional subgroups including , , , and . Two downregulated genes related to and , respectively. The genes either associated with an or an pathway. From the overexpressed genes, 18 genes were subsequently processed with a Custom Array microfluidic card system in order to assess respective expression status at the mRNA level relative to 5 reference genes (Gapdh, Est1, Rlp3, Mdh-1, Rpl37) selected upon a constitutive and stable expression level. The results generated by the two independent tools used for the assessment of gene expression, i.e., microarray and microfluidic card system, exhibited a high level of congruency (Spearman correlation rho = 0.81, p = 7.87e-5) and thus validated the applied methods. CONCLUSIONS/SIGNIFICANCE: Based on this set of biomarkers, new diagnostic targets have been made available to predict disease status and progression. These biomarkers may also offer new targets for immuno-therapeutic intervention.