Discovery of a Novel Potent and Selective HSD17B13 Inhibitor, BI-3231, a Well-Characterized Chemical Probe Available for Open Science.

Genome-wide association studies in patients revealed HSD17B13 as a potential new target for the treatment of nonalcoholic steatohepatitis (NASH) and other liver diseases. However, the physiological function and the disease-relevant substrate of HSD17B13 remain unknown. In addition, no suitable chemical probe for HSD17B13 has been published yet. Herein, we report the identification of the novel potent and selective HSD17B13 inhibitor BI-3231. Through high-throughput screening (HTS), using estradiol as substrate, compound 1 was identified and selected for subsequent optimization resulting in compound 45 (BI-3231). In addition to the characterization of compound 45 for its functional, physicochemical, and drug metabolism and pharmacokinetic (DMPK) properties, NAD+ dependency was investigated. To support Open Science, the chemical HSD17B13 probe BI-3231 will be available to the scientific community for free via the opnMe platform, and thus can help to elucidate the pharmacology of HSD17B13.

Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis.

As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.

Chemical Derivatization Enables MALDI-TOF-Based High-Throughput Screening for Microbial Trimethylamine (TMA)-Lyase Inhibitors.

Microbial-dependent trimethylamine (TMA) generation from dietary precursors such as choline was recently linked to cardiovascular diseases (CVDs) as well as chronic kidney disease (CKD). Inhibition of TMA-generating enzymes in gut bacteria would be an innovative approach to treat these diseases. The potential to accurately quantify secreted TMA levels highlights the capacity of mass spectrometry (MS) for tracking microbial TMA-lyase activity. However, high-throughput screening (HTS) by conventional MS instrumentation is hampered by limited sample throughput. Recent advancement in liquid handling and instrumentation of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS provides an HTS-compatible MS technology. The deciphering of enzymatic reactions using this label-free readout has been successfully applied but has thus far been limited to peptide/protein-centric activity assays. Here, we demonstrate the versatile applicability of MALDI-TOF by tracking a small molecule within a highly complex sample background. The key to success for this concept was chemical derivatization of the target molecule enabling quantitative assessment of microbial TMA formation. Further, its potential was demonstrated in a side-by-side comparison to RapidFire-MS in a primary screen and subsequent dose-response experiments. Overall, the established assay enables the screening for microbial TMA-lyase inhibitors and serves as a proof of concept for the applicability of MALDI-TOF for demanding assay concepts per se.

RapidFire BLAZE-Mode Is Boosting ESI-MS Toward High-Throughput-Screening.

Label-free in vitro potency assays are an emerging field in drug discovery to enable more physiological conditions, to improve the readout quality, and to save time. For this approach mass spectrometry (MS) is a powerful technology to directly follow physiological processes. The speed of this methodology, however, was for a long time not compatible with chemiluminescence- or fluorescence-based assays. Recent advances in matrix-assisted laser desorption/ionization (MALDI) instrumentation paved the way for high-throughput MS analysis of label-free assays for large compound libraries, whereas electrospray ionization (ESI)-based mass spectrometers equipped with RapidFire autosamplers were limited to medium throughput. Here we present a technological advancement of the RapidFire device to enable cycle times of 2.5 s per sample. This newly developed BLAZE-mode substantially boosted the ESI-MS analysis speed, providing an alternative technology for label-free high-throughput screening.

Allosteric Activation of Striatal-Enriched Protein Tyrosine Phosphatase (STEP, PTPN5) by a Fragment-like Molecule.

Protein tyrosine phosphatase non-receptor type 5 (PTPN5, STEP) is a brain specific phosphatase that regulates synaptic function and plasticity by modulation of N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking. Dysregulation of STEP has been linked to neurodegenerative and neuropsychiatric diseases, highlighting this enzyme as an attractive therapeutic target for drug discovery. Selective targeting of STEP with small molecules has been hampered by high conservation of the active site among protein tyrosine phosphatases. We report the discovery of the first small molecule allosteric activator for STEP that binds to the phosphatase domain. Allosteric binding is confirmed by both X-ray and 15N NMR experiments, and specificity has been demonstrated by an enzymatic test cascade. Molecular dynamics simulations indicate stimulation of enzymatic activity by a long-range allosteric mechanism. To allow the scientific community to make use of this tool, we offer to provide the compound in the course of an open innovation initiative.

HIV-1 diversity, transmission dynamics and primary drug resistance in Angola.

OBJECTIVES: To assess HIV-1 diversity, transmission dynamics and prevalence of transmitted drug resistance (TDR) in Angola, five years after ART scale-up. METHODS: Population sequencing of the pol gene was performed on 139 plasma samples collected in 2009 from drug-naive HIV-1 infected individuals living in Luanda. HIV-1 subtypes were determined using phylogenetic analysis. Drug resistance mutations were identified using the Calibrated Population Resistance Tool (CPR). Transmission networks were determined using phylogenetic analysis of all Angolan sequences present in the databases. Evolutionary trends were determined by comparison with a similar survey performed in 2001. RESULTS: 47.1% of the viruses were pure subtypes (all except B), 47.1% were recombinants and 5.8% were untypable. The prevalence of subtype A decreased significantly from 2001 to 2009 (40.0% to 10.8%, P = 0.0019) while the prevalence of unique recombinant forms (URFs) increased > 2-fold (40.0% to 83.1%, P < 0.0001). The most frequent URFs comprised untypable sequences with subtypes H (U/H, n = 7, 10.8%), A (U/A, n = 6, 9.2%) and G (G/U, n = 4, 6.2%). Newly identified U/H recombinants formed a highly supported monophyletic cluster suggesting a local and common origin. TDR mutation K103N was found in one (0.7%) patient (1.6% in 2001). Out of the 364 sequences sampled for transmission network analysis, 130 (35.7%) were part of a transmission network. Forty eight transmission clusters were identified; the majority (56.3%) comprised sequences sampled in 2008-2010 in Luanda which is consistent with a locally fuelled epidemic. Very low genetic distance was found in 27 transmission pairs sampled in the same year, suggesting recent transmission events. CONCLUSIONS: Transmission of drug resistant strains was still negligible in Luanda in 2009, five years after the scale-up of ART. The dominance of small and recent transmission clusters and the emergence of new URFs are consistent with a rising HIV-1 epidemics mainly driven by heterosexual transmission.

Antiretroviral drug resistance surveillance among treatment-naive human immunodeficiency virus type 1-infected individuals in Angola: evidence for low level of transmitted drug resistance.

The prevalence of transmitted human immunodeficiency virus type 1 drug resistance in Angola in 2001 in 196 untreated patients was investigated. All subtypes were detected, along with unclassifiable and complex recombinant strains. Numerous new polymorphisms were identified in the reverse transcriptase and protease. Two (1.6%) unrelated patients harbored nucleoside reverse transcriptase inhibitor- and nonnucleoside reverse transcriptase inhibitor-resistant viruses (mutations: M41L, D67N, M184V, L210W, T215Y or T215F, and K103N). Continued surveillance of drug resistance is required for maximization of ART efficacy in Angola.

Ectoparasite infestations of hedgehogs (Erinaceus europaeus) are associated with small-scale landscape structures in an urban-suburban environment.

Animals that exploit heterogeneous and patchy environments encounter different local habitat conditions that influence their interaction with the environment, such as the acquisition of parasites. How and at which scales interaction processes between parasites, hosts, and the environment are realized remains largely unknown. We examined the infestation patterns of 56 hedgehogs (Erinaceus europaeus) with fleas and ticks at a small spatial scale within a 12 km(2) area along a suburban-urban gradient in southwestern Germany. The structure and type of habitats surrounding hedgehog capture locations were estimated from digital land cover data within radii of 20, 50, and 100 m. These were assumed to match the ranging area and underlying heterogeneous landscape matrix in which host-parasite interactions take place. Landscape-based models suggested that flea burdens significantly decreased with the diversity and heterogeneity of land cover, as well as with the areal coverage of roads within radii of 50 and 100 m. Overall tick infestation levels were mostly explained by the number of arable patches and the areal coverage of roads within radii of 50 and 100 m, as well as date of capture. Examination of the semivariance in model residuals revealed no evident spatial structure in any of the models with flea or tick infestation patterns as response variables. Our results, which are based on a sampling scheme within a relatively small spatio-temporal window, suggest that heterogeneous landscape matrices affect parasitization rates of animals in urban environments, with clear differences at the individual level.

Characterization of a new sensitive PCR assay for quantification of viral DNA isolated from patients with hepatitis B virus infections.

Sensitive and accurate quantification of hepatitis B virus (HBV) DNA is necessary for monitoring patients with chronic hepatitis receiving antiviral therapy in order to determine treatment response and to adapt therapy in case of inadequate virologic control. The development of quantitative PCR assays has been crucial in meeting these needs. The objective of this study was to compare the performance of a new real-time PCR assay (Abbott RealTime) for HBV DNA with that of three other commercial assays for the detection of HBV DNA. These were the Versant 3.0 branched-chain DNA assay, the Cobas Amplicor HBV Monitor test, and the Cobas AmpliPrep-Cobas TaqMan hepatitis B virus assay (CAP-CTM). HBV DNA was measured in blood samples taken from two cohorts of patients with chronic hepatitis. HBV DNA levels measured with the Abbott RealTime assay were highly correlated with those measured with the other three tests over their respective dynamic ranges (r, 0.88 to 0.96). The sensitivity (detection limit, 10 IU/ml) and dynamic range of the Abbott RealTime assay (10(1) to 10(9) IU/ml) was superior to that of the Versant assay. The RealTime assay recognized both HBV strains belonging to genotypes A to G and those bearing polymerase gene mutations equivalently. In conclusion, this study demonstrates the utility of the Abbott RealTime assay for monitoring HBV DNA levels in patients with chronic hepatitis B. Its sensitivity and wide dynamic range should allow optimal monitoring of antiviral therapy and timely treatment adaptation.

Cytomegalovirus quantification in plasma by an automated real-time PCR assay.

BACKGROUND: Sensitive quantitation of cytomegalovirus (CMV) DNA in blood is helpful for the diagnosis of CMV infection or reactivation and the monitoring of transplanted patients. OBJECTIVES: We compared a new PCR assay coupled with an automated extraction system (CMV real-time PCR, Abbott Molecular, Des Plaines, IL, USA) to a previously validated method (ultrasensitive Cobas Amplicor CMV DNA Monitor, Roche Molecular, Indianapolis, IN, USA). RESULTS: Using limiting dilutions of CMV DNA positive plasma, the two assays had a similar detection threshold ranging between 20 and 45 copies/ml. Coefficients of variation of CMV real-time PCR assay varied from 1 to 12% for CMV DNA levels between 10,000 and 20 copies/ml. Viral loads assessed by the two methods on 179 clinical samples showed an overall concordance of 89% and an excellent correlation (R=0.94). Discrepancies were only observed for samples with low CMV DNA levels (<300 copies/ml); 18 samples were positive by CMV real-time PCR only, and 2 samples by ultrasensitive Cobas CMV only. Values obtained by CMV real-time PCR were on average 0.4 log higher than those of ultrasensitive Cobas CMV. Successive samples of transplanted patients with evidence of CMV infection/reactivation revealed that CMV real-time PCR assay was positive earlier and for a longer period of time after treatment initiation. CONCLUSIONS: Although both assays had similar analytical performances, the CMV real-time PCR assay has the advantages of automated extraction and higher dynamic range, and shows a trend for an improved sensitivity that might impact on clinical decisions.

Comparison of four commercial quantitative HIV-1 assays for viral load monitoring in clinical daily routine.

BACKGROUND: Quantification of viral load (VL) is standard for monitoring HIV-1 therapy and is crucial before deciding whether to switch or to continue a current antiretroviral regimen. METHODS: We compared the performance of the four most widely used commercial viral-load assays, COBAS Amplicor Monitor v1.5, Versant HIV-1 RNA 3.0, Abbott RealTime HIV-1 and Cobas AmpliPrep/Cobas TaqMan HIV-1 (CAP/CTM), in terms of intra- and inter-assay variability, as well as hands-on-time, specificity and ability to quantify group M subtypes. RESULTS: Although linearity and correlation were confirmed for the assays and comparable sensitivity and specificity were verified for genetically diverse HIV-1 subtypes, demonstrating suitability for monitoring of HIV group M isolates, the viral loads obtained showed variations, with a mean difference of 0.1-0.4 log, depending on the system used. CONCLUSIONS: Although sensitivity and precision were confirmed for all the systems, differences between them should be taken into account when viral load monitoring of the same person is performed using different systems.

Performance of the automated Abbott RealTime HIV-1 assay on a genetically diverse panel of specimens from London: comparison to VERSANT HIV-1 RNA 3.0, AMPLICOR HIV-1 MONITOR v1.5, and LCx HIV RNA Quantitative assays.

Automated RNA extraction and quantitation of HIV-1 by real-time PCR offer potential advantages of efficient sample processing, improved sensitivity, expanded dynamic range and reduced contamination risk. In this study, plasma was collected from 100 HIV-1 infected patients visiting The Courtyard Clinic of St. George's Hospital in London, United Kingdom (UK). Viral loads measured using the automated Abbott RealTime HIV-1 assay (m2000sp sample preparation and m2000rt amplification and detection instruments) were compared to results obtained with Versant HIV-1 RNA 3.0 (bDNA), AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) and LCx HIV RNA Quantitative (LCx HIV) assays. Based on gag p24, pol integrase, and env gp41 sequences, the panel included 26 subtype A, 20 B, 27 C, 10 D, 1 CRF01_AE, 3 CRF02_AG and 13 recombinant viruses. RealTime HIV-1, bDNA, Monitor v1.5 and LCx HIV quantitated 82, 74, 82, and 83% of samples, respectively, with 82, 71, 69 and 80 of the 100 samples measured within the dynamic ranges. Viral loads were highly correlated with 99% of values within 1 log(10) copies/ml between tests. The automated m2000 system and RealTime HIV-1 assay can increase laboratory throughput, enhance overall efficiency and reduce operator-associated errors while providing reliable quantitation of genetically diverse strains of HIV-1.