MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

Heterogeneous distribution of kinesin-streptavidin complexes revealed by mass photometry.

Kinesin-streptavidin complexes are widely used in microtubule-based active-matter studies. The stoichiometry of the complexes is empirically tuned but experimentally challenging to determine. Here, mass photometry measurements reveal heterogenous distributions of kinesin-streptavidin complexes. Our binding model indicates that heterogeneity arises from both the kinesin-streptavidin mixing ratio and the kinesin-biotinylation efficiency.

Chemoselective Labeling and Immobilization of Phosphopeptides with Phosphorimidazolide Reagents.

Protein phosphorylation is one of the most ubiquitous post-translational modifications, regulating numerous essential processes in cells. Accordingly, the large-scale annotation of phosphorylation sites continues to provide central insight into the regulation of signaling networks. The global analysis of the phosphoproteome typically relies on mass spectrometry analysis of phosphopeptides, with an enrichment step necessary due to the sub-stoichiometric nature of phosphorylation. Several affinity-based methods and chemical modification strategies have been developed to date, but the choice of enrichment method can have a considerable impact on the results. Here, we show that a biotinylated, photo-cleavable phosphorimidazolide reagent permits the immobilization and subsequent cleavage of phosphopeptides. The method is capable of the capture and release of phosphopeptides of varying characteristics, and this mild and selective strategy expands the current repertoire for phosphopeptide chemical modification with the potential to enrich and identify new phosphorylation sites in the future.

Affinity-based proteomics reveals novel targets of inositol pyrophosphate (5-IP7 )-dependent phosphorylation and binding in Trypanosoma cruzi replicative stages.

Diphosphoinositol-5-pentakisphosphate (5-PP-IP5 ), also known as inositol heptakisphosphate (5-IP7 ), has been described as a high-energy phosphate metabolite that participates in the regulation of multiple cellular processes through protein binding or serine pyrophosphorylation, a posttranslational modification involving a ß-phosphoryl transfer. In this study, utilizing an immobilized 5-IP7 affinity reagent, we performed pull-down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5-IP7 -regulated processes in the two proliferative stages of the unicellular parasite Trypanosoma cruzi. Our protein screen clearly defined two cohorts of putative targets either in the presence of magnesium ions or in metal-free conditions. We endogenously tagged four protein candidates and immunopurified them to assess whether 5-IP7 -driven phosphorylation is conserved in T. cruzi. Among the most interesting targets, we identified a choline/o-acetyltransferase domain-containing phosphoprotein that undergoes 5-IP7 -mediated phosphorylation events at a polyserine tract (Ser578-580 ). We also identified a novel SPX domain-containing phosphoribosyltransferase [EC 2.7.6.1] herein termed as TcPRPPS4. Our data revealed new possible functional roles of 5-IP7 in this divergent eukaryote, and provided potential new targets for chemotherapy.

Comparison of Bioorthogonal ß-Lactone Activity-Based Probes for Selective Labeling of Penicillin-Binding Proteins.

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of ß-lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal ß-lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity-based probes for PBPs will facilitate the activity-based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.

Salmonella Effectors SseK1 and SseK3 Target Death Domain Proteins in the TNF and TRAIL Signaling Pathways.

Strains of Salmonella utilize two distinct type three secretion systems to deliver effector proteins directly into host cells. The Salmonella effectors SseK1 and SseK3 are arginine glycosyltransferases that modify mammalian death domain containing proteins with N-acetyl glucosamine (GlcNAc) when overexpressed ectopically or as recombinant protein fusions. Here, we combined Arg-GlcNAc glycopeptide immunoprecipitation and mass spectrometry to identify host proteins GlcNAcylated by endogenous levels of SseK1 and SseK3 during Salmonella infection. We observed that SseK1 modified the mammalian signaling protein TRADD, but not FADD as previously reported. Overexpression of SseK1 greatly broadened substrate specificity, whereas ectopic co-expression of SseK1 and TRADD increased the range of modified arginine residues within the death domain of TRADD. In contrast, endogenous levels of SseK3 resulted in modification of the death domains of receptors of the mammalian TNF superfamily, TNFR1 and TRAILR, at residues Arg376 and Arg293 respectively. Structural studies on SseK3 showed that the enzyme displays a classic GT-A glycosyltransferase fold and binds UDP-GlcNAc in a narrow and deep cleft with the GlcNAc facing the surface. Together our data suggest that salmonellae carrying sseK1 and sseK3 employ the glycosyltransferase effectors to antagonise different components of death receptor signaling.

Antiviral Activity, Safety, and Pharmacokinetics of Capsid Assembly Modulator NVR 3-778 in Patients with Chronic HBV Infection.

BACKGROUND & AIMS: NVR 3-778 is a first-in-class hepatitis B virus (HBV) capsid assembly modulator that can inhibit HBV replication. We performed a proof-of-concept study to examine the safety, pharmacokinetics, and antiviral activity of NVR 3-778 in patients with chronic HBV infection. METHODS: We performed a phase 1 study in 73 hepatitis B envelope antigen (HBeAg)-positive patients with chronic HBV infection without cirrhosis. In a 2-part study (part 1 in New Zealand and part 2 in Hong Kong, Singapore, Taiwan, Korea, and the United States), patients were randomly assigned to groups that were given oral NVR 3-778 (100 mg, 200 mg, or 400 mg daily or 600 mg or 1000 mg twice daily) or placebo for 4 weeks. Additional groups received combination treatment with pegylated interferon (pegIFN) and NVR 3-778 (600 mg twice daily) or pegIFN with placebo. RESULTS: Reductions in serum levels of HBV DNA and HBV RNA were observed in patients receiving ≥1200 mg/d NVR 3-778. The largest mean reduction in HBV DNA was observed in the group given NVR 3-778 plus pegIFN (1.97 log10 IU/mL), compared with the groups given NVR 3-778 or pegIFN alone (1.43 log10 IU/mL and 1.06 log10 IU/mL, respectively). The mean reduction in HBV RNA was also greatest in the group given NVR 3-778 plus pegIFN (2.09 log10 copies/mL), compared with the groups given NVR 3-778 or pegIFN alone (1.42 log10 copies/mL and 0.89 log10 copies/mL, respectively). There was no significant mean reduction in HBsAg during the 4-week treatment period. There were no discontinuations and no pattern of dose-related adverse effects with NVR 3-778. CONCLUSIONS: In a phase 1 study of HBeAg-positive patients with chronic HBV infection without cirrhosis, NVR 3-778 was well tolerated and demonstrated antiviral activity. The agent reduced serum levels of HBV DNA and HBV RNA, to the greatest extent in combination with pegIFN. The observed reductions in HBV RNA confirmed the novel mechanism of NVR 3-778. Clinicaltrials.gov no. NCT02112799 (single-center) and NCT02401737 (multicenter).

Liver safety assessment in clinical trials of new agents for chronic hepatitis B.

Investigational agents that reduce or eliminate covalently closed circular DNA (cccDNA) or enhance host immunity against hepatitis B virus (HBV)-infected hepatocytes are intended to induce a durable off-treatment clearance of hepatitis B surface antigen (HBsAg) (referred to as functional cure). The aim of this paper was to highlight challenges in interpreting liver safety data in clinical trials of these agents when given alone or in combination regimens. The incidence, grading and management of spontaneous serum ALT flares in untreated chronic HBV patients are reviewed along with a summary of serum ALT flares observed during the registration trials for peginterferon and nucleos(t)ide reverse transcriptase inhibitors. Recommendations regarding the detection, management and interpretation of liver safety biomarker data in future clinical trials as well as suggested inclusion and exclusion criteria for phase 1/2 vs phase 3 studies are provided. Criteria to help classify liver safety signals as being due to the intended therapeutic response, emergence of drug-resistant HBV virions, or idiosyncratic drug-induced liver injury are provided along with a review of the role of an expert hepatic adjudication panel in assessing a compound's hepatotoxicity profile. Finally, an algorithmic approach to the differential diagnosis and recommended medical evaluation and management of individual clinical trial patients that develop a liver safety signal is provided along with the rationale to collect and test research blood samples for future mechanistic studies.

On-chip electrochemical detection of glucose towards the miniaturised quality control of carbohydrate-based radiotracers.

The miniaturisation of positron emission tomography (PET) radiotracer production is facilitating a move towards a dose-on-demand strategy that would enable a stratified approach to patient diagnostics, but while the on-chip synthesis steps have been demonstrated, the subsequent quality control (QC) testing steps have received much less attention. As part of the development of an integrated QC platform for PET tracers, we have developed two microfluidic electrochemical detectors for the pulsed amperometric detection (PAD) of carbohydrate-based radiotracers, with a particular view to the QC testing of the most important tracer, [18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG). The first device employed a commercial screen-printed electrode (SPE) to enable a single-use format, while the second device incorporated wire electrodes for use as a more permanent fixture in a QC instrument. A flow-injection analysis (FIA)-style setup was used to inject boluses of d-glucose into the chips in a proxy for intended chromatographic separations prior to PAD. In proof-of-concept testing of the devices, the chips featuring the SPE and the wire electrodes yielded limits of detection of 0.1 ppm and 9 ppm, respectively, each below the required limits for [18F]FDG, and thus making both methodologies viable for the QC testing of PET radiotracers in a dose-on-demand format.

Right nostril biases to experimental scents in Canis familiaris.

Although olfactory laterality in canids has been demonstrated experimentally, the extent to which nostril bias occurs in "nature" is not well known. We tested whether there was olfactory laterality of untrained dogs in various off-leash dog parks within Victoria, British Columbia to manipulated scents placed at the tail base of full-size dog replica. Using video-playback, we found that of 192 separate approaches (N = 119 different subjects), dogs used the right nostril first greater than 66% of the time and for longer periods when investigating estrous dog secretions, deer urine and coyote urine. Similar trends were observed when using scents on a similar-sized box rather than the dog model. There was no side preference for the scent of commercial pet food. These results support right hemisphere control of the sympathetic-hypothalamic-pituitary-adrenal axis and encourage more detailed evaluations of olfactory laterality in wild canids and other carnivores where olfaction is the major sensory modality.

Plastic Scintillator-Based Microfluidic Devices for Miniaturized Detection of Positron Emission Tomography Radiopharmaceuticals.

A miniaturized radio-HPLC detector has been developed comprising a microfluidic device fabricated from plastic scintillator in combination with a silicon photomultiplier light sensor, and tested with samples containing a positron-emitting radionuclide, [18 F]fluoride. This cost-effective, small footprint analytical tool is ideal for incorporation into integrated quality control systems for the testing of positron emission tomography (PET) radiopharmaceuticals to good manufacturing practice (GMP) standards.

Stapling of two PEGylated side chains increases the conformational stability of the WW domain via an entropic effect.

Hydrocarbon stapling and PEGylation are distinct strategies for enhancing the conformational stability and/or pharmacokinetic properties of peptide and protein drugs. Here we combine these approaches by incorporating asparagine-linked O-allyl PEG oligomers at two positions within the ß-sheet protein WW, followed by stapling of the PEGs via olefin metathesis. The impact of stapling two sites that are close in primary sequence is small relative to the impact of PEGylation alone and depends strongly on PEG length. In contrast, stapling of two PEGs that are far apart in primary sequence but close in tertiary structure provides substantially more stabilization, derived mostly from an entropic effect. Comparison of PEGylation + stapling vs. alkylation + stapling at the same positions in WW reveals that both approaches provide similar overall levels of conformational stability.

Effectiveness of ravidasvir plus sofosbuvir in interferon-naïve and treated patients with chronic hepatitis C genotype-4.

BACKGROUND & AIMS: Although treatment of hepatitis C virus (HCV) and HCV-genotype-4 (GT4) has become very effective, it remains very expensive, and affordable options are needed, especially in limited resource countries. The aim of this study was to assess the efficacy and safety of the combination of ravidasvir (an NS5A inhibitor) and sofosbuvir to treat patients with chronic HCV-GT4 infection. METHODS: A total of 300 patients with HCV-GT4 infection were recruited in three groups: treatment-naïve patients with or without compensated Child-A cirrhosis (Group 1); interferon-experienced patients without cirrhosis (Group 2); and interferon-experienced patients with cirrhosis (Group 3). Groups 1 and 2 received ravidasvir 200 mg QD plus sofosbuvir 400 mg QD for 12 weeks and were randomized 1:1 to treatment with or without weight-based ribavirin. Group 3 patients received ravidasvir plus sofosbuvir with ribavirin and were randomized 1:1 to a treatment duration of 12 weeks or 16 weeks. The primary endpoint was sustained virologic response at 12 weeks post-treatment (SVR12). RESULTS: A total of 298 patients were enrolled: 149 in Group 1, 79 in Group 2 and 70 in Group 3. SVR12 was achieved in 95.3% of all patients who started the study, including 98% of patients without cirrhosis and 91% of patients with cirrhosis, whether treatment-naïve or interferon-experienced. Ribavirin intake and history of previous interferon therapy did not affect SVR12 rates. No virologic breakthroughs were observed and the study treatment was well tolerated. CONCLUSIONS: Treatment with ravidasvir plus sofosbuvir, with or without ribavirin, was well tolerated and associated with high sustained virologic response rate for HCV-GT4 infected patients with and without cirrhosis, regardless of previous interferon-based treatments. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT02371408. LAY SUMMARY: This study evaluated efficacy and safety of the new oral hepatitis C drug ravidasvir in combination with the approved oral drug sofosbuvir in 298 patients infected with hepatitis C type 4. Our results showed that treatment with ravidasvir plus sofosbuvir, with or without ribavirin, was well tolerated and associated with high response rate in patients with and without cirrhosis.

Polyethylene Glycol Based Changes to ß-Sheet Protein Conformational and Proteolytic Stability Depend on Conjugation Strategy and Location.

The development of chemical strategies for site-specific protein modification now enables researchers to attach polyethylene glycol (PEG) to a protein drug at one or more specific locations (i.e., protein PEGylation). However, aside from avoiding enzyme active sites or protein-binding interfaces, distinguishing the optimal PEGylation site from the available alternatives has conventionally been a matter of trial and error. As part of a continuing effort to develop guidelines for identifying optimal PEGylation sites within proteins, we show here that the impact of PEGylation at various sites within the ß-sheet model protein WW depends strongly on the identity of the PEG-protein linker. The PEGylation of Gln or of azidohomoalanine has a similar impact on WW conformational stability as does Asn-PEGylation, whereas the PEGylation of propargyloxyphenylalanine is substantially stabilizing at locations where Asn-PEGylation was destabilizing. Importantly, we find that at least one of these three site-specific PEGylation strategies leads to substantial PEG-based stabilization at each of the positions investigated, highlighting the importance of considering conjugation strategy as an important variable in selecting optimal PEGylation sites. We further demonstrate that using a branched PEG oligomer intensifies the impact of PEGylation on WW conformational stability and also show that PEG-based increases to conformational stability are strongly associated with corresponding increases in proteolytic stability.

Chemical tools for interrogating inositol pyrophosphate structure and function.

The inositol pyrophosphates (PP-InsPs) are a unique group of intracellular messengers that represent some of the most highly phosphorylated molecules in nature. Genetic perturbation of the PP-InsP biosynthetic network indicates a central role for these metabolites in maintaining cellular energy homeostasis and in controlling signal transduction networks. However, despite their discovery over two decades ago, elucidating their physiologically relevant isomers, the biochemical pathways connecting these molecules to their associated phenotypes, and their modes of signal transduction has often been stymied by technical challenges. Many of the advances in understanding these molecules to date have been facilitated by the total synthesis of the various PP-InsP isomers and by the development of new methods that are capable of identifying their downstream signalling partners. Chemical tools have also been developed to distinguish between the proposed PP-InsP signal transduction mechanisms: protein binding, and a covalent modification of proteins termed protein pyrophosphorylation. In this article, we review these recent developments, discuss how they have helped to illuminate PP-InsP structure and function, and highlight opportunities for future discovery.

Cellular Cations Control Conformational Switching of Inositol Pyrophosphate Analogues.

The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions.