Protein kinase affinity reagents based on a 5-aminoindazole scaffold

Affinity reagents that target protein kinases are powerful tools for signal transduction research. Here, we describe a general set of kinase ligands based on a 5-aminoindazole scaffold. This scaffold can readily be derivatized with diverse binding elements and immobilized analogs allow selective enrichment of protein kinases from complex mixtures.     

LaPO4 nanoparticles doped with actinium-225 that partially sequester daughter radionuclides

Nanoscale materials have been envisioned as carriers for various therapeutic drugs, including radioisotopes. Inorganic nanoparticles (NPs) are particularly appealing vehicles for targeted radiotherapy because they can package several radioactive atoms into a single carrier and can potentially retain daughter radioisotopes produced by in vivo generators such as actinium-225 ((225)Ac, t(1/2) = 10 d). Decay of this radioisotope to stable bismuth-209 proceeds through a chain of short-lived daughters accompanied by the emission of four α-particles that release >27 MeV of energy. The challenge in realizing the enhanced cytotoxic potential of in vivo generators lies in retaining the daughter nuclei at the therapy site. When (225)Ac is attached to targeting agents via standard chelate conjugation methods, all of the daughter radionuclides are released after the initial α-decay occurs. In this work, (225)Ac was incorporated into lanthanum phosphate NPs to determine whether the radioisotope and its daughters would be retained within the dense mineral lattice. Further, the (225)Ac-doped NPs were conjugated to the monoclonal antibody mAb 201B, which targets mouse lung endothelium through the vasculature, to ascertain the targeting efficacy and in vivo retention of radioisotopes. Standard biodistribution techniques and microSPECT/CT imaging of (225)Ac as well as the daughter radioisotopes showed that the NPs accumulated rapidly in mouse lung after intravenous injection. By showing that excess, competing, uncoupled antibodies or NPs coupled to control mAbs are deposited primarily in the liver and spleen, specific targeting of NP-mAb 201B conjugates was demonstrated. Biodistribution analysis showed that ～30% of the total injected dose of La((225)Ac)PO(4) NPs accumulated in mouse lungs 1 h postinjection, yielding a value of % ID/g >200. Furthermore, after 24 h, 80% of the (213)Bi daughter produced from (225)Ac decay was retained within the target organ and (213)Bi retention increased to ～87% at 120 h. In vitro analyses, conducted over a 1 month interval, demonstrated that ～50% of the daughters were retained within the La((225)Ac)PO(4) NPs at any point over that time frame. Although most of the γ-rays from radionuclides in the (225)Ac decay chain are too energetic to be captured efficiently by SPECT detectors, appropriate energy windows were found that provided dramatic microSPECT images of the NP distribution in vivo. We conclude that La((225)Ac)PO(4)-mAb 201B conjugates can be targeted efficiently to mouse lung while partially retaining daughter products and that targeting can be monitored by biodistribution techniques and microSPECT imaging.     

Mass spectrometric detection of targeting peptide bioconjugation to Red clover necrotic mosaic virus

Plant virus nanoparticle (PVN) formulations constructed from Red clover necrotic mosaic virus by drug infusion and targeting peptide conjugation can be employed as drug delivery tools. In this investigation, we studied the cross-linked structures formed by application of sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) and succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester (SMPEG) as heterobifunctional linkers in the bioconjugation process. The plant virus formulations using several targeting peptides cross-linked to the plant virus capsid were characterized by LC/MS(E) analysis, which produced at least 69% sequence coverage using trypsin and chymotrypsin digestion. The results showed evidence for several types of modification located in three domains of the capsid protein. Extensive linker modifications on lysines or cysteines were detected in all the domains, including both intended peptide-capsid cross-links and unintended intracapsid cross-links. Surprisingly, the most extensive peptide modification was observed in the R domain, which is thought to be quite inaccessible to peptides and cross-linking reagents in solution, since it is on the interior of the virus. These results show that heterobifunctional linkers may not be the most efficient method for attachment of peptides to plant virus capsids. As an alternative conjugation strategy, maleimide peptides were used to conjugate with the virus in a one-step reaction. Analysis by LC/MS(E) showed that these one-step maleimide coupling reactions were more specific, such as modifications of C154 and to a lesser extent C267, and provide a means for achieving more effective PVN formulations.     

Dynamic imaging of the effector immune response to listeria infection in vivo

Host defense against the intracellular pathogen Listeria monocytogenes (Lm) requires innate and adaptive immunity. Here, we directly imaged immune cell dynamics at Lm foci established by dendritic cells in the subcapsular red pulp (scDC) using intravital microscopy. Blood borne Lm rapidly associated with scDC. Myelomonocytic cells (MMC) swarmed around non-motile scDC forming foci from which blood flow was excluded. The depletion of scDC after foci were established resulted in a 10-fold reduction in viable Lm, while graded depletion of MMC resulted in 30-1000 fold increase in viable Lm in foci with enhanced blood flow. Effector CD8+ T cells at sites of infection displayed a two-tiered reduction in motility with antigen independent and antigen dependent components, including stable interactions with infected and non-infected scDC. Thus, swarming MMC contribute to control of Lm prior to development of T cell immunity by direct killing and sequestration from blood flow, while scDC appear to promote Lm survival while preferentially interacting with CD8+ T cells in effector sites.     

Boltzmann energy-based image analysis demonstrates that extracellular domain size differences explain protein segregation at immune synapses

Immune synapses formed by T and NK cells both show segregation of the integrin ICAM1 from other proteins such as CD2 (T cell) or KIR (NK cell). However, the mechanism by which these proteins segregate remains unclear; one key hypothesis is a redistribution based on protein size. Simulations of this mechanism qualitatively reproduce observed segregation patterns, but only in certain parameter regimes. Verifying that these parameter constraints in fact hold has not been possible to date, this requiring a quantitative coupling of theory to experimental data. Here, we address this challenge, developing a new methodology for analysing and quantifying image data and its integration with biophysical models. Specifically we fit a binding kinetics model to 2 colour fluorescence data for cytoskeleton independent synapses (2 and 3D) and test whether the observed inverse correlation between fluorophores conforms to size dependent exclusion, and further, whether patterned states are predicted when model parameters are estimated on individual synapses. All synapses analysed satisfy these conditions demonstrating that the mechanisms of protein redistribution have identifiable signatures in their spatial patterns. We conclude that energy processes implicit in protein size based segregation can drive the patternation observed in individual synapses, at least for the specific examples tested, such that no additional processes need to be invoked. This implies that biophysical processes within the membrane interface have a crucial impact on cell:cell communication and cell signalling, governing protein interactions and protein aggregation.     

Wing-dimorphism and population expansion of Pterostichus melanarius (Illiger, 1798) at small and large scales in central Alberta, Canada (Coleoptera, Carabidae, Pterostichini)

A study spanning ten years revealed changes in wing-morph ratios corroborating the hypothesis that the wing-dimorphic introduced carabid, Pterostichus melanarius Ill.,is spreading through flight, from the city of Edmonton, Canada and establishing populations in natural aspen forest of more rural areas 45-50 km to the East. Comparison of wing-morph ratios between Pterostichus melanarius and the native wing dimorphic species Agonum retractum LeConte suggests that the spatial variation in ratios for Pterostichus melanarius does not reflect underlying environmental variation, but instead the action of selective forces on this wing-dimorphic species. About ten years after its earliest detection in some rural sites the frequency of macropterous individuals in Pterostichus melanarius has decreased c. five-fold, but it is still above the level seen in European populations in which the two wing-morphs are thought to exist in equilibrium. Pterostichus melanarius is expanding its range in native aspen forest much faster than three other introduced species Clivina fossor L.), Carabus granulatus O.F. Müllerand Clivina fossor L also encountered in this study. The two Carabus species are flightless, but Carabus fossor can be dimorphic. Although these four non-native ground beetle species comprise >85% of the carabids collected at sites in urban Edmonton, activity-density of native carabids was similar across the urban-rural gradient, suggesting little direct impact of introduced species on the local abundance of native species. In a second study conducted at a smaller scale near George Lake, Alberta, macropterous individuals of Pterostichus melanarius have penetrated furthest and most rapidly into native aspen forest. Furthermore, the percentage of micropterous individuals has increased markedly in areas first colonized a decade previously. Overall, these studies support the idea that macropterous beetles in wing-d dimorphic species are important vanguards for early colonization of unexploited territory, but that flightless individuals replace the flying morph relatively rapidly once populations are established.     

Epigenetic regulation of flowering time in polyploids

Polyploidy plays a significant role in the evolution of flowering plants. Understanding the effects of polyploidy on the epigenetic regulation of adaptive traits may resolve questions about the success of polyploids. One such trait, flowering time, has been the subject of several gene expression studies because it has one of the best characterized genetic networks and because polyploidy has a significant impact on generating variation in flowering time. Future research on the epigenetic consequences of polyploidy on flowering time should begin to examine natural variation in an ecological context, while continuing to make use of resynthesized polyploids.     

Bivalent inhibitors of the tyrosine kinases ABL and SRC: determinants of potency and selectivity

We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.