Effect of Modulating FcRn Binding on Direct and Pretargeted Tumor Uptake of Full-length Antibodies.

Full-length antibodies lack ideal pharmacokinetic properties for rapid targeted imaging, prompting the pursuit of smaller peptides and fragments. Nevertheless, studying the disposition properties of antibody-based imaging agents can provide critical insight into the pharmacology of their therapeutic counterparts, particularly for those coupled with potent payloads. Here, we evaluate modulation of binding to the neonatal Fc receptor (FcRn) as a protein engineering-based pharmacologic strategy to minimize the overall blood pool background with directly labeled antibodies and undesirable systemic click reaction of radiolabeled tetrazine with circulating pretargeted trans-cyclooctene (TCO)-modified antibodies. Noninvasive SPECT imaging of mice bearing HER2-expressing xenografts was performed both directly (111In-labeled antibody) and indirectly (pretargeted TCO-modified antibody followed by 111In-labeled tetrazine). Pharmacokinetic modulation of antibodies was achieved by two distinct methods: Fc engineering to reduce binding affinity to FcRn, and delayed administration of an antibody that competes with binding to FcRn. Tumor imaging with directly labeled antibodies was feasible in the absence of FcRn binding, rapidly attaining high tumor-to-blood ratios, but accompanied by moderate liver and spleen uptake. Pretargeted imaging of tumors with non-FcRn-binding antibody was also feasible, but systemic click reaction still occurred, albeit at lower levels than with parental antibody. Our findings demonstrate that FcRn binding impairment of full-length IgG antibodies moderately lowers tumor accumulation of radioactivity, and shifts background activity from blood pool to liver and spleen. Furthermore, reduction of FcRn binding did not eliminate systemic click reaction, but yielded greater improvements in tumor-to-blood ratio when imaging with directly labeled antibodies than with pretargeting.

Site-specific conjugation allows modulation of click reaction stoichiometry for pretargeted SPECT imaging.

Antibody pretargeting is a promising strategy for improving molecular imaging, wherein the separation in time of antibody targeting and radiolabeling can lead to rapid attainment of high contrast, potentially increased sensitivity, and reduced patient radiation exposure. The inverse electron demand Diels-Alder 'click' reaction between trans-cyclooctene (TCO) conjugated antibodies and radiolabeled tetrazines presents an ideal platform for pretargeted imaging due to rapid reaction kinetics, bioorthogonality, and potential for optimization of both slow and fast clearing components. Herein, we evaluated a series of anti-human epidermal growth factor receptor 2 (HER2) pretargeting antibodies containing distinct molar ratios of site-specifically incorporated TCO. The effect of stoichiometry on tissue distribution was assessed for pretargeting TCO-modified antibodies (monitored by 125I) and subsequent accumulation of an 111In-labeled tetrazine in a therapeutically relevant HER2+tumor-bearing mouse model. Single photon emission computed tomography (SPECT) imaging was also employed to assess tumor imaging at various TCO-to-monoclonal antibody (mAb) ratios. Increasing TCO-to-mAb molar ratios correlated with increased in vivo click reaction efficiency evident by increased tumor distribution and systemic exposure of 111In-labeled tetrazines. The pharmacokinetics of TCO-modified antibodies did not vary with stoichiometry. Pretargeted SPECT imaging of HER2-expressing tumors using 111In-labeled tetrazine demonstrated robust click reaction with circulating antibody at ~2 hours and good tumor delineation for both the 2 and 6 TCO-to-mAb ratio variants at 24 hours, consistent with a limited cell-surface pool of pretargeted antibody and benefit from further distribution and internalization. To our knowledge, this represents the first reported systematic analysis of how pretargeted imaging is affected solely by variation in click reaction stoichiometry through site-specific conjugation chemistry.

Extracellular Toxoplasma gondii tachyzoites metabolize and incorporate unnatural sugars into cellular proteins.

Toxoplasma gondii is an obligate intracellular parasite that infects all nucleated cell types in diverse warm-blooded organisms. Many of the surface antigens and effector molecules secreted by the parasite during invasion and intracellular growth are modified by glycans. Glycosylated proteins in the nucleus and cytoplasm have also been reported. Despite their prevalence, the complete inventory and biological significance of glycosylated proteins in Toxoplasma remain unknown. In this study, we aimed to globally profile parasite glycoproteins using a bioorthogonal chemical reporter strategy. This strategy involves the metabolic incorporation of unnatural functional groups (i.e., "chemical reporters") into Toxoplasma glycans, followed by covalent labeling with visual probes or affinity tags. The two-step approach enables the visualization and identification of newly biosynthesized glycoconjugates in the parasite. Using a buffer that mimics intracellular conditions, extracellular Toxoplasma tachyzoites were found to metabolize and incorporate unnatural sugars (equipped with bioorthogonal functional groups) into diverse proteins. Covalent chemistries were used to visualize and retrieve these labeled structures. Subsequent mass spectrometry analysis revealed 89 unique proteins. This survey identified novel proteins as well as previously characterized proteins from lectin affinity analyses.

Finding the right (bioorthogonal) chemistry.

Bioorthogonal chemistries can be used to tag diverse classes of biomolecules in cells and other complex environments. With over 20 unique transformations now available, though, selecting an appropriate reaction for a given experiment is challenging. In this article, we compare and contrast the most common classes of bioorthogonal chemistries and provide a framework for matching the reactions with downstream applications. We also discuss ongoing efforts to identify novel biocompatible reactions and methods to control their reactivity. The continued expansion of the bioorthogonal toolkit will provide new insights into biomolecule networks and functions and thus refine our understanding of living systems.

Isomeric cyclopropenes exhibit unique bioorthogonal reactivities.

Bioorthogonal chemistries have provided tremendous insight into biomolecule structure and function. However, many popular bioorthogonal transformations are incompatible with one another, limiting their utility for studies of multiple biomolecules in tandem. We identified two reactions that can be used concurrently to tag biomolecules in complex environments: the inverse electron-demand Diels-Alder reaction of tetrazines with 1,3-disubstituted cyclopropenes, and the 1,3-dipolar cycloaddition of nitrile imines with 3,3-disubstituted cyclopropenes. Remarkably, the cyclopropenes used in these transformations differ by the placement of a single methyl group. Such orthogonally reactive scaffolds will bolster efforts to monitor multicomponent processes in cells and organisms.

Borrelia burgdorferi sensu lato prevalence in tick populations in Estonia.

BACKGROUND: Estonia is located in a unique area of co-distribution of Ixodes ricinus and I. persulcatus, which are the main tick vectors of Borrelia burgdorferi sensu lato. In the last decade, the incidence rate of Lyme borreliosis in Estonia has increased dramatically up to 115.4 per 100,000 in 2012. Here we present the first survey of the presence, the prevalence and genetic characteristics of B. burgdorferi s.l. complex spirochetes in the tick population in Estonia. METHODS: During the years 2006-2009, 2833 unfed Ixodes ricinus and I. persulcatus were collected from 43 sites in 7 counties in mainland Estonia as well as in 10 sites on the Saaremaa Island. DNA samples from ticks were analyzed individually using nested PCR of the ribosomal 5S-23S spacer region followed by bidirectional sequencing. RESULTS: The overall estimated prevalence of B. burgdorferi s.l was 9.7% and varied from 4.9% to 24.2% on the mainland and to 10.7% in Saaremaa Island. Ixodes persulcatus ticks showed significantly higher prevalence rates compared to that in I. ricinus-16.3% and 8.2%, respectively. The most prevalent genospecies was B. afzelii which was detected in 53.5% of Borrelia-positive ticks, followed by B. garinii and B. valaisiana with 26.2% and 5.5%, respectively. Also, B. bavariensis and B. burgdorferi s.s. DNA in single I. ricinus ticks were detected. Borrelia afzelii, B. garinii and B. valaisiana were detected in both tick species. Two genetic subgroups of B. garinii (NT29 and 20047) and two genetic subgroups of B. afzelii (NT28 and VS461) were found to be circulating in all studied regions as well as in both tick species, except B. garinii subgroup NT29, which was found only in I. persulcatus ticks. CONCLUSIONS: In the current study we detected the circulation of five B. burgdorferi s.l. genospecies and estimated the prevalence in ticks in different regions of Estonia. Detection and genetic characterization of Borrelia genospecies, especially those of public health importance, in the natural foci may help assessing high risk areas of human exposure to B. burgdorferi s.l.

Tick-borne pathogens in ticks feeding on migratory passerines in Western part of Estonia.

During southward migration in the years 2006-2009, 178 migratory passerines of 24 bird species infested with ticks were captured at bird stations in Western Estonia. In total, 249 nymphal ticks were removed and analyzed individually for the presence of Borrelia burgdorferi sensu lato (s.l.), tick-borne encephalitis virus (TBEV), and Anaplasma phagocytophilum. The majority of ticks were collected from Acrocephalus (58%), Turdus (13%), Sylvia (8%), and Parus (6%) bird species. Tick-borne pathogens were detected in nymphs removed from Acrocephalus, Turdus, and Parus bird species. TBEV of the European subtype was detected in 1 I. ricinus nymph removed from A. palustris. B. burgdorferi s.l. DNA was found in 11 ticks (4.4%) collected from Turdus and Parus species. Bird-associated B. garinii and B. valaisiana were detected in I. ricinus nymphs removed from T. merula. Rodent-associated B. afzelii was detected in 3 I. ricinus nymphs from 2 P. major birds. One of the B. afzelii-positive nymphs was infected with a mix of 2 B. afzelii strains, whereas 1 of these strains was also detected in another nymph feeding on the same great tit. The sharing of the same B. afzelii strain by 2 nymphs indicates a possible transmission of B. afzelii by co-feeding on a bird. A. phagocytophilum DNA was detected in 1 I. ricinus nymph feeding on a T. iliacus. The results of the study confirm the possible role of migratory birds in the dispersal of ticks infected with tick-borne pathogens along the southward migration route via Estonia.

Detection and genetic characterization of relapsing fever spirochete Borrelia miyamotoi in Estonian ticks.

During the years 2008-2010 I. ricinus and I. persulcatus ticks were collected from 64 sites in mainland Estonia and on the island Saaremaa. Presence of B. miyamotoi was found in 0.9% (23/2622) of ticks. The prevalence in I. persulcatus and I. ricinus ticks differed significantly, 2.7% (15/561) and 0.4% (8/2061), respectively. The highest prevalence rates were in found South-Eastern Estonia in an area of I. persulcatus and I. ricinus sympatry and varied from 1.4% (1/73) to 2.8% (5/178). Co-infections with B. burgdorferi s.l. group spirochetes and tick-borne encephalitis virus were also revealed. Genetic characterization of partial 16S rRNA, p66 and glpQ genes demonstrated that Estonian sequences belong to two types of B. miyamotoi and cluster with sequences from Europe and the European part of Russia, as well as with sequences from Siberia, Asia and Japan, here designated as European and Asian types, respectively. Estonian sequences of the European type were obtained from I. ricinus ticks only, whereas the Asian type of B. miyamotoi was shown for both tick species in the sympatric regions.

Functionalized cyclopropenes as bioorthogonal chemical reporters.

Chemical reporters are unique functional groups that can be used to label biomolecules in living systems. Only a handful of broadly applicable reporters have been identified to date, owing to the rigorous demands placed on these functional groups in biological settings. We describe here a new chemical reporter-cyclopropene-that can be used to target biomolecules in vitro and in live cells. A variety of substituted cyclopropene scaffolds were synthesized and found to be stable in aqueous solution and in the presence of biological nucleophiles. Furthermore, some of the cyclopropene units were metabolically introduced into cell surface glycans and subsequently detected with covalent probes. The small size and selective reactivity of cyclopropenes will facilitate efforts to tag diverse collections of biomolecules in vivo.

Synthesis of sansalvamide A peptidomimetics: triazole, oxazole, thiazole, and pseudoproline containing compounds.

Peptidomimetic-based macrocycles typically have improved pharmacokinetic properties over those observed with peptide analogs. Described are the syntheses of 13 peptidomimetic derivatives that are based on active Sansalvamide A structures, where these analogs incorporate heterocycles (triazoles, oxazoles, thiazoles, or pseudoprolines) along the macrocyclic backbone. The syntheses of these derivatives employ several approaches that can be applied to convert a macrocyclic peptide into its peptidomimetic counterpart. These approaches include peptide modifications to generate the alkyne and azide for click chemistry, a serine conversion into an oxazole, a Hantzsch reaction to generate the thiazole, and protected threonine to generate the pseudoproline derivatives. Furthermore, we show that two different peptidomimetic moieties, triazoles and thiazoles, can be incorporated into the macrocyclic backbone without reducing cytotoxicity: triazole and thiazole.

Histone Deacetylase Inhibitors: Synthesis of Cyclic Tetrapeptides and their Triazole Analogues.

Synthesis of nine macrocyclic peptide HDAC inhibitors and three triazole derivatives are described. HDAC inhibitory activity of these compounds against HeLa cell lysate is evaluated. The biological data demonstrates that incorporation of a triazole unit improves the HDAC inhibitory activity.

Macrocyclic inhibitors of hsp90.

Heat shock proteins (HSP) are a family of highly conserved proteins, whose expression increases in response to stresses that may threaten cell survival. Over the past decade, heat shock protein 90 (Hsp90) has emerged as a potential therapeutic target for cancer as it plays a vital role in normal cell maturation and acts as a molecular chaperone for proper folding, assembly, and stabilization of many oncogenic proteins. To date, a majority of Hsp90 inhibitors that have been discovered are macrocycles. The relatively rigid conformation provided by the macrocyclic scaffold allows for a selective interaction with a biological target such as Hsp90. This review highlights the discovery and development of nine macrocycles that inhibit the function of Hsp90, detailing their potency and the client proteins affected by Hsp90 inhibition.

Inhibition of calpain but not caspase activity by spectrin fragments.

Calpains and caspases are ubiquitous cysteine proteases that are associated with a variety of cellular pathways. Calpains are involved in processes such as long term potentiation, cell motility and apoptosis, and have been shown to cleave non-erythroid (brain) alpha- and beta-spectrin and erythroid beta-spectrin. The cleavage of erythroid alpha-spectrin by calpain has not been reported. Caspases play an important role in the initiation and execution of apoptosis, and have been shown to cleave non-erythroid but not erythroid spectrin. We have studied the effect of spectrin fragments on calpain and caspase activities. The erythroid and non-erythroid spectrin fragments used were from the N-terminal region of alpha-spectrin, and C-terminal region of beta-spectrin, both consisting of regions involved in spectrin tetramer formation. We observed that the all spectrin fragments exhibited a concentration-dependent inhibitory effect on calpain, but not caspase activity. It is clear that additional studies are warranted to determine the physiological significance of calpain inhibition by spectrin fragments. Our findings suggest that calpain activity is modulated by the presence of spectrin partial domains at the tetramerization site. It is not clear whether the inhibitory effect is substrate specific or is a general effect. Further studies of this inhibitory effect may lead to the identification and development of new therapeutic agents specifically for calpains, but not for caspases. Proteins/peptides with a coiled coil helical conformation should be studied for potential inhibitory effects on calpain activity.