Minimal Binding Research Articles

Perspectives on the Optimal Genetically Engineered Pig in 2018 for Initial Clinical Trials of Kidney or Heart Xenotransplantation.

For a clinical trial today, what might realistically be the optimal pig among those currently available? Deletion of expression of the 3 pig carbohydrate antigens, against which humans have natural (preformed) antibodies (triple-knockout pigs), should form the basis of any clinical trial. However, because both complement and coagulation can be activated in the absence of antibody, the expression of human complement- and coagulation-regulatory proteins is likely to be important in protecting the graft further. Any genetic manipulation that might reduce inflammation of the graft, for example, expression of hemeoxygenase-1 or A20, may also be beneficial to the long-term survival of the graft. The transgene for human CD47 is likely to have a suppressive effect on monocyte/macrophage and T-cell activity. Furthermore, deletion of xenoantigen expression and expression of a human complement-regulatory protein are both associated with a reduced T-cell response. Although there are several other genetic manipulations that may reduce the T-cell response further, it seems likely that exogenous immunosuppressive therapy, particularly if it includes costimulation blockade, will be sufficient. We would therefore suggest that, with our present knowledge and capabilities, the optimal pig might be a triple-knockout pig that expressed 1 or more human complement-regulatory proteins, 1 or more human coagulation-regulatory proteins, a human anti-inflammatory transgene, and CD47. Absent or minimal antibody binding is important, but we suggest that the additional insertion of protective human transgenes will be beneficial, and may be essential.

Selection and Characterization of Anti-Dengue NS1 Single Domain Antibodies

Reliable detection and diagnosis of dengue virus (DENV) is important for both patient care and epidemiological control. Starting with a llama immunized with a mixture of recombinant nonstructural protein 1 (NS1) antigen from the four DENV serotypes, a phage display immune library of single domain antibodies was constructed and binders selected which exhibited specificity and affinity for DENV NS1. Each of these single domain antibodies was evaluated for its binding affinity to NS1 from the four serotypes, and incorporated into a sandwich format for NS1 detection. An optimal pair was chosen that provided the best combination of sensitivity for all four DENV NS1 antigens spiked into 50% human serum while showing no cross reactivity to NS1 from Zika virus, yellow fever virus, tick-borne encephalitis virus, and minimal binding to NS1 from Japanese encephalitis virus and West Nile virus. These rugged and robust recombinant binding molecules offer attractive alternatives to conventional antibodies for implementation into immunoassays destined for resource limited locals.

Characterization of the Interaction of Activated FVII with the Endothelial Protein C Receptor (EPCR) in the Rat

Activated FVII (FVIIa) has been shown to interact with EPCR resulting in long-term extravascular tissue presence in mice, well beyond its short circulating half-life. In addition, data in hemophilia patients on short term FVIIa prophylaxis indicate that clinical improvements can persist even in the post-prophylaxis period. Taken together, these data suggest that EPCR may sequester administered FVIIa in the extravascular space that retains activity, potentially explaining the hemostatic improvements that persist long after its infusion has ceased. As such, the EPCR-FVIIa interaction may have mechanistic and translational ramifications in the treatment of bleeds in hemophilia which needs to be further investigated. Unfortunately, hemophilic mice do not model the human disorder in terms of bleeding diathesis. In contrast, the novel rat model of hemophilia A (Nielsen LN et al, 2014) exhibits spontaneous bleeds that, if untreated, can be fatal. Therefore, this model can be utilized to better understand the FVIIa-EPCR interaction in the on-demand and prophylactic action of FVIIa administration in hemophilia. As an initial step towards that goal, here we characterize for the first time the rat FVIIa-EPCR interaction in vitro. We generated rat FVIIa by introduction of a PACE/Furin intracellular cleavage site (RKRRKR) between the light and heavy chains of rat FVII, an approach we have previously shown to result in secretion of two-chain activated FVII protease of various species (human, mouse and canine) in vitro. Recombinant rat FVIIa was purified in a two-chain form using conditioned medium from a HEK-293 stable cell line. The affinity of rat FVIIa to rat EPCR was assessed by incubation of the protease on engineered CHO-K1 cells that stably expressed full length rat EPCR. We found that rat FVIIa exhibited minimal binding to rat EPCR; in contrast human FVIIa bound rat EPCR with a Kd of ~400 nM. The lack of EPCR binding of rat FVIIa, similar to what we previously observed with mouse FVIIa (Pavani G et al., 2014 and 2016), complicates studies aimed at deciphering the hemostatic effects of the FVIIa-EPCR interaction in hemophilia using the rat. Therefore, we decided to engineer a rat FVIIa molecule with EPCR binding capacity so that it can be compared to rat FVIIa (non-EPCR binder) in subsequent in vivo experiments. For this, a rat FVIIa chimeric protease was generated using the first 11 amino acids of the Gla domain of rat protein C (rat FVIIa-FVRAG, with substitutions L4F, L8V, W9R, S10A, S11G). Using CHO-K1 expressing rat EPCR, we found that purified recombinant rat FVIIa-FVRAG bound rat EPCR with a Kd of ~500 nM, comparable to human FVIIa. Considering that the Gla domain, where the modifications are located, is essential for protease function, we also determined the procoagulant activity of rat FVIIa-FVRAG vs. FVIIa. We performed a thrombin generation assay in which each protease was added at increasing concentration (0-500 nM) and we measured the lag time, peak thrombin, endogenous thrombin potential. We found that rat FVIIa, rat FVIIa-FVRAG and human FVIIa (control) behaved similarly in all measured parameters for each concentration tested. In conclusion, we have defined the lack of EPCR binding of rat FVIIa and characterized a recombinant rat FVIIa molecule that can bind rat EPCR in vitro as a gain of function, but is otherwise similar to rat FVIIa. This molecule can now be used to probe the FVIIa-EPCR interaction in the novel and relevant hemophilia A rat model. DisclosuresMargaritis:Bayer Hemophilia Awards: Research Funding; Bristol Myers Squibb: Other: Salary (spouse); Novo Nordisk A/S: Research Funding.

Plasma-polymerized antifouling biochips for label-free measurement of protease activity in cell culture media

We report polyethylene glycol (PEG)-grafting antifouling surfaces using a plasma copolymerized (PcP) technique to monitor protease activity in complex media. By varying the mixing ratio of the PEG and ethylenediamine (EDA) precursors, the PcP-PEG-EDA (PcP-PE) film was able to easily control surface amine density with good preservation of the internal PEG structure. We found that nonspecific protein adsorption was dramatically reduced in serum-containing media on the PcP-PE films, as opposed to that on plasma polymerized-EDA (PP-E) films without PEG. When SPR sensor chips coated with PcP-PE film were employed to detect protease activity, biotinylated luciferase probes (luciferase-peptide-biotin) on streptavidin-conjugated SPR chips enabled real-time and label-free measurement of matrix metalloproteinase activity in cell culture media. Owing to its excellent antifouling ability, this newly developed method boasts minimal nonspecific binding and can serve as a biochip platform to promote a wide range of applications in the biological field.

A comparative analysis of symmetric diketopyrrolopyrrole-cored small conjugated molecules with aromatic flanks: From geometry to charge transport.

Diketopyrrolopyrrole (DPP) derivatives are promising compounds for application in organic electronics. Here, we investigate several symmetrical N-unsubstituted and N-methyl substituted DPPs which differ in the heteroatom in the aromatic flanks. The conformational, electronic, and optical properties are characterized for single molecules in vacuum or a solvent. The intermolecular interactions are evaluated for interacting dimers. Here, a number of stacking geometries is tested, and dimers with mutual orientation of the molecules corresponding to the minimal binding energies are determined. The predicted charge carrier mobilities for stacks having minimal binding energies corroborate experimentally measured values. We conclude that DFT prediction of such stacks is a promising and computationally inexpensive approach to a rough estimation of transport properties. Additionally, the super-cell of the experimentally resolved crystal structure is used to study the dynamics and to compute the charge transport along the hopping pathways. We discuss obtained high mobilities and relate them to the symmetry of DPP core. © 2018 Wiley Periodicals, Inc.

Induction of oligoclonal CD8 T cell responses against pulmonary metastatic cancer by a phospholipid-conjugated TLR7 agonist

Recent advances in cancer immunotherapy have improved patient survival. However, only a minority of patients with pulmonary metastatic disease respond to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T1 breast cancer models, as well as in B16 melanoma, and Lewis lung carcinoma models. In the 4T1 breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist compared with the unconjugated agonist. 1V270 exhibited a wide therapeutic window and minimal off-target receptor binding. The 1V270 therapy inhibited colonization by tumor cells in the lungs in an NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-specific CD8+ cell-dependent adaptive immune responses that suppressed late-stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analyses showed that 1V270 therapy induced oligoclonal T cells in the lungs and mediastinal lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy. Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor-specific adaptive immune responses. These results indicate that systemic 1V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers and that TCR repertoire analyses can be used to monitor, and to predict, the response to therapy.

Abstract 2789: Development of highly potent T-cell receptor bispecifics with picomolar activity against tumor-specific HLA ligands

Abstract T-cell receptor (TCR)-based immunotherapy has emerged as a promising treatment modality for malignant diseases. TCRs naturally recognize human leucocyte antigen (HLA)-bound peptides from foreign and endogenous antigens regardless of their source proteins' extracellular or intracellular location. Using its proprietary discovery engine XPRESIDENT®, Immatics can identify, quantify, and validate Tumor-Associated Peptides (TUMAPs) exclusively presented in cancer tissues. Immatics has established state-of-the-art technology to discover and affinity maturate TUMAP-specific TCRs originating from human T-cell repertoire. The maturated single-chain TCRs (scTv) are used to build a pipeline of highly potent T-cell engaging bispecific TCR molecules directed against TUMAPs. In brief, we use artificial antigen-presenting cells to selectively expand TUMAP-specific T-cells from which the coding TCR sequence is retrieved by 5'RACE after highly sensitive flow cytometry-based single cell sorting. About 50-150 TCRs per TUMAP are transiently re-expressed on human T-cells and extensively characterized for their functional properties. TCRs exhibiting highly active and specific TUMAP recognition are selected for yeast surface display to generate stabilized and affinity maturated scTv. The maturated scTv are engineered into Immatics' proprietary bispecific TCR scaffold comprising a humanized T-cell recruiting antibody domain for potent redirection and activation of T-cells against TUMAPs and an effector function-silenced IgG1 Fc domain. Here we present data of our bispecific TCR program targeting the TUMAP Ag008-01 bound to HLA-A*02. We confirmed the abundant presence of Ag008-01 in several cancer indications and its absence in human normal tissues by using XPRESIDENT® technology combining quantitative mass spectrometry, transcriptomics and bioinformatics. Based on the parental TCR showing highly active and specific recognition of Ag008-01, we generated stabilized and affinity maturated scTv variants with significant improvement in binding affinity towards Ag008-01 in the range of 1000-fold and higher. The maturated scTv variants showed no or minimal binding to off-target peptides selected from the XPRESIDENT® normal tissue database based on the criteria of highest sequence similarity to Ag008-01. By incorporating the maturated scTv into our proprietary bispecific TCR format, which outperformed five alternative TCR bispecific format designs, we obtained highly potent bispecific TCR molecules with picomolar activity. We observed half-maximal lysis of Ag008-01 expressing tumor cell lines at TCR bispecific concentrations below 100 picomolar while no reactivity was observed towards a panel of cell lines lacking Ag008-01 expression. Our data support proof-of-concept for the design of our novel class of T-cell engaging bispecific TCR-antibody fusion proteins. Citation Format: Sebastian Bunk, Martin Hofmann, Felix Unverdorben, Leonie Alten, Meike Hutt, Claudia Wagner, Oliver Schoor, Mathias Ferber, Jens Fritsche, Toni Weinschenk, Harpreet Singh-Jasuja, Dominik Maurer, Carsten Reinhardt. Development of highly potent T-cell receptor bispecifics with picomolar activity against tumor-specific HLA ligands [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2789.

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs.

Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

2516 Ultra-low Na18F tracer dosing for preclinical skeletal imaging enables new concepts in digital PET/CT

OBJECTIVES/SPECIFIC AIMS: The aim of this study was to assess the ultra-dose Na18F dPET protocol feasibility for skeleton imaging in a canine model with reduced radiation dose and preserved quantitative characteristics. We hypothesized that administering an ultra-low Na18F dose would provide suitable image quality while reducing subject’s exposure to radiation. METHODS/STUDY POPULATION: In total, 13 adult male beagles [weight (kg) mean±SD; 14.3±2.2] were scanned. The dogs were administered 3 different Na18F doses: 3 (standard dose/SD), 1 (low dose/LD), and 0.05 (ultra-low dose/ULD) mCi. Imaging started ≃45 minutes post injection for ≃ 33 minute total acquisition time. Covering the whole body, 11 bed positions, acquiring 120 (3 mCi) and 180 (1, 0.05 mCi) seconds per bed position. All imaging was performed on a digital photon counting system (Philips Vereos, pre-commercial release). PET list mode data were reconstructed using Time-of-flight with 4, 2, and 1 mm3 voxel volumes. Point spread function, and Gaussian filtering were applied. Two experienced blinded readers evaluated image sets overall quality, tissue characterization, and quality of background in the whole body skeleton. Three-dimensional (3D) regions of interest (ROI) were traced over the distal femur, first lumbar vertebra, and a portion of the liver, recording standard uptake values (SUVmax and SUVmean). RESULTS/ANTICIPATED RESULTS: All the scans and reconstructions were successfully completed in all subjects. Decreasing Na18F dose from the standard dose (3 mCi) to the ultra-low dose/ULDO (0.05 mCi), demonstrated acceptable image quality and quantification. Ultra-low dose Na18F SUVmean values for the 3D ROIs reported (mean±SD) 2.6±0.7, 2.5±1.1, 9±1.6, and 0.6±0.3 from the right and left distal femur, first lumbar vertebra, and a portion of the liver, respectively. When compared the SD with the LD and ULD, dPET demonstrated acceptable image quality and definition for qualitative overall assessment. This was also found for the overall quantitative ROI assessment of the healthy canine skeletons. DISCUSSION/SIGNIFICANCE OF IMPACT: Ultra-low dose Na18F at a level of 50 μCi for a 14 kg canine appears to be diagnostically feasible and a robust option to reduce (60-fold) radiotracer doses in a translational animal model using a dPET system. Furthermore, it allows us to move preclinical nuclear medicine imaging forward with substantial reduced exposure levels while preserving image quality. Both visual and quantitative results indicate that the standard-dose bone Na18F dPET can be decreased with a satisfactory diagnostic image quality. Ultra-low Na18F dose is indeed important for younger populations, control patients, and nononcological diseases/conditions. Favorable pharmacokinetics of Na18F (such as high bone uptake, minimal binding to serum proteins, rapid single-pass extraction, and fast clearance from the soft tissues) in addition to the technological capabilities of dPET/CT demonstrated feasibility enabling dose reduction strategies. Ultra-low dose has diagnostic reproducibility and lower radiation burden compared with higher fixed dose techniques in current available guidelines [Society of Nuclear Medicine and Molecular Imaging; SNMMI (5–10 mCi)]. Na18F dPET/CT provides higher sensitivity and diagnostic accuracy, which enables high-quality images with lower tracer activity in this translational animal model. Future research will apply the same methodology to other anatomical targets as well as to the use of different tracers. Preclinical nuclear medicine imaging using ultra-low tracer doses, demonstrated the potential to obtain reasonable quality images and diminishing radiation surveillance in accordance with as low as reasonably achievable tracer levels.

In Vivo Characterization and Quantification of Neurofibrillary Tau PET Radioligand 18F-MK-6240 in Humans from Alzheimer Disease Dementia to Young Controls.

Tau PET imaging has potential for elucidating changes in the deposition of neuropathological tau aggregates that are occurring during the progression of Alzheimer disease (AD). This work investigates in vivo kinetics, quantification strategies, and imaging characteristics of a novel tau PET radioligand 18F-MK-6240 in humans. Methods: Fifty-one individuals ranging from cognitively normal young controls to persons with dementia underwent T1-weighted MRI as well as 11C-PiB and 18F-MK-6240 PET imaging. PET data were coregistered to the MRI, and time-activity curves were extracted from regions of interest to assess 18F-MK-6240 kinetics. The pons and inferior cerebellum were investigated as potential reference regions. Reference tissue methods (Logan graphical analysis [LGA] and multilinear reference tissue method [MRTM2]) were investigated for quantification of 18F-MK-6240 distribution volume ratios (DVRs) in a subset of 19 participants. Stability of DVR methods was evaluated using truncated scan durations. SUV ratio (SUVR) estimates were compared with DVR estimates to determine the optimal timing window for SUVR analysis. Parametric SUVR images were used to identify regions of potential off-target binding and to compare binding patterns with neurofibrillary tau staging established in neuropathology literature. Results: SUVs in the pons and the inferior cerebellum indicated consistent clearance across all 51 subjects. LGA and MRTM2 DVR estimates were similar, with LGA slightly underestimating DVR compared with MRTM2. DVR estimates remained stable when truncating the scan duration to 60 min. SUVR determined 70-90 min after injection of 18F-MK-6240 indicated linearity near unity when compared with DVR estimates and minimized potential spill-in from uptake outside the brain. 18F-MK-6240 binding patterns in target regions were consistent with neuropathological neurofibrillary tau staging. Off-target binding regions included the ethmoid sinus, clivus, meninges, substantia nigra, but not the basal ganglia or choroid plexus. Conclusion:18F-MK-6240 is a promising PET radioligand for in vivo imaging of neurofibrillary tau aggregates in AD with minimal off-target binding in the human brain.

A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183-383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

A novel retinoid X receptor agonist, UAB30, inhibits rhabdomyosarcoma cells in vitro

BackgroundWhile patients with early-stage rhabdomyosarcoma (RMS) have seen steady improvement in prognosis over the last 50 y, those with advanced-stage or high-grade disease continue to have a dismal prognosis. Retinoids have been shown to cause growth suppression and terminal differentiation in RMS cells, but the toxicities associated with retinoic acid limit its use. Rexinoids provide an alternative treatment approach to retinoic acid. Rexinoids primarily bind the retinoid X receptor with minimal retinoic acid receptor binding, the entity responsible for many of the toxicities of retinoid therapies. UAB30 is a novel rexinoid with limited toxicities. We hypothesized that UAB30 would lead to decreased cell survival in RMS. Materials and methodsTwo RMS cell lines, one embryonal (RD) subtype and one alveolar (St. Jude Cancer Research Hospital 30) subtype, were used. Cells were treated with UAB30, and cytotoxicity, proliferation, mobility, and apoptosis were evaluated. ResultsUAB30 significantly decreased RMS tumor cell viability and proliferation. Invasion, migration, and attachment-independent growth were reduced following UAB30 treatment. UAB30 also resulted in apoptosis and G1 cell cycle arrest. UAB30 affected both the alveolar and embryonal RMS cell lines in a similar fashion. ConclusionsThe results of these studies suggest a potential therapeutic role for the low-toxicity synthetic retinoid X receptor selective agonist, UAB30, in RMS treatment.

A Ligand System for the Flexible Functionalization of Quantum Dots via Click Chemistry

AbstractWe present a novel ligand, 5‐norbornene‐2‐nonanoic acid, which can be directly added during established quantum dot (QD) syntheses in organic solvents to generate “clickable” QDs at a few hundred nmol scale. This ligand has a carboxyl group at one terminus to bind to the surface of QDs and a norbornene group at the opposite end that enables straightforward phase transfer of QDs into aqueous solutions via efficient norbornene/tetrazine click chemistry. Our ligand system removes the traditional ligand‐exchange step and can produce water‐soluble QDs with a high quantum yield and a small hydrodynamic diameter of approximately 12 nm at an order of magnitude higher scale than previous methods. We demonstrate the effectiveness of our approach by incubating azido‐functionalized CdSe/CdS QDs with 4T1 cancer cells that are metabolically labeled with a dibenzocyclooctyne‐bearing unnatural sugar. The QDs exhibit high targeting efficiency and minimal nonspecific binding.

A Ligand System for the Flexible Functionalization of Quantum Dots via Click Chemistry.

We present a novel ligand, 5-norbornene-2-nonanoic acid, which can be directly added during established quantum dot (QD) syntheses in organic solvents to generate "clickable" QDs at a few hundred nmol scale. This ligand has a carboxyl group at one terminus to bind to the surface of QDs and a norbornene group at the opposite end that enables straightforward phase transfer of QDs into aqueous solutions via efficient norbornene/tetrazine click chemistry. Our ligand system removes the traditional ligand-exchange step and can produce water-soluble QDs with a high quantum yield and a small hydrodynamic diameter of approximately 12 nm at an order of magnitude higher scale than previous methods. We demonstrate the effectiveness of our approach by incubating azido-functionalized CdSe/CdS QDs with 4T1 cancer cells that are metabolically labeled with a dibenzocyclooctyne-bearing unnatural sugar. The QDs exhibit high targeting efficiency and minimal nonspecific binding.

Treatment of Tardive Dyskinesia: A General Overview with Focus on the Vesicular Monoamine Transporter 2 Inhibitors.

Tardive dyskinesia (TD) encompasses the spectrum of iatrogenic hyperkinetic movement disorders following exposure to dopamine receptor-blocking agents (DRBAs). Despite the advent of atypical or second- and third-generation antipsychotics with a presumably lower risk of complications, TD remains a persistent and challenging problem. Prevention is the first step in mitigating the risk of TD, but early recognition, gradual withdrawal of offending medications, and appropriate treatment are also critical. As TD is often a persistent and troublesome disorder, specific antidyskinetic therapies are often needed for symptomatic relief. The vesicular monoamine transporter 2 (VMAT2) inhibitors, which include tetrabenazine, deutetrabenazine, and valbenazine, are considered the treatment of choice for most patients with TD. Deutetrabenazine-a deuterated version of tetrabenazine-and valbenazine, the purified parent product of one of the main tetrabenazine metabolites, are novel VMAT2 inhibitors and the only drugs to receive approval from the US FDA for the treatment of TD. VMAT2 inhibitors deplete presynaptic dopamine and reduce involuntary movements in many hyperkinetic movement disorders, particularly TD, Huntington disease, and Tourette syndrome. The active metabolites of the VMAT2 inhibitors have high affinity for VMAT2 and minimal off-target binding. Compared with tetrabenazine, deutetrabenazine and valbenazine have pharmacokinetic advantages that translate into less frequent dosing and better tolerability. However, no head-to-head studies have compared the various VMAT2 inhibitors. One of the major advantages of VMAT2 inhibitors over DRBAs, which are still being used by some clinicians in the treatment of some hyperkinetic disorders, including TD, is that they are not associated with the development of TD. We also briefly discuss other treatment options for TD, including amantadine, clonazepam, Gingko biloba, zolpidem, botulinum toxin, and deep brain stimulation. Treatment of TD and other drug-induced movement disorders must be individualized and based on the severity, phenomenology, potential side effects, and other factors discussed in this review.

Predicting the Effect of Mutations in the KRas/c-Raf-RBD Protein-Protein Interface

KRas is a small GTPase commonly implicated in several difficult-to-treat cancers such as pancreatic ductal adenocarcinoma (PDAC). KRas normally cycles between an active, GTP-bound form and an inactive, GDP-bound form. Active KRas functions by forming protein-protein interactions (PPIs) with multiple effector proteins in order to regulate various important signal transduction pathways. However, when KRas is mutated it is constitutively active which leads to signal transduction pathway dysregulation that subsequently increases and sustains tumorigenicity and invasiveness. KRas has long been considered an “undruggable” target due to its picomolar affinity for its substrate. However, blocking the PPIs between KRas and its effectors eliminates harmful downstream effects. The tightest known binder of KRas is c-Raf, an enzyme in the ERK1/2 pathway. The Ras-binding domain (RBD) is the minimal binding domain of c-Raf that selectively binds to active, GTP-bound KRas. Previous work has measured Kd for various mutations in the KRas/c-Raf-RBD interface [1, 2]. We use OSPREY [3] (Open Source Protein REdesign for You), a state-of-the-art software package for computational structure-based protein design (CSPD), along with K∗ [4], an algorithm that estimates the binding constant for a given protein complex, to computationally predict the effect of these mutations. We compared our computational predictions to the experimental measurements and found that we can accurately predict the effect of these mutations. These results validate the accuracy of CSPD with OSPREY to target protein-protein interfaces and give us confidence that we can accurately redesign the KRas/c-Raf-RBD interface for future work towards targeting “undruggable” proteins. [1] M. Fridman, et al. Journal of Biological Chemistry, 275(39):30363-30371, 2000. [2] C. Kiel, et al. Journal of Biological Chemistry, 284(46):31893-31902, 2009. [3] P. Gainza, et al. Methods in enzymology, 523:87-107, 2013. [4] I. Georgiev, et al. Journal of computational chemistry, 29(10):1527-1542, 2008.

Production and characterization of a novel Delta-like 1 functional unit as a tool for Notch pathway activation and generation of a specific antibody

Notch signalling is an evolutionary conserved cell-to-cell communication pathway crucial for development and tissue homeostasis. Abnormal Notch signalling by mutations or deregulated expression of its receptors and/or ligands can lead to cancer making it a potential therapeutic target. Delta-like1 (DLL1) is a ligand of the Notch pathway implicated in different types of cancer, including breast cancer. Herein, we produced rhDLL1-DE3, a novel soluble form of DLL1 protein, which contains the DSL domain and EGF1-3 repeats critical for Notch pathway activation. cDNA fragments of human DLL1, encoding truncated versions of DLL1 with regions required to activate Notch receptors, were cloned and expressed as histidine-fused proteins in bacterial and mammalian cells. Expression tests in mammalian cells showed almost exclusively expression of the rhDLL1-DE3 protein form comprising the minimal binding regions DSL to EGF3 to Notch receptors. The highest yield of rhDLL1-DE3 was obtained from E. coli inclusion bodies. The produced protein, with purity higher than 95% bound to human Notch1 recombinant protein, by both Biolayer interferometry and ELISA assays. Cellular assays revealed rhDLL1-DE3 was biologically active as it increased expression of Notch-dependent genes in inducible pluripotent and breast cancer cells. Moreover, rhDLL1-DE3 allowed the generation of polyclonal antibodies by immunization that efficiently recognized DLL1 proteins by immunoblot, and caused a significant decrease of Notch1 expression in MCF7 breast cancer cells. The rhDLL1-DE3 protein might thus be used for Notch pathway activation and to generate anti-DLL1 monoclonal antibodies by immunization or phage display technology to unveil the effect of DLL1 in breast cancer.

Biophysical insights from a single chain camelid antibody directed against the Disrupted-in-Schizophrenia 1 protein.

Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. VHH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a VHH domain derived from an immunized Lama glama, displaying high affinity for the human DISC1 C region (aa 691–836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The VHH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our VHH antibody.

Anti-Tau Antibodies Based Electrochemical Sensor for Detection of Tau Protein Biomarkers

Neuronal tau protein is one of the biomarkers of neurodegeneration which remains without a cure and differential diagnosis. Hence, novel methods are needed for detection of tau protein. Toward this aim, the detection of full-length tau441 protein was achieved using the electrochemical immunoassay based on direct immobilization of anti-tau antibodies on Au surface by using an active N-hydroxysuccinimide ester coupling chemistry (Ab-Au). The electrochemical impedance spectroscopy was used to monitor tau441 binding to Ab-Au in the presence [Fe(CN)6]3−/4− redox probe. The reduction in the charge-transfer resistance, Rct, was observed upon tau441 binding to Ab-Au. Detection of tau441 was observed in μM to nM range with high specificity and selectivity. The electrochemical immunosensor exhibited minimal non-specific binding to bovine serum albumin. Tau441 did not bind to Au surface functionalized with non-tau-specific antibody MOPC-21. The electrochemical biosensor was optimized by screening various anti-tau antibodies. The tau441 binding to anti-tau antibodies was also measured using Enzyme-Linked Immunosorbent Assay (ELISA). The label-free electrochemical impedance spectroscopy immunosensor may be used for selective detection of tau protein in biological fluids, as a biomarker of neurodegeneration.

Pharmacokinetics, biodistribution and receptor mediated endocytosis of a natural Angelica sinensis polysaccharide

The interest in developing new drug carriers for delivery to the liver using natural polysaccharides with a high galactose content has necessitated the study of the pharmacokinetics and tissue distribution of these polysaccharides. In this paper, a new method was established for the microanalysis of Angelica sinensis polysaccharide (ASP) in biosamples. Fluorescein-labelled ASP (FA) was rapidly eliminated from the bloodstream and distributed to the liver with high specificity following intravenous injection. The analysis of the hepatocellular localization demonstrated that FA was predominantly endocytosed by the parenchymal cells, an observation consistent with the results obtained from microscopy studies. Additionally, FA showed a high affinity for asialoglycoprotein receptor-rich cells, while minimal binding of FA to asialoglycoprotein receptor-poor cells was observed. Moreover, the absorption of FA was markedly inhibited by the co-administration of neogalactosylalbumin (NGA) both in vivo and in vitro. To allow for the visualization of the systemic circulation of ASP, 99mTc-DTPA-ASP was synthesized and in vivo imaging was performed with single photon emission computed tomography (SPECT). It also showed a high aggregation of 99mTc-DTPA-ASP in liver. These results suggest that the distribution of ASP to the liver occurs via asialoglycoprotein receptor (ASGPR) mediated endocytosis and ASP could potentially be applied as a new carrier for delivering drugs to the liver.