Tissue Binding Research Articles

Monomethyl Auristatin E (MMAE), a Payload for Multiple Antibody Drug Conjugates (ADCs), Demonstrates Differential Red Blood Cell Partitioning Across Human and Animal Species

Background: Monomethyl auristatin E (MMAE) has been used as a payload for several Food and Drug Administration (FDA) approved antibody-drug conjugates (ADCs). It is known that MMAE is released from the ADC following binding, internalization and proteolytic degradation in target tissues. A striking discrepancy in systemic MMAE levels has been observed across species with 50-fold higher MMAE levels in human than that in rodents when normalized by ADC dose with unknown mechanism. Hypothesis and purpose: Multiple factors could affect systemic MMAE levels such as production and elimination of unconjugated MMAE following ADC dosing. In this study, we have explored whether MMAE displays differential red blood cell (RBC) partitioning across species that may contribute to the different MMAE levels seen between human and animals. Experiments: To determine MMAE RBC partitioning, tritium labeled MMAE ([3H]-MMAE) was incubated in whole blood from mice, rats, monkeys and humans in vitro, then RBC partitioning was determined and compared across species. To test whether MMAE released from the ADC would show any difference in RBC partitioning, pinatuzumab vedotin or polatuzumab vedotin was administered to mice, rats, and monkeys. MMAE levels were measured in both blood and plasma, and the ratios of MMAE levels were calculated as blood-to-plasma ratio (in vivo RBC partitioning). Results: Our in vitro data showed that unconjugated MMAE has a species-dependent RBC partitioning with strong RBC partitioning in mouse, rat, followed by monkey blood, whereas minimal RBC partitioning was seen in human blood. Incubation of 2 nM of MMAE in mouse blood resulted in a blood-to-plasma ratio of 11.8 ± 0.291, followed by rat, monkey, and human at 2.36 ± 0.0825, 1.57 ± 0.0250, and 0.976 ± 0.0620, respectively. MMAE RBC partitioning is also concentration-dependent, with an inverse relationship between RBC partitioning and MMAE concentration (higher RBC partitioning at lower concentration). In vivo dosing of pinatuzumab vedotin in mouse displayed systemic MMAE at about a 5-fold higher blood concentration compared to plasma concentration once MMAE reached a pseudo-equilibrium, while systemic MMAE from blood and plasma concentration showed a 1.65-fold difference in rat. Implication and conclusion: These data demonstrated that MMAE has a distinct RBC partitioning across different species, which may contribute to, at least in part, to the differential in the systemic MMAE levels observed in vivo between preclinical and clinical studies. These findings highlight the importance of fully characterizing the ADME properties of both the ADC and its payload, to enable better translation from animals to human for ADC development.

Predictions of Bedaquiline Central Nervous System Exposure in Patients with Tuberculosis Meningitis Using Physiologically based Pharmacokinetic Modeling.

The use of bedaquiline as a treatment option for drug-resistant tuberculosis meningitis (TBM) is of interest to address the increased prevalence of resistance to first-line antibiotics. To this end, we describe a whole-body physiologically based pharmacokinetic (PBPK) model for bedaquiline to predict central nervous system (CNS) exposure. A whole-body PBPK model was developed for bedaquiline and its metabolite, M2. The model included compartments for brain and cerebrospinal fluid (CSF). Model predictions were evaluated by comparison to plasma PK time profiles following different dosing regimens and sparse CSF concentrations data from patients. Simulations were then conducted to compare CNS and lung exposures to plasma exposure at clinically relevant dosing schedules. The model appropriately described the observed plasma and CSF bedaquiline and M2 concentrations from patients with pulmonary tuberculosis (TB). The model predicted a high impact of tissue binding on target site drug concentrations in CNS. Predicted unbound exposures within brain interstitial exposures were comparable with unbound vascular plasma and unbound lung exposures. However, unbound brain intracellular exposures were predicted to be 7% of unbound vascular plasma and unbound lung intracellular exposures. The whole-body PBPK model for bedaquiline and M2 predicted unbound concentrations in brain to be significantly lower than the unbound concentrations in the lung at clinically relevant doses. Our findings suggest that bedaquiline may result in relatively inferior efficacy against drug-resistant TBM when compared with efficacy against drug-resistant pulmonary TB.

Abstract 744: Novel conditionally active biologic (CAB) tetravalent T-cell engagers targeting solid tumors

Abstract The use of T-cell engagers (TCEs) has great therapeutic potential in oncology. This potential, however, is diminished due to severe toxicity (cytokine release effects, as well as on-target off-tumor toxicities). We leveraged BioAtla’s Conditionally Active Biologic (CAB) technology (1) to develop bispecific antibodies which have no or very little binding to CD3 and to the tumor target antigen (TAA) in healthy tissue (normal physiological conditions), yet have strong binding in diseased tissues (i.e. tumor microenvironment, TME) derived from glycolytic tumor metabolism, which supports tumor growth (Warburg effect). The conditional activity in the TME is achieved by optimizing the sequences of the binding domains and does not require masking and subsequent activation of the binding domain as used in pro-drugs. We have developed dual-CAB T-cell engagers targeting serval well-established tumor associated antigens. Data demonstrating superior potency with reduced off-target binding through in vitro and in vivo characterization of three new DualCAB TCEs, targeting PSMA, Trop2, and Tissue Factor (TF) will be presented.(1) Chang HW, Frey G, Liu H, Xing C, Steinman L, Boyle WJ and Short, JM Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches. Proc Natl Acad Sci U S A 2021;118. Citation Format: Ana Paula Cugnetti, Haizhen Liu, Patricia McNeeley, Charles Xing, Kathryn Woodard, Hwai Chang, Gerhard Frey, William J. Boyle, Jay M. Short. Novel conditionally active biologic (CAB) tetravalent T-cell engagers targeting solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 744.

O95: The role of the complement system in mediating ischaemic cholangiopathy in DCD liver transplantation

Abstract Background The discrepancy between available organs and the number of patients on the waiting list have led to an increasing number of donor after circulatory death (DCD) liver grafts being used. DCD liver grafts suffer from a higher risk of ischaemic cholangiopathy, the pathophysiology of which is poorly understood. We hypothesized that the complement system mediates damage to the arterial endothelium, thereby affecting the ability of cholangiocytes to regenerate. Methods Livers declined for clinical transplantation were perfused for up to 10 hours with anti-coagulated packed red cells at 37° with or without complement inhibition. Levels of circulating complement components, tissue binding of complement proteins, and organ damage markers were measured. Results 5 DBD and 5 DCD livers were perfused, and 5 further DCD livers being perfused with eculizumab. C3 production was significantly higher in the untreated DCD cohort compared to DBD livers at 10 hours (52.68 mg/ml vs 8.62 mg/ml, p<0.005). C5b-9 deposition on the arterial endothelium of the portal tracts was significantly higher in the untreated DCD cohort compared to the treated DCD livers (mean 0.9 R.U vs 0.4 R.U, p<0.05). There was a non-significant difference in bile production and composition between the treated DCD and untreated DCD cohort. Conclusions There is evidence that the complement system mediates the damage to the arterial endothelium within the portal tracts of the liver. This damage could be ameliorated by complement inhibition. Further work to correlate this findings with clinical outcomes will be required.

Radiosynthesis, structural identification and in vitro tissue binding study of [18F]FNA-S-ACooP, a novel radiopeptide for targeted PET imaging of fatty acid binding protein 3

BackgroundFatty acid binding protein 3 (FABP3) is a target with clinical relevance and the peptide ligand ACooP has been identified for FABP3 targeting. ACooP is a linear decapeptide containing a free amino and thiol group, which provides opportunities for conjugation. This work is to develop methods for radiolabeling of ACooP with fluorine-18 (18F) for positron emission tomography (PET) applications, and evaluate the binding of the radiolabeled ACooP in human tumor tissue sections with high FABP3 expression.ResultsThe prosthetic compound 6-[18F]fluoronicotinic acid 4-nitrophenyl ester was conveniently prepared with an on-resin 18F-fluorination in 29.9% radiochemical yield and 96.6% radiochemical purity. Interestingly, 6-[18F]fluoronicotinic acid 4-nitrophenyl ester conjugated to ACooP exclusively by S-acylation instead of the expected N-acylation, and the chemical identity of the product [18F]FNA-S-ACooP was confirmed. In the in vitro binding experiments, [18F]FNA-S-ACooP exhibited heterogeneous and high focal binding in malignant tissue sections, where we also observed abundant FABP3 positivity by immunofluorescence staining. Blocking study further confirmed the [18F]FNA-S-ACooP binding specificity.ConclusionsFABP3 targeted ACooP peptide was successfully radiolabeled by S-acylation using 6-[18F]fluoronicotinic acid 4-nitrophenyl ester as the prosthetic compound. The tissue binding and blocking studies together with anti-FABP3 immunostaining confirmed [18F]FNA-S-ACooP binding specificity. Further preclinical studies of [18F]FNA-S-ACooP are warranted.

The Effects of Structural & Materials Design on the Mechanisms of Tissue Integration with the 3D Printed Polyether-Ether-Ketone Cranial Implants in Vivo

3D printed polyether-ether-ketone (PEEK) implant has become a popular clinical alternative to implants made of titanium alloy for cranial bone substitutes due to its bone-match properties, however its biological inert hindered the progress of clinical applications of such material. To enhance the tissue integration capability of PEEK implants and promote their long-term stability, cranial implants of gradient porous structures were designed and manufactured via fused filament fabrication (FFF) 3D printing technology in both PEEK and PEEK/HA composites materials, then functionally evaluation of the implants on the tissue in-growth and the osteointegration mechanisms was conducted via animal tests. The 3D printed PEEK scaffold was found to have 2–5 folds of the compressive strength to those of the natural cranial bone. The in vivo studies verified that the porous PEEK/HA scaffold could effectively induce the bone ingrowth to form a stable biointegration boundary surrounding the host bone tissue after 4 weeks of implantation. Moreover, the PEEK/HA scaffold showed no significant advantages in improving the soft tissue in-growth, only making its distribution more evenly. It is also interestingly to find out that the vertically connective pores throughout the implant did not enhance the tissue binding force even though it did promote the nutrient transportation. In conclusion, the use of PEEK/HA composite material and a well-designed porous structure was proved to be an effective approach to improve the biointegration between the implant and host tissues.

Novel melanin‐binding carbonic anhydrase II inhibitor for long‐acting topical glaucoma treatment

Aims/Purpose: We are developing new eye drop treatment for glaucoma with dosing frequency once a week by utilizing drug binding to ocular melanin. Carbonic anhydrase (CA) II enzyme inhibitors decrease intraocular pressure (IOP) and thus halt the progression of retinal damage by reducing the secretion of aqueous humour in the ciliary body. Since ciliary body is a melanin‐rich tissue, we hypothesize that long‐lasting CAII inhibition and IOP‐lowering effect may be achieved due to gradual drug release from melanin‐bound drug depot.Methods: Melanin binding affinity for about 30 novel CAII inhibitors was studied with microscale thermophoresis, and three structurally rather similar and equipotent molecules with different melanin binding properties were selected for in vivo efficacy studies. IOP‐lowering effect of these new molecules was studied with albino and pigmented rabbits with iCare® PRO rebound tonometer. For pharmacokinetic studies, the high melanin binding new CAI (molecule 184) was labelled with tritium, and the study was conducted with albino and pigmented rats with conventional methods. Bound drug was analysed from iris‐ciliary body pellet and unbound fraction from supernatants.Results: Melanin‐binding affinity of new CAII inhibitors varies from low or non‐binders (Kd > 650 μM) to high melanin binders (Kd < 65 μM). In vivo efficacy results show that the molecules decrease IOP in both albino and pigmented eyes at the level comparable with dorzolamide (Trusopt®). In pigmented eyes, melanin binding prolongs the effect of compound 184 even up to 2 weeks after single eye drop. In the pharmacokinetic rat study, molecule 184 shows remarkably prolonged tissue binding and high iris‐ciliary body exposure in pigmented eyes.Conclusions: After topical administration IOP‐lowering effect of high melanin‐binding CA II inhibitor is significantly prolonged in pigmented eyes.

Evaluation of an Image-Derived Input Function for Kinetic Modeling of Nicotinic Acetylcholine Receptor-Binding PET Ligands in Mice.

Positron emission tomography (PET) radioligands that bind with high-affinity to α4β2-type nicotinic receptors (α4β2Rs) allow for in vivo investigations of the mechanisms underlying nicotine addiction and smoking cessation. Here, we investigate the use of an image-derived arterial input function and the cerebellum for kinetic analysis of radioligand binding in mice. Two radioligands were explored: 2-[18F]FA85380 (2-FA), displaying similar pKa and binding affinity to the smoking cessation drug varenicline (Chantix), and [18F]Nifene, displaying similar pKa and binding affinity to nicotine. Time-activity curves of the left ventricle of the heart displayed similar distribution across wild type mice, mice lacking the β2-subunit for ligand binding, and acute nicotine-treated mice, whereas reference tissue binding displayed high variation between groups. Binding potential estimated from a two-tissue compartment model fit of the data with the image-derived input function were higher than estimates from reference tissue-based estimations. Rate constants of radioligand dissociation were very slow for 2-FA and very fast for Nifene. We conclude that using an image-derived input function for kinetic modeling of nicotinic PET ligands provides suitable results compared to reference tissue-based methods and that the chemical properties of 2-FA and Nifene are suitable to study receptor response to nicotine addiction and smoking cessation therapies.

Sea-ATI unravels novel vocabularies of plant active cistrome.

The cistrome consists of all cis-acting regulatory elements recognized by transcription factors (TFs). However, only a portion of the cistrome is active for TF binding in a specific tissue. Resolving the active cistrome in plants remains challenging. In this study, we report the assay sequential extraction assisted-active TF identification (sea-ATI), a low-input method that profiles the DNA sequences recognized by TFs in a target tissue. We applied sea-ATI to seven plant tissues to survey their active cistrome and generated 41 motif models, including 15 new models that represent previously unidentified cis-regulatory vocabularies. ATAC-seq and RNA-seq analyses confirmed the functionality of thecis-elements from the new models, in that they are actively bound in vivo, located near the transcription start site, and influence chromatin accessibility and transcription. Furthermore, comparing dimeric WRKY CREs between sea-ATI and DAP-seq libraries revealed that thermodynamics and genetic drifts cooperatively shaped their evolution. Notably, sea-ATI can identify not only positive but also negative regulatory cis-elements, thereby providing unique insights into the functional non-coding genome of plants.

A New Version of the Tissue Composition-Based Model for Improving the Mechanism-Based Prediction of Volume of Distribution at Steady-State for Neutral Drugs

In-vitro models are available in the literature for predicting the volume of distribution at steady-state (Vdss) of drugs. The mechanistic model refers to the tissue composition-based model (TCM), which includes important factors that govern Vdss such as drug physiochemistry and physiological data. The recognized TCM published by Rodgers and Rowland (TCM-RR) and a subsequent adjustment made by Simulations Plus Inc. (TCM-SP) have been shown to be generally less accurate with neutral compared to ionized drugs. Therefore, improving these models for neutral drugs becomes necessary. The objective of this study was to propose a new TCM for improving the prediction of Vdss for neutral drugs. The new TCM included two modifications of the published models (i) accentuate the effect of the blood-to-plasma ratio (BPR) that should cover permeated molecules across the biomembranes, which is lacking in these models for neutral compounds, and (ii) use a different approach to estimate the binding in tissues. The new TCM was validated with a large dataset of 202 commercial and proprietary compounds including preclinical and clinical data. All scenario datasets were predicted more accurately with the TCM-New, whereas all statistical parameters indicate that the TCM-New showed significant improvements in terms of accuracy over the TCM-RR and TCM-SP. Predictions of Vdss were frequently more accurate for the TCM-new with 83% within twofold error versus only 50% for the TCM-RR. And more than 95% of the predictions were within threefold error and patient interindividual differences can be predicted with the TCM-New, greatly exceeding the accuracy of the published models. Overall, the new TCM incorporating BPR significantly improved the Vdss predictions in animals and humans for neutral drugs, and, hence, has the potential to better support the drug discovery and facilitate the first-in-human predictions.

Synthesis, Characterization, and Applications of a Superior Dendrimer-Based Polymer for Multiplexed Ion Beam Imaging Time-of-Flight Analysis.

High-dimensional single-cell mass spectrometric imaging techniques such as multiplexed ion beam imaging by time-of-flight mass spectrometry (MIBI-TOF), imaging mass cytometry (IMC), and flow cytometry-based CyTOF utilize antibodies conjugated to linear metal-chelating polymers. Here, we report on the synthesis and characterization of a dendrimer-based polymer and its utilization in tissue imaging using MIBI-TOF. We compared the staining performance in FFPE tissue of antibodies for lineage-specific immune proteins (CD20, CD3, CD45, FoxP3) that were conjugated with dendrimer or linear polymer. Staining of serial tissue sections with dendron-conjugated and linear-polymer-conjugated antibodies revealed comparable avidities of dendrons and linear polymers with log2 (ratio of mean positive pixel intensity of staining for linear polymers to dendrons) within the range ±0.25. Interestingly, dendron-conjugated antibodies were observed to have some advantages over linear polymer-conjugated antibodies. For example, tissue staining of a nuclear protein, FoxP3 with dendron-conjugated antibodies showed notably less background staining than that of linear-polymer-conjugated antibodies. Additionally, dendron-conjugated antibodies did not exhibit off-target cytosolic binding in neural tissue typically observed when using linear polymer conjugates. Taken together, this work provides a versatile framework for using third-generation dendron-conjugated antibodies with improved staining over conventional linear polymers.

Blood- and Urine-Based Liquid Biopsy for Early-Stage Cancer Investigation: Taken Clear Renal Cell Carcinoma as a Model

Liquid biopsy is a noninvasive technique that can provide valuable information for disease characterization by using biofluids as a source of biomarkers. Proteins found in biofluids can offer a wealth of information for understanding pathological processes. In this study, we used early-stage clear cell renal cell carcinoma (ccRCC) as a model to explore the proteomic relationships among tissue, plasma, and urine. We analyzed samples of tumor tissue, plasma, and urine from a cohort of 27 ccRCC patients with T1-2 stage and 27 matched healthy controls, using liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis. We integrated the differential proteins found in the three types of samples to explore ccRCC-associated molecular changes. Our results showed that both plasma and urine proteomes could reflect functional changes in tumor tissue. In plasma, cytoskeletal proteins and metabolic enzymes were differentially expressed, while in urine, adhesion molecules and defense proteins showed differential levels. The differential proteins found in plasma and urine both reflect the binding and catalytic activity of tumor tissue. Additionally, proteins only changed in biofluids could reflect body immune response changes, with plasma proteins involved in actin cytoskeleton and oxidative stress, and urine proteins involved in granulocyte adhesion and leukocyte extravasation signaling. Plasma and urine proteins could effectively distinguish RCC from control, with good performances (plasma/urine: 92.6%/92.6% specificity, 96.3%/92.6% sensitivity, and an area under the curve of 0.981/0.97). In conclusion, biofluids could not only reflect functional changes in tumor tissue but also reflect changes in the body's immune response. These findings will benefit the understanding of body biomarkers in tumors and the discovery of potential disease biomarkers.

Synaptic vesicle glycoprotein 2A (SV2A) levels in the cerebral cortex in patients with Alzheimer's disease: a radioligand binding study in postmortem brains

Histological and biochemical analyses in postmortem tissues have demonstrated neurodegenerative changes in the cerebral cortex in patients with Alzheimer's disease (AD), and it has been suggested that this represents a loss of synapses. PET imaging of the (pre)synaptic vesicular glycoprotein 2A (SV2A) has demonstrated a reduction in synapse density in AD in the hippocampus but not consistently in the neocortex. This investigation examines the level of [3H]UCB-J binding in postmortem cortical tissue from patients with AD and matched healthy controls using autoradiography. Among the neocortical areas examined, the binding was significantly lower only in the middle frontal gyrus in AD compared to matched controls. No differences were observed in the parietal, temporal, or occipital cortex. The binding levels in the frontal cortex in the AD cohort displayed large variability among subjects, and this revealed a highly significant negative association with the age of the patient. These results demonstrate low UCB-J binding in the frontal cortex of patients with AD, and this biomarker correlates negatively with age, which may further indicate that SV2A could be an important biomarker in AD patients.

Computational design and molecular modeling of morphine derivatives for preferential binding in inflamed tissue.

The opioid epidemic has impacted over 10million Americans in 2019. Opioids, like morphine, bind non-selectively in both peripheral tissue, leading to effective pain relief, and central tissue, resulting in dangerous side effects and addiction. The inflamed conditions of injured tissues have a lower pH (pH=6-6.5) environment than healthy tissue (pH=7.4). We aim to design a morphine derivative that binds selectively within inflamed tissue using molecular extension and dissection techniques. Morphine binds to the μ-opioid receptor (MOR) when the biochemically active amine group is protonated. Fluorination of a β-carbon from the tertiary amine group led to a reduced pKa of the derivative through induction. Through a decrease in the pKa, protonation is still statistically favored in lower pH environments of inflamed tissue but primarily deprotonated in healthy tissue. The cyclohexenol and N-methyl-piperidine rings of morphine are removed to increase conformational flexibility when binding while still maintaining the interactions required for analgesia. Electronic structure calculations were performed with Gaussian16 using the Keck Computational Research Cluster at Chapman University to determine the pKa. The theoretical pKa values are determined at the M06-2X(SMD)/aug-cc-pVDZ level of theory to calculate the ΔG°aq values for the amine deprotonation reactions. Fluoromorphine β-C2 was designed computationally and modeled within the MOR using Maestro: Schrödinger. This derivative exhibits a pKa reduction and enhanced ligand-protein interactions within the MOR. β-fluorination decreased the overall pKa values of the morphine derivatives (pKa: 6.1-7.83) relative to morphine, reducing binding within healthy, central tissue.

UNUSUAL PRESENTATION OF LUPUS NEPHRITIS IN A 22 YEAR OLD MALE : A CASE REPORT

Systemic lupus erythematosus (SLE) is an autoimmune disease in which organ and cell undergo damage initially mediated by tissue binding autoantibodies & immune complexes.In most patients, autoantibodies are present for a few years before the rst clinical symptoms appears. It is ve to six times more prevalent in women than in men .Nephritis is usually the most serious manifestation of SLE, particularly because nephritis and infection are the leading cause of mortality in the rst decade of disease. We present a case of a 22 yr old male who came with complaint of bilateral pedal oedema and pufness of face since 1 month . On examination, patient was stable hemodynamically with pulse of 88 per minute, blood pressure of 110/80 millimeter of mercury with room air saturation of 99 percent and jugular venous pressure of 6-7 millimeters of water. He also had pallor in lower palpebral conjunctiva and bilateral pitting pedal oedema.There was no clubbing, icterus,cyanosis or lymphadenopathy. His routine blood investigations showed anaemia , leucocytosis , elevated creatinine, hyperkalaemia , hypoalbuminemia, iron deciency ,urine routine microscopy suggestive of RBC cast 60-70/high power eld . Complement C3 and C4 were low, ANA positive grade 3(1:1000) homogeneous and cytoplasmic speckled, ANA BLOT positive for dsDNA, nucleosome, Histones, Sm/RNP,Mi2b.His ultrasound (USG) revealed bilateral bulky kidney and heterogeneous with raised cortical echogenicity and normal size. His renal biopsy was done which showed diffuse lupus nephritis(ISN/RPS2018 modication)class IV and indices(modied NIH) of activity 15/24 and chronicity 2/12, necrotizing vascular lesions along with evidence of vascular immune deposits in diffuse immunoorescence studies and associated inammatory cells are noted. Patient received intravenous pulse therapy of methyl prednisolone 1gram for 5 days with monitoring of kidney function test. Patient was put on cyclophosphamide with hemodialysis for immunosuppression because patient was not responding to steroids, as seen by rising serum creatinine and urea levels and declining urine output. The patient improved dramatically, urine output increased and serum creatinine and urea decreased, therefore haemodialysis was discontinued, and the patient was managed conservatively and discharged on oral prednisolone with a monthly cyclophospmide injection.

Identification of a Putative α-synuclein Radioligand Using an in silico Similarity Search.

Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates. Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson's disease (PD) and Alzheimer's disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21. In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aβ42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.). Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aβ and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies.

Feasibility Study to Assess Canagliflozin Distribution and Sodium-Glucose Co-Transporter 2 Occupancy Using [18 F]Canagliflozin in Patients with Type 2 Diabetes.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors, including canagliflozin, reduce the risk of cardiovascular and kidney outcomes in patients with and without type 2 diabetes, albeit with a large interindividual variation. The underlying mechanisms for this variation in response might be attributed to differences in SGLT2 occupancy, resulting from individual variation in plasma and tissue drug exposure and receptor availability. We performed a feasibility study for the use of [18 F]canagliflozin positron emission tomography (PET) imaging to determine the association between clinical canagliflozin doses and SGLT2 occupancy in patients with type 2 diabetes. We obtained two 90-minute dynamic PET scans with diagnostic intravenous [18 F]canagliflozin administration and a full kinetic analysis in 7 patients with type 2 diabetes. Patients received 50, 100, or 300 mg oral canagliflozin (n = 2:4:1) 2.5 hours before the second scan. Canagliflozin pharmacokinetics and urinary glucose excretion were measured. The apparent SGLT2 occupancy was derived from the difference between the apparent volume of distribution of [18 F]canagliflozin in the baseline and post-drug PET scans. Individual canagliflozin area under the curve from oral dosing until 24-hours (AUCP0-24h ) varied largely (range 1,715-25,747 μg/L*hour, mean 10,580 μg/L*hour) and increased dose dependently with mean values of 4,543, 6,525, and 20,012 μg/L*hour for 50, 100, and 300 mg, respectively (P = 0.046). SGLT2 occupancy ranged between 65% and 87%, but did not correlate with canagliflozin dose, plasma exposure, or urinary glucose excretion. We report the feasibility of [18 F]canagliflozin PET imaging to determine canagliflozin kidney disposition and SGLT2 occupancy. This suggests the potential of [18 F]canagliflozin as a tool to visualize and quantify clinically SGLT2 tissue binding.

Expanding the apelin receptor pharmacological toolbox using novel fluorescent ligands.

The apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a class A GPCR, targeting the apelin receptor is an attractive therapeutic strategy. With improvements in imaging techniques, and the stability and brightness of dyes, fluorescent ligands are becoming increasingly useful in studying protein targets. Here, we describe the design and validation of four novel fluorescent ligands; two based on [Pyr1]apelin-13 (apelin488 and apelin647), and two based on ELA-14 (ELA488 and ELA647). Fluorescent ligands were pharmacologically assessed using radioligand and functional in vitro assays. Apelin647 was validated in high content imaging and internalisation studies, and in a clinically relevant human embryonic stem cell-derived cardiomyocyte model. Apelin488 and ELA488 were used to visualise apelin receptor binding in human renal tissue. All four fluorescent ligands retained the ability to bind and activate the apelin receptor and, crucially, triggered receptor internalisation. In high content imaging studies, apelin647 bound specifically to CHO-K1 cells stably expressing apelin receptor, providing proof-of-principle for a platform that could screen novel hits targeting this GPCR. The ligand also bound specifically to endogenous apelin receptor in stem cell-derived cardiomyocytes. Apelin488 and ELA488 bound specifically to apelin receptor, localising to blood vessels and tubules of the renal cortex. Our data indicate that the described novel fluorescent ligands expand the pharmacological toolbox for studying the apelin receptor across multiple platforms to facilitate drug discovery.

Revealing the Specificity of Human H1 Influenza A Viruses to Complex N-Glycans.

Influenza virus infection remains a threat to human health since viral hemagglutinins are constantly drifting, escaping infection and vaccine-induced antibody responses. Viral hemagglutinins from different viruses display variability in glycan recognition. In this context, recent H3N2 viruses have specificity for α2,6 sialylated branched N-glycans with at least three N-acetyllactosamine units (tri-LacNAc). In this work, we combined glycan arrays and tissue binding analyses with nuclear magnetic resonance experiments to characterize the glycan specificity of a family of H1 variants, including the one responsible for the 2009 pandemic outbreak. We also analyzed one engineered H6N1 mutant to understand if the preference for tri-LacNAc motifs could be a general trend in human-type receptor-adapted viruses. In addition, we developed a new NMR approach to perform competition experiments between glycans with similar compositions and different lengths. Our results point out that pandemic H1 viruses differ from previous seasonal H1 viruses by a strict preference for a minimum of di-LacNAc structural motifs.

Dual specificity and target gene selection by the MADS-domain protein FRUITFULL.

How transcription factors attain their target gene specificity and how this specificity may be modulated, acquiring different regulatory functions through the development of plant tissues, is an open question. Here we characterized different regulatory roles of the MADS-domain transcription factor FRUITFULL (FUL) in flower development and mechanisms modulating its activity. We found that the dual role of FUL in regulating floral transition and pistil development is associated with its different in vivo patterns of DNA binding in both tissues. Characterization of FUL protein complexes by liquid chromatography-tandem mass spectrometry and SELEX-seq experiments shows that aspects of tissue-specific target site selection can be predicted by tissue-specific variation in the composition of FUL protein complexes with different DNA binding specificities, without considering the chromatin status of the target region. This suggests a role for dynamic changes in FUL TF complex composition in reshaping the regulatory functions of FUL during flower development.