Low-affinity Binders Research Articles

Single-chain nanobody inhibition of Notch and avidity enhancement utilizing the β-pore forming toxin Aerolysin.

Notch plays critical roles in developmental processes and disease pathogenesis, which has led to numerous efforts to modulate its function with small molecules and antibodies. Here we present a nanobody inhibitor of Notch signaling, derived from a synthetic phage-display library targeting the notch Negative Regulatory Region (NRR). The nanobody inhibits Notch signaling in a luciferase reporter assay and in Notch-dependent hematopoietic progenitor cell differentiation assay, despite a modest 19uM affinity for Notch. We addressed the low affinity by fusion to a membrane-associating domain derived from the β-Pore forming toxin Aerolysin, resulting in a significantly improved IC50 for Notch inhibition. The nanobody-aerolysin fusion inhibits proliferation of T-ALL cell lines with similar efficacy to other Notch pathway inhibitors. Overall, this study reports the development of a Notch inhibitory antibody, and demonstrates a proof-of-concept for a generalizable strategy to increase the efficacy and potency of low-affinity antibody binders.

High-affinity biomolecular interactions are modulated by low-affinity binders

The strength of molecular interactions is characterized by their dissociation constants (KD). Only high-affinity interactions (KD ≤ 10−8 M) are extensively investigated and support binary on/off switches. However, such analyses have discounted the presence of low-affinity binders (KD > 10−5 M) in the cellular environment. We assess the potential influence of low-affinity binders on high-affinity interactions. By employing Gillespie stochastic simulations and continuous methods, we demonstrate that the presence of low-affinity binders can alter the kinetics and the steady state of high-affinity interactions. We refer to this effect as ‘herd regulation’ and have evaluated its possible impact in two different contexts including sex determination in Drosophila melanogaster and in signalling systems that employ molecular thresholds. We have also suggested experiments to validate herd regulation in vitro. We speculate that low-affinity binders are prevalent in biological contexts where the outcomes depend on molecular thresholds impacting homoeostatic regulation.

Probing Protein-Ligand Methyl-π Interaction Geometries through Chemical Shift Measurements of Selectively Labeled Methyl Groups.

Fragment-based drug design is heavily dependent on the optimization of initial low-affinity binders. Herein we introduce an approach that uses selective labeling of methyl groups in leucine and isoleucine side chains to directly probe methyl-π contacts, one of the most prominent forms of interaction between proteins and small molecules. Using simple NMR chemical shift perturbation experiments with selected BRD4-BD1 binders, we find good agreement with a commonly used model of the ring-current effect as well as the overall interaction geometries extracted from the Protein Data Bank. By combining both interaction geometries and chemical shift calculations as fit quality criteria, we can position dummy aromatic rings into an AlphaFold model of the protein of interest. The proposed method can therefore provide medicinal chemists with important information about binding geometries of small molecules in fast and iterative matter, even in the absence of high-resolution experimental structures.

Cyclosporin A-Based PROTACs Can Deplete Abundant Cellular Cyclophilin A without Suppressing T Cell Activation.

Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future.

Translocator protein (TSPO) genotype does not change cerebrospinal fluid levels of glial activation, axonal and synaptic damage markers in early Alzheimer’s disease

BackgroundPET imaging of the translocator protein (TSPO) is used to assess in vivo brain inflammation. One of the main methodological issues with this method is the allelic dependence of the radiotracer affinity. In Alzheimer’s disease (AD), previous studies have shown similar clinical and patho-biological profiles between TSPO genetic subgroups. However, there is no evidence regarding the effect of the TSPO genotype on cerebrospinal-fluid biomarkers of glial activation, and synaptic and axonal damage. MethodWe performed a trans-sectional study in early AD to compare cerebrospinal-fluid levels of GFAP, YKL-40, sTREM2, IL-6, IL-10, NfL and neurogranin between TSPO genetic subgroups. ResultsWe recruited 33 patients with early AD including 16 (48%) high affinity binders, 13 (39%) mixed affinity binders, and 4/33 (12%) low affinity binders. No difference was observed in terms of demographics, and cerebrospinal fluid levels of each biomarker for the different subgroups. ConclusionTSPO genotype is not associated with a change in glial activation, synaptic and axonal damage in early AD. Further studies with larger numbers of participants will be needed to confirm that the inclusion of specific TSPO genetic subgroups does not introduce selection bias in studies and trials of AD that combine TSPO imaging with cerebrospinal fluid biomarkers.

An acetyltranferase moonlights as a regulator of the RNA binding repertoire of the RNA chaperone Hfq in Escherichia coli.

Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, a mediator for sRNA-mRNA annealing. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was previously identified as a silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and investigated its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppressed all tested Hfq-dependent sRNA signaling. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked the interaction interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, and HqbA interacted with Hfq at a site far from the active site. This suggests that HqbA may be bifunctional, with separate roles for regulating via Hfq interaction and for acetylation of undefined substrates. Gel shift assays revealed that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs but not high-affinity RNAs. Comparative RNA immunoprecipitation of Hfq and sequencing showed enrichment of two tRNA precursors, metZWV and proM, by Hfq in mutants that lost the HqbA-Hfq interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

NK1 receptor binding of a few low molecular weight 3,5-bistrifluoromethylbenzene derivatives

We were interested in finding smallest possible ligands of the NK1 receptor (NK1R). Based on structural considerations and molecular docking (scoring) we selected six simple (low molecular weight, MW) 3,5-bistrifluoromethylbenzene derivatives and tested them for human NK1R affinity. The compounds turned out to have detectable but very low affinity for the receptor (IC50 > 100 μM). The discrepancy between the docking scores and the experimental affinity provoked us to perform additional tests of AutoDock Vina scoring function performance against NK1R. The scoring function did not differentiate low MW-low affinity from low MW-high affinity binders, while it underestimated affinity of low MW-high affinity in comparison to high MW-high affinity compounds. The scoring function did not achieve satisfactory performance in discerning low MW-low affinity and low MW-high affinity binders from the sets of decoys. Analysis of molecular dynamics simulation of one of the studied compounds with NK1R suggests that docking might overestimate strength of the H-bond interaction.

18F]DPA-714: Effect of co-medications, age, sex, BMI and TSPO polymorphism on the human plasma input function.

We aimed to assess the effect of concomitant medication, age, sex, body mass index and 18-kDa translocator protein (TSPO) binding affinity status on the metabolism and plasma pharmacokinetics of [18F]DPA-714 and their influence on the plasma input function in a large cohort of 201 subjects who underwent brain and whole-body PET imaging to investigate the role of neuroinflammation in neurological diseases. The non-metabolized fraction of [18F]DPA-714 was estimated in venous plasma of 138 patients and 63 healthy controls (HCs; including additional arterial sampling in 16 subjects) during the 90min brain PET acquisition using a direct solid-phase extraction method. The mean fraction between 70 and 90min post-injection ([18F]DPA-71470-90) and corresponding normalized plasma concentration (SUV70-90) were correlated with all factors using a multiple linear regression model. Differences between groups (arterial vs venous measurements; HCs vs patients; high- (HAB), mixed- (MAB) and low-affinity binders (LAB); subjects with vs without co-medications, females vs males were also assessed using the non-parametric Mann-Whitney or Kruskal-Wallis ANOVA tests. Finally, the impact of co-medications on the brain uptake of [18F]DPA-714 at equilibrium was investigated. As no significant differences were observed between arterial and venous [18F]DPA-71470-90 and SUV70-90, venous plasma was used for correlations. [18F]DPA-71470-90 was not significantly different between patients and HCS (59.7 ± 12.3% vs 60.2 ± 12.9%) despite high interindividual variability. However, 47 subjects exhibiting a huge increase or decrease of [18F]DPA-71470-90 (up to 88% or down to 23%) and SUV70-90 values (2-threefold) were found to receive co-medications identified as inhibitors or inducers of CYP3A4, known to catalyse [18F]DPA-714 metabolism. Comparison between cortex-to-plasma ratios using individual input function (VTIND) or population-based input function derived from untreated HCs (VTPBIF) indicated that non-considering the individual metabolism rate led to a bias of about 30% in VT values. Multiple linear regression model analysis of subjects free of these co-medications suggested significant correlations between [18F]DPA-71470-90 and age, BMI and sex while TSPO polymorphism did not influence the metabolism of the radiotracer. [18F]DPA-714 metabolism fell with age and BMI and was significantly faster in females than in males. Whole-body PET/CT exhibited a high uptake of the tracer in TSPO-rich organs (heart wall, spleen, kidneys…) and those involved in metabolism and excretion pathways (liver, gallbladder) in HAB and MAB with a strong decrease in LAB (-89% and -85%) resulting in tracer accumulation in plasma (4.5 and 3.3-fold increase). Any co-medication that inhibits or induces CYP3A4 as well as TSPO genetic status, age, BMI and sex mostly contribute to interindividual variations of the radiotracer metabolism and/or concentration that may affect the input function of [18F]DPA-714 and consequently its human brain and peripheral uptake. INFLAPARK, NCT02319382, registered December 18, 2014, retrospectively registered; IMABIO 3, NCT01775696, registered January 25, 2013, retrospectively registered; INFLASEP, NCT02305264, registered December 2, 2014, retrospectively registered; EPI-TEP, EudraCT 2017-003381-27, registered September 24, 2018.

When weak is strong: a plea for low affinity binders for optical microscopy.

The exploitation of low-affinity molecular interactions in protein labeling is an emerging topic in optical microscopy. Such non-covalent and low-affinity interactions can be realized with various concepts from chemistry and for different molecule classes, and lead to a constant renewal of fluorescence signals at target sites. Further benefits are a versatile use across microscopy methods, in 3D, live and many-target applications. In recent years, several classes of low-affinity labels were developed and a variety of powerful applications demonstrated. Still, this research field is underdeveloped, while the potential is huge.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscles and Structure-Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 μM.

Facile Display of Homomultivalent Proteins for In Vitro Selections.

Low-affinity protein binders are emerging as valuable domains for therapeutic applications because of their higher specificity when presented in multivalent ligands that increase the overall strength and selectivity of receptor binding. De novo discovery of low-affinity binders would be enhanced by the large library sizes attainable with in vitro selection systems, but these platforms generally maximize recovery of high-affinity monovalent binders. Here, we present a facile technology that uses rolling circle amplification to create homomultivalent libraries. We show proof of principle of this approach in ribosome display with off-rate selections of a bivalent ligand against monovalent and bivalent targets, thereby demonstrating high enrichment (up to 166-fold) against a low-affinity target that is bivalent but not monovalent. This approach to homomultivalent library construction can be applied to any binder tolerant of N- and C-terminal fusions and provides a platform for performing in vitro display selections with controlled protein valency and orientation.

Systematic Study of in Vitro Selection Stringency Reveals How To Enrich High-Affinity Aptamers.

Aptamers are oligonucleotide receptors with great potential for sensing and therapeutic applications. They are isolated from random libraries through an in vitro method termed systematic evolution of ligands by exponential enrichment (SELEX). Although SELEX-based methods have been widely employed over several decades, many aspects of the experimental process remain poorly understood in terms of how to adjust the selection conditions to obtain aptamers with the desired set of binding characteristics. As a result, SELEX is often performed with arbitrary parameters that tend to produce aptamers with insufficient affinity and/or specificity. Having a better understanding of these basic principles could increase the likelihood of obtaining high-quality aptamers. Here, we have systematically investigated how altering the selection stringency in terms of target concentration─which is essentially the root source of selection pressure for aptamer isolation─affects the outcome of SELEX. By performing four separate trials of SELEX for the same small-molecule target, we experimentally prove that the use of excessively high target concentrations promotes enrichment of low-affinity binders while also suppressing the enrichment of high-affinity aptamers. These findings should be broadly applicable across SELEX methods, given that they share the same core operating principle, and will be crucial for guiding selections to obtain high-quality aptamers in the future.

Humanized Nanobody Anti-CD72 CAR-T Cells Efficiently Eliminate B-Cell Malignancies Via Improved Affinity for CD72 but Induce Persistent Antigen Downregulation In Vivo

Background: Approximately 50% of pediatric B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin lymphoma patients treated with CD19 chimeric antigen receptor (CAR)-T cells relapse. CD22 CAR-T cells are curative only rarely. New immunotherapeutic targets are urgently needed. Our laboratory recently identified CD72 as a target on B-cell malignancies and developed a fully synthetic nanobody against CD72 (NbD4), demonstrating that NbD4 CAR-T cells (nanoCARs) are potent in vitro and in vivo (Nix et al., Cancer Discovery (2021)). We previously described the development of a humanized version of this nanobody binder (H24) that had improved efficacy in preliminary studies (Nix et al., ASH 2021). As we move CD72 nanoCARs toward clinical translation, in this follow-up study we aimed to compare H24 vs. NbD4 by 1) tumor clearance kinetics; 2) T-cell immunophenotypes; 3) in vivo mechanism of relapse; and 4) binding affinity to CD72. Methods: CD4/CD8 CAR-T cells were generated by lentiviral transduction. Single time point (24 hr) in vitro cytotoxicity was assessed across a range of effector:tumor (E:T) ratios. CAR-T elimination kinetics was determined by IncuCyte live cell imaging. CD45RA and CD62L flow cytometry was used to assess T-cell immunophenotypes. In vivo studies were performed by implanting 5e5 luciferase-labeled Jeko cells into NSG mice on Day -4, and administration of 3e6 CAR-T cells on Day 0. At sacrifice, flow cytometry was performed to evaluate CD72 expression on relapsed tumors. Relapsed Jeko was re-implanted into naïve NSG mice for 31 days. Binding affinity of recombinant NbD4 or H24 to biotinylated CD72 extracellular domain was determined by bio-layer interferometry. Results: In single time point assays, H24 nanoCARs eliminated SEM B-ALL cells with similar efficacy to CD19 CAR-Ts (not significant) and were more potent than NbD4 nanoCARs at a 1:10 E:T ratio (72% vs 44% cytotoxicity; p = 3e-4). Against a Jeko mantle cell lymphoma model, at a 1:1 E:T ratio, H24 nanoCARs were more potent than both CD19 CAR-Ts (96% vs 87%; p < 1e-4) and NbD4 nanoCARs (96% vs 70%; p < 1e-4). In live cell imaging assays, H24 nanoCARs eliminated SEM faster than CD19 CAR-Ts (Fig. 1a). After SEM co-culture, H24 nanoCARs secreted more IL-2 and IFNγ compared to NbD4 (IL-2 p < 1e-4; IFNγ p = 4e-4) and CD19 CAR-Ts (IL-2 p = 4e-4; IFNγ p = 3e-4). Versus SEM, both pre- and post-tumor proportions of naïve T cells (CD45RA+/CD62L+) were similar for H24, NbD4, and CD19 CAR-Ts. Bulk RNA-seq after exposure to SEM revealed that H24 nanoCARs upregulate genes relevant to T cell activation and effector T cell function, compared to CD19 CAR-Ts. In in vivo Jeko studies, we treated mice with NbD4 or H24 nanoCARs and extracted tumors after relapse. We found that both nanoCARs led to CD72 loss on tumors (Fig. 1b). Surprisingly, however, upon re-implantation into a new cohort of NSG mice, H24 relapse Jeko continued to maintain low CD72 expression, while re-implanted NbD4 relapse Jeko had restoration of CD72 in the absence of anti-CD72 immune pressure (H24 vs NbD4 CD72 expression p = 4e-2). Given more potent anti-tumor efficacy and more persistent CD72 suppression in murine models, we hypothesized that H24 has a higher binding affinity for CD72. Indeed, H24 demonstrated a dissociation constant (KD) versus CD72 of 33.0 ± 0.04 nM, while NbD4 was 50.4 ± 0.05 nM. These results support the notion that relatively low-affinity binders can still generate potent CAR-Ts, when compared to published values of KD <1 nM for the anti-CD19 scFv FMC63. Conclusions: We have developed a new, humanized anti-CD72 cellular therapy known as H24 nanoCARs. Humanizing the framework regions, while keeping the complementary determining regions (CDRs) identical to NbD4, was intended to reduce immunogenicity. We were surprised that eight amino acid substitutions in the framework regions, with no changes to CDRs, improved antitumor efficacy and binding affinity for CD72. These improved characteristics motivate our planned clinical translation of H24-based nanoCARs. We also found that CD72 downregulation occurs after CD72 nanoCAR treatment in mice, and that increased potency of H24 correlates with longer-term CD72 suppression on tumors. These findings will inform expected outcomes in future clinical trials and motivate further studies to prevent CD72 downregulation after nanoCAR treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

InVivo Evaluation of 6 Analogs of 11C-ER176 as Candidate 18F-Labeled Radioligands for 18-kDa Translocator Protein.

Because of its excellent ratio of specific to nondisplaceable uptake, the radioligand 11C-ER176 can successfully image 18-kDa translocator protein (TSPO), a biomarker of inflammation, in the human brain and accurately quantify target density in homozygous low-affinity binders. Our laboratory sought to develop an 18F-labeled TSPO PET radioligand based on ER176 with the potential for broader distribution. This study used generic 11C labeling and invivo performance in the monkey brain to select the most promising among 6 fluorine-containing analogs of ER176 for subsequent labeling with longer-lived 18F. Methods: Six fluorine-containing analogs of ER176-3 fluoro and 3 trifluoromethyl isomers-were synthesized and labeled by 11C methylation at the secondary amide group of the respective N-desmethyl precursor. PET imaging of the monkey brain was performed at baseline and after blockade by N-butan-2-yl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide (PK11195). Uptake was quantified using radiometabolite-corrected arterial input function. The 6 candidate radioligands were ranked for performance on the basis of 2 invivo criteria: the ratio of specific to nondisplaceable uptake (i.e., nondisplaceable binding potential [BPND]) and the time stability of total distribution volume (VT), an indirect measure of lack of radiometabolite accumulation in the brain. Results: Total TSPO binding was quantified as VT corrected for plasma free fraction (VT/fP) using Logan graphical analysis for all 6 radioligands. VT/fP was generally high at baseline (222 ± 178 mL·cm-3) and decreased by 70%-90% after preblocking with PK11195. BPND calculated using the Lassen plot was 9.6 ± 3.8; the o-fluoro radioligand exhibited the highest BPND (12.1), followed by the m-trifluoromethyl (11.7) and m-fluoro (8.1) radioligands. For all 6 radioligands, VT reached 90% of the terminal 120-min values by 70 min and remained relatively stable thereafter, with excellent identifiability (SEs < 5%), suggesting that no significant radiometabolites accumulated in the brain. Conclusion: All 6 radioligands had good BPND and good time stability of VT Among them, the o-fluoro, m-trifluoromethyl, and m-fluoro compounds were the 3 best candidates for development as radioligands with an 18F label.

Preclinical evaluation of (S)-[18F]GE387, a novel 18-kDa translocator protein (TSPO) PET radioligand with low binding sensitivity to human polymorphism rs6971

PurposePositron emission tomography (PET) studies with radioligands for 18-kDa translocator protein (TSPO) have been instrumental in increasing our understanding of the complex role neuroinflammation plays in disorders affecting the brain. However, (R)-[11C]PK11195, the first and most widely used TSPO radioligand has limitations, while the next-generation TSPO radioligands have suffered from high interindividual variability in binding due to a genetic polymorphism in the TSPO gene (rs6971). Herein, we present the biological evaluation of the two enantiomers of [18F]GE387, which we have previously shown to have low sensitivity to this polymorphism.MethodsDynamic PET scans were conducted in male Wistar rats and female rhesus macaques to investigate the in vivo behaviour of (S)-[18F]GE387 and (R)-[18F]GE387. The specific binding of (S)-[18F]GE387 to TSPO was investigated by pre-treatment with (R)-PK11195. (S)-[18F]GE387 was further evaluated in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation. Sensitivity to polymorphism of (S)-GE387 was evaluated in genotyped human brain tissue.Results(S)-[18F]GE387 and (R)-[18F]GE387 entered the brain in both rats and rhesus macaques. (R)-PK11195 blocked the uptake of (S)-[18F]GE387 in healthy olfactory bulb and peripheral tissues constitutively expressing TSPO. A 2.7-fold higher uptake of (S)-[18F]GE387 was found in the inflamed striatum of LPS-treated rodents. In genotyped human brain tissue, (S)-GE387 was shown to bind similarly in low affinity binders (LABs) and high affinity binders (HABs) with a LAB to HAB ratio of 1.8.ConclusionWe established that (S)-[18F]GE387 has favourable kinetics in healthy rats and non-human primates and that it can distinguish inflamed from normal brain regions in the LPS model of neuroinflammation. Crucially, we have reconfirmed its low sensitivity to the TSPO polymorphism on genotyped human brain tissue. Based on these factors, we conclude that (S)-[18F]GE387 warrants further evaluation with studies on human subjects to assess its suitability as a TSPO PET radioligand for assessing neuroinflammation.

Investigating Complex Samples with Molograms of Low-Affinity Binders.

In vitro diagnostics relies on the quantification of minute amounts of a specific biomolecule, called biomarker, from a biological sample. The majority of clinically relevant biomarkers for conditions beyond infectious diseases are detected by means of binding assays, where target biomarkers bind to a solid phase and are detected by biochemical or physical means. Nonspecifically bound biomolecules, the main source of variation in such assays, need to be washed away in a laborious process, restricting the development of widespread point-of-care diagnostics. Here, we show that a diffractometric assay provides a new, label-free possibility to investigate complex samples, such as blood plasma. A coherently arranged sub-micron pattern, that is, a peptide mologram, is created to demonstrate the insensitivity of this diffractometric assay to the unwanted masking effect of nonspecific interactions. In addition, using an array of low-affinity binders, we also demonstrate the feasibility of molecular profiling of blood plasma in real time and show that individual patients can be differentiated based on the binding kinetics of circulating proteins.

Synthesis and pharmacological evaluation of [18F]PBR316: a novel PET ligand targeting the translocator protein 18 kDa (TSPO) with low binding sensitivity to human single nucleotide polymorphism rs6971.

Radiopharmaceuticals that target the translocator protein 18 kDa (TSPO) have been investigated with positron emission tomography (PET) to study neuroinflammation, neurodegeneration and cancer. We have developed the novel, achiral, 2-phenylimidazo[1,2-a]pyridine, PBR316 that targets the translocator protein 18 kDa (TSPO) that addresses some of the limitations inherent in current TSPO ligands; namely specificity in binding, blood brain barrier permeability, metabolism and insensitivity to TSPO binding in subjects as a result of rs6971 polymorphism. PBR316 has high nanomolar affinity (4.7-6.0 nM) for the TSPO, >5000 nM for the central benzodiazepine receptor (CBR) and low sensitivity to rs6971 polymorphism with a low affinity binders (LABs) to high affinity binders (HABs) ratio of 1.5. [18F]PBR316 was prepared in 20 ± 5% radiochemical yield, >99% radiochemical purity and a molar activity of 160-400 GBq μmol-1. Biodistribution in rats showed high uptake of [18F]PBR316 in organs known to express TSPO such as heart (3.9%) and adrenal glands (7.5% ID per g) at 1 h. [18F]PBR316 entered the brain and accumulated in TSPO-expressing regions with an olfactory bulb to brain ratio of 3 at 15 min and 7 at 4 h. Radioactivity was blocked by PK11195 and Ro 5-4864 but not Flumazenil. Metabolite analysis showed that radioactivity in adrenal glands and the brain was predominantly due to the intact radiotracer. PET-CT studies in mouse-bearing prostate tumour xenografts indicated biodistribution similar to rats with radioactivity in the tumour increasing with time. [18F]PBR316 shows in vitro binding that is insensitive to human polymorphism and has specific and selective in vivo binding to the TSPO. [18F]PBR316 is suitable for further biological and clinical studies.

Could cross-reactivity rescue Foxp3+ regulatory T cell precursors from thymic deletion?

Thymocytes that bind with high affinity to peptides displayed by MHC class II (pMHC-II) are deleted while low-affinity binders differentiate into naive CD4+ T cells. However, Foxp3+ regulatory T cells (Tregs) seem to defy this binary choice as their precursors require high-affinity interaction with pMHC-II for maturation in the thymus. Here, we rely on the antigen-specific interpretive framework, SPIRAL (Specific ImmunoRegulatory Algorithm), to propose that Tregs escape thymic deletion by forming dyads with IL-2-producing T cells via antigen cross-reactivity. This interpretation reconciles contradictions related to Treg ontogeny in the thymus and their role in modulating antigen-specific immune responses.

Positron emission tomography to image cerebral neuroinflammation in ischaemic stroke: a pilot study

Background Activated microglia play a complex role in neuroinflammation associated with acute ischaemic stroke. As a potential target for anti-inflammatory therapy, it is crucial to understand the association between intensity, extent and the clinical outcome of a stroke. The 18-kDa translocator protein is a marker of cerebral microglial activation and of macrophage infiltration after damage to the brain. It can be imaged by positron emission tomography. Therefore, the recently developed radiopharmaceutical [18F]-GE180 was used in patients after a mild to moderate stroke and compared with [11C]-(R)-PK11195, which has already been established in research but cannot be used in routine clinical settings because of its very short half-life. Objectives Objectives for phase 1 were to evaluate the tolerability of positron emission tomography scanning, to assess the technical feasibility of imaging the 18-kDa translocator protein using [18F]-GE180 as radiopharmaceutical, to compare [18F]-GE180 with [11C]-(R)-PK11195 as reference. Objectives for phase 2 were examining the relation of positron emission tomography imaging with clinical outcome, magnetic resonance imaging and systemic inflammation. However, the study was ended after phase 1 because of the results obtained in that phase and did not enter phase 2. Methods Ten participants (aged 24–89 years, median 68 years) (eight male and two female) with a history of recent ischaemic stroke of mild to moderate severity (modified Rankin scale score of 2–3) in the middle cerebral artery territory were scanned 18 to 63 days (median 34.5 days) after the stroke by magnetic resonance imaging (Philips 1.5 T; Philips, Amsterdam, the Netherlands), [18F]-GE180 (200 MBq, 30-minute dynamic scan) and [11C]-(R)-PK11195 (740 MBq, 60-minute dynamic scan) positron emission tomography (Siemens HRRT; Siemens, Munich, Germany). The two positron emission tomography scans were performed on 2 separate days (mean 3.4 days apart). Five patients were randomised to receive the [18F]-GE180 scan at the first session and five patients were randomised to receive it at the second session. Participants were genotyped for the rs6971 18-kDa translocator protein polymorphism, which is known to affect binding of [18F]-GE180 but not of [11C]-(R)-PK11195. All positron emission tomography and magnetic resonance data sets were co-registered with T1-weighted magnetic resonance image scans. Binding of [18F]-GE180 was compared with [11C]-(R)-PK11195 for the infarct and contralateral reference regions. Spearman’s rank-order correlation was used to compare tracers, t-tests to compare patient subgroups. Results Tolerability of scans was rated as 4.36 (range 4–5) out of a maximum of 5 by participants, and there were no serious adverse events. There was a close correlation between [18F]-GE180 and [11C]-(R)-PK11195 (r = 0.79 to 0.84). The 18-kDa translocator protein polymorphism had a significant impact on the uptake of [18F]-GE180, which was very low in normal cortex. Ischaemic lesions with contrast enhancement on magnetic resonance as an indicator of blood–brain barrier damage showed a significantly higher uptake of [18F]-GE180 than the lesions without enhancement, even in low-affinity binders. Conclusions [18F]-GE180 was safe and well tolerated. However, strong dependency of uptake on blood–brain barrier damage and a genetic 18-kDa translocator protein polymorphism, as well as a high contribution of vascular signal to the uptake and evidence of non-specific binding in ischaemic lesions with blood–brain barrier damage, limits the clinical applicability of [18F]-GE180 as a diagnostic marker of neuroinflammation. Limitations As the study was ended after phase 1, this was only a small pilot trial. Further studies are warranted to fully understand the influence of blood–brain barrier damage on positron emission tomography microglia imaging. Trial registration Registered as a clinical trial with EudraCT 2014-000591-26. Funding This project was funded by the Efficacy and Mechanism Evaluation programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership, and will be published in full in Efficacy and Mechanism Evaluation; Vol. 7, No. 1. See the NIHR Journals Library website for further information. It was also supported by GE Healthcare (Chicago, IL, USA) by free production and delivery of [18F]-GE180 and by supply of regulatory documents (Investigational Medical Product Dossier, Investigator’s Brochure). There was partial support by the European Commission (INMiND, grant #278850) and the NIHR Sheffield Biomedical Research Centre.

Human Serum Albumin Binding in a Vial: A Novel UV-pH Titration Method To Assist Drug Design.

The knowledge on human serum albumin (HSA) binding is of utmost importance as it affects pharmacokinetic behavior and bioavailability of drugs. In this article, we report a novel method to screen for ionizable molecules with high HSA binding affinity based on pKa shifts using UV-pH titration. We investigated the HSA binding of 27 drugs and compared the results to experimental data from conventional methods. In most cases, significant shifts (ΔpKa > 0.1) were observed for drugs with high HSA binding, while no change could be detected for low-affinity binders. We showed the pivotal role of ionization centers in the formation of strong interactions between drug and HSA using molecular docking studies. We also verified our findings by testing five modified analogues designed by structural considerations. Significant decreases in their HSA binding proved that the UV-pH titration method combined with an in silico support can be used as a medicinal chemistry tool to assist rational molecular design.