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The two tandem bromodomains of BET (bromodomain and extra-terminal domain) proteins (BD1 and BD2) may play distinct and critical roles in neurological diseases. To better understand the underlying mechanisms of the BD1 bromodomain and facilitate brain permeable domain-selective inhibitor development, we describe here the development of the first BET BD1 positron emission tomography (PET) radioligand [(11)C]1a. Compound 1a was tested to possess potent binding affinities and good selectivity (>20-fold over BD2) for BD1 bromodomains of BRD2 (K(d) = 25 nM), BRD3 (K(d) = 24 nM), and BRD4 (K(d) = 19 nM). Physicochemical characterization of 1a indicated the brain permeability and specific binding. [(11)C]1a was radiosynthesized in a good radiochemical yield (RCY: 25–30%) and molar activity (258 GBq μmol(−1)). The PET imaging studies of [(11)C]1a in mice showed moderate brain uptake (with peak SUV = 0.7) and binding specificity. Furthermore, [(11)C]1a demonstrated translational potential in the non-human primate (NHP) PET imaging study, which sets the stage for clinical translation.

Demyelination causes slowed or failed neuronal conduction and is a driver of disability in multiple sclerosis and other neurological diseases. Currently, the gold standard for imaging demyelination is MRI, but despite its high spatial resolution and sensitivity to demyelinated lesions, it remains challenging to obtain specific and quantitative measures of molecular changes involved in demyelination. To understand the contribution of demyelination in different diseases and to assess the efficacy of myelin-repair therapies, it is critical to develop new in vivo imaging tools sensitive to changes induced by demyelination. Upon demyelination, axonal K(+) channels, normally located underneath the myelin sheath, become exposed and increase in expression, causing impaired conduction. Here, we investigate the properties of the K(+) channel PET tracer [(18)F]3F4AP in primates and its sensitivity to a focal brain injury that occurred three years prior to imaging. [(18)F]3F4AP exhibited favorable properties for brain imaging including high brain penetration, high metabolic stability, high plasma availability, high reproducibility, high specificity, and fast kinetics. [(18)F]3F4AP showed preferential binding in areas of low myelin content as well as in the previously injured area. Sensitivity of [(18)F]3F4AP for the focal brain injury was higher than [(18)F]FDG, [(11)C]PiB, and [(11)C]PBR28, and compared favorably to currently used MRI methods.

SUMMARY It is well understood that fatty acids can be synthesized, imported, and modified to meet requisite demands in cells. However, following the movement of fatty acids through the multiplicity of these metabolic steps has remained difficult. To better address this problem, we developed Fatty Acid Source Analysis (FASA), a model that defines the contribution of synthesis, import, and elongation pathways to fatty acid homeostasis in saturated, monounsaturated, and polyunsaturated fatty acid pools. Application of FASA demonstrated that elongation can be a major contributor to cellular fatty acid content and showed that distinct pro-inflammatory stimuli (e.g., Toll-like receptors 2, 3, or 4) specifically reprogram homeostasis of fatty acids by differential utilization of synthetic and elongation pathways in macrophages. In sum, this modeling approach significantly advances our ability to interrogate cellular fatty acid metabolism and provides insight into how cells dynamically reshape their lipidomes in response to metabolic or inflammatory signals., Graphical Abstract, In Brief Argus et al. developed Fatty Acid Source Analysis (FASA), a model that quantifies cellular fatty acid synthesis, elongation, and import. FASA is used to demonstrate that elongation can be a major contributor to cellular fatty acid content and that different stimuli reprogram macrophage fatty acid elongation pathways in distinct ways.

Feraheme (FH) nanoparticles (NPs) have been used extensively for treatment of iron anemia (due to their slow release of ionic iron in acidic environments). In addition, injected FH NPs are internalized by monocytes and function as MRI biomarkers for the pathological accumulation of monocytes in disease. We have recently expanded these applications by radiolabeling FH NPs for positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging using a heat-induced radiolabeling (HIR) strategy. Imaging FH NPs using PET/SPECT has important advantages over MRI due to lower iron doses and improved quantitation of tissue NP concentrations. HIR of FH NPs leaves the physical and biological properties of the NPs unchanged and allows researchers to build on the extensive knowledge obtained about the pharmacokinetic and safety aspects of FH NPs. In this protocol, we present the step-by-step procedures for heat (120 °C)-induced bonding of three widely employed radiocations (89Zr4 + or 64Cu2 + for PET, and 111In3 + for SPECT) to FH NPs using a chelateless radiocation surface adsorption (RSA) approach. In addition, we describe the conversion of FH carboxyl groups into amines and their reaction with an N-hydroxysuccinimide (NHS) of a Cy5.5 fluorophore. This yields Cy5.5-FH, a fluorescent FH that enables the cells internalizing Cy5.5-FH to be examined using flow cytometry. Finally, we describe procedures for in vivo and ex vivo uptake of Cy5.5-FH by monocytes and for in vivo microPET/CT imaging of HIR-FH NPs. Synthesis of HIR-FH requires experience with working with radioactive cations and can be completed within < 4 h. Synthesis of Cy5.5-FH NPs takes ~17 h.

Heat-induced radiolabeling (HIR) yielded (89) Zr-Feraheme (FH) nanoparticles (NPs) that were used to determine NP pharmacokinetics (PK) by positron emission tomography (PET). Standard uptake values indicated a fast hepatic uptake that corresponded to blood clearance, and a second, slow uptake process by lymph nodes and spleen. By cytometry, NPs were internalized by circulating monocytes and monocytes in vitro. Using an IV injection of HIR (89) Zr-FH (rather than in vitro cell labeling), PET/PK provided a view of monocyte trafficking, a key component of the immune response.

The iron chelator deferoxamine (DFO), approved for the treatment of iron overload, has been examined as a therapeutic in a variety of conditions which iron may exacerbate. To evaluate the potential of DFO-bearing PEG-like nanoprobes (DFO-PNs) as therapeutics, we determined their pharmacokinetics (PK) in normal mice, and imaged their accumulation in a tumor model and in models of transient brain ischemia and inflammation. DFO-PNs consist of a DFO, a Cy5.5, and PEG (5 kDa or 30 kDa) attached to Lys-Cys scaffold. Tumor uptake of a [(89)Zr]:DFO-PN(10) (30 kDa PEG, diameter 10 nm) was imaged by PET, surface fluorescence, and fluorescence microscopy. DFO-PN(10) was internalized by tumor cells (fluorescence microscopy) and by cultured cells (by FACS). [(89)Zr]:DFO-PN(4.3) (5 kDa PEG, diameter 4.3 nm) concentrated at incision generated inflammations but not at sites of transient brain ischemia. DFO-PNs are fluorescent, PK tunable forms of DFO that might be investigated as antitumor or anti-inflammatory agents.

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer's disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease.

Large-molecule tracers, such as labeled antibodies, have shown success in immuno-PET for imaging of specific cell surface biomarkers. However, previous work has shown that localization of such tracers shows high levels of heterogeneity in target tissues, due to both the slow diffusion and the high affinity of these compounds. In this work, we investigate the effects of subvoxel spatial heterogeneity on measured time-activity curves in PET imaging and the effects of ignoring diffusion limitation on parameter estimates from kinetic modeling.Partial differential equations (PDE) were built to model a radially symmetric reaction-diffusion equation describing the activity of immuno-PET tracers. The effects of slower diffusion on measured time-activity curves and parameter estimates were measured in silico, and a modified Levenberg-Marquardt algorithm with Bayesian priors was developed to accurately estimate parameters from diffusion-limited data. This algorithm was applied to immuno-PET data of mice implanted with prostate stem cell antigen-overexpressing tumors and injected with (124)I-labeled A11 anti-prostate stem cell antigen minibody.Slow diffusion of tracers in linear binding models resulted in heterogeneous localization in silico but no measurable differences in time-activity curves. For more realistic saturable binding models, measured time-activity curves were strongly dependent on diffusion rates of the tracers. Fitting diffusion-limited data with regular compartmental models led to parameter estimate bias in an excess of 1,000% of true values, while the new model and fitting protocol could accurately measure kinetics in silico. In vivo imaging data were also fit well by the new PDE model, with estimates of the dissociation constant (Kd) and receptor density close to in vitro measurements and with order of magnitude differences from a regular compartmental model ignoring tracer diffusion limitation.Heterogeneous localization of large, high-affinity compounds can lead to large differences in measured time-activity curves in immuno-PET imaging, and ignoring diffusion limitations can lead to large errors in kinetic parameter estimates. Modeling of these systems with PDE models with Bayesian priors is necessary for quantitative in vivo measurements of kinetics of slow-diffusion tracers.

Co-targeting of both de novo and salvage pathways for dCTP biosynthesis shows efficacy in T-ALL and B-ALL., Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.

The sterol regulatory element-binding proteins (SREBP) are key transcriptional regulators of lipid metabolism and cellular growth. It has been proposed that SREBP signaling regulates cellular growth through its ability to drive lipid biosynthesis. Unexpectedly, we find that loss of SREBP activity inhibits cancer cell growth and viability by uncoupling fatty acid synthesis from desaturation. Integrated lipid profiling and metabolic flux analysis revealed that cancer cells with attenuated SREBP activity maintain long-chain saturated fatty acid synthesis, while losing fatty acid desaturation capacity. We traced this defect to the uncoupling of fatty acid synthase activity from stearoyl-CoA desaturase 1 (SCD1)–mediated desaturation. This deficiency in desaturation drives an imbalance between the saturated and monounsaturated fatty acid pools resulting in severe lipotoxicity. Importantly, replenishing the monounsaturated fatty acid pool restored growth to SREBP-inhibited cells. These studies highlight the importance of fatty acid desaturation in cancer growth and provide a novel mechanistic explanation for the role of SREBPs in cancer metabolism. Cancer Res; 73(9); 2850–62. ©2013 AACR.