Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. The lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. We used a diversity-oriented synthetic approach and developed a library of PLD inhibitors with considerable pharmacological characterization. Here we report the rigorous evaluation of that library, which contains highly potent inhibitors, including the first isoform-selective PLD inhibitors. Specific members of this series inhibit isoforms with > 100-fold selectivity both in vitro and in cells. A subset of inhibitors was shown to block invasiveness in metastatic breast cancer models. These findings demonstrate the power of diversity-oriented synthesis combined with biochemical assays and mass spectrometric lipid profiling of cellular responses to develop the first isoform-selective PLD inhibitors—a new class of antimetastatic agents.

There is a critical need to develop and rigorously validate molecular imaging biomarkers to aid diagnosis and characterization of primary brain tumors. Elevated expression of translocator protein (TSPO) has been shown to predict disease progression and aggressive, invasive behavior in a variety of solid tumors. Thus, noninvasive molecular imaging of TSPO expression could form the basis of a novel, predictive cancer imaging biomarker. In quantitative preclinical PET studies, we evaluated a high-affinity pyrazolopyrimidinyl-based TSPO imaging ligand, N,N-diethyl-2-(2-(4-(2-(18F)-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ([18F]DPA-714), as a translational probe for quantification of TSPO levels in glioma. Methods Glioma-bearing rats were imaged with [18F]DPA-714 in a microPET system. Dynamic images were acquired simultaneously upon injection of [18F]DPA-714 (130 – 200 MBq/0.2 mL). Arterial blood was collected to derive the input function (AIF), with HPLC radiometabolite analysis performed upon select samples for AIF correction. Compartmental modeling was performed using the corrected AIF. Specific tumor cell binding of DPA-714 was evaluated by radioligand displacement of [3H]PK 11195 with DPA-714 in vitro and displacement of [18F]DPA-714 with excess DPA-714 in vivo. Immediately following imaging, tumor and healthy brain tissues were harvested for validation by western blotting and immunohistochemistry. Results [18F]DPA-714 was found to preferentially accumulate in tumors with modest uptake in contralateral brain. Infusion with DPA-714 (10 mg/kg) displaced [18F]DPA-714 binding by greater than 60% on average. Tumor uptake of [18F]DPA-714 was similar to another high-affinity TSPO imaging ligand, [18F]PBR06, and agreed with ex vivo assay of TSPO protein levels in tumor and healthy brain. Conclusions These studies illustrate the feasibility of using [18F]DPA-714 for visualization of TSPO-expressing brain tumors. Importantly, [18F]DPA-714 appears suitable for quantitative assay of tumor TSPO levels in vivo. Given the relationship between elevated TSPO levels and poor outcome in oncology, these studies suggest the potential of [18F]DPA-714 PET to serve as a novel predictive cancer imaging modality.

This Letter describes the synthesis and structure–activity-relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of alternative halogenated piperidinyl benzimidazolone privileged structures, in combination with a key (S)-methyl group, novel PLD inhibitors with low nM potency and unprecedented levels of PLD1 isoform selectivity (~1700-fold) over PLD2 were developed.

Focused library synthesis and structure-activity relationship development of 5,6,7-substituted pyrazolopyrimidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (6b), a novel translocator protein (TSPO) ligand exhibiting a 36-fold enhancement in affinity compared to another pyrazolopyrimidine-based TSPO ligand, 6a (DPA-714). Radiolabeling with fluorine-18 (18F) facilitated production of 2-(5,7-diethyl-2-(4-(2-[18F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (18F-6b) in high radiochemical yield and specific activity. In vivo studies of 18F-6b were performed which illuminated this agent as an improved probe for molecular imaging of TSPO-expressing cancers.

Purpose: To evaluate noninvasive imaging methods as predictive biomarkers of response to trastuzumab in mouse models of HER2-overexpressing breast cancer. The correlation between tumor regression and molecular imaging of apoptosis, glucose metabolism, and cellular proliferation was evaluated longitudinally in responding and nonresponding tumor-bearing cohorts.

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Representing an enormous health care and socioeconomic challenge, breast cancer is the second most common cancer in the world and the second most common cause of cancer-related death. Although many of the challenges associated with preventing, treating, and ultimately curing breast cancer are addressable in the laboratory, successful translation of groundbreaking research to clinical populations remains an important barrier. Particularly when compared with research on other types of solid tumors, breast cancer research is hampered by a lack of tractable in vivo model systems that accurately recapitulate the relevant clinical features of the disease. A primary objective of this article was to provide a generalizable overview of the types of in vivo model systems, with an emphasis primarily on murine models, that are widely deployed in preclinical breast cancer research. Major opportunities to advance precision cancer medicine facilitated by molecular imaging of preclinical breast cancer models are discussed.

Purpose Alanine-serine-cysteine transporter 2 (ASCT2) expression has been demonstrated as a promising lung cancer biomarker. (2S,4R)-4-[18F]Fluoroglutamine (4-[18F]fluoro-Gln) positron emission tomography (PET) was evaluated in preclinical models of non-small cell lung cancer as a quantitative, non-invasive measure of ASCT2 expression. Procedures In vivo microPET studies of 4-[18F]fluoro-Gln uptake were undertaken in human cell line xenograft tumor-bearing mice of varying ASCT2 levels, followed by a genetically engineered mouse model of epidermal growth factor receptor (EGFR)-mutant lung cancer. The relationship between a tracer accumulation and ASCT2 levels in tumors was evaluated by IHC and immunoblotting. Result 4-[18F]Fluoro-Gln uptake, but not 2-deoxy-2-[18F]fluoro-D-glucose, correlated with relative ASCT2 levels in xenograft tumors. In genetically engineered mice, 4-[18F]fluoro-Gln accumulation was significantly elevated in lung tumors, relative to normal lung and cardiac tissues. Conclusions 4-[18F]Fluoro-Gln PET appears to provide a non-invasive measure of ASCT2 expression. Given the potential of ASCT2 as a lung cancer biomarker, this and other tracers reflecting ASCT2 levels could emerge as precision imaging diagnostics in this setting.