The PET radiotracer 18 F-(2S,4R)4-Fluoroglutamine ( 18 F-Gln) reflects glutamine transport and can be used to infer glutamine metabolism. Mouse xenograft studies have demonstrated that 18 F-Gln uptake correlates directly with glutamine pool size and is inversely related to glutamine metabolism through the glutaminase enzyme. To provide a framework for the analysis of 18 F-Gln-PET, we have examined 18 F-Gln uptake kinetics in mouse models of breast cancer at baseline and after inhibition of glutaminase. We describe results of the pre-clinical analysis and computer simulations with the goal of model validation and performance assessment in anticipation of human breast cancer patient studies. Methods: Triplenegative breast cancer and receptor-positive xenografts were implanted in athymic mice. PET mouse imaging was performed at baseline and after treatment with a glutaminase inhibitor or a vehicle solution for a total of four mouse groups. Dynamic PET images were obtained for one hour beginning at the time of intravenous injection of 18 F-FGln. Kinetic analysis and computer simulations were performed on a representative time-activity curves (TAC), testing 1-and 2-compartment models to describe kinetics.Results: Dynamic imaging for one hour captured blood and tumor TACs indicative of largely reversible uptake of 18 F-FGln in tumors. Consistent with this observation, a two-compartment model indicated a relatively low estimate of trapping (k 3 ) , suggesting that the one-compartment model is preferable. Logan plot graphical analysis demonstrated late linearity, supporting reversible kinetics and modeling with a single compartment. Analysis of the mouse data and simulations suggests that estimates of glutamine pools size, specifically the V D for 18 F-FGln, were more reliably estimated using the one-compartment reversible model compared to the two-compartment irreversible model. Tumor-to-blood ratios, a more practical potential proxy of V D , demonstrated good correlation with volume of distribution from singlecompartment models and Logan analyses. Conclusions: Kinetic analysis of dynamic 18 F-Gln-PET images demonstrated the ability to measure V D to estimate glutamine pool size, a key indicator of cellular glutamine metabolism, by both a one-compartment model and Logan analysis. Changes in V D with glutaminase inhibition supports the ability to assess response to glutamine metabolism-targeted therapy.Concordance of kinetic measures with tumor-to-blood ratios provides a clinically feasible approach for human imaging.

Using a microwave-assisted protocol, we synthesized 16 peptoid-capped HDAC inhibitors (HDACi) with fluorinated linkers and identified two hit compounds. In biochemical and cellular assays, 10h stood out as a potent unselective HDACi with remarkable cytotoxic potential against different therapy-resistant leukemia cell lines. 10h demonstrated prominent antileukemic activity with low cytotoxic activity toward healthy cells. Moreover, 10h exhibited synergistic interactions with the DNA methyltransferase inhibitor decitabine in AML cell lines. The comparison of crystal structures of HDAC6 complexes with 10h and its nonfluorinated counterpart revealed a similar occupation of the L1 loop pocket but slight differences in zinc coordination. The substitution pattern of the acyl residue turned out to be crucial in terms of isoform selectivity. The introduction of an isopropyl group onto the phenyl ring provided the highly HDAC6-selective inhibitor 10p, which demonstrated moderate synergy with decitabine and exceeded the HDAC6 selectivity of tubastatin A.

Histone deacetylases (HDAC’s) are key regulatory enzymes in gene transcription and cellular motility through the deacetylation of lysine residues. These enzymes bear a distinct clinical significance, as the upregulation of HDACs has been associated with oncogenesis for many hematological malignancies and proliferation of other neurodegenerative or immune disorders. There are four different classes of HDACs, three of which require zinc for catalysis. Among the zinc dependent isozymes, HDAC6 is thought to be a particularly desirable therapeutic target, as its selective inhibition in malignant cells is accompanied by fewer associated toxicities in comparison to pan‐HDAC inhibition. Therefore, optimizing HDAC6 inhibitor selectivity has the potential to yield great clinical significance. This selectivity is often conferred by designing inhibitors with bulky capping groups and a hydrophobic linker region, which make favorable interactions in the hydrophobic region of the HDAC6 active site. Many inhibitors contain a hydroxamate zinc‐binding group, which can coordinate with bidentate or monodentate geometry. In efforts to optimize HDAC6 selectivity by exploring alternative zinc‐binding groups, a unique inhibitor containing an oxadiazole ring was discovered. Surprisingly, the crystal structure of its complex with HDAC6 reveals that the oxadiazole undergoes a ring opening reaction to yield an acylhydrazide that binds with an extended conformation in the active site.

Bavarostat (EKZ-001) is a selective inhibitor of histone deacetylase 6 (HDAC6) that contains a meta-fluorophenylhydroxamate Zn 2+ -binding group. The recently determined crystal structure of its complex with HDAC6 from Danio rerio (zebrafish) revealed that the meta-fluoro substituent binds exclusively in an aromatic crevice defined by F583 and F643 rather than being oriented out toward solvent. To explore the binding of inhibitor C−F groups in this fluorophilic crevice, we now report a series of 10 simple fluorophenylhydroxamates bearing one or more fluorine atoms with different substitution patterns. Inhibitory potencies against human and zebrafish HDAC6 range widely from 121 to >30,000 nM. The best inhibitory potency is measured for meta-difluorophenylhydroxamate (5) with IC 50 = 121 nM against human HDAC6; the worst inhibitory potencies are measured for ortho-fluorophenylhydroxamate (1) as well as fluorophenylhydroxamates 4, 7, 9, and 10, although there are some variations in activity trends against human and zebrafish HDAC6. These studies show that aromatic ring fluorination at the meta position(s) does not improve inhibitory activity against human HDAC6 relative to the nonfluorinated parent compound phenylhydroxamate (IC 50 = 120 nM), but meta-fluorination does not seriously compromise inhibitory activity either. Crystal structures of selected zebrafish HDAC6−fluorophenylhydroxamate complexes reveal that the fluoroaromatic ring is uniformly accommodated in the F583−F643 aromatic crevice, so ring fluorination does not perturb the inhibitor binding conformation. However, hydroxamate−Zn 2+ coordination is bidentate for some inhibitors and monodentate for others. These studies will inform design strategies underlying the design of 18 F-labeled HDAC6 inhibitors intended for positron emission tomography.