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1.  A red-shifted fluorescent substrate for aldehyde dehydrogenase 
Nature communications  2014;5:3662.
Selection of cells positive for aldehyde dehydrogenase (ALDH) activity from a green fluorescent background is difficult with existing reagents. Here we report a red-shifted fluorescent substrate for ALDH, AldeRed 588-A, for labeling viable ALDHpos cells. We demonstrate that AldeRed 588-A successfully isolates ALDHhi human hematopoietic stem cells from heterogeneous cord blood mononuclear cells. AldeRed 588-A can be used for multi-color applications to fractionate ALDHpos cells in the presence of green fluorophores including the ALDEFLUOR™ reagent and cells expressing eGFP. AldeRed 588-A stains ALDHpos murine pancreatic centroacinar and terminal duct cells, as visualized by fluorescent microscopy. AldeRed588-A provides a useful tool to select stem cells or study ALDH within a green fluorescent background.
PMCID: PMC4063304  PMID: 24759454
2.  Synthesis and Biological Evaluation of Substrate-Based Imaging Agents for the Prostate-Specific Membrane Antigen 
Macromolecular research  2013;21(5):565-573.
Prostate-specific membrane antigen (PSMA) is an attractive target for the imaging of prostate cancer (PCa) due to the elevated expression on the surface of prostate tumor cells. Most PSMA-targeted low molecular weight imaging agents are inhibitors of PSMA. We have synthesized a series of substrate-based PSMA-targeted imaging agents by mimicking poly-γ-glutamyl folic acid, an endogenous substrate of PSMA. In vitro the γ-linked polyglutamate conjugates proved to be better substrates than the corresponding α-linked glutamates. However, in vivo imaging studies of γ-ray-emitting and γ-linked glutamates did not demonstrate selective uptake in PSMA-pos-itive over PSMA-negative tumors. Subsequent chromatographic studies and in silico molecular dynamics simulations indicated that hydrolysis of the substrates is slow and access to the enzymatic active site is limited. These results inform the design of future substrate-based imaging agents for PSMA.
PMCID: PMC4198338  PMID: 25328507
molecular imaging; prodrug; prostate cancer; SPECT; PSMA
3.  Synthesis and Biological Evaluation of Low Molecular Weight Fluorescent Imaging Agents for the Prostate-Specific Membrane Antigen 
Bioconjugate chemistry  2012;23(12):2377-2385.
Targeted near-infrared (NIR) optical imaging can be used in vivo to detect specific tissues, including malignant cells. A series of NIR fluorescent ligands targeting the prostate-specific membrane antigen (PSMA) was synthesized and each compound was tested for its ability to image PSMA+ tissues in experimental models of prostate cancer. The agents were prepared by conjugating commercially available active esters of NIR dyes, including IRDye800CW, IRDye800RS, Cy5.5, Cy7, or a derivative of indocyanine green (ICG) to the terminal amine group of (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)pentanedioic acid 1, (14S,18S)-1-amino-8,16-dioxo-3,6-dioxa-9,15,17-triazaicosane-14,18,20-tricarboxylic acid 2 and (3S,7S)-26-amino-5,13,20-trioxo-4,6,12,21-tetraaza-hexacosane-1,3,7,22-tetracarboxylic acid 3. The Ki values for the dye-inhibitor conjugates ranged from 1 to 700 pM. All compounds proved capable of imaging PSMA+ tumors selectively to varying degrees depending on the choice of fluorophore and linker. The highest tumor uptake was observed with IRDye800CW employing a poly(ethylene glycol) or lysine-suberate linker, as in 800CW-2 and 800CW-3, while the highest tumor to nontarget tissue ratios were obtained for Cy7 with these same linkers, as in Cy7-2 and Cy7-3. Compounds 2 and 3 provide useful scaffolds for targeting of PSMA+ tissues in vivo and should be useful for preparing NIR dye conjugates designed specifically for clinical intraoperative optical imaging devices.
PMCID: PMC4131203  PMID: 23157641
4.  Effect of Chelators on the Pharmacokinetics of 99mTc-Labeled Imaging Agents for the Prostate-Specific Membrane Antigen (PSMA) 
Journal of medicinal chemistry  2013;56(15):6108-6121.
Technetium-99m, the most commonly used radionuclide in nuclear medicine, can be attached to biologically important molecules through a variety of chelating agents, the choice of which depends upon the imaging application. The prostate-specific membrane antigen (PSMA) is increasingly recognized as an important target for imaging and therapy of prostate cancer (PCa). Three different 99mTc-labeling methods were employed to investigate the effect of the chelator on the biodistribution and PCa tumor uptake profiles of 12 new urea based PSMA-targeted radiotracers. This series includes hydrophilic ligands for radiolabeling with the [99mTc(CO)3]+ core (L8-10), traditional NxSy-based chelating agents with varying charge and polarity for the 99mTc-oxo core (L11-18), and a 99mTc-organohydrazine-labeled radioligand (L19). 99mTc(I)-Tricarbonyl-labeled [99mTc]L8 produced the highest PSMA+ PC3 PIP to PSMA− PC3 flu tumor ratios, and demonstrated the lowest retention in normal tissues including kidney after 2 h. These results suggest that choice of chelator is an important pharmacokinetic consideration in the development of 99mTc-labeled radiopharmaceuticals targeting PSMA.
PMCID: PMC3773988  PMID: 23799782
5.  Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II 
Journal of biomolecular screening  2012;17(8):1030-1040.
Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000–compound library. The novel assay presented here is robust, highly reproducible (Z′ = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.
PMCID: PMC4112551  PMID: 22751730
fluorescence polarization; high-throughput screening; glutamate carboxypeptidase II; prostate-specific membrane antigen; metallopeptidase
6.  The Novel PPAR α/γ Dual Agonist MHY 966 Modulates UVB–Induced Skin Inflammation by Inhibiting NF-κB Activity 
PLoS ONE  2013;8(10):e76820.
Ultraviolet B (UVB; 290~320nm) irradiation-induced lipid peroxidation induces inflammatory responses that lead to skin wrinkle formation and epidermal thickening. Peroxisome proliferator-activated receptor (PPAR) α/γ dual agonists have the potential to be used as anti-wrinkle agents because they inhibit inflammatory response and lipid peroxidation. In this study, we evaluated the function of 2-bromo-4-(5-chloro-benzo[d]thiazol-2-yl) phenol (MHY 966), a novel synthetic PPAR α/γ dual agonist, and investigated its anti-inflammatory and anti-lipid peroxidation effects. The action of MHY 966 as a PPAR α/γ dual agonist was also determined in vitro by reporter gene assay. Additionally, 8-week-old melanin-possessing hairless mice 2 (HRM2) were exposed to 150 mJ/cm2 UVB every other day for 17 days and MHY 966 was simultaneously pre-treated every day for 17 days to investigate the molecular mechanisms involved. MHY 966 was found to stimulate the transcriptional activities of both PPAR α and γ. In HRM2 mice, we found that the skins of mice exposed to UVB showed significantly increased pro-inflammatory mediator levels (NF-κB, iNOS, and COX-2) and increased lipid peroxidation, whereas MHY 966 co-treatment down-regulated these effects of UVB by activating PPAR α and γ. Thus, the present study shows that MHY 966 exhibits beneficial effects on inflammatory responses and lipid peroxidation by simultaneously activating PPAR α and γ. The major finding of this study is that MHY 966 demonstrates potential as an agent against wrinkle formation associated with chronic UVB exposure.
PMCID: PMC3793945  PMID: 24130794
7.  Synthesis, biological evaluation, and radioiodination of halogenated closo-carboranyl thymidine analogues1 
Inorganic Chemistry  2011;51(1):629-639.
The synthesis and initial biological evaluation of 3-carboranyl thymidine analogues (3CTAs) that are (radio)halogenated at the closo-carborane cluster is described. Radiohalogenated 3CTAs have the potential to be used in the radiotherapy and imaging of cancer, as they may be selectively entrapped in tumor cells through monophosphorylation by human thymidine kinase 1 (hTK1). Two strategies for the synthesis of a 127I-labeled form of a specific 3CTA, previously designated as N5, are described: 1) direct iodination of N5 with iodine monochloride (ICl) and aluminum chloride (AlCl3) to obtain N5-127I and 2) initial monoiodination of o-carborane to 9-iodo-o-carborane followed by its functionalization to N5-127I. The former strategy produced N5-127I in low yields along with di-, tri-, and tetra-iodinated N5 as well as decomposition products, whereas the latter method produced only N5-127I in high yields. N5-127I was subjected to nucleophilic halogen- and isotope exchange reactions using Na79/81Br and Na125I, respectively, in the presence of Herrmann's catalyst to obtain N5-79/81Br and N5-125I, respectively. Two intermediate products formed using the second strategy, 1-(tert-butyldimethylsilyl)-9-iodo-o-carborane and 1-(tert-butyldimethylsilyl)-12-iodo-o-carborane, were subjected to x-ray diffraction studies to confirm that substitution at a single carbon atom of 9-iodo-o-carborane resulted in the formation of two structural isomers. To the best of our knowledge, this is the first report of halogen and isotope exchange reactions of B-halocarboranes that have been conjugated to a complex biomolecule. Human TK1 phosphorylation rates of N5, N5-127I, and N5-79/81Br ranged from 38.0 % to 29.6% relative to that of thymidine, the endogenous hTK1 substrate. The in vitro uptake of N5, N5-127I, and N5-79/81Br in L929 TK1 (+) cells was 2.0 ×, 1.8 ×, and 1.4 × greater than that in L929 TK1 (−) cells.
PMCID: PMC3257809  PMID: 22175713
3-Carboranyl thymidine analogues (3CTAs); (radio)halogenated carboranes; (radio)halogenated 3CTAs; thymidine kinase; nucleophilic isotope exchange; selective monoiodination at the carborane cage; tetraiodination at the carborane cage; phosphoryl transfer assay; cancer therapy and imaging
8.  2-(3-{1-Carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [18F]DCFPyL, a PSMA-based PET Imaging Agent for Prostate Cancer 
We have synthesized and evaluated in vivo 2-(3-{1-carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pentanedioic acid, [18F]DCFPyL, as a potential imaging agent for the prostate-specific membrane antigen, PSMA. PSMA is upregulated in prostate cancer epithelia as well as in the neovasculature of most solid tumors.
Experimental Design
[18F]DCFPyL was synthesized in two steps from the p-methoxybenzyl (PMB) protected lys-C(O)-glu urea precursor using 6-[18F]fluoronicotinic acid tetrafluorophenyl ester ([18F]F-Py-TFP) for introduction of 18F. Radiochemical synthesis was followed by biodistribution and imaging with PET in immunocompromised mice using isogenic PC3 PSMA+ and PSMA− xenograft models. Human radiation dosimetry estimates were calculated using OLINDA/EXM 1.0.
DCFPyL displays a Ki value of 1.1 ± 0.1 nM for PSMA. [18F]DCFPyL was produced in radiochemical yields of 36-53% (decay corrected) and specific radioactivities of 340 – 480 Ci/mmol (12.6 – 17.8 GBq/μmol, n = 3). In an immunocompromised mouse model [18F]DCFPyL clearly delineated PSMA+ PC3 PIP prostate tumor xenografts on imaging with PET. At 2 h post-injection, 39.4 ± 5.4 percent injected dose per gram of tissue (%ID/g) was evident within the PIP tumor, with a ratio of 358:1 of uptake within PIP to PSMA− PC3 flu tumor placed in the opposite flank. At or after 1 h post-injection, minimal non-target tissue uptake of [18F]DCFPyL was observed. The bladder wall is the dose-limiting organ.
These data suggest [18F]DCFPyL as a viable, new positron-emitting imaging agent for PSMA-expressing tissues.
PMCID: PMC3243762  PMID: 22042970
PSMA; PET; molecular imaging; prostate cancer; PC3
9.  Novel substrate-based inhibitors of human glutamate carboxypeptidase II with enhanced lipophilicity 
Journal of medicinal chemistry  2011;54(21):7535-7546.
Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring non-polar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of non-polar interactions governing GCPII affinity towards novel substrates as well as formerly unnoticed plasticity of the S1′ specificity pocket. Based on those data, we designed, synthesized and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.
PMCID: PMC3222833  PMID: 21923190
PSMA; NAALADase; GCPII; zinc peptidase; folate hydrolase; inhibition; quantum mechanics/molecular mechanics (QM/MM)
11.  68Ga-Labeled Inhibitors of Prostate-Specific Membrane antigen (PSMA) for Imaging Prostate Cancer 
Journal of Medicinal Chemistry  2010;53(14):5333-5341.
Gallium-68 is a generator-produced radionuclide for positron emission tomography (PET) that is being increasingly used for radiolabeling of tumor-targeting peptides. Compounds [68Ga]3 and [68Ga]6 are high-affinity, urea-based inhibitors of the prostate-specific membrane antigen (PSMA) that were synthesized in decay-uncorrected yields ranging from 60 – 70% and radiochemical purities of more than 99%. Compound [68Ga]3 demonstrated 3.78 ± 0.90 percent injected dose per gram of tissue (%ID/g) within PSMA+ PIP tumor at 30 min post-injection, while [68Ga]6 showed a two hour PSMA+ PIP tumor uptake value of 3.29 ± 0.77%ID/g. Target (PSMA+ PIP) to non-target (PSMA− flu) ratios were 4.6 and 18.3, respectively, at those time points. Both compounds delineated tumor clearly by small animal PET. The urea series of imaging agents for PSMA can be radiolabeled with 68Ga, a cyclotron-free isotope useful for clinical PET studies, with maintenance of target specificity.
PMCID: PMC3341619  PMID: 20568777
gallium; molecular imaging; positron emission tomography; prostate-specific membrane antigen; radiopharmaceutical
12.  Synthesis and Evaluation of Technetium-99m- and Rhenium-Labeled Inhibitors of the Prostate-Specific Membrane Antigen (PSMA) 
Journal of Medicinal Chemistry  2008;51(15):4504-4517.
The prostate-specific membrane antigen (PSMA) is increasingly recognized as a viable target for imaging and therapy of cancer. We prepared seven 99mTc/Re-labeled compounds by attaching known Tc/Re chelating agents to an amino-functionalized PSMA inhibitor (lys-NHCONH-glu) with or without a variable length linker moiety. Ki values ranged from 0.17 to 199 nM. Ex vivo biodistribution and in vivo imaging demonstrated the degree of specific binding to engineered PSMA+ PC3 PIP tumors. PC3-PIP cells are derived from PC3 that have been transduced with the gene for PSMA. Despite demonstrating nearly the lowest PSMA inhibitory potency of this series, [99mTc(CO)3(L1)]+ (L1 = (2-pyridylmethyl)2N(CH2)4CH(CO2H)-NHCO-(CH2)6CO-NH-lys-NHCONH-glu) showed the highest, most selective PIP tumor uptake, at 7.9 ± 4.0% injected dose per gram of tissue at 30 min postinjection. Radioactivity cleared from nontarget tissues to produce a PIP to flu (PSMA-PC3) ratio of 44:1 at 120 min postinjection. PSMA can accommodate the steric requirements of 99mTc/Re complexes within PSMA inhibitors, the best results achieved with a linker moiety between the ε amine of the urea lysine and the chelator.
PMCID: PMC3336105  PMID: 18637669
13.  Bioisosterism of Urea-Based GCPII Inhibitors: Synthesis and Structure-Activity Relationships Studies 
We report a strategy based on bioisosterism to improve the physicochemical properties of existing hydrophilic, urea-based GCPII inhibitors. Comprehensive structure-activity relationship studies of the P1’ site of ZJ-43- and DCIBzL-based compounds identified several glutamate-free inhibitors with Ki values below 20 nM. Among them, compound 32d (Ki = 11 nM) exhibited selective uptake in GCPII-expressing tumors by SPECT-CT imaging in mice. A novel conformational change of amino acids in the S1’ pharmacophore pocket was observed in the X-ray crystal structure of GCPII complexed with 32d.
PMCID: PMC2818328  PMID: 19897367
PSMA; glutamate carboxypeptidase II; molecular imaging; radiopharmaceutical; SPECT
14.  A Low Molecular Weight PSMA-Based Fluorescent Imaging Agent for Cancer 
We synthesized YC-27 3 to provide a fluorescent imaging probe for the prostate-specific membrane antigen (PSMA), a marker for hormone-independent prostate cancer and tumor neovasculature, with suitable pharmacokinetics for use in vivo. Immediate precursor trifluoroacetate salt of 2-(3-{5-[7-(5-amino-1-carboxy-pentylcarbamoyl)-heptanoylamino]-1-carboxy-pentyl}-ureido)-pentanedioic acid 2 was conjugated with a commercially available near-infrared light emitting dye (IRDye 800CW) to provide 3 in 72% yield. YC-27 3 demonstrated a PSMA inhibitory activity of 0.37 nM and was capable of generating target-to-nontarget ratios of at least 10 in PSMA-expressing PC3-PIP vs. PSMA-negative PC3-flu tumors in vivo. YC-27 3 may be useful for study of PSMA-expressing tissue in preclinical models or for intraoperative guidance.
PMCID: PMC2787846  PMID: 19818734
Prostate-specific membrane antigen; NAALADase; fluorescence; urea; molecular imaging
15.  Identification of Inhibitors of ABCG2 by a Bioluminescence Imaging-based High-throughput Assay 
Cancer research  2009;69(14):5867-5875.
ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)-based assay was developed to allow high-throughput compound screening. This assay exploits our finding that D-luciferin, the substrate of firefly luciferase (fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of D-luciferin and enhance bioluminescence signal by increasing intracellular D-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the US Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty seven compounds demonstrated BLI enhancement, a measure of anti-ABCG2 activity, of five-fold or greater, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g. mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from an FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing.
PMCID: PMC2711991  PMID: 19567678
multidrug resistance; ABC transporter; cancer chemotherapy; BCRP; drug screen
16.  Radiohalogenated Prostate-Specific Membrane Antigen (PSMA)-Based Ureas as Imaging Agents for Prostate Cancer 
Journal of medicinal chemistry  2008;51(24):7933-7943.
To extend our development of new imaging agents targeting the prostate-specific membrane antigen (PSMA), we have used the versatile intermediate 2-[3-(5-amino-1-carboxy-pentyl)-ureido]-pentanedioic acid (Lys-C(O)-Glu), which allows ready incorporation of radiohalogens for single photon emission computed tomography (SPECT) and positron emission tomography (PET). We prepared 2-[3-[1-carboxy-5-(4-[125I]iodo-benzoylamino)-pentyl]-ureido]-pentanedioic acid ([125I]3), 2-[3-[1-carboxy-5-(4-[18F]fluoro-benzoylamino)-pentyl]-ureido]-pentanedioic acid ([18F]6) and 2-(3-[1-carboxy-5-[(5-[125I]iodo-pyridine-3-carbonyl)-amino]-pentyl]-ureido)-pentanedioic acid ([125I]8) in 65 - 80% (non-decay-corrected), 30 - 35% (decay corrected) and 59 - 75% (non-decay-corrected) radiochemical yields. Compound [125I]3 demonstrated 8.8 ± 4.7 percent injected dose per gram (%ID/g) within PSMA+ PC-3 PIP tumor at 30 min postinjection, which persisted, with clear delineation of the tumor by SPECT. Similar tumor uptake values at early time points were demonstrated for [18F]6 (using PET) and [125I]8. Because of the many radiohalogenated moieties that can be attached via the ε amino group, the intermediate Lys-C(O)-Glu is an attractive template upon which to develop new imaging agents for prostate cancer.
PMCID: PMC2631656  PMID: 19053825
NAALADase; GCPII; radiopharmaceutical; SPECT; PET; small animal imaging

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