A series of phosphoramidate-based prostate specific membrane antigen (PSMA) inhibitors of increasing lipophilicity were synthesized (4, 5, and 6), and their fluorine-18 analogs were evaluated for use as positron emission tomography (PET) imaging agents for prostate cancer. To gain insight into their modes of binding, they were also cocrystallized with the extracellular domain of PSMA. All analogs exhibited irreversible binding to PSMA with IC50 values ranging from 0.4 to 1.3 nM. In vitro assays showed binding and rapid internalization (80-95%, 2 h) of the radiolabeled ligands in PSMA(+) cells. In vivo distribution demonstrated significant uptake in CWR22Rv1 (PSMA(+)) tumor, with tumor to blood ratios of 25.6:1, 63.6:1, and 69.6:1 for [(18)F]4, [(18)F]5, and [(18)F]6, respectively, at 2 h postinjection. Installation of aminohexanoic acid (AH) linkers in the phosphoramidate scaffold improved their PSMA binding and inhibition and was critical for achieving suitable in vivo imaging properties, positioning [(18)F]5 and [(18)F]6 as favorable candidates for future prostate cancer imaging clinical trials.

Cancer Targeted Technology Woodinville Washington 98072 United States

Department of Chemistry Washington State University Pullman Washington 99164 4630 United States

Department of Radiology and Biomedical Imaging University of California San Francisco 185 Berry Street San Francisco California 94107 United States

Institute of Biotechnology 252 50 Prague Czech Republic

Zobrazit více v PubMed

Foss CA, Mease RC, Fan H, Wang Y, Ravert HT, Dannals RF, Olszewski RT, Heston WD, Kozikowski AP, Pomper MG. Radiolabeled small-molecule ligands for prostate-specific membrane antigen: in vivo imaging in experimental models of prostate cancer. Clin Cancer Res. 2005;11:4022–4028. PubMed

Gao X, Cui Y, Levenson RM, Chung LW, Nie S. In vivo cancer targeting and imaging with semiconductor quantum dots. Nat Biotechnol. 2004;22:969–976. PubMed

Humblet V, Lapidus R, Williams LR, Tsukamoto T, Rojas C, Majer P, Hin B, Ohnishi S, De Grand AM, Zaheer A, Renze JT, Nakayama A, Slusher BS, Frangioni JV. High-affinity near-infrared fluorescent small-molecule contrast agents for in vivo imaging of prostate-specific membrane antigen. Mol Imaging. 2005;4:448–462. PubMed

Pomper MG, Musachio JL, Zhang J, Scheffel U, Zhou Y, Hilton J, Maini A, Dannals RF, Wong DF, Kozikowski AP. 11C-MCG: synthesis, uptake selectivity, and primate PET of a probe for glutamate carboxypeptidase II (NAALADase) Mol Imaging. 2002;1:96–101. PubMed

Smith-Jones PM, Vallabhajosula S, Navarro V, Bastidas D, Goldsmith SJ, Bander NH. Radiolabeled monoclonal antibodies specific to the extracellular domain of prostate-specific membrane antigen: preclinical studies in nude mice bearing LNCaP human prostate tumor. J Nucl Med. 2003;44:610–617. PubMed

Tasch J, Gong M, Sadelain M, Heston WD. A unique folate hydrolase, prostate-specific membrane antigen (PSMA): a target for immunotherapy? Crit Rev Immunol. 2001;21:249–261. PubMed

Liu T, Wu LY, Choi JK, Berkman CE. Targeted photodynamic therapy for prostate cancer: inducing apoptosis via activation of the caspase-8/-3 cascade pathway. Int J Oncol. 2010;36:777–784. PubMed

Fracasso G, Bellisola G, Cingarlini S, Castelletti D, Prayer-Galetti T, Pagano F, Tridente G, Colombatti M. Anti-tumor effects of toxins targeted to the prostate specific membrane antigen. Prostate. 2002;53:9–23. PubMed

Lu J, Celis E. Recognition of prostate tumor cells by cytotoxic T lymphocytes specific for prostate-specific membrane antigen. Cancer Res. 2002;62:5807–5812. PubMed

Mease RC, Foss CA, Pomper MG. PET imaging in prostate cancer: focus on prostate-specific membrane antigen. Curr Top Med Chem. 2013;13:951–962. PubMed PMC

Chen Y, Foss CA, Byun Y, Nimmagadda S, Pullambhatla M, Fox JJ, Castanares M, Lupold SE, Babich JW, Mease RC, Pomper MG. Radiohalogenated prostate-specific membrane antigen (PSMA)-based ureas as imaging agents for prostate cancer. J Med Chem. 2008;51:7933–7943. PubMed PMC

Banerjee SR, Foss CA, Castanares M, Mease RC, Byun Y, Fox JJ, Hilton J, Lupold SE, Kozikowski AP, Pomper MG. Synthesis and evaluation of technetium-99m- and rhenium-labeled inhibitors of the prostate-specific membrane antigen (PSMA) J Med Chem. 2008;51:4504–4517. PubMed PMC

Banerjee SR, Pullambhatla M, Shallal H, Lisok A, Mease RC, Pomper MG. A modular strategy to prepare multivalent inhibitors of prostate-specific membrane antigen (PSMA) Oncotarget. 2011;2:1244–1253. PubMed PMC

Banerjee SR, Pullambhatla M, Byun Y, Nimmagadda S, Green G, Fox JJ, Horti A, Mease RC, Pomper MG. 68Ga-labeled inhibitors of prostate-specific membrane antigen (PSMA) for imaging prostate cancer. J Med Chem. 2010;53:5333–5341. PubMed PMC

Chen Y, Pullambhatla M, Foss CA, Byun Y, Nimmagadda S, Senthamizhchelvan S, Sgouros G, Mease RC, Pomper MG. 2-(3-{1-Carboxy-5-[(6-[18F]fluoro-pyridine-3-carbonyl)-amino]-pentyl}-ureido)-pen tanedioic acid, [18F]DCFPyL, a PSMA-based PET imaging agent for prostate cancer. Clin Cancer Res. 2011;17:7645–7653. PubMed PMC

Cho SY, Gage KL, Mease RC, Senthamizhchelvan S, Holt DP, Jeffrey-Kwanisai A, Endres CJ, Dannals RF, Sgouros G, Lodge M, Eisenberger MA, Rodriguez R, Carducci MA, Rojas C, Slusher BS, Kozikowski AP, Pomper MG. Biodistribution, tumor detection, and radiation dosimetry of 18F-DCFBC, a low-molecular-weight inhibitor of prostate-specific membrane antigen, in patients with metastatic prostate cancer. J Nucl Med. 2012;53:1883–1891. PubMed PMC

Szabo Z, Mena E, Rowe SP, Plyku D, Nidal R, Eisenberger MA, Antonarakis ES, Fan H, Dannals RF, Chen Y, Mease RC, Vranesic M, Bhatnagar A, Sgouros G, Cho SY, Pomper MG. Initial Evaluation of [(18)F]DCFPyL for Prostate-Specific Membrane Antigen (PSMA)-Targeted PET Imaging of Prostate Cancer. Mol Imaging Biol. 2015;17:565–574. PubMed PMC

Mease RC, Dusich CL, Foss CA, Ravert HT, Dannals RF, Seidel J, Prideaux A, Fox JJ, Sgouros G, Kozikowski AP, Pomper MG. N-[N-[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-[18F]-fluorobenzyl-L-cysteine, [18F]DCFBC: a new imaging probe for prostate cancer. Clin Cancer Res. 2008;14:3036–3043. PubMed PMC

Liu T, Toriyabe Y, Kazak M, Berkman CE. Pseudoirreversible inhibition of prostate-specific membrane antigen by phosphoramidate peptidomimetics. Biochemistry. 2008;47:12658–12660. PubMed

Liu T, Nedrow-Byers JR, Hopkins MR, Berkman CE. Spacer length effects on in vitro imaging and surface accessibility of fluorescent inhibitors of prostate specific membrane antigen. Bioorg Med Chem Lett. 2011;21:7013–7016. PubMed PMC

Nedrow-Byers JR, Jabbes M, Jewett C, Ganguly T, He H, Liu T, Benny P, Bryan JN, Berkman CE. A phosphoramidate-based prostate-specific membrane antigen-targeted SPECT agent. Prostate. 2012;72:904–912. PubMed

Lapi SE, Wahnishe H, Pham D, Wu LY, Nedrow-Byers JR, Liu T, Vejdani K, VanBrocklin HF, Berkman CE, Jones EF. Assessment of an 18F-labeled phosphoramidate peptidomimetic as a new prostate-specific membrane antigen-targeted imaging agent for prostate cancer. J Nucl Med. 2009;50:2042–2048. PubMed PMC

Nedrow-Byers JR, Moore AL, Ganguly T, Hopkins MR, Fulton MD, Benny PD, Berkman CE. PSMA-targeted SPECT agents: mode of binding effect on in vitro performance. Prostate. 2013;73:355–362. PubMed PMC

Ganguly T, Dannoon S, Hopkins MR, Murphy S, Cahaya H, Blecha JE, Jivan S, Drake CR, Barinka C, Jones EF, VanBrocklin HF, Berkman CE. A high-affinity [(18)F]-labeled phosphoramidate peptidomimetic PSMA-targeted inhibitor for PET imaging of prostate cancer. Nucl Med Biol. 2015;42:780–787. PubMed PMC

Wu LY, Anderson MO, Toriyabe Y, Maung J, Campbell TY, Tajon C, Kazak M, Moser J, Berkman CE. The molecular pruning of a phosphoramidate peptidomimetic inhibitor of prostate-specific membrane antigen. Bioorg Med Chem. 2007;15:7434–7443. PubMed PMC

Maung J, Mallari JP, Girtsman TA, Wu LY, Rowley JA, Santiago NM, Brunelle AN, Berkman CE. Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates. Bioorg Med Chem. 2004;12:4969–4979. PubMed

Anderson MO, Wu LY, Santiago NM, Moser JM, Rowley JA, Bolstad ES, Berkman CE. Substrate specificity of prostate-specific membrane antigen. Bioorg Med Chem. 2007;15:6678–6686. PubMed PMC

Liu T, Toriyabe Y, Kazak M, Berkman CE. Pseudoirreversible inhibition of prostate-specific membrane antigen by phosphoramidate peptidomimetics. Biochemistry. 2008;47:12658–12660. PubMed

Barinka C, Mlcochova P, Sacha P, Hilgert I, Majer P, Slusher BS, Horejsi V, Konvalinka J. Amino acids at the N- and C-termini of human glutamate carboxypeptidase II are required for enzymatic activity and proper folding. Eur J Biochem. 2004;271:2782–2790. PubMed

Barinka C, Rovenska M, Mlcochova P, Hlouchova K, Plechanovova A, Majer P, Tsukamoto T, Slusher BS, Konvalinka J, Lubkowski J. Structural insight into the pharmacophore pocket of human glutamate carboxypeptidase II. J Med Chem. 2007;50:3267–3273. PubMed

Pavlicek J, Ptacek J, Barinka C. Glutamate carboxypeptidase II: an overview of structural studies and their importance for structure-based drug design and deciphering the reaction mechanism of the enzyme. Curr Med Chem. 2012;19:1300–1309. PubMed

Zhang AX, Murelli RP, Barinka C, Michel J, Cocleaza A, Jorgensen WL, Lubkowski J, Spiegel DA. A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules. J Am Chem Soc. 2010;132:12711–12716. PubMed PMC

Nedrow JR, Latoche JD, Day KE, Modi J, Ganguly T, Zeng D, Kurland BF, Berkman CE, Anderson CJ. Targeting PSMA with a Cu-64 labeled phosphoramidate inhibitor for PET/CT imaging of variant PSMA-expressing xenografts in mouse models of prostate cancer. Mol Imaging Biol. 2016;18:402–410. PubMed

Liu T, Wu LY, Kazak M, Berkman CE. Cell-Surface labeling and internalization by a fluorescent inhibitor of prostate-specific membrane antigen. Prostate. 2008;68:955–964. PubMed

Hillier SM, Maresca KP, Femia FJ, Marquis JC, Foss CA, Nguyen N, Zimmerman CN, Barrett JA, Eckelman WC, Pomper MG, Joyal JL, Babich JW. Preclinical evaluation of novel glutamate-urea-lysine analogues that target prostate-specific membrane antigen as molecular imaging pharmaceuticals for prostate cancer. Cancer Res. 2009;69:6932–6940. PubMed PMC

Bacich DJ, Pinto JT, Tong WP, Heston WD. Cloning, expression, genomic localization, and enzymatic activities of the mouse homolog of prostate-specific membrane antigen/NAALADase/folate hydrolase. Mamm Genome. 2001;12:117–123. PubMed

Ghosh A, Wang X, Klein E, Heston WDW. Novel role of prostate-specific membrane antigen in suppressing prostate cancer invasiveness. Cancer Res. 2005;65:727–731. PubMed

Krug M, Weiss MS, Heinemann U, Mueller U. XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS. J Appl Crystallogr. 2012;45:568–572.

Klusak V, Barinka C, Plechanovova A, Mlcochova P, Konvalinka J, Rulisek L, Lubkowski J. Reaction mechanism of glutamate carboxypeptidase II revealed by mutagenesis, X-ray crystallography, and computational methods. Biochemistry. 2009;48:4126–4138. PubMed PMC

Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr, Sect D: Biol Crystallogr. 2010;66:486–501. PubMed PMC

Murshudov GN, Skubak P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr, Sect D: Biol Crystallogr. 2011;67:355–367. PubMed PMC

Schuttelkopf AW, van Aalten DM. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr, Sect D: Biol Crystallogr. 2004;60:1355–1363. PubMed

Chen VB, Arendall WB, 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr, Sect D: Biol Crystallogr. 2010;66:12–21. PubMed PMC

Nedrow-Byers JR, Jabbes M, Jewett C, Ganguly T, He H, Liu T, Benny P, Bryan JN, Berkman CE. A phosphoramidate-based prostate-specific membrane antigen-targeted SPECT agent. Prostate. 2012;72:904–912. PubMed

Kuppuswamy D, Pike LJ. Ligand-induced desensitization of 125I-epidermal growth factor internalization. J Biol Chem. 1989;264:3357–3363. PubMed