BACKGROUND: Metabolomic analyses from our group and others have shown that tumors treated with glutamine antagonists (GA) exhibit robust accumulation of formylglycinamide ribonucleotide (FGAR), an intermediate in the de novo purine synthesis pathway. The increase in FGAR is attributed to the inhibition of the enzyme FGAR amidotransferase (FGAR-AT) that catalyzes the ATP-dependent amidation of FGAR to formylglycinamidine ribonucleotide (FGAM). While perturbation of this pathway resulting from GA therapy has long been recognized, no study has reported systematic quantitation and analyses of FGAR in plasma and tumors. OBJECTIVE: Herein, we aimed to evaluate the efficacy of our recently discovered tumor-targeted GA prodrug, GA-607 (isopropyl 2-(6-acetamido-2-(adamantane-1-carboxamido)hexanamido)-6-diazo-5-oxohexanoate), and demonstrate its target engagement by quantification of FGAR in plasma and tumors. METHODS: Efficacy and pharmacokinetics of GA-607 were evaluated in a murine EL4 lymphoma model followed by global tumor metabolomic analysis. Liquid chromatography-mass spectrometry (LC-MS) based methods employing the ion-pair chromatography approach were developed and utilized for quantitative FGAR analyses in plasma and tumors. RESULTS: GA-607 showed preferential tumor distribution and robust single-agent efficacy in a murine EL4 lymphoma model. While several metabolic pathways were perturbed by GA-607 treatment, FGAR showed the highest increase qualitatively. Using our newly developed sensitive and selective LC-MS method, we showed a robust >80- and >10- fold increase in tumor and plasma FGAR levels, respectively, with GA-607 treatment. CONCLUSION: These studies describe the importance of FGAR quantification following GA therapy in cancer and underscore its importance as a valuable pharmacodynamic marker in the preclinical and clinical development of GA therapies.

Drug Metabolism Covance Laboratories Inc Madison WI 53704 United States

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i Prague 166 10 Czech Republic

Jiangxi Science and Technology Normal University Nanchang Jiangxi 330013 China

Johns Hopkins Drug Discovery Johns Hopkins School of Medicine Baltimore MD 21205 United States

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Pedley A.M., Benkovic S.J. A new view into the regulation of purine metabolism: The purinosome. Trends Biochem. Sci. 2017;42(2):141–154. doi: 10.1016/j.tibs.2016.09.009. PubMed DOI PMC

Yin J., Ren W., Huang X., Deng J., Li T., Yin Y. Potential mechanisms connecting purine metabolism and cancer therapy. Front. Immunol. 2018;9:1697. doi: 10.3389/fimmu.2018.01697. PubMed DOI PMC

Wang Y., Wang W., Xu L., Zhou X., Shokrollahi E., Felczak K., van der Laan L.J., Pankiewicz K.W., Sprengers D., Raat N.J., Metselaar H.J., Peppelenbosch M.P., Pan Q. Cross talk between nucleotide synthesis pathways with cellular immunity in constraining hepatitis E virus replication. Antimicrob. Agents Chemother. 2016;60(5):2834–2848. doi: 10.1128/AAC.02700-15. PubMed DOI PMC

Aleo F. The hormonal regulation of purine biosynthesis: basal levels of different hormones in primary gout. 1984. PubMed

Hershfield M.S., Seegmiller J.E. Gout and the regulation of purine biosynthesis. Horiz. Biochem. Biophys. 1976;2:134–162. PubMed

Grayzel A.I., Seegmiller J.E., Love E. Suppression of uric acid synthesis in the gouty human by the use of 6-diazo-5-oxo-L-norleucine. J. Clin. Invest. 1960;39:447–454. doi: 10.1172/JCI104057. PubMed DOI PMC

Hoskins A.A., Anand R., Ealick S.E., Stubbe J. The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation. Biochemistry. 2004;43(32):10314–10327. doi: 10.1021/bi049127h. PubMed DOI

Levenberg B., Melnick I., Buchanan J.M. Biosynthesis of the purines. XV. The effect of aza-L-serine and 6-diazo-5-oxo-L-norleucine on inosinic acid biosynthesis de novo. J. Biol. Chem. 1957;225(1):163–176. doi: 10.1016/S0021-9258(18)64919-1. PubMed DOI

Lyons S.D., Sant M.E., Christopherson R.I. Cytotoxic mechanisms of glutamine antagonists in mouse L1210 leukemia. J. Biol. Chem. 1990;265(19):11377–11381. doi: 10.1016/S0021-9258(19)38603-X. PubMed DOI

Mádrová L., Krijt M., Barešová V., Václavík J., Friedecký D., Dobešová D., Součková O., Škopová V., Adam T., Zikánová M. Mass spectrometric analysis of purine de novo biosynthesis intermediates. PLoS One. 2018;13(12):e0208947. doi: 10.1371/journal.pone.0208947. PubMed DOI PMC

Tenora L., Alt J., Dash R.P., Gadiano A.J., Novotná K., Veeravalli V., Lam J., Kirkpatrick Q.R., Lemberg K.M., Majer P., Rais R., Slusher B.S. Tumor-targeted delivery of 6-diazo-5-oxo-l-norleucine (DON) using substituted acetylated lysine prodrugs. J. Med. Chem. 2019;62(7):3524–3538. doi: 10.1021/acs.jmedchem.8b02009. PubMed DOI PMC

Wang S.Z., Poore B., Alt J., Price A., Allen S.J., Hanaford A.R., Kaur H., Orr B.A., Slusher B.S., Eberhart C.G., Raabe E.H., Rubens J.A. Unbiased metabolic profiling predicts sensitivity of high MYC-expressing atypical teratoid/rhabdoid tumors to glutamine inhibition with 6-diazo-5-oxo-l-norleucine. Clin. Cancer Res. 2019;25(19):5925–5936. doi: 10.1158/1078-0432.CCR-19-0189. PubMed DOI PMC

Leone R.D., Zhao L., Englert J.M., Sun I.M., Oh M.H., Sun I.H., Arwood M.L., Bettencourt I.A., Patel C.H., Wen J., Tam A., Blosser R.L., Prchalova E., Alt J., Rais R., Slusher B.S., Powell J.D. Glutamine blockade induces divergent metabolic programs to overcome tumor immune evasion. Science. 2019;366(6468):1013–1021. doi: 10.1126/science.aav2588. PubMed DOI PMC

Sharma N.S., Gupta V.K., Garrido V.T., Hadad R., Durden B.C., Kesh K., Giri B., Ferrantella A., Dudeja V., Saluja A., Banerjee S. Targeting tumor-intrinsic hexosamine biosynthesis sensitizes pancreatic cancer to anti-PD1 therapy. J. Clin. Invest. 2020;130(1):451–465. doi: 10.1172/JCI127515. PubMed DOI PMC

Lemberg K.M., Zhao L., Wu Y., Veeravalli V., Alt J., Aguilar J.M.H., Dash R.P., Lam J., Tenora L., Rodriguez C., Nedelcovych M.T., Brayton C., Majer P., Blakeley J.O., Rais R., Slusher B.S. The novel glutamine antagonist prodrug JHU395 has antitumor activity in malignant peripheral nerve sheath tumor. Mol. Cancer Ther. 2020;19(2):397–408. doi: 10.1158/1535-7163.MCT-19-0319. PubMed DOI PMC

Tcyganov E., Mastio J., Chen E., Gabrilovich D.I. Plasticity of myeloid-derived suppressor cells in cancer. Curr. Opin. Immunol. 2018;51:76–82. doi: 10.1016/j.coi.2018.03.009. PubMed DOI PMC

Oh M-H., Sun I.H., Zhao L., Leone R.D., Sun I.M., Xu W., Collins S.L., Tam A.J., Blosser R.L., Patel C.H., Englert J.M., Arwood M.L., Wen J., Chan-Li Y., Tenora L., Majer P., Rais R., Slusher B.S., Horton M.R., Powell J.D. Targeting glutamine metabolism enhances tumor-specific immunity by modulating suppressive myeloid cells. J. Clin. Invest. 2020;130(7):3865–3884. doi: 10.1172/JCI131859. PubMed DOI PMC

Cervantes-Madrid D., Romero Y., Dueñas-González A. Reviving lonidamine and 6-diazo-5-oxo-l-norleucine to be used in combination for metabolic cancer therapy. BioMed Res. Int. 2015;2015:690492. doi: 10.1155/2015/690492. PubMed DOI PMC

Alt J., Potter M.C., Rojas C., Slusher B.S. Bioanalysis of 6-diazo-5-oxo-l-norleucine in plasma and brain by ultra-performance liquid chromatography mass spectrometry. Anal. Biochem. 2015;474:28–34. doi: 10.1016/j.ab.2015.01.001. PubMed DOI PMC

Clasquin M.F., Melamud E., Rabinowitz J.D. 2012. PubMed PMC

Thakare R., Chhonker Y.S., Gautam N., Alamoudi J.A., Alnouti Y. Quantitative analysis of endogenous compounds. J. Pharm. Biomed. Anal. 2016;128:426–437. doi: 10.1016/j.jpba.2016.06.017. PubMed DOI

Strassburg K., Huijbrechts A.M., Kortekaas K.A., Lindeman J.H., Pedersen T.L., Dane A., Berger R., Brenkman A., Hankemeier T., van Duynhoven J., Kalkhoven E., Newman J.W., Vreeken R.J. Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis: application in cardiac surgery. Anal. Bioanal. Chem. 2012;404(5):1413–1426. doi: 10.1007/s00216-012-6226-x. PubMed DOI PMC

Magill G.B., Myers W.P., Reilly H.C., Putnam R.C., Magill J.W., Sykes M.P., Escher G.C., Karnofsky D.A., Burchenal J.H. Pharmacological and initial therapeutic observations on 6-diazo-5-oxo-1-norleucine (DON) in human neoplastic disease. Cancer. 1957;10(6):1138–1150. doi: 10.1002/1097-0142(195711/12)10:6<1138:AID-CNCR2820100608>3.0.CO;2-K. PubMed DOI

Earhart R.H., Amato D.J., Chang A.Y., Borden E.C., Shiraki M., Dowd M.E., Comis R.L., Davis T.E., Smith T.J. Phase II trial of 6-diazo-5-oxo-L-norleucine versus aclacinomycin-A in advanced sarcomas and mesotheliomas. Invest. New Drugs. 1990;8(1):113–119. doi: 10.1007/BF00216936. PubMed DOI

Rahman A., Smith F.P., Luc P.T., Woolley P.V. Phase I study and clinical pharmacology of 6-diazo-5-oxo-L-norleucine (DON). Invest. New Drugs. 1985;3(4):369–374. doi: 10.1007/BF00170760. PubMed DOI

DeWald H.A., Moore A.M. 6-diazo-5-oxo-l-norleucine, a new tumor-inhibitory substance.1a preparation of l-, d- and dl-forms1b. J. Am. Chem. Soc. 1958;80(15):3941–3945. doi: 10.1021/ja01548a036. DOI

Li M.C., Whitmore W.F., Jr, Golbey R., Grabstald H. Effects of combined drug therapy on metastatic cancer of the testis. JAMA. 1960;174:1291–1299. doi: 10.1001/jama.1960.03030100059013. PubMed DOI

A clinical study of the comparative effect of nitrogen mustard and DON in patients with bronchogenic carcinoma, Hodgkin’s disease, lymphosarcoma, and melanoma. J. Natl. Cancer Inst. 1959;22(2):433–439. doi: 10.1093/jnci/22.2.433. PubMed DOI

Sullivan M.P., Beatty E.C., Jr, Hyman C.B., Murphy M.L., Pierce M.I., Severo N.C. A comparison of the effectiveness of standard dose 6-mercaptopurine, combination 6-mercaptopurine and DON, and high-loading 6-mercaptopurine therapies in treatment of the acute leukemias of childhood: results of a coperative study. Cancer Chemother. Rep. 1962;18:83–95. PubMed

Eagan R.T., Frytak S., Nichols W.C., Creagan E.T., Ingle J.N. Phase II study on DON in patients with previously treated advanced lung cancer. Cancer Treat. Rep. 1982;66(8):1665–1666. PubMed

Lemberg K.M., Vornov J.J., Rais R., Slusher B.S. We’re not “don” yet: Optimal dosing and prodrug delivery of 6-diazo-5-oxo-l-norleucine. Mol. Cancer Ther. 2018;17(9):1824–1832. doi: 10.1158/1535-7163.MCT-17-1148. PubMed DOI PMC

Yamashita A.S., da Costa Rosa M., Stumpo V., Rais R., Slusher B.S., Riggins G.J. The glutamine antagonist prodrug JHU-083 slows malignant glioma growth and disrupts mTOR signaling. Neurooncol. Adv. 2020;3(1):a149. doi: 10.1093/noajnl/vdaa149. PubMed DOI PMC

Hanaford A.R., Alt J., Rais R., Wang S.Z., Kaur H., Thorek D.L.J., Eberhart C.G., Slusher B.S., Martin A.M., Raabe E.H. Orally bioavailable glutamine antagonist prodrug JHU-083 penetrates mouse brain and suppresses the growth of MYC-driven medulloblastoma. Transl. Oncol. 2019;12(10):1314–1322. doi: 10.1016/j.tranon.2019.05.013. PubMed DOI PMC

Baresova V., Krijt M., Skopova V., Souckova O., Kmoch S., Zikanova M. CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enzyme substrates and affect purinosome formation. Mol. Genet. Metab. 2016;119(3):270–277. doi: 10.1016/j.ymgme.2016.08.004. PubMed DOI

Krijt M., Souckova O., Baresova V., Skopova V., Zikanova M. Metabolic tools for identification of new mutations of enzymes engaged in purine synthesis leading to neurological impairment. Folia Biol. (Praha) 2019;65(3):152–157. PubMed

Bagnara A.S., Letter A.A., Henderson J.F. Multiple mechanisms of regulation of purine biosynthesis de novo in intact tumor cells. Biochim. Biophys. Acta. 1974;374(3):259–270. doi: 10.1016/0005-2787(74)90247-0. PubMed DOI

Jones B.R., Schultz G.A., Eckstein J.A., Ackermann B.L. Surrogate matrix and surrogate analyte approaches for definitive quantitation of endogenous biomolecules. Bioanalysis. 2012;4(19):2343–2356. doi: 10.4155/bio.12.200. PubMed DOI

Ho S., Gao H. Surrogate matrix: opportunities and challenges for tissue sample analysis. Bioanalysis. 2015;7(18):2419–2433. doi: 10.4155/bio.15.161. PubMed DOI

Fan T.W.M., Lorkiewicz P.K., Sellers K., Moseley H.N., Higashi R.M., Lane A.N. Stable isotope-resolved metabolomics and applications for drug development. Pharmacol. Ther. 2012;133(3):366–391. doi: 10.1016/j.pharmthera.2011.12.007. PubMed DOI PMC

Parker S.L., Pandey S., Sime F.B., Stuart J., Lipman J., Roberts J.A., Wallis S.C. A validated LC-MS/MS method for the simultaneous quantification of the novel combination antibiotic, ceftolozane-tazobactam, in plasma (total and unbound), CSF, urine and renal replacement therapy effluent: application to pilot pharmacokinetic studies. Clin. Chem. Lab. Med. 2020;59(5):921–933. doi: 10.1515/cclm-2020-1196. PubMed DOI

Tan A., Awaiye K., Trabelsi F. Impact of calibrator concentrations and their distribution on accuracy of quadratic regression for liquid chromatography-mass spectrometry bioanalysis. Anal. Chim. Acta. 2014;815:33–41. doi: 10.1016/j.aca.2014.01.036. PubMed DOI

Gu H., Liu G., Wang J., Aubry A.F., Arnold M.E. Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance. Anal. Chem. 2014;86(18):8959–8966. doi: 10.1021/ac5018265. PubMed DOI

Park H.Y., Kim M.J., Lee S., Jin J., Lee S., Kim J.G., Choi Y.K., Park K.G. Inhibitory effect of a glutamine antagonist on proliferation and migration of VSMCs via simultaneous attenuation of glycolysis and oxidative phosphorylation. Int. J. Mol. Sci. 2021;22(11):5602. doi: 10.3390/ijms22115602. PubMed DOI PMC

Pham K., Maxwell M.J., Sweeney H., Alt J., Rais R., Eberhart C.G., Slusher B.S., Raabe E.H. Novel glutamine antagonist JHU395 suppresses MYC-driven medulloblastoma growth and induces apoptosis. J. Neuropathol. Exp. Neurol. 2021;80(4):336–344. doi: 10.1093/jnen/nlab018. PubMed DOI PMC

Lemberg K.M. Abstract 1293: A novel protide purine antimetabolite combines with the prodrug glutamine antagonist JHU395 in preclinical models of Ras-driven sarcomas. Cancer Res. 2021;81(13) Suppl.:1293. PubMed

Johnson M.L. Phase 1 and phase 2a, first-in-human (FIH) study, of DRP-104, a broad glutamine antagonist, in adult patients with advanced solid tumors. J. Clin. Oncol. 2021;39(15) Suppl.:TPS3149–TPS3149. doi: 10.1200/JCO.2021.39.15_suppl.TPS3149. DOI

Pro-905, a Novel Purine Antimetabolite, Combines with Glutamine Amidotransferase Inhibition to Suppress Growth of Malignant Peripheral Nerve Sheath Tumor

Discovery of tert-Butyl Ester Based 6-Diazo-5-oxo-l-norleucine Prodrugs for Enhanced Metabolic Stability and Tumor Delivery

Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug