Evaluation of the reducing potential of PSMA-containing endosomes by FRET imaging

Abstract

Aim: Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues. For many ligand-targeted drug conjugates, a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells. Because disulfide bond reduction is frequently exploited to ensure intracellular drug release, an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design. While the redox properties of folate receptor trafficking endosomes have been previously reported, little is known about the trafficking of prostate-specific membrane antigen (PSMA), a receptor that is experiencing increasing use for drug targeting in humans.

Methods: To obtain this information, we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green.

Results: We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells, and that reduction of its disulfide bond proceeds steadily but incompletely following internalization. The fact that maximal disulfide reduction reaches only ~50%, even after 24 h incubation, suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity.

Conclusion: As the level of disulfide reduction differs between PSMA trafficked and previously published folate trafficked conjugates, it also follows that not all internalizing receptors are translocated through similar intracellular compartments. Taken together, these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.

Keywords

DUPA / prostate-specific membrane antigen / endosomes / endocytosis

References

[1] Allen TM.Ligand-targeted therapeutics in anticancer therapy..Nat Rev Cancer2002;2:750-63

[2] Deneka AY,Beck T.Tumor-targeted drug conjugates as an emerging novel therapeutic approach in small cell lung cancer (SCLC)..Cancers2019;11:1297 PMCID:PMC6769513

[3] Srinivasarao M,Low PS.Principles in the design of ligand-targeted cancer therapeutics and imaging agents..Nat Rev Drug Discov2015;14:203-19

[4] Srinivasarao M.Ligand-targeted drug delivery..Chem Rev2017;117:12133-64

[5] Ojima I.Guided molecular missiles for tumor-targeting chemotherapy - case studies using the second-generation toxoids as warheads..Acc Chem Res2008;41:108-19

[6] Riber CF,Zelikin AN.Self-immolative linkers literally bridge disulfide chemistry and the realm of thiol-free drugs..Adv Healthc Mater2015;4:1887-90

[7] Lee MH,Kim JS.Disulfide-based multifunctional conjugates for targeted theranostic drug delivery..Acc Chem Res2015;48:2935-46

[8] Go Y.Redox compartmentalization in eukaryotic cells..Biochim Biophys Acta2008;1780:1273-90 PMCID:PMC2601570

[9] Elkin SR,Schmid SL.Endocytic pathways and endosomal trafficking: a primer..Wien Med wochenschr2016;166:196-204 PMCID:PMC4873410

[10] Doherty GJ.Mechanisms of endocytosis..Annu Rev Biochem2009;78:857-902

[11] Yang J,Vlahov IR,Low PS.Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging..Proc Natl Acad Sci USA2006;103:13872-7 PMCID:PMC1564263

[12] Low PS.Folate-targeted therapeutic and imaging agents for cancer..Curr Opin Chem Biol2009;13:256-62

[13] Low PS,Doorneweerd DD.Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases..Acc Chem Res2008;41:120-9

[14] Lv Q,Zhang R.Prostate-specific membrane antigen targeted therapy of prostate cancer using a DUPA-paclitaxel conjugate..Mol Pharm2018;15:1842-52

[15] Leamon CP,Bloomfield A.Prostate-specific membrane antigen-specific antitumor activity of a self-immolative tubulysin conjugate..Bioconjug Chem2019;30:1805-13

[16] Kularatne SA,Santhapuram HR.Prostate-specific membrane antigen targeted imaging and therapy of prostate cancer using a PSMA inhibitor as a homing ligand..Mol Pharm2009;6:780-9

[17] Kratochwil C,Stefanova M.PSMA-targeted radionuclide therapy of metastatic castration-resistant prostate cancer with 177Lu-labeled PSMA-617..J Nucl Med2016;57:1170-6

[18] Hoff DDV,Ramanathan RK.Phase I study of PSMA-targeted docetaxel-containing nanoparticle BIND-014 in patients with advanced solid tumors..Clin Cancer Res2016;22:3157-63

[19] Giesel FL,Lawal I.Intraindividual comparison of 18F-PSMA-1007 and 18F-DCFPyL PET/CT in the prospective evaluation of patients with newly diagnosed prostate carcinoma: a pilot stud..J Nucl Med2018;59:1076-80

[20] Kozikowski AP,Nan F.Synthesis of urea-based inhibitors as active site probes of glutamate carboxypeptidase II: efficacy as analgesic agents..J Med Chem2004;47:1729-38

[21] Kozikowski AP,Conti P.Design of remarkably simple, yet potent urea-based inhibitors of glutamate carboxypeptidase II (NAALADase)..J Med Chem2001;44:298-301

[22] Wüstemann T,Babich J.Targeting prostate cancer: prostate-specific membrane antigen based diagnosis and therapy..Med Res Rev2019;39:40-69

[23] Lu Y,Kleindl PJ.Antiinflammatory activity of a novel folic acid targeted conjugate of the mTOR inhibitor everolimus..Mol Med2015;21:584-96 PMCID:PMC4656201

[24] Lu Y,Westrick E,Gehrke MA.Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid-aminopterin conjugate..Arthritis Res Ther2011;13:R56 PMCID:PMC3132048

[25] Patil Y,Amitay Y,Kumar S.Targeting of folate-conjugated liposomes with co-entrapped drugs to prostate cancer cells via prostate-specific membrane antigen (PSMA)..Nanomedicine2018;14:1407-16

[26] Liu H,Moy P.Constitutive and antibody-induced internalization of prostate-specific membrane antigen..Cancer Res1998;58:4055-60