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Choosing the right molar activity of an 123I-labeled irreversible inhibitor of transglutaminase 2 for quantitative expression profiling in tissues

Donat, C.; Laube, M.; Kopka, K.; Pietzsch, H.-J.; Pietzsch, J.; Löser, R.; Wodtke, R.



The Ca2+-dependent transamidase activity of transglutaminase 2 (TGase2) is tightly regulated in healthy cells but can be utilized by various cancer cells to support their survival and progression. Therefore, molecules targeting this enzyme are promising candidates for the functional characterization of TGase2 in tumors. Recently, we developed an 18F-labeled irreversible inhibitor and highlighted its potential as radiometric tool for the in vitro characterization of TGase2. Herein, we report on the kinetic characterization of a 123I-labeled Nε-acryloyllysine, [123I]1, and its use for quantifying the functional expression of TGase2 in tissues
[123I]1 was synthesized as recently presented [1]. The inhibitory potency of [123I]1 by means of its kinact/KI value was determined by a radio-TLC method using recombinant human TGase2. In vitro autoradiography was performed with fresh-frozen sections (12 µm) of several organs (heart, kidney, liver, spleen, and muscle), extracted from healthy NMRI nude mice. Binding experiments with [123I]1 were conducted at 0.7 MBq/mL in MOPS buffer at pH 7.4 containing 3 mM CaCl2 and 5 mM DTT. Non-specific binding was assessed in the presence of the TGase2 inhibitor Z006. Different molar activities (Am) were adjusted by the addition of compound 1.
[123I]1 was reliably obtained in high (radio)chemical purities of >99% and radiochemical yields of 79±6% (n=8). The Am was determined to be >6 TBq/µmol and the kinact/KI value to be 10,200 M-1s-1 (±1,000). Association (Figure 1) of n.c.a. [123I]1 at 37°C over 4 h to tissue sections furnished a high binding capacity and excellent ratios of total binding (TB) to non-specific binding (NSB). However, assessment of the quantitative TGase2 expression is limited as the inhibition rate at n.c.a. level is too low to achieve complete radioligand binding. Therefore, Am values of 70, 14, 7, and 1 GBq/µmol were adjusted to increase the association rates. A value of 7 GBq/µmol appeared to be optimal based on the extent of binding and the TB/NSB ratios. Higher Am values of 70 and 14 GBq/µmol still led to incomplete reaction and thus a lower apparent TGase2 concentration. In contrast, a Am value of 1 GBq/µmol resulted in extensive self-block, as indicated by an increased NSB (Figure 1). The highest TGase2 concentration has been observed in the heart and was lowest in muscle, with values of 1.3 and 0.2 pmol/mm3, respectively. Dissociation of [123I]1 under similar conditions proved the irreversible binding to TGase2 as only a minimal amount (<10%) of total bound radioligand dissociates over 4 h.
A detailed in vitro and ex vivo evaluation of the TGase2-inhibitor [123I]1 proved its applicability as radiometric tool for quantifying the functional expression of that enzyme. The observed low reaction rate of [123I]1 at high Am values was compensated by standard addition which might also have implications for the in vivo application of this compound.
The authors thank ROTOP Radiopharmacy for continuously providing [123I]iodide. Financial support by European Regional Development Fund (EFRE) for ML, HJP and RW is gratefully acknowledged.
[1] Laube et al, Nucl. Med. Biol., 2021, 96–97S, S79-S80,

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