Characterization of Structurally Diverse ¹⁸F‑Labeled d‑TCO Derivatives as a PET Probe for Bioorthogonal Pretargeted Imaging

Background: The pretargeted imaging strategy using inverse electron demand Diels–Alder (IEDDA) cycloaddition between a trans-cyclooctene (TCO) and tetrazine (Tz) has emerged and rapidly grown as a promising concept to improve radionuclide imaging and therapy in oncology. This strategy has mostly relied on the use of radiolabeled Tz together with TCO-modified targeting vectors leading to a rapid growth of the number of available radiolabeled tetrazines, while only a few radiolabeled TCOs are currently reported. Here, we aim to develop novel and structurally diverse 18F-labeled cis-dioxolane-fused TCO (d-TCO) derivatives to further expand the bioorthogonal toolbox for in vivo ligation and evaluate their potential for positron emission tomography (PET) pretargeted imaging. Results: A small series of d-TCO derivatives were synthesized and tested for their reactivity against tetrazines, with all compounds showing fast reaction kinetics with tetrazines. A fluorescence-based pretargeted blocking study was developed to investigate the in vivo ligation of these compounds without labor-intensive prior radiochemical development. Two compounds showed excellent in vivo ligation results with blocking efficiencies of 95 and 97%. Two novel 18F-labeled d-TCO radiotracers were developed, from which [18F]MICA-214 showed good in vitro stability, favorable pharmacokinetics, and moderate in vivo stability. Micro-PET pretargeted imaging with [18F]MICA-214 in mice bearing LS174T tumors treated with tetrazine-modified CC49 monoclonal antibody (mAb) (CC49-Tz) showed significantly higher uptake in tumor tissue in the pretargeted group (CC49-Tz 2.16 ± 0.08% ID/mL) when compared to the control group with nonmodified mAb (CC49 1.34 ± 0.07% ID/mL). Conclusions: A diverse series of fast-reacting fluorinated d-TCOs were synthesized. A pretargeted blocking approach in tumor-bearing mice allowed the choice of a lead compound with fast reaction kinetics with Tz. A novel 18F-labeled d-TCO tracer was developed and used in a pretargeted PET imaging approach, allowing specific tumor visualization in a mouse model of colorectal cancer. Although further optimization of the radiotracer is needed to enhance the tumor-to-background ratios for pretargeted imaging, we anticipate that the 18F-labeled d-TCO will find use in studies where increased hydrophilicity and fast bioconjugation are required

Effect of RNAi on <i>T</i>. <i>brucei</i> cell growth <i>in vivo</i>.

<p>ICR mice were inoculated with RNAi cell lines targeting the two OPB-like genes (a), two POP-like genes (b), dipeptidyl peptidase-8 (c), or the type-I signal peptidase (d). Two mice in each experiment were left untreated (filled symbols) and two were given doxycycline (open symbols) to induce RNAi. The arrow indicates doxycycline administration. Parasitaemia in infected mice was counted at the times indicated.</p

Rescue of <i>SPP1</i> RNAi growth defect by expression of recoded SPPI.

<p>(a) Expression of recoded HA-tagged SPP1 detected by Western blot. Cell lysates from RNAi cell line (-), the RNAi cell line expressing SPP1 from the recoded gene (<i>SPP1</i>^<i>R</i>), or the RNAi cell line expressing inactive SPP1 from a recodedgene (<i>SPP1</i>^{<i>R</i>,<i>I</i>}) were probed with anti-HA antibody (Roche). Detection of EF-1α was used as a loading control. (b, c and e) Parasite growth was measured in cell lines after inducing <i>SPP1</i> RNAi with tetracycline (open squares) or without treatment (closed squares) in the <i>SPP1</i> RNAi cell line expressing <i>SPP1</i> from: the recoded gene <i>SPP1</i>^<i>R</i> (b), the parental <i>SPP1</i> RNAi cell line (c), or the RNAi cell line expressing inactive SPP1 from a recoded gene, <i>SPP1</i>^{<i>R</i>,<i>I</i>} (e). (d) Quantitative PCR showing relative quantification (RQ) of endogenous <i>SPP1</i> transcript in the RNAi cell line expressing active SPP1 from the <i>SPP1</i>^<i>R</i> gene either without induction (control, black bars) or after induction by tetracycline (+ Tet, white bars).</p

Circulating Stromal Cell-Derived Factor 1α Levels in Heart Failure: A Matter of Proper Sampling

<div><p>Background</p><p>The chemokine Stromal cell-derived factor 1α (SDF1α, CXCL12) is currently under investigation as a biomarker for various cardiac diseases. The correct interpretation of SDF1α levels is complicated by the occurrence of truncated forms that possess an altered biological activity.</p><p>Methodology</p><p>We studied the immunoreactivities of SDF1α forms and evaluated the effect of adding a DPP4 inhibitor in sampling tubes on measured SDF1α levels. Using optimized sampling, we measured DPP4 activity and SDF1α levels in patients with varying degrees of heart failure.</p><p>Results</p><p>The immunoreactivities of SDF1α and its degradation products were determined with three immunoassays. A one hour incubation of SDF1α with DPP4 at 37°C resulted in 2/3 loss of immunoreactivity in each of the assays. Incubation with serum gave a similar result. Using appropriate sampling, SDF1α levels were found to be significantly higher in those heart failure patients with a severe loss of left ventricular function. DPP4 activity in serum was not altered in the heart failure population. However, the DPP4 activity was found to be significantly decreased in patients with high SDF1α levels</p><p>Conclusions</p><p>We propose that all samples for SDF1α analysis should be collected in the presence of at least a DPP4 inhibitor. In doing so, we found higher SDF1α levels in subgroups of patients with heart failure. Our work supports the need for further research on the clinical relevance of SDF1α levels in cardiac disease.</p></div

Recombinant TbDPP8 activity and inhibition.

<p>(a) Rate of cleavage by TbDPP8 of H-Gly-Pro-AMC (grey bars) and Z-Gly-Pro-AMC (white bars). (b) Structures of inhibitors used against TbDPP8. (c) Inhibitory activity of compounds against <i>T</i>. <i>brucei</i> DPP8, human DPP IV, human DPP8, and bloodstream form <i>T</i>. <i>brucei</i> 427.</p

SDF1α concentrations in patient samples collected in tubes with DPP4-I.

<p>(A) No difference was found between patients with a different type of LV dysfunction (none 1096 ± 47 pg/ml; HfpEF 1043 ± 55 pg/ml; decompensated HfrEF 1201 ± 145 pg/ml; recompensated HfrEF 1109 ± 51 pg/ml). (B) For the different severities of LV dysfunction a significant difference was found in patients with a severe loss of LV function (normal 1076 ± 36 pg/ml; slight loss 1002 ± 62 pg/ml; loss 1090 ± 55 pg/ml; severe loss 1705 ± 447 pg/ml; *p < 0.05).</p

The average SDF1α immunoreactivity of healthy plasma with the RnD SDF1α duoset when immediately analyzed (no incubation, n = 13, range [749–1776 pg/ml]) or after an incubation of 1 h at 37°C (n = 7, range [832–1776 pg/ml]).

<p>Blood was collected in tubes with or without DPP4-I. A significantly lower immunoreactivity was found in regular tubes versus the DPP4-I containing tubes (no incubation: 67.6 ± 3.5%; incubation: 27.8 ± 2.8%; *p < 0.05; results ± SEM).</p

The SDF1α immunoreactivity measured by commercially available kits.

<p>(A) SDF1α (500pg/ml) incubated in PBS for 1 h at 37°C was set at 100% immunoreactivity. Incubation in the presence of DPP4 (25 U/l) resulted in a significantly lower immunoreactivity compared to intact SDF1α (RnD lot 1: 40.5 ± 2.8%; RnD lot 2: 32.7 ± 1.5%; Raybiotech: 27.1 ± 2.4%; Peprotech 32.5 ± 1.2%; *p < 0.05; results ± SEM; n = 5). (B) The immunoreactivity of pure SDF1α (500pg/ml) spiked into serum and incubated for 1 h at 25°C or 37°C was measured with different commercial kits. As the 100% reference, SDF1α spiked into inhibited serum (DPP4-I and Roche protease inhibitor cocktail) was chosen. <i>Ex vivo</i> degradation in serum significantly decreased the immunoreactivity of SDF1α (25°C: RnD lot 1: 54.3 ± 4.1%; RnD lot 2: 31.4 ± 6.4%; Raybiotech: 48.3 ± 5.1%; Peprotech: 62.4 ± 2.3% and 37°C: RnD lot 1: 46.6 ± 0.4%; RnD lot 2: 23.5 ± 1.7%; Raybiotech: 25.9 ± 2.7%; Peprotech 47.8 ± 1.9%; *p < 0.05; results ± SEM; n = 5).</p

Discovery and SAR of Novel and Selective Inhibitors of Urokinase Plasminogen Activator (uPA) with an Imidazo[1,2‑<i>a</i>]pyridine Scaffold

Urokinase plasminogen activator (uPA) is a biomarker and therapeutic target for several cancer types. Its inhibition is regarded as a promising, noncytotoxic approach in cancer therapy by blocking growth and/or metastasis of solid tumors. Earlier, we reported the modified substrate activity screening (MSAS) approach and applied it for the identification of fragments with affinity for uPA’s S1 pocket. Here, these fragments are transformed into a novel class of uPA inhibitors with an imidazo­[1,2-<i>a</i>]­pyridine scaffold. The SAR for uPA inhibition around this scaffold is explored, and the best compounds in the series have nanomolar uPA affinity and selectivity with respect to the related trypsin-like serine proteases (thrombin, tPA, FXa, plasmin, plasma kallikrein, trypsin, FVIIa). Finally, the approach followed for translating fragments into small molecules with a decorated scaffold architecture is conceptually straightforward and can be expected to be broadly applicable in fragment-based drug design

Comparison of cardiovascular parameters between SDF1α tertiles (Low = First Tertile, Medium = Second Tertile and High = Third Tertile).

<p>No significant differences were observed in the Heart Rate (A), Ejection Fraction (B), End-Diastolic Volume Index (C), End-Systolic Volume Index (D) and End-Diastolic Pressure (E). The first and third tertile had significantly different DPP4 activities (F). *p < 0.05.</p