Timo Stellfeld

Synthesis, 18F-Labeling, and in Vitro and in Vivo Studies of Bombesin Peptides Modified with Silicon-Based Building Blocks

The gastrin-releasing peptide receptor (GRPr) is overexpressed on various human tumors. The goal of our study was the synthesis of new 18F-labeled bombesin analogues for the PET imaging of GRPr expression in prostate tumor using a silicon-based one-step n. c. a. radiolabeling method. The silicon-containing building blocks were efficiently coupled to the N-terminus of the peptides via solid-phase synthesis. Radiolabeling of the obtained peptide precursors proceeded smoothly under acidic conditions (34-85% conversion). Using the di-tert-butyl silyl building block as labeling moiety, products containing a hydrolytically stable 18F-label were obtained. In in vitro receptor binding experiments 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH 2 ( 4b, IC50 = 22.9 nM) displayed a 12-fold higher binding affinity than 2-(4-(di-tert-butylfluorosilyl)phenyl)acetyl-Arg-Ava-Gln-Trp-Ala-Val-Gly-His(3Me)-Sta-Leu-NH2 ( 3b, IC50 = 276.6 nM), and 4b was therefore chosen for further evaluation. In vitro and ex vivo metabolite studies of [18F]4b showed no significant degradation. In biodistribution experiments, tumor uptake of [18F]4b was low and unspecific, whereas the GRPr-rich pancreas revealed a high and specific accumulation of the radiotracer. This study demonstrates the applicability of our silicon-based one-step n. c. a. radiolabeling method for the synthesis of new 18F-labeled bombesin derivatives. This innovative approach represents a general, straightforward access to radiolabeled peptides as PET imaging probes.

Direct One-Step18F-Labeling of Peptides via Nucleophilic Aromatic Substitution

Methods for the radiolabeling molecules of interest with [18F]-fluoride need to be rapid, convenient, and efficient. Numerous [18F]-labeled prosthetic groups, e.g., N-succinimidyl 4 [18F]-fluorobenzoate ([18F]-SFB), 4-azidophenacyl-[18F]-fluoride ([18F]-APF), and 1-(3-(2-[18F]fluoropyridin-3-yloxy)propyl)pyrrole-2,5-dione ([18F]-FpyMe), for conjugating to biomolecules have been developed. As the synthesis of these prosthetic groups usually requires multistep procedures, there is still a need for direct methods for the nucleophilic [18F]-fluorination of biomolecules. We report here on the development of a procedure based on the trimethylammonium (TMA) leaving group attached to an aromatic ring and activated with different electron-withdrawing groups (EWGs). A series of model compounds containing different electron-withdrawing substituents, a trimethylammonium leaving group, and carboxylic functionality for subsequent coupling to peptides were designed and synthesized. The optimal model compound, 2-cyano-4-(methoxycarbonyl)-N,N,N-trimethylbenzenaminium trifluoromethanesulfonate, was converted to carboxylic acid and coupled to peptides. The results of the one-step [18F]-fluorination of tetrapeptides and bombesin peptides show that the direct 18F-labeling of peptides is feasible under mild conditions and in good radiochemical yields.

18F-Labeled Bombesin Analog for Specific and Effective Targeting of Prostate Tumors Expressing Gastrin-Releasing Peptide Receptors

Bombesin is a peptide exhibiting high affinity for the gastrin-releasing peptide receptor (GRPr), which is highly overexpressed on prostate cancer cells. In the present study, we developed an 18F-labeled bombesin analog, 18F-BAY 86-4367, which is currently being clinically tested for use in PET of prostate cancer. Methods: In vitro pharmacologic studies were performed to characterize the nonradioactive (19F) standard of the bombesin analog for binding affinity and selectivity for GRPr. The stability of 18F-BAY 86-4367 was determined in murine and human plasma. In vivo, the tumor-targeting potential and pharmacokinetic profile of the 18F tracer were analyzed with biodistribution experiments and PET studies of prostate tumor–bearing mice. Results: The nonradioactive (19F) standard of the bombesin analog showed subnanomolar and GRPr-selective binding affinity. The stability of the tracer in murine and human plasma was found to be high. In 2 prostate cancer xenograft models (PC-3 and LNCaP), 18F-BAY 86-4367 showed more specific and effective GRPr-based targeting in vivo than the benchmark radiotracers 18F-fluoroethylcholine and 18F-FDG. In addition, rapid tumor targeting and fast renal excretion (∼70%) and hepatobiliary excretion (∼10%) were identified in both xenograft models. Furthermore, PET studies provided clear and specific visualization of PC-3 tumors in mice. Conclusion: Favorable preclinical data showing specific and effective tumor targeting by 18F-BAY 86-4367 suggest that a clinical trial be undertaken to test its diagnostic utility in PET for prostate carcinoma patients.

In vitro and in vivo characterization of novel 18F-labeled bombesin analogues for targeting GRPR-positive tumors.

The gastrin-releasing peptide receptor (GRPR) is overexpressed on a number of human tumors and has been targeted with radiolabeled bombesin analogues for the diagnosis and therapy of these cancers. Seven bombesin analogues containing various linkers and peptide sequences were designed, synthesized, radiolabeled with (18)F, and characterized in vitro and in vivo as potential PET imaging agents. Binding studies displayed nanomolar binding affinities toward human GRPR for all synthesized bombesin analogues. Two high-affinity peptide candidates 6b (K(i) = 0.7 nM) and 7b (K(i) = 0.1 nM) were chosen for further in vivo evaluation. Both tracers revealed specific uptake in GRPR-expressing PC-3 tumors and the pancreas. Compared to [(18)F]6b, compound [(18)F]7b was characterized by superior tumor uptake, higher specificity of tracer uptake, and more favorable tumor-to-nontarget ratios. In vivo PET imaging allowed for the visualization of PC-3 tumor in nude mice suggesting that [(18)F]7b is a promising PET tracer candidate for the diagnosis of GRPR-positive tumors in humans.

Studies toward the Development of New Silicon-Containing Building Blocks for the Direct 18F-Labeling of Peptides

Silicon-containing prosthetic groups have been conjugated to peptides to allow for a single-step labeling with (18)F radioisotope. The fairly lipophilic di-tert-butylphenylsilane building block contributes unfavorably to the pharmacokinetic profile of bombesin conjugates. In this article, theoretical and experimental studies toward the development of more hydrophilic silicon-based building blocks are presented. Density functional theory calculations were used to predict the hydrolytic stability of di-tert-butylfluorosilanes 2-23 with the aim to improve the in vivo properties of (18)F-labeled silicon-containing biomolecules. As a further step toward improving the pharmacokinetic profile, hydrophilic linkers were introduced between the lipophilic di-tert-butylphenylsilane building block and the bombesin congeners. Increased tumor uptake was shown with two of these peptides in xenograft-bearing mice using positron emission tomography and biodistribution studies. The introduction of a hydrophilic linker is thus a viable approach to improve the tumor uptake of (18)F-labeled silicon-bombesin conjugates.

Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability

INTRODUCTION Atherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques. METHODS Human carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging. RESULTS Gene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques. CONCLUSIONS MMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively.

Design, synthesis, and initial evaluation of a high affinity positron emission tomography probe for imaging matrix metalloproteinases 2 and 9.

The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions. Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor for imaging active MMP2/MMP9 in vivo. We synthesized a series of N-sulfonylamino acid derivatives with low to high nanomolar inhibitory potencies. (R)-2-(4-(4-Fluorobenzamido)phenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (7) exhibited the best in vitro binding properties: MMP2 IC50 = 1.8 nM, MMP9 IC50 = 7.2 nM. Radiolabeling of 7 with no carrier added (18)F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly depended on the iodonium counteranion (ClO4(-) > Br(-) > TFA(-) > tosylate). (18)F-7 was obtained in up to 20% radiochemical yield (decay corrected), high radiochemical purity, and >90 GBq/μmol specific radioactivity. The radiolabeled compound showed excellent stability in vitro and in mice in vivo.

Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2

Protein lysine methyltransferases have recently emerged as a new target class for the development of inhibitors that modulate gene transcription or signaling pathways. SET and MYND domain containing protein 2 (SMYD2) is a catalytic SET domain containing methyltransferase reported to monomethylate lysine residues on histone and nonhistone proteins. Although several studies have uncovered an important role of SMYD2 in promoting cancer by protein methylation, the biology of SMYD2 is far from being fully understood. Utilization of highly potent and selective chemical probes for target validation has emerged as a concept which circumvents possible limitations of knockdown experiments and, in particular, could result in an improved exploration of drug targets with a complex underlying biology. Here, we report the development of a potent, selective, and cell-active, substrate-competitive inhibitor of SMYD2, which is the first reported inhibitor suitable for in vivo target validation studies in rodents.

Single-step radiofluorination of peptides using continuous flow microreactor

18F radiolabelling of peptides bearing two different prosthetic groups was successfully conducted in a continuous flow microfluidic device for the first time. Radiochemical yields were dependent on precursor concentration, reaction temperature and flow rate. The choice of leaving group had a dramatic influence on the reaction outcome. Rapid reaction optimization was possible.

Abstract 5239: Probing the cancer epigenome: empowering target validation by open innovation

Low reproducibility of published target validation studies as well as the frequent failure of genetic knock-down effects to phenocopy those of small molecule inhibitors have been recognized as road blocks for cancer drug discovery. Academic and industrial institutions have started to address these issues by providing access to high quality small molecular probes for novel targets of interest. Here we discuss probe discovery challenges and quality criteria based on the generation of three novel inhibitors for epigenetic targets. ATAD2 (ATPase family AAA-domain containing protein 2) is an epigenetic regulator that binds to chromatin through its bromodomain (BD). ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors such as ERα and Myc. A more thorough validation of ATAD2 as a therapeutic target has been hampered by the lack of appropriate ATAD2 inhibitors. Here we disclose a structurally unprecedented series of ATAD2 BD inhibitors identified from a DNA-encoded library screen. Optimization delivered BAY-850, a highly potent and exceptionally selective ATAD2 BD inhibitor, which fully recapitulates effects seen by genetic mutagenesis studies in a cellular assay. The three BD and PHD-finger (BRPF) family members are found in histone acetyltransferase complexes. Whereas bromodomain inhibitors with dual activity against BRPF1 and 2 have been described before, we now disclose BAY-299, the first nanomolar inhibitor of the BRPF2 BD with high selectivity against its paralogs. Isoform selectivity was confirmed in cellular protein-protein interaction assays and rationalized based on X-Ray structures. BAY-598, a highly selective, cellularly active and orally bioavailable inhibitor of the protein lysine methyl transferase SMYD2, had been disclosed previously (Stresemann et al., AACR 2015). Development of BAY-598 allowed the identification of new methylation targets of SMYD2 as well as a proposed role of SMYD2 in pancreatic cancer. These results support further development of small molecule inhibitors as research tools to probe the functional role of novel epigenetic targets and underscore the power of open innovation for advancing our understanding of cancer target biology. Citation Format: Ingo V. Hartung, Cheryl Arrowsmith, Volker Badock, Naomi Barak, Markus Berger, Peter J. Brown, Clara D. Christ, Erik Eggert, Ursula Egner, Oleg Fedorov, Amaury E. Fernandez-Montalvan, Matyas Gorjanacz, Andrea Haegebarth, Bernard Haendler, Roman C. Hillig, Simon H. Holton, Kilian V. Huber, Seong J. Koo, Antonius ter Laak, Susanne Mueller, Anke Mueller-Fahrnow, Cora Scholten, Stephan Siegel, Timo Stellfeld, Detlef Stoeckigt, Carlo Stresemann, Masoud Vedadi, Joerg Weiske, Hilmar Weinmann. Probing the cancer epigenome: empowering target validation by open innovation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5239. doi:10.1158/1538-7445.AM2017-5239