A miniaturized mode-of-action profiling platform enables high throughput characterization of the molecular and cellular dynamics of EZH2 inhibition.

The market approval of Tazemetostat (TAZVERIK) for the treatment of follicular lymphoma and epithelioid sarcoma has established "enhancer of zeste homolog 2" (EZH2) as therapeutic target in oncology. Despite their structural similarities and common mode of inhibition, Tazemetostat and other EZH2 inhibitors display differentiated pharmacological profiles based on their target residence time. Here we established high throughput screening methods based on time-resolved fluorescence energy transfer, scintillation proximity and high content analysis microscopy to quantify the biochemical and cellular binding of a chemically diverse collection of EZH2 inhibitors. These assays allowed to further characterize the interplay between EZH2 allosteric modulation by methylated histone tails (H3K27me3) and inhibitor binding, and to evaluate the impact of EZH2's clinically relevant mutant Y641N on drug target residence times. While all compounds in this study exhibited slower off-rates, those with clinical candidate status display significantly slower target residence times in wild type EZH2 and disease-related mutants. These inhibitors interact in a more entropy-driven fashion and show the most persistent effects in cellular washout and antiproliferative efficacy experiments. Our work provides mechanistic insights for the largest cohort of EZH2 inhibitors reported to date, demonstrating that-among several other binding parameters-target residence time is the best predictor of cellular efficacy.

Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists.

PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders.

Discovery and Structure-Based Design of Potent Covalent PPARγ Inverse-Agonists BAY-4931 and BAY-0069.

The ligand-activated nuclear receptor peroxisome-proliferator-activated receptor-γ (PPARG or PPARγ) represents a potential target for a new generation of cancer therapeutics, especially in muscle-invasive luminal bladder cancer where PPARγ is a critical lineage driver. Here we disclose the discovery of a series of chloro-nitro-arene covalent inverse-agonists of PPARγ that exploit a benzoxazole core to improve interactions with corepressors NCOR1 and NCOR2. In vitro treatment of sensitive cell lines with these compounds results in the robust regulation of PPARγ target genes and antiproliferative effects. Despite their imperfect physicochemical properties, the compounds showed modest pharmacodynamic target regulation in vivo. Improvements to the in vitro potency and efficacy of BAY-4931 and BAY-0069 compared to those of previously described PPARγ inverse-agonists show that these compounds are novel tools for probing the in vitro biology of PPARγ inverse-agonism.

Rational Design and Synthesis of Selective PRMT4 Inhibitors: A New Chemotype for Development of Cancer Therapeutics*.

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates the arginine residues of histone H3 and nonhistone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and, potentially, therapeutics. Although several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Herein, we report the structure-based design of a new class of alanine-containing 3-arylindoles as potent and selective PRMT4 inhibitors, and describe key structure-activity relationships for this class of compounds.

Potent and Selective Human Prostaglandin F (FP) Receptor Antagonist (BAY-6672) for the Treatment of Idiopathic Pulmonary Fibrosis (IPF).

Idiopathic pulmonary fibrosis (IPF) is a rare and devastating chronic lung disease of unknown etiology. Despite the approved treatment options nintedanib and pirfenidone, the medical need for a safe and well-tolerated antifibrotic treatment of IPF remains high. The human prostaglandin F receptor (hFP-R) is widely expressed in the lung tissue and constitutes an attractive target for the treatment of fibrotic lung diseases. Herein, we present our research toward novel quinoline-based hFP-R antagonists, including synthesis and detailed structure-activity relationship (SAR). Starting from a high-throughput screening (HTS) hit of our corporate compound library, multiple parameter improvements-including increase of the relative oral bioavailability Frel from 3 to ≥100%-led to a highly potent and selective hFP-R antagonist with complete oral absorption from suspension. BAY-6672 (46) represents-to the best of our knowledge-the first reported FP-R antagonist to demonstrate in vivo efficacy in a preclinical animal model of lung fibrosis, thus paving the way for a new treatment option in IPF.

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.

Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal form of cancer with few therapeutic options. We found that levels of the lysine methyltransferase SMYD2 (SET and MYND domain 2) are elevated in PDAC and that genetic and pharmacological inhibition of SMYD2 restricts PDAC growth. We further identified the stress response kinase MAPKAPK3 (MK3) as a new physiologic substrate of SMYD2 in PDAC cells. Inhibition of MAPKAPK3 impedes PDAC growth, identifying a potential new kinase target in PDAC. Finally, we show that inhibition of SMYD2 cooperates with standard chemotherapy to treat PDAC cells and tumors. These findings uncover a pivotal role for SMYD2 in promoting pancreatic cancer.

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.

Studies toward the development of new silicon-containing building blocks for the direct (18)F-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.

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.

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.

18F-labeled bombesin analog for specific and effective targeting of prostate tumors expressing gastrin-releasing peptide receptors.

UNLABELLED: 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 (18)F-labeled bombesin analog, (18)F-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 ((19)F) standard of the bombesin analog for binding affinity and selectivity for GRPr. The stability of (18)F-BAY 86-4367 was determined in murine and human plasma. In vivo, the tumor-targeting potential and pharmacokinetic profile of the (18)F tracer were analyzed with biodistribution experiments and PET studies of prostate tumor-bearing mice. RESULTS: The nonradioactive ((19)F) 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), (18)F-BAY 86-4367 showed more specific and effective GRPr-based targeting in vivo than the benchmark radiotracers (18)F-fluoroethylcholine and (18)F-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 (18)F-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.

Direct one-step 18F-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.

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.