OBJECTIVES:Gadoquatrane is a tetrameric extracellular gadolinium-based contrast agent (GBCA) with a T1 relaxivity of 11.8 L/(mmol Gd*s) at 1.41 T in human plasma, which is currently in Phase 3 clinical development. In the current study, the stability of gadoquatrane was assessed in comparison with approved macrocyclic GBCAs in several in vitro and in vivo assays. MATERIALS AND METHODS:Kinetic inertness, a key determinant of complex stability, was assessed for gadoquatrane, gadoteridol, gadobutrol, gadoterate, and gadopiclenol at equimolar Gd concentrations by measuring the time course of dissociation at pH 1.2 and 37°C using a complexometric assay. Kinetic inertness was also determined in human plasma at pH 7.4 and 37°C using ion exchange chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS). The binding of gadoquatrane, gadobutrol, and gadopiclenol to synthetic hydroxyapatite, the inorganic component of bone, was investigated in vitro and the Gd content in histological bone slices 1 week after a single injection of these 3 selected GBCAs in rats (0.6 mmol Gd/kg, equivalent to a human dose of 0.1 mmol Gd/kg) was analyzed using laser ablation coupled to ICP-MS. RESULTS:The dissociation half-lives at pH 1.2 (mean, 95% confidence interval in parenthesis) were 28.6 (28.1, 29.1) days for gadoquatrane, 14.2 (13.8, 14.6) days for gadopiclenol, 2.7 (2.6, 2.8) days for gadoterate, 14.1 (13.1, 15.1) hours for gadobutrol, and 2.2 (2.0, 2.4) hours for gadoteridol. After 15 days of incubation in human plasma at pH 7.4, no released Gd 3+ ions above the lower limit of quantification (LLOQ, 0.01% of total Gd) were observed for gadoquatrane and gadoterate, while for gadobutrol, gadopiclenol and gadoteridol the concentrations of released Gd 3+ ions reached 0.12 (0.11, 0.13)%, 0.20 (0.19, 0.21)%, and 0.20 (0.20, 0.21)%, respectively. The rates of dissociation for gadopiclenol and gadoteridol were similar. For gadoquatrane, gadobutrol, and gadopiclenol, the binding to hydroxyapatite was examined. It was very low (< 0.02% of total Gd) for all 3 GBCAs. The Gd concentration 1 week after the injection of 0.6 mmol Gd/kg of the 3 GBCAs in bone marrow were in a comparable range of 2.3-3.0 nmol Gd/g tissue. In the epiphysis the Gd concentrations for gadoquatrane (1.2 (1.0, 1.4)) and gadobutrol (1.2 (1.0, 1.4)) were lower compared to gadopiclenol (2.2 (1.9, 2.6)). In the diaphysis the respective values were 0.5 (0.4, 0.7) nmol Gd/g, 1.0 (0.8, 1.3) nmol Gd/g, and 2.7 (2.1, 3.5) nmol Gd/g. Elemental imaging of the femur obtained in this in vivo study revealed no Gd containing structures in the mineralized bone for gadoquatrane (< 1 nmol Gd/g). For gadopiclenol, a visible thin layer of Gd concentration (interquartile range [IQR]: 17-38 nmol Gd/g, maximum value ~80 nmol Gd/g) in the subcortical layer of the bone was observed. The same layer contained a lower Gd concentration for gadobutrol (IQR: 1.2-3.5 nmol Gd/g, maximum value ~12 nmol Gd/g). CONCLUSIONS:The investigations demonstrated that gadoquatrane has the highest kinetic inertness towards release of Gd 3+ ions in strong acidic environment compared to all approved macrocyclic GBCAs. In human plasma at pH 7.4 no release of Gd 3+ ions was observed from gadoquatrane, similar to gadoterate and kinetic inertness was higher than for gadoteridol, gadobutrol and gadopiclenol. The high stability of gadoquatrane was supported by very low Gd concentrations in mineralized bone in an in vivo study in rats.

Objectives The aim of this report was to characterize the key physicochemical, pharmacokinetic (PK), and magnetic resonance imaging (MRI) properties of gadoquatrane (BAY 1747846), a newly designed tetrameric, macrocyclic, extracellular gadolinium-based contrast agent (GBCA) with high relaxivity and stability. Materials and Methods The r1-relaxivities of the tetrameric gadoquatrane at 1.41 and 3.0 T were determined in human plasma and the nuclear magnetic relaxation dispersion profiles in water and plasma. The complex stability was analyzed in human serum over 21 days at pH 7.4 at 37 degrees C and was compared with the linear GBCA gadodiamide and the macrocyclic GBCA (mGBCA) gadobutrol. In addition, zinc transmetallation assay was performed to investigate the kinetic inertness. Protein binding and the blood-to-plasma ratio were determined in vitro using rat and human plasma. The PK profile was evaluated in rats (up to 7 days postinjection). Magnetic resonance imaging properties were investigated using a glioblastoma (GS9L) rat model. Results The new chemical entity gadoquatrane is a macrocyclic tetrameric Gd complex with one inner sphere water molecule per Gd (q = 1). Gadoquatrane showed high solubility in buffer (1.43 mol Gd/L, 10 mM Tris-HCl, pH 7.4), high hydrophilicity (logP -4.32 in 1-butanol/water), and negligible protein binding. The r1-relaxivity of gadoquatrane in human plasma per Gd of 11.8 mM(-1)center dot s(-1) (corresponding to 47.2 mM(-1)center dot s(-1) per molecule at 1.41 T at 37 degrees C, pH 7.4) was more than 2-fold (8-fold per molecule) higher compared with established mGBCAs. Nuclear magnetic relaxation dispersion profiles confirmed the more than 2-fold higher r1-relaxivity in human plasma for the clinically relevant magnetic field strengths from 0.47 to 3.0 T. The complex stability of gadoquatrane at physiological conditions was very high. The observed Gd release after 21 days at 37 degrees C in human serum was below the lower limit of quantification. Gadoquatrane showed no Gd3+ release in the presence of zinc in the transmetallation assay. The PK profile (plasma elimination, biodistribution, recovery) was comparable to that of gadobutrol. In MRI, the quantitative evaluation of the tumor-to-brain contrast in the rat glioblastoma model showed significantly improved contrast enhancement using gadoquatrane compared with gadobutrol at the same Gd dose administered (0.1 mmol Gd/kg body weight). In comparison to gadoterate meglumine, similar contrast enhancement was reached with gadoquatrane with 75% less Gd dose. In terms of the molecule dose, this was reduced by 90% when compared with gadoterate meglumine. Because of its tetrameric structure and hence lower number of molecules per volume, all prepared formulations of gadoquatrane were iso-osmolar to blood. Conclusions The tetrameric gadoquatrane is a novel, highly effective mGBCA for use in MRI. Gadoquatrane provides favorable physicochemical properties (high relaxivity and stability, negligible protein binding) while showing essentially the same PK profile (fast extracellular distribution, fast elimination via the kidneys in an unchanged form) to established mGBCAs on the market. Overall, gadoquatrane is an excellent candidate for further clinical development.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody-drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure-activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate─to our knowledge for the first time─the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.

Objectives The aim of this report was to characterize the key physicochemical, pharmacokinetic (PK), and magnetic resonance imaging (MRI) properties of gadoquatrane (BAY 1747846), a newly designed tetrameric, macrocyclic, extracellular gadolinium-based contrast agent (GBCA) with high relaxivity and stability. Materials and Methods The r1-relaxivities of the tetrameric gadoquatrane at 1.41 and 3.0 T were determined in human plasma and the nuclear magnetic relaxation dispersion profiles in water and plasma. The complex stability was analyzed in human serum over 21 days at pH 7.4 at 37°C and was compared with the linear GBCA gadodiamide and the macrocyclic GBCA (mGBCA) gadobutrol. In addition, zinc transmetallation assay was performed to investigate the kinetic inertness. Protein binding and the blood-to-plasma ratio were determined in vitro using rat and human plasma. The PK profile was evaluated in rats (up to 7 days postinjection). Magnetic resonance imaging properties were investigated using a glioblastoma (GS9L) rat model. Results The new chemical entity gadoquatrane is a macrocyclic tetrameric Gd complex with one inner sphere water molecule per Gd ( q = 1). Gadoquatrane showed high solubility in buffer (1.43 mol Gd/L, 10 mM Tris-HCl, pH 7.4), high hydrophilicity (logP −4.32 in 1-butanol/water), and negligible protein binding. The r1-relaxivity of gadoquatrane in human plasma per Gd of 11.8 mM −1 ·s −1 (corresponding to 47.2 mM −1 ·s −1 per molecule at 1.41 T at 37°C, pH 7.4) was more than 2-fold (8-fold per molecule) higher compared with established mGBCAs. Nuclear magnetic relaxation dispersion profiles confirmed the more than 2-fold higher r1-relaxivity in human plasma for the clinically relevant magnetic field strengths from 0.47 to 3.0 T. The complex stability of gadoquatrane at physiological conditions was very high. The observed Gd release after 21 days at 37°C in human serum was below the lower limit of quantification. Gadoquatrane showed no Gd 3+ release in the presence of zinc in the transmetallation assay. The PK profile (plasma elimination, biodistribution, recovery) was comparable to that of gadobutrol. In MRI, the quantitative evaluation of the tumor-to-brain contrast in the rat glioblastoma model showed significantly improved contrast enhancement using gadoquatrane compared with gadobutrol at the same Gd dose administered (0.1 mmol Gd/kg body weight). In comparison to gadoterate meglumine, similar contrast enhancement was reached with gadoquatrane with 75% less Gd dose. In terms of the molecule dose, this was reduced by 90% when compared with gadoterate meglumine. Because of its tetrameric structure and hence lower number of molecules per volume, all prepared formulations of gadoquatrane were iso-osmolar to blood. Conclusions The tetrameric gadoquatrane is a novel, highly effective mGBCA for use in MRI. Gadoquatrane provides favorable physicochemical properties (high relaxivity and stability, negligible protein binding) while showing essentially the same PK profile (fast extracellular distribution, fast elimination via the kidneys in an unchanged form) to established mGBCAs on the market. Overall, gadoquatrane is an excellent candidate for further clinical development.

Eukaryotes have evolved two major pathways to repair potentially lethal DNA double-strand breaks. Homologous recombination represents a precise, DNA-template-based mechanism available during the S and G2 cell cycle phase, whereas non-homologous end joining, which requires DNA-dependent protein kinase (DNA-PK), allows for fast, cell cycle-independent but less accurate DNA repair. Here, we report the discovery of BAY-8400, a novel selective inhibitor of DNA-PK. Starting from a triazoloquinoxaline, which had been identified as a hit from a screen for ataxia telangiectasia and Rad3-related protein (ATR) inhibitors with inhibitory activity against ATR, ATM, and DNA-PK, lead optimization efforts focusing on potency and selectivity led to the discovery of BAY-8400. In in vitro studies, BAY-8400 showed synergistic activity of DNA-PK inhibition with DNA damage-inducing targeted alpha therapy. Combination of PSMA-targeted thorium-227 conjugate BAY 2315497 treatment of human prostate tumor-bearing mice with BAY-8400 oral treatment increased antitumor efficacy, as compared to PSMA-targeted thorium-227 conjugate monotherapy.

Introduction/ Purpose of study Targeting M2 macrophages as an immunosuppressive cell population within the tumor-microenvironment has become an important effort within preclinical research to achieve and enhance anti-tumor efficacy. In contrast to reprogramming approaches, we focused on effective and specific depletion of M2 macrophages (Mphs) by a new antibody-drug-conjugate (ADC) comprising a CD206-binding antibody conjugated to a nicotinamide phosphoribosyl transferase (NAMPT) toxophore which was proved to be very potent in cell killing. Description of procedures Human (hu) and mouse (ms) cross-reactive CD206 antibodies were identified by phage display using recombinant hu/ms CD206-extracellular domains. Selected clones showing strong CD206 binding by ELISA were reformatted into huIgG1 format and cellular targeting confirmed by FACS binding and internalization using CD206-transfected HEK cells. Antibodies were conjugated to a NAMPT toxophore by Cys-conjugation using two different non-cleavable linkers. Murine peritoneal Mphs collected after thioglycolate stimulation were polarized to M1 (LPS + IFNγ) or M2 (IL4 + IL13 + PGE2) within 24h and characterized for cytokines and typical markers via FACS (CD206, CD163, CD80/MHCII) and qPCR (iNOS, TGFβ, ARG1). After 96h treatment with either CD206- or isotype-NAMPT-ADCs, viability of Mphs was determined. In vivo pharmacokinetics (PK) of unconjugated antibodies were analyzed in plasma of female BALB/c mice for up to 72h. Summary of data Of the resulting 57 hu/ms cross-reactive CD206-binding antibodies, TPP-17829 and TPP-17836 were identified with single-digit nanomolar binding affinity and strong internalization in CD206-transfected cells as a prerequisite for an ADC approach. Conjugation of the antibodies to NAMPT toxophores yielded drug-antibody-ratios of 4-7 as determined by SEC-UV without significant aggregation. These two CD206-NAMPT-ADCs successfully depleted polarized M2-Mphs characterized by high CD206 expression, whereas isotype-NAMPT-ADC controls had no effect. While the unconjugated NAMPT toxophore was potent in depleting primary low-proliferating M1/M2 Mphs, by comparison, the anti-proliferative toxophore KSP (kinesin-spindle-protein inhibitor) was completely ineffective. PK analysis of TPP-17829 and TPP-17836, however, revealed a rapid plasma clearance within the first hours, most likely target-mediated via the liver, rendering them unacceptable for in vivo efficacy testing as NAMPT-ADCs. Conclusions CD206-NAMPT-ADCs demonstrated proof of concept for specific depletion of M2-Mphs in vitro. However, specific in vivo depletion of M2 Mphs from tumors will be challenging with respect to fast plasma clearance observed for anti-CD206 antibodies. Citation Format: Sandra Berndt, Katharina Filarsky, Patrick Smith, Niels Boehnke, Markus Berger, Fionnuala McAleese Eser, Hans-Georg Lerchen, Phillip Ellinger, Mathias Gehrmann, Jim Wu, Dominic Hildebrand, Bertolt Kreft, Uwe Gritzan. Effective depletion of M2 macrophages by CD206-NAMPT-ADCs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 677.

Abstract Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a differentiated mode-of-action for tumor-targeting antibody-drug conjugates (ADCs) independent from cell proliferation. This opens up the possibility to target slowly growing tumors as well as resting antigen-positive tumor cells in addition to highly proliferative tumors. We developed a novel structural class of NAMPT inhibitors as ADC payloads to complement the currently available effector chemistries. An SAR-driven approach supported by available structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffold and linker led to highly potent effector chemistries which were conjugated to anti-C4.4a (LYPD3) or anti-B7H3 (CD276) antibodies and tested on antigen-positive and -negative cancer cell lines derived from solid and hematological tumor indications. Furthermore, tuning of the hydrophilicity of the linker and the conjugation method ensured low aggregation of the NAMPT inhibitor ADCs. Pharmacokinetic studies were performed in human plasma to assess the stability of the linkage of the NAMPT inhibitor payload to the antibody. Moreover, permeability studies of the payload metabolites helped to evaluate potential by-stander effects of the ADCs. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile versus the corresponding isotype control ADCs. In depth in vitro and in vivo studies on the internalization and the metabolism allowed analysis of the intracellular fate of the payload metabolites and revealed the formation of the phosphoribosylated catabolite adducts in C4.4a expressing A549-cells. Taken together, we hereby present the development of a new NAMPT inhibitor-based payload class applicable for conjugation to diverse antibodies with a good technical profile and high potency and selectivity in antigen-positive cancer models. Citation Format: Niels Böhnke, Markus Berger, Nils Griebenow, Anja Giese, Judith Günther, Anette Sommer, Stefanie Hammer, Sandra Berndt, Antje M. Wengner, Rudolf Beier, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Antje Rottmann, Michael Erkelenz, Naomi Barak, Ulf Bömer, Dominik Mumber, Bertolt Kreft, Lars Linden, Carl Friedrich Nising, Hilmar Weinmann. Identification and optimization of a novel NAMPT inhibitor-based ADC payload class for cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2907.

Abstract Nicotinamide phosphoribosyl-transferase (NAMPT) is the rate-limiting enzyme in the salvage pathway, generating nicotinamide adenine dinucleotide (NAD) from nicotinamide (NAM). Inhibition of intracellular NAMPT activity represents a differentiated mode-of-action for tumor-targeting antibody-drug conjugates (ADCs) as it is not dependent on cell proliferation. Thus, NAMPT inhibitor-based ADCs have the potential to target both proliferating and resting tumor cells. We therefore developed a novel structural class of NAMPT inhibitors (NAMPTi) as a potent ADC payload class and characterized NAMPTi and NAMPTi-ADCs in vitro and in vivo in preclinical tumor models. We profiled the small molecule NAMPTi BAY-346 in comparison to the kinesin spindle protein inhibitor (KSPi) BAY-331 on a panel of 350 cancer cell lines from various tumor indications showing a differential sensitivity profile of BAY-346 vs BAY-331, with cell lines characterized by low NAMPT mRNA levels as being very sensitive to BAY-346. NAMPTi BAY-346, but not KSPi BAY-331, reduced the viability of quiescent HaCat cells as well as of serum starved non-proliferating NCI-N87 cells. In vitro treatment with NAMPTi BAY-346, which bears a pyridine warhead that can be phosphoribosylated by NAMPT, resulted in IC50 values in the nanomolar to subnanomolar range (2.8 nM to 0.01 nM) in cell lines derived from solid and hematologic tumor indications (e.g., THP-1, MV-4-11, U-251, NCI-H292, MDA-MB-453, LoVo, KPL4, HT1197 and BxPC3). BAY-346 was 100-fold more potent than the small molecule NAMPTi BAY-248, which cannot be phosphoribosylated. A BAY-346 derived NAMPTi was conjugated as a payload to a series of antibodies targeting different tumor-associated antigens: C4.4a (LYPD3), HER2, B7H3 (CD276), and TWEAKR (Fn14/ TNFRSF10A). The resulting NAMPTi-ADCs were tested in proliferation and cellular mechanistic in vitro assays. NAMPTi-ADCs depleted NAD+ in tumor cells and showed potent growth inhibitory activity with IC50 values in the subnanomolar- to nanomolar range in a target-dependent manner. A C4.4a-NAMPTi-ADC and a HER2-NAMPTi-ADC were tested In the C4.4a- and HER2-expressing MDA-MB-453 cell line derived subcutaneous breast cancer model xenografted on NOD/SCID mice. Both NAMPTi-ADCs showed highly potent anti-tumor Efficacy: The C4.4a-NAMPTi-ADC induced complete responses (in 3 of 8 mice) and stable diseases (in 5 of 8 mice) and the HER2-NAMPTi-ADC achieved complete tumor regression in all treated animals. In addition, in the THP-1 acute myeloid leukemia (AML) subcutaneous in vivo model, a B7H3-NAMPTi-ADC induced complete tumor responses in 7 of 8 treated animals. Taken together, we identified a new series of NAMPT inhibitors as a novel class of ADC payloads exhibiting strong in vivo efficacy in various preclinical xenograft models. Citation Format: Anette Sommer, Stefanie Hammer, Sandra Berndt, Antje M. Wengner, Niels Boehnke, Markus Berger, Nils Griebenow, Andreas Steffen, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Lisa Dietz, Hannah Joerissen, Anja Giese, Maria Quanz, Zhengzheng Bao, Xiuli Wu, Hilmar Weinmann, Lars Linden, Bertolt Kreft, Dominik Mumberg. Anti-tumor activity of a novel structural class of NAMPT inhibitor-based ADCs in models of hematologic and solid tumor indications [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1807.

We report on the discovery of the new clinical candidate BAY 1003803 as glucocorticoid receptor agonist for the topical treatment of psoriasis or severe atopic dermatitis. In the course of optimizing the amino alcohol series as a highly potent new non-steroidal lead structure, considerations were made as to how physicochemical properties and safety concerns relate to structural motifs. BAY 1003803 demonstrates strong anti-inflammatory activity in vitro paired with a pharmacokinetic profile suitable for topical application.

Synthetic glucocorticoids (GC) are essential for the treatment of a broad range of inflammatory diseases. However, their use is limited by target related adverse effects on, e.g., glucose homeostasis and bone metabolism. Starting from a nonsteroidal GR ligand (4) that is a full agonist in reporter gene assays, we exploited key functional triggers within the receptor, generating a range of structurally diverse partial agonists. Of these, only a narrow subset exhibited full anti-inflammatory efficacy and a significantly reduced impact on adverse effect markers in human cell assays compared to prednisolone. This led to the discovery of AZD9567 (15) with excellent in vivo efficacy when dosed orally in a rat model of joint inflammation. Compound 15 is currently being evaluated in clinical trials comparing the efficacy and side effect markers with those of prednisolone.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

Abstract ATAD2 (ATPase family AAA-domain containing protein 2, also called ANCCA) is an epigenetic regulator that binds to chromatin through its bromodomain (BD), a motif specialized for acetyl-lysine recognition. ATAD2 directly associates with multiple transcription factors such as ERα, AR, E2F, and Myc; hence, ATAD2 has been proposed to act as a co-factor for oncogenic transcription factors. Furthermore, we have recently reported a novel role for ATAD2 during DNA replication, uncovering interactions between ATAD2 and histone acetylation marks on newly synthesized histone H4. High expression of ATAD2 strongly correlates with poor patient prognosis in multiple tumor types, including gastric, endometrial, hepatocellular, ovarian, breast and lung cancers. However, the exact function of ATAD2 in these tumor types remains unclear. A more thorough validation of ATAD2 as a therapeutic target is hampered by the lack of isoform-selective, potent and cellularly active ATAD2 inhibitors. A systematic assessment of crystal structures of BD-containing protein family predicted that development of selective inhibitors of ATAD2 would be challenging. In line with this prediction, only limited progress in developing lead compounds targeting ATAD2 has been reported so far. A few notable exceptions relied on fragments as starting points, however, their weak potency, insufficient selectivity against other BDs, permeability limitations or modest cellular activity have curbed their further development towards drug candidates. Here we embarked on a novel strategy to identify ATAD2 inhibitors: 11 different DNA-encoded libraries adding up to 67 billion unique encoded compounds were combined and incubated with ATAD2 BD followed by two rounds of affinity-mediated selection. This approach provided with several series of binders, for which specific target engagement of their SMOL moiety upon off-DNA synthesis was confirmed in biochemical and biophysical assays. Several rounds of potency optimization led to the identification of BAY-850, a highly potent and ATAD2 (isoform A) mono-selective inhibitor, which holds an amine substituted 3-(2-furyl)benzamide core. This compound shows - as revealed by size exclusion chromatography and native mass spectrometry - a novel mode of action for a BD inhibitor based on specific target dimerization. In a cellular fluorescence recovery after photobleaching (FRAP) assay BAY-850 displaced wild-type ATAD2 from the chromatin to the same extent as the genetic mutagenesis of ATAD2 BD. In contrast, chemically very similar inactive control compounds showed no major effects on ATAD2 association with the chromatin. These results qualify BAY-850 as the first biologically active ATAD2 isoform A-specific chemical probe, which will enable further elucidation of the cancer biology of this intriguing protein. Citation Format: Amaury E. Fernández-Montalván, Markus Berger, Benno Kuropka, Seong Joo Koo, Volker Badock, Joerg Weiske, Simon J. Holton, Apirat Chaikuad, Laura Díaz-Sáez, James Bennett, Oleg Federov, Kilian Huber, Paolo Centrella, Matthew A. Clark, Christoph E. Dumelin, Eric A. Sigel, Holly S. Soutter, Dawn M. Troast, Ying Zhang, John W. Cuozzo, Anthony D. Keefe, Didier Roche, Vincent Rodeschini, Jan Hübner, Hilmar Weinmann, Ingo V. Hartung, Matyas Gorjanacz. Potent and isoform-selective ATAD2 bromodomain inhibitor with unprecedented chemical structure and mode of action [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 5084. doi:10.1158/1538-7445.AM2017-5084

ATAD2 (ANCCA) is an epigenetic regulator and transcriptional cofactor, whose overexpression has been linked to the progress of various cancer types. Here, we report a DNA-encoded library screen leading to the discovery of BAY-850, a potent and isoform selective inhibitor that specifically induces ATAD2 bromodomain dimerization and prevents interactions with acetylated histones in vitro, as well as with chromatin in cells. These features qualify BAY-850 as a chemical probe to explore ATAD2 biology.

Heavy-metal-based contrast agents (CAs) offer enhanced X-ray absorption for X-ray computed tomography (CT) compared to the currently used iodinated CAs. We report the discovery of new lanthanide and hafnium azainositol complexes and their optimization with respect to high water solubility and stability. Our efforts culminated in the synthesis of BAY-576, an uncharged hafnium complex with 3:2 stoichiometry and broken complex symmetry. The superior properties of this asymmetrically substituted hafnium CA were demonstrated by a CT angiography study in rabbits that revealed excellent signal contrast enhancement.

Abstract 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

A structure-based design approach led to the identification of a novel class of indazole ether based, non-steroidal glucocorticoid receptor (GR) modulators. Several examples were identified that displayed cell potency in the picomolar range, inhibiting LPS-induced TNF-α release by primary peripheral blood mononuclear cells (PBMCs). Additionally, an improved steroid hormone receptor binding selectivity profile, compared to classical steroidal GR agonists, was demonstrated. The indazole ether core tolerated a broad range of substituents allowing for modulation of the physiochemical parameters. A small sub-set of indazole ethers, with pharmacokinetic properties suitable for oral administration, was investigated in a rat antigen-induced joint inflammation model and demonstrated excellent anti-inflammatory efficacy.

We report on the discovery of two new lead series for the development of glucocorticoid receptor agonists. Firstly, the discovery of tetrahydronaphthalenes led to metabolically stable and dissociated compounds. Their binding mode to the glucocorticoid receptor could be elucidated through an X-ray structure. Closer inspection into the reaction path and analyses of side products revealed a new amino alcohol series also addressing the glucocorticoid receptor and demonstrating strong anti-inflammatory activity in vitro.

Objective: Characterization of BAY-576, a new x-ray contrast agent which is not based on iodine, but rather on the heavy metal hafnium. Compared with iodine, hafnium provides better x-ray absorption in the energy range of computed tomography (CT) and allows images of comparable quality to be acquired at a significantly reduced radiation dose.Materials and Methods: A range of standard methods were used to explore the physicochemistry of BAY-576 as well as its tolerability in in vitro assays, its pharmacokinetics and toxicology in rats, and its performance in CT imaging in rabbits.Results: BAY-576 is an extraordinarily stable chelate with a metal content of 42% (wt/wt) and with excellent water solubility. Formulations of 300 mg Hf/mL exhibited viscosity (3.3-3.6 mPa) and osmolality (860-985 mOsm/kg) in the range of nonionic x-ray agents. No relevant effects on erythrocytes, the coagulation, or complement system or on a panel of 87 potential biological targets were observed. The compound did not bind to plasma proteins of a number of species investigated. After intravenous injection in rats, it was excreted fast and mainly via the kidneys. Its pharmacokinetics was comparable to known extracellular contrast agents. A dose of 6000 mg Hf/kg, approximately 10 to 20 times the expected diagnostic dose, was well tolerated by rats with only moderate adverse effects. Computed tomography imaging in rabbits bearing a tumor in the liver demonstrated excellent image quality when compared with iopromide at the same contrast agent dose in angiography during the arterial phase. At 70% of the radiation dose, BAY-576 provided a contrast-to-noise ratio of the tumor, which was equivalent to iopromide at 100% radiation dose.Conclusions: The profile of BAY-576 indicates its potential as the first compound in a new class of noniodine x-ray contrast agents, which can contribute to the reduction of the radiation burden in contrast-enhanced CT imaging.

In recognition of the seminal contributions of F. A. Cotton and A. Bino to the field of aqueous chemistry of organometallic, trinuclear cluster compounds of tungsten, we describe their modifications and use as contrast agents for X-ray computed tomography. To enable their fundamental work for an advantageous diagnostic application in medicine a new generation of polydentate W3O2 complexes with improved hydrolytical stability has been synthesized and characterized. The applicability as new metal based contrast agent has been demonstrated in a computed tomography angiography animal study with increased signal intensity. Especially the bis tridentate W3O2 complexes with their reduced stereochemical complexity represent a promising new class in the field of X-ray contrast agents.

A continuous-flow process for the synthesis of triaminophloroglucinol has been developed. The synthetic procedure is based on a sequential nitration/reduction protocol which uses phloroglucinol as an inexpensive substrate. During the initial exothermic nitration step employing a combination of ammonium nitrate and sulfuric acid, the temperature was controlled through the enhanced heat transfer derived from the high surface-to-volume ratio of the utilized capillary tubing. Clogging of the tubing due to precipitation of trinitrophloroglucinol (TNPG) was avoided by immersing the tubular reactor in an ultrasound bath during the process. The nitration mixture was diluted with water and immediately subjected to catalytic hydrogenation of the nitro groups using a commercially available continuous-flow reactor and PtO2 as heterogeneous catalyst, thus avoiding the isolation of the highly unstable and explosive TNPG intermediate.