Heiner Apeler

NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle

Soluble guanylyl cyclase (sGC) is the only proven receptor for the ubiquitous biological messenger nitric oxide (NO) and is intimately involved in many signal transduction pathways, most notably in regulating vascular tone and platelet function. sGC is a heterodimeric (α/ß) protein that converts GTP to cyclic GMP; NO binds to its prosthetic haem group. Here, we report the discovery of a novel sGC activating compound, its interaction with a previously unrecognized regulatory site and its therapeutic implications. Through a high‐throughput screen we identified BAY 58‐2667, an amino dicarboxylic acid which potently activates sGC in an NO‐independent manner. In contrast to NO, YC‐1 and BAY 41‐2272, the sGC stimulators described recently, BAY 58‐2667 activates the enzyme even after it has been oxidized by the sGC inhibitor ODQ or rendered haem deficient. Binding studies with radiolabelled BAY 58‐2667 show a high affinity site on the enzyme. Using photoaffinity labelling studies we identified the amino acids 371 (α‐subunit) and 231 – 310 (ß‐subunit) as target regions for BAY 58‐2667. sGC activation by BAY 58‐2667 results in an antiplatelet activity both in vitro and in vivo and a potent vasorelaxation which is not influenced by nitrate tolerance. BAY 58‐2667 shows a potent antihypertensive effect in conscious spontaneously hypertensive rats. In anaesthetized dogs the hemodynamic effects of BAY 58‐2667 and GTN are very similar on the arterial and venous system. This novel type of sGC activator is a valuable research tool and may offer a new approach for treating cardiovascular diseases.

Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide.

Abstract Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide, a messenger molecule with multiple clinical implications. Understanding the activation of sGC is an important step for establishing new therapeutic principles. We have now overexpressed sGC in a baculovirus/Sf9 system optimized for high protein yields to facilitate spectral and kinetic studies of the activation mechanisms of this enzyme. It was expressed in a batch fermenter using a defined mixture of viruses encoding the α1 and β1 subunits of the rat lung enzyme. The expressed enzyme was purified from the cytosolic fraction by anion exchange chromatography, hydroxyapatite chromatography, and size exclusion chromatography. By use of this new method 2.5 l culture yielded about 1 mg of apparently homogeneous sGC with a content of about one heme per heterodimer without the need of a heme reconstitution step. The enzyme did not contain stoichiometric amounts of copper. The basal activities of the purified enzyme were 153 and 1259 nmol min–1 mg–1 in the presence of Mg2+ and Mn2+, respectively. The nitric oxide releasing agent 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) stimulated the enzyme 160-fold with Mg2+, whereas the NO-independent activator 3-(5’-hydroxymethyl-2’-furyl)-1- benzylindazole (YC-1) induced an increase in the activity of 101-fold at a concentration of 300 µM. The combination of DEA/NO (10 µM) and YC-1 (100 µM) elicited a dose-dependent synergistic stimulation with a maximum of a 792-fold increase over the basal activity in the presence of Mg2+, resulting in a specific activity of 121 µmol min–1 mg–1. The synergistic stimulation of DEA/NO and YC-1 was attenuated by the sGC inhibitor 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ) (10 µM) by 94%. In a different experimental setup a saturated carbon monoxide solution in the absence of ambient oxygen or NO stimulated the enzyme 15-fold in the absence and 1260-fold in the presence of YC-1 compared to an argon control. The heme spectra of the enzyme showed a shift of the Soret peak from 432 to 399 and 424 nm in the presence of DEA/NO or carbon monoxide, respectively. The heme spectra were not affected by YC-1 in the absence or in the presence of DEA/NO or of carbon monoxide, which reflects the fact that YC-1 does not interact directly with the heme group of the enzyme. In summary, this study shows that our expression/purification procedure is suitable for producing large amounts of highly pure sGC which contains one heme per heterodimer without a reconstitution step. The activator experiments show that in a synergistic stimulation with YC-1 sGC can be activated maximally both by nitric oxide and by carbon monoxide and that YC-1 does not directly act via heme. The described method should help to facilitate the investigation of the new therapeutic principle of NO-independent guanylyl cyclase activators.

Pharmacological actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41-8543: in vitro studies.

BAY 41‐8543 is a novel, highly specific and so far the most potent NO‐independent stimulator of sGC. Here we report the effects of BAY 41‐8543 on the isolated enzyme, endothelial cells, platelets, isolated vessels and Langendorff heart preparation. BAY 41‐8543 stimulates the recombinant sGC concentration‐dependently from 0.0001 μM to 100 μM up to 92‐fold. In combination, BAY 41‐8543 and NO have synergistic effects over a wide range of concentrations. Similar results are shown in implying that BAY 41‐8543 stimulates the sGC directly and furthermore makes the enzyme more sensitive to its endogenous activator NO. In vitro, BAY 41‐8543 is a potent relaxing agent of aortas, saphenous arteries, coronary arteries and veins with IC50‐values in the nM range. In the rat heart Langendorff preparation, BAY 41‐8543 potently reduces coronary perfusion pressure from 10−9 to 10−6 g ml−1 without any effect on left ventricular pressure and heart rate. BAY 41‐8543 is effective even under nitrate tolerance conditions proved by the same vasorelaxing effect on aortic rings taken either from normal or nitrate‐tolerant rats. BAY 41‐8543 is a potent inhibitor of collagen‐mediated aggregation in washed human platelets (IC50=0.09 μM). In plasma, BAY 41‐8543 inhibits collagen‐mediated aggregation better than ADP‐induced aggregation, but has no effect on the thrombin pathway. BAY 41‐8543 is also a potent direct stimulator of the cyclic GMP/PKG/VASP pathway in platelets and synergizes with NO over a wide range of concentrations. These results suggest that BAY 41‐8543 is on the one hand an invaluable tool for studying sGC signaling in vitro and on the other hand its unique profile may offer a novel approach for treating cardiovascular diseases.

NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272

BackgroundThe most important receptor for nitic oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.ResultsWe developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the α1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236–290 of the α1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.ConclusionsOur data demonstrate that the region surrounding the cysteines 238 and 243 in the α1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

Preclinical Antitumor Efficacy of BAY 1129980—a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody–Drug Conjugate for the Treatment of Non–Small Cell Lung Cancer

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non–small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody–drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893–904. ©2017 AACR.

Abstract 46: Preclinical activity of novel antibody-drug conjugates with pyrrole-based kinesin spindle protein inhibitors targeting different tumor antigens

Antibody-drug conjugates (ADCs) are promising agents that are developed for targeted delivery of cytotoxic payloads to tumor cells. ADCs share a common design of antibody, linker, and cytotoxic payload. Despite significant efforts, the number of available payload classes with a differentiated mode-of-action that can successfully be employed to generate antibody-drug conjugates (ADCs) is still rather limited. So far, only ADCs with microtubule depolymerizing or DNA binding payloads have been approved. The identification of ADC payload classes with a novel mode-of-action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of the kinesin spindle protein (KSP/Eg5/KIF11) have generated interest due to their high anti-tumor activity. However, the transfer of the potency of small molecule KSP inhibitors (KSPis) to highly efficient clinical regimens with a sufficient therapeutic window remains challenging. Through the conjugation of a novel pyrrole subclass of KSPis to antibodies targeting different cancer antigens, we generated a panel of ADCs and characterized them both in vitro and in vivo. ADCs targeting either EGFR or TWEAKR/Fn14 showed strong and specific internalization and displayed specific and potent anti-proliferative efficacy in vitro. In cytotoxicity assays, these ADCs exhibited sub-nanomolar potency in antigen-positive cancer cell lines (EGFR/TWEAKR-pos. NCI-H292; TWEAKR-pos. BxPC3, LoVo) and more than 100-fold selectivity versus non-targeted control-ADC containing the same linker and the same payload. Furthermore, selective anti-tumor efficacy of EGFR- and TWEAKR-KSPi-ADCs was demonstrated in vivo using both cancer cell line-derived models of NSCLC (NCI-H292), urothelial cell carcinoma (UCC) (KU-19-19), and renal cell carcinoma (A498), as well as in the TWEAKR-positive patient-derived xenograft UCC model BFX469. At doses of 5-10 mg/kg qw or bw potent anti-tumor efficacy with treated-to-control ratios (T/C) between 0.16 to 0.28 as well as complete regressions were observed. In summary, KSP inhibitors have been established as a promising new payload class allowing the generation of highly potent and selective ADCs for the treatment of solid tumors. Citation Format: Anette Sommer, Sandra Berndt, Hans-Georg Lerchen, Beatrix Stelte-Ludwig, Sven Wittrock, Anne-Sophie Rebstock, Lisa Dietz, Christoph Mahlert, Simone Greven, Nils Griebenow, Yolanda Cancho-Grande, Rolf Jautelat, Heiner Apeler, Bertolt Kreft. Preclinical activity of novel antibody-drug conjugates with pyrrole-based kinesin spindle protein inhibitors targeting different tumor antigens [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 46. doi:10.1158/1538-7445.AM2017-46

Abstract 3234: Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class

The number of cytotoxic payload classes with different modes-of-action which have been successfully employed in antibody-drug conjugates (ADC) is still rather limited. So far, only ADCs with microtubule inhibitors, DNA binding payloads or topoisomerase I inhibitors have been advanced into clinical testing. To this end, the identification of ADC payload classes with a novel mode of action will increase therapeutic options and potentially help to overcome resistance. Inhibitors of kinesin spindle protein (KSP/Eg5) have generated interest due to their high antitumor potency. However, transferring the preclinical potency of small molecule KSP inhibitors (KSPis) into highly efficient clinical regimens with a sufficient therapeutic window has remained challenging. We have investigated a new pyrrole subclass of KSPis which showed subnanomolar potency against a large panel of tumor cell lines for their utility as a novel payload class in ADCs. Towards this goal different attachment sites for linkers have been explored in the KSPi molecule which were found compatible with cleavable and/or non-cleavable linkers. Subnanomolar potency and selectivity of ADCs with antibodies targeting either HER2, EGFR or TWEAKR could be demonstrated in vitro. For selected ADCs, the intracellular trafficking and metabolite formation was investigated and KSP inhibition was confirmed as the ADC mode of action. Depending on the linker composition differential profiles of the ADC metabolites with regard to efflux, cellular permeation, and bystander effect have been achieved. Moreover, specific accumulation in the tumor versus other tissues was demonstrated in biodistribution studies in vivo. In conclusion, KSP inhibitors have been established as a versatile new payload class for the generation of highly potent and selective ADCs. Citation Format: Hans-Georg Lerchen, Sven Wittrock, Nils Griebenow, Mario Lobell, Anne-Sophie Rebstock, Yolanda Cancho-Grande, Beatrix Stelte-Ludwig, Christoph Mahlert, Simone Greven, Anette Sommer, Sandra Berndt, Carsten Terjung, Heiner Apeler, Bertolt Kreft, Rolf Jautelat. Development of potent and selective antibody-drug conjugates with pyrrole-based KSP inhibitors as novel payload class [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 3234. doi:10.1158/1538-7445.AM2017-3234

Abstract 1210: Preclinical pharmacology and repeated dose toxicity of the novel agonistic TWEAK receptor binding antibody BAY-356

TWEAK receptor (TWEAKR, FN14) is a member of the tumor necrosis factor receptor superfamiliy and is highly expressed in a variety of human solid tumor types, and its overexpression is associated with poor prognosis and metastasis. To explore targeting of TWEAKR for cancer therapy we have generated the novel, anti-TWEAKR antibody BAY-356. Its potent agonistic activity leads to TWEAKR hyperactivation and subsequent induction of cell death in vitro and tumor growth inhibition in vivo. BAY-356 is a fully human aglycosylated antibody (Kd ∼ 10nM) that binds to a novel epitope within the TWEAKR ectodomain of various species as determined by BiaCore. In vitro, BAY-356 showed strong agonistic activity on TWEAKR-positive tumor cells, including activation of NFκB- and STAT1 pathways, increase of TWEAKR protein expression, increased IL-8 secretion, caspase 3/7 activation, and proliferation inhibition in a dose-dependent manner. BAY-356 inhibited tumor growth in several TWEAKR-positive tumor models (NCI-H1975, WiDr, ScaBER, and HN10321) with growth inhibition rates of 49-71% when treated with 3-10 mg/kg BAY-356 twice weekly for up to 3 weeks. The activity of BAY-356 was independent of ADCC activation. In a preventative syngeneic CT26-tumor model in Balb/c mice, BAY-356 induced complete responses. Anti-tumor activity of BAY-356 was associated with high tumor levels of TNF alpha protein. To investigate the toxicity of BAY-356, a repeated dose-toxicity study was performed in Cynomolgus monkeys. Animals were dosed with 10, 20, and 40 mg/kg by weekly intravenous injection for 4 weeks. Compound-related clinical findings consisted of an increase of the serum markers amylase and lipase from 10 mg/kg onwards, urea and creatinine from 20 mg/kg onwards and the transaminases ALT and GDPH at 40 mg/kg. Histopathological evaluation revealed focal ductular epithelial hyperplasia with periductular fibrosis in the exocrine pancreas (at 10 & 20 mg/kg), renal tubular hyperplasia and degeneration, Bowman capsule hyperplasia, and glomerulosclerosis in the kidney starting at 10 mg/kg and bile duct hyperplasia in liver at 20 mg/kg and higher. The HNSTD was set as the highest tested dose of 40 mg/kg. Immunohistochemical analysis of TWEAKR expression in these organs demonstrated a dose dependent induction and increase when compared to untreated controls which correlated with the histopathological findings. From these data it can be concluded that hyperactivation of TWEAKR signaling by BAY-356 leading to strong anti-tumor efficacy in various mouse models is invariably accompanied by target-mediated side-effects originating from enhanced TWEAKR induction in in particular in kidneys, pancreas, and liver of sensitive species such as Cynomolgus monkeys. Citation Format: Sandra Berndt, Christian Votsmeier, Ruprecht Zierz, Jakob Walter, Anna-Lena Frisk, Stefanie Hammer, Heiner Apeler, Bertolt Kreft. Preclinical pharmacology and repeated dose toxicity of the novel agonistic TWEAK receptor binding antibody BAY-356. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1210.