Design and Preclinical Characterization Program toward Asundexian (BAY 2433334), an Oral Factor XIa Inhibitor for the Prevention and Treatment of Thromboembolic Disorders.

Activated coagulation factor XI (FXIa) is a highly attractive antithrombotic target as it contributes to the development and progression of thrombosis but is thought to play only a minor role in hemostasis so that its inhibition may allow for decoupling of antithrombotic efficacy and bleeding time prolongation. Herein, we report our major efforts to identify an orally bioavailable, reversible FXIa inhibitor. Using a protein structure-based de novo design approach, we identified a novel micromolar hit with attractive physicochemical properties. During lead modification, a critical problem was balancing potency and absorption by focusing on the most important interactions of the lead series with FXIa while simultaneously seeking to improve metabolic stability and the cytochrome P450 interaction profile. In clinical trials, the resulting compound from our extensive research program, asundexian (BAY 2433334), proved to possess the desired DMPK properties for once-daily oral dosing, and even more importantly, the initial pharmacological hypothesis was confirmed.

BAY-7081: A Potent, Selective, and Orally Bioavailable Cyanopyridone-Based PDE9A Inhibitor.

Despite advances in the treatment of heart failure in recent years, options for patients are still limited and the disease is associated with considerable morbidity and mortality. Modulating cyclic guanosine monophosphate levels within the natriuretic peptide signaling pathway by inhibiting PDE9A has been associated with beneficial effects in preclinical heart failure models. We herein report the identification of BAY-7081, a potent, selective, and orally bioavailable PDE9A inhibitor with very good aqueous solubility starting from a high-throughput screening hit. Key aspect of the optimization was a switch in metabolism of our lead structures from glucuronidation to oxidation. The switch proved being essential for the identification of compounds with improved pharmacokinetic profiles. By studying a tool compound in a transverse aortic constriction mouse model, we were able to substantiate the relevance of PDE9A inhibition in heart diseases.

Pharmacological profile of asundexian, a novel, orally bioavailable inhibitor of factor XIa.

BACKGROUND: Activated coagulation factor XI (FXIa) contributes to the development and propagation of thrombosis but plays only a minor role in hemostasis; therefore, it is an attractive antithrombotic target. OBJECTIVES: To evaluate the pharmacology of asundexian (BAY 2433334), a small molecule inhibitor targeting FXIa, in vitro and in various rabbit models. METHODS: The effects of asundexian on FXIa activity, selectivity versus other proteases, plasma thrombin generation, and clotting assays were evaluated. Antithrombotic effects were determined in FeCl2 - and arterio-venous (AV) shunt models. Asundexian was administered intravenously or orally, before or during thrombus formation, and with or without antiplatelet drugs (aspirin and ticagrelor). Potential effects of asundexian on bleeding were evaluated in ear-, gum-, and liver injury models. RESULTS: Asundexian inhibited human FXIa with high potency and selectivity. It reduced FXIa activity, thrombin generation triggered by contact activation or low concentrations of tissue factor, and prolonged activated partial thromboplastin time in human, rabbit, and various other species, but not in rodents. In the FeCl2 -injury models, asundexian reduced thrombus weight versus control, and in the arterial model when added to aspirin and ticagrelor. In the AV shunt model, asundexian reduced thrombus weight when administered before or during thrombus formation. Asundexian alone or in combination with antiplatelet drugs did not increase bleeding times or blood loss in any of the models studied. CONCLUSIONS: Asundexian is a potent oral FXIa inhibitor with antithrombotic efficacy in arterial and venous thrombosis models in prevention and intervention settings, without increasing bleeding.

Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase.

DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

Design, Synthesis, and Pharmacological Characterization of a Neutral, Non-Prodrug Thrombin Inhibitor with Good Oral Pharmacokinetics.

Despite extensive research on small molecule thrombin inhibitors for oral application in the past decades, only a single double prodrug with very modest oral bioavailability has reached human therapy as a marketed drug. We have undertaken major efforts to identify neutral, non-prodrug inhibitors. Using a holistic analysis of all available internal data, we were able to build computational models and apply these for the selection of a lead series with the highest possibility of achieving oral bioavailability. In our design, we relied on protein structure knowledge to address potency and identified a small window of favorable physicochemical properties to balance absorption and metabolic stability. Protein structure information on the pregnane X receptor helped in overcoming a persistent cytochrome P450 3A4 induction problem. The selected compound series was optimized to a highly potent, neutral, non-prodrug thrombin inhibitor by designing, synthesizing, and testing derivatives. The resulting optimized compound, BAY1217224, has reached first clinical trials, which have confirmed the desired pharmacokinetic properties.

Druggability Assessment for Selected Serine Proteases in a Pharmaceutical Industry Setting.

Target druggability assessment is an integral part of the early target characterization and selection process in pharmaceutical industry. Here, we investigate a set of five different serine proteases from the blood coagulation cascade. The aim of this study is twofold. Firstly, leveraging the wealth of available in-house high-throughput screening (HTS) data, we analyze HTS hit rates and discuss their predictive value for the development of small molecule (SMOL) candidates. Purely structure-activity relationship (SAR) based druggability ratings are compared with computational protein-structure based druggability assessments. Secondly, we evaluate the impact of using conformational ensembles from molecular dynamics (MD) simulations instead of single static crystal structures as basis for computational druggability assessments. Based on this study, we recommend incorporating molecular dynamics routinely into the early target characterization process, especially if only a single X-ray structure is available.

Freezing the Bioactive Conformation to Boost Potency: The Identification of BAY†85-8501, a Selective and Potent Inhibitor of Human Neutrophil Elastase for Pulmonary Diseases.

Human neutrophil elastase (HNE) is a key protease for matrix degradation. High HNE activity is observed in inflammatory diseases. Accordingly, HNE is a potential target for the treatment of pulmonary diseases such as chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchiectasis (BE), and pulmonary hypertension (PH). HNE inhibitors should reestablish the protease-anti-protease balance. By means of medicinal chemistry a novel dihydropyrimidinone lead-structure class was identified. Further chemical optimization yielded orally active compounds with favorable pharmacokinetics such as the chemical probe BAY-678. While maintaining outstanding target selectivity, picomolar potency was achieved by locking the bioactive conformation of these inhibitors with a strategically positioned methyl sulfone substituent. An induced-fit binding mode allowed tight interactions with the S2 and S1 pockets of HNE. BAY 85-8501 ((4S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-3,6-dimethyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile) was shown to be efficacious in a rodent animal model related to ALI. BAY 85-8501 is currently being tested in clinical studies for the treatment of pulmonary diseases.

Characterization of the cellular activity of PDE 4 inhibitors using two novel PDE 4 reporter cell lines.

We report here the generation and pharmacological characterization of two novel PDE 4B1 and PDE 4D3 reporter cell lines. Intracellular cAMP levels are monitored in these cells by a cAMP-sensitive biosensor. We used the recombinant PDE 4B1 and PDE 4D3 reporter cell lines to characterize the cellular effects of various competitive and allosteric PDE 4 inhibitors. In addition, we compared the cellular activity of these PDE 4 inhibitors with the in vitro inhibition of full-length PDE 4D3 and a truncated enzyme comprising the PDE 4D3 catalytic domain. Two different groups of PDE 4 inhibitors could be identified. The first group, including competitive inhibitors like roflumilast, cilomilast and piclamilast, shows similar in vitro activity on full-length and truncated PDE 4D3 and comparably low cellular activity. The second group, including the allosteric inhibitors PMNPQ, D159153, and D159404, shows much better inhibition of full-length versus truncated PDE 4D3. In addition, these compounds show high cellular activity. Our data obtained with the prototype PDE 4 inhibitor rolipram show that rolipram has properties intermediate between the two groups. The results imply that these novel PDE 4 reporter cell lines are well-suited for the characterization of the cellular activity of PDE 4 inhibitors and may also support a better understanding of the complex PDE 4 pharmacology.

The novel selective PDE9 inhibitor BAY 73-6691 improves learning and memory in rodents.

The present study investigated the putative pro-cognitive effects of the novel selective PDE9 inhibitor BAY 73-6691. The effects on basal synaptic transmission and long-term potentiation (LTP) were investigated in rat hippocampal slices. Pro-cognitive effects were assessed in a series of learning and memory tasks using rodents as subjects. BAY 73-6691 had no effect on basal synaptic transmission in hippocampal slices prepared from young adult (7- to 8-week-old) Wistar rats. A dose of 10 microM, but not 30 microM, BAY 73-6691 enhanced early LTP after weak tetanic stimulation. The dose effective in young adult Wistar rats did not affect LTP in hippocampal slices prepared from young (7- to 8-week-old) Fischer 344 X Brown Norway (FBNF1) rats, probably reflecting strain differences. However, it increased basal synaptic transmission and enhanced early LTP after weak tetanic stimulation in hippocampal slices prepared from very old (31- to 35-month-old) FBNF1 rats. BAY 73-6691 enhanced acquisition, consolidation, and retention of long-term memory (LTM) in a social recognition task and tended to enhance LTM in an object recognition task. Bay 73-6691 attenuated the scoplamine-induced retention deficit in a passive avoidance task, and the MK-801-induced short-term memory deficits in a T-maze alternation task. The mechanism of action, possibly through modulation of the NO/cGMP-PKG/CREB pathway, is discussed. Our findings support the notion that PDE9 inhibition may be a novel target for treating memory deficits that are associated with aging and neurodegenerative disorders such as Alzheimer's disease.

PDE-10A inhibitors as insulin secretagogues.

Modulation of cAMP levels has been linked to insulin secretion in preclinical animal models and in humans. The high expression of PDE-10A in pancreatic islets suggested that inhibition of this enzyme may provide the necessary modulation to elicit increased insulin secretion. Using an HTS approach, we have identified quinoline-based PDE-10A inhibitors as insulin secretagogues in vitro. Optimized compounds were evaluated in vivo where improvements in glucose tolerance and increases in insulin secretion were measured.

Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels.

ROS are a risk factor of several cardiovascular disorders and interfere with NO/soluble guanylyl cyclase/cyclic GMP (NO/sGC/cGMP) signaling through scavenging of NO and formation of the strong oxidant peroxynitrite. Increased oxidative stress affects the heme-containing NO receptor sGC by both decreasing its expression levels and impairing NO-induced activation, making vasodilator therapy with NO donors less effective. Here we show in vivo that oxidative stress and related vascular disease states, including human diabetes mellitus, led to an sGC that was indistinguishable from the in vitro oxidized/heme-free enzyme. This sGC variant represents what we believe to be a novel cGMP signaling entity that is unresponsive to NO and prone to degradation. Whereas high-affinity ligands for the unoccupied heme pocket of sGC such as zinc-protoporphyrin IX and the novel NO-independent sGC activator 4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino) methyl [benzoic]acid (BAY 58-2667) stabilized the enzyme, only the latter activated the NO-insensitive sGC variant. Importantly, in isolated cells, in blood vessels, and in vivo, BAY 58-2667 was more effective and potentiated under pathophysiological and oxidative stress conditions. This therapeutic principle preferentially dilates diseased versus normal blood vessels and may have far-reaching implications for the currently investigated clinical use of BAY 58-2667 as a unique diagnostic tool and highly innovative vascular therapy.

Characterization of the first potent and selective PDE9 inhibitor using a cGMP reporter cell line.

We report here the in vitro characterization of 1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one (BAY 73-6691), the first potent and selective inhibitor of phosphodiesterase 9 (PDE9), which is currently under preclinical development for the treatment of Alzheimer's disease. This compound selectively inhibits human (IC50 = 55 nM) and murine (IC50 = 100 nM) PDE9 activity in vitro and shows only moderate activity against other cyclic nucleotide-specific phosphodiesterases. We also report the generation and characterization of a stably transfected PDE9 Chinese hamster ovary cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca2+ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system was used for the characterization of the cellular activity of the new PDE9 inhibitor. BAY 73-6691 alone did not significantly increase basal cGMP levels in this experimental setting. However, in combination with submaximal stimulating concentrations of the sGC activator 4-[((4-carboxybutyl)[2-[(4-phenethyl-benzyl)oxy]phenethyl]amino)methyl] benzoic acid (BAY 58-2667), the compound induced concentration-dependent luminescence signals and intracellular cGMP accumulation. The PDE9 inhibitor significantly potentiated the cGMP signals generated by sGC activating compounds such as BAY 58-2667 or 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and induced leftward shifts of the corresponding concentration-response curves. Using our newly generated PDE9 reporter cell line, we could show that BAY 73-6691 is able to efficiently penetrate cells and to inhibit intracellular PDE9 activity.