Pharmacological characterization of a novel 5-hydroxybenzothiazolone-derived b2-adrenoceptor agonist with functional selectivity for anabolic effects on skeletal muscle resulting in a wider cardiovascular safety window in preclinical studies

Copyright ª 2019 by The Author(s) The anabolic effects of b2-adrenoceptor (b2-AR) agonists on skeletal muscle have been demonstrated in various species. However, the clinical use of b2-AR agonists for skeletal muscle wasting conditions has been limited by their undesired cardiovascular effects. Here, we describe the preclinical pharmacological profile of a novel 5-hydroxybenzothiazolone (5-HOB) derived b2-AR agonist in comparison with formoterol as a representative b2-AR agonist that have been well characterized. In vitro, 5-HOB has nanomolar affinity for the human b2-AR and selectivity over the b1-AR and b3-AR. 5-HOB also shows potent agonistic activity at the b2-AR in primary skeletal muscle myotubes and induces hypertrophy of skeletal muscle myotubes. Compared with formoterol, 5-HOB demonstrates comparable full-agonist activity on cAMP production in skeletal muscle cells and skeletal muscle tissue–derived membranes. In contrast, a greatly reduced intrinsic activity was determined in cardiomyocytes and cell membranes prepared from the rat heart. In addition, 5-HOB shows weak effects on chronotropy, inotropy, and vascular relaxation compared with formoterol. In vivo, 5-HOB significantly increases hind limb muscle weight in rats with attenuated effects on heart weight and ejection fraction, unlike formoterol. Furthermore, changes in cardiovascular parameters after bolus subcutaneous treatment in rats and rhesus monkeys are significantly lower with 5-HOB compared with formoterol. In conclusion, the pharmacological profile of 5-HOB indicates superior tissue selectivity compared with the conventional b2-AR agonist formoterol in preclinical studies and supports the notion that such tissue-selective agonists should be investigated for the safe treatment of muscle-wasting conditions without cardiovascular limiting effects

Two-dimensional liquid chromatography/mass spectrometry set-up for structural elucidation of metabolites in complex biological matrices.

For absorption, distribution, metabolism and excretion (ADME) studies of drug candidates, mass spectrometry (MS) has become an indispensable tool for the characterization of biotransformation pathways. Samples from in vivo animal studies such as plasma, tissue extracts or excreta contain vast amounts of endogenous compounds. Therefore, the generation of metabolite patterns requires dedicated sample pre-treatment and sophisticated separation methods. Methodologies used for metabolite separation are often inappropriate for structure elucidation. Therefore, a two-dimensional liquid chromatography (LC) approach in combination with MS was developed. Study samples were analyzed using high-performance liquid chromatography (HPLC) for the generation of a qualitative and quantitative metabolite pattern (first dimension) with high reproducibility and recovery without extensive sample pre-treatment. Selected radioactive metabolite fractions were then applied to micro-HPLC with off-line radioactivity monitoring and subsequent MS detection (second dimension). Applying the two-dimensional HPLC/MS approach not only major metabolites could be identified, even minor and trace metabolites were characterized. The usage of sampled metabolite fractions allowed also the re-analysis of specific metabolites for additional investigations (e.g. H/D exchange experiments or product ion scanning experiments). It could be clearly shown that the two-dimensional HPLC/MS approach showed mass spectra with higher sensitivity and selectivity significantly improving the characterization of minor and trace metabolites in in vivo ADME studies

Fixing clearance as early as lead optimization using high throughput in-vitro incubations in combination with exact mass detection and automatic structure elucidation of metabolites

New enabling MS technologies have made it possible to elucidate metabolic pathways present in ex-vivo (blood, bile and/or urine) or in vitro (liver microsomes, hepatocytes and/or S9) samples. When investigating samples from high throughput assays the challenge that the user is facing now is to extract the appropriate information and compile it so that it is understandable to all. Medicinal chemist may then design the next generation of (better) drug candidates combining the needs for potency and metabolic stability and their synthetic creativity. This review focuses on the comparison of these enabling MS technologies and the IT tools developed for their interpretation

A first-in-class small molecule inhibitor of the COP9 signalosome subunit 5 for the treatment of cancer

The COP9 signalosome (CSN) is the platform for assembly and disassembly of cullin-RING E3 ubiquitin ligases (CRL), which comprise the largest enzyme family of the ubiquitin proteasome system (UPS) in humans1-4 . Over 200 CRL complexes are implicated in the regulation of almost all cellular processes including cell cycle progression, transcription, and apoptosis5, and aberrant CRL activity is frequently associated with cancer6-8. Since CSN functions as the protease which cleaves the ubiquitin-like protein Nedd8 from CRLs and thereby initiates their remodelling, inhibitors of the catalytic subunit CSN59 may have therapeutic potential for the treatment of tumours10,11. Here we present CSN5i- 3, a potent, selective and orally available small molecule inhibitor of CSN5. The compound traps CRLs in the neddylated, active state leading to the autoinactivation of a subset of CRLs, e.g. SCFSkp2, by inducing the degradation of their substrate recognition module (SRM). As a result, the corresponding CRL substrates are stabilized, e.g. tumour suppressors p21 and p27. Surprisingly, we also found CRLs, e.g. SCFβTrCP, whose SRMs are not degraded upon CSN5 inhibition, and their substrates remain unaffected. CSN5i-3 differentially affected tumour cell lines and suppressed growth of a human xenograft in mice. Our results provide insights into how CSN regulates CRLs and suggest that CSN5 inhibition has therapeutic potential for the treatment of cancer

New potent DOT1L inhibitors for in vivo evaluation in mouse

In MLL-rearranged cancer cells, disruptor of telomeric silencing 1-like protein (DOT1L) is aberrantly recruited to ectop-ic loci leading to local hypermethylation of H3K79 and consequently misexpression of leukemogenic genes. A struc-ture-guided optimization of a HTS hit led to the discovery of DOT1L inhibitors with subnanomolar potency, allowing to test the therapeutic principle of DOT1L inhibition in a preclinical mouse tumor xenograft model. Compounds dis-playing good exposure in mouse and nanomolar inhibition of target gene expression in cell were obtained and tested in viv

Discovery of Amino Alcohols as Highly Potent, Selective, and Orally Efficacious Inhibitors of Leukotriene A4 Hydrolase.

The discovery of chiral amino alcohols derived from our previously disclosed clinical LTA4H inhibitor LYS006 is described. In a biochemical assay, their optical antipodes showed similar potencies, which could be rationalized by the cocrystal structures of these compounds bound to LTA4H. Despite comparable stabilities in liver microsomes, they showed distinct in vivo PK properties. Selective O-phosphorylation of the (R)-enantiomers in blood led to clearance values above the hepatic blood flow, whereas the (S)-enantiomers were unaffected and exhibited satisfactory metabolic stabilities in vivo. Introduction of two pyrazole rings led to compound (S)-2 with a more balanced distribution of polarity across the molecule, exhibiting high selectivity and excellent potency in vitro and in vivo. Furthermore, compound (S)-2 showed favorable profiles in 16-week IND-enabling toxicology studies in dogs and rats. Based on allometric scaling and potency in whole blood, compound (S)-2 has the potential for a low oral efficacious dose administered once daily

The Discovery of Novel Dot1L Inhibitors through a Structure-Based Fragmentation Approach

Oncogenic MLL fusion proteins aberrantly recruit Dot1L, a histone methyltransferase, to ectopic loci, leading to local hypermethylation of H3K79 and misexpression of HoxA genes driving MLL-rearranged leukemias. Inhibition of the methyltransferase activity of Dot1L in this setting is predicted to reverse aberrant H3K79 methylation, leading to re-pression of leukemogenic genes and tumor growth inhibition. In the context of our Dot1L drug discovery program, high-throughput screening led to the identification of 2, a weak Dot1L inhibitor with an unprecedented, induced pocket binding mode. A medicinal chemistry campaign, strongly guided by structure-based consideration and ligand-based morphing, enabled the discovery of 12 and 13, potent, selective, and structurally completely novel Dot1L inhibitors

Identification of NVP-CLR457 as an Orally Bioavailable Non-CNS-Penetrant pan-Class IA Phosphoinositol-3-Kinase Inhibitor.

Balanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor 40 (NVP-CLR457) was identified with what was considered to be a best-in-class profile. Key to the optimization to achieve this profile was eliminating a microtubule stabilizing off-target activity, balancing the pan-class I PI3K inhibition profile, minimizing CNS penetration, and developing an amorphous solid dispersion formulation. A rationale for the poor tolerability profile of 40 in a clinical study is discussed

Efficacy and safety of MAS825 (anti-IL-1ꞵ/IL-18) in COVID-19 patients with pneumonia and impaired respiratory function.

MAS825, a bispecific IL-1⍰/IL-18 monoclonal antibody, could improve clinical outcomes in COVID19 pneumonia by reducing inflammasome-mediated inflammation. Hospitalized nonventilated patients with COVID-19 pneumonia (n=138) were randomized (1:1) to receive MAS825 (10 mg/kg single i.v.) or placebo in addition to standard of care (SoC). The primary endpoint was the composite Acute Physiology and Chronic Health Evaluation II (APACHE II) score on Day 15 or on day of discharge (whichever was earlier) with worst case imputation for death. Other study endpoints included safety, Creactive protein (CRP), SARS-CoV2 presence and inflammatory markers. On Day 15, the APACHE II score was 14.5±1.87 and 13.5±1.8 in the MAS825 and placebo groups, respectively (P=0.33). MAS825 + SoC led to 33% relative reduction in intensive care unit (ICU) admissions, ~1 day reduction in ICU stay, reduction in mean duration of oxygen support (13.5 versus 14.3 days) and earlier clearance of virus on Day 15 versus placebo + SoC group. On Day 15, compared with placebo group, patients treated with MAS825 + SoC showed a 51% decrease in CRP levels, 42% lower IL-6 levels, 19% decrease in neutrophil levels and 16% lower interferon-γ levels, indicative of IL-1β and IL-18 pathway engagement. MAS825 + SoC did not improve APACHE II score in hospitalized patients with severe COVID19 pneumonia; however, it inhibited relevant clinical and inflammatory pathway biomarkers and resulted in faster virus clearance versus placebo + SoC. MAS825 used in conjunction with SoC was well tolerated. None of the adverse events (AEs) or serious AEs were treatment-related