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.

Linker-Dependent Folding Rationalizes PROTAC Cell Permeability.

Proteolysis-targeting chimeras (PROTACs) must be cell permeable to reach their target proteins. This is challenging as the bivalent structure of PROTACs puts them in chemical space at, or beyond, the outer limits of oral druggable space. We used NMR spectroscopy and molecular dynamics (MD) simulations independently to gain insights into the origin of the differences in cell permeability displayed by three flexible cereblon PROTACs having closely related structures. Both methods revealed that the propensity of the PROTACs to adopt folded conformations with a low solvent-accessible 3D polar surface area in an apolar environment is correlated to high cell permeability. The chemical nature and the flexibility of the linker were essential for the PROTACs to populate folded conformations stabilized by intramolecular hydrogen bonds, π-π interactions, and van der Waals interactions. We conclude that MD simulations may be used for the prospective ranking of cell permeability in the design of cereblon PROTACs.

Discovery of the SMYD3 Inhibitor BAY-6035 Using Thermal Shift Assay (TSA)-Based High-Throughput Screening.

SMYD3 (SET and MYND domain-containing protein 3) is a protein lysine methyltransferase that was initially described as an H3K4 methyltransferase involved in transcriptional regulation. SMYD3 has been reported to methylate and regulate several nonhistone proteins relevant to cancer, including mitogen-activated protein kinase kinase kinase 2 (MAP3K2), vascular endothelial growth factor receptor 1 (VEGFR1), and the human epidermal growth factor receptor 2 (HER2). In addition, overexpression of SMYD3 has been linked to poor prognosis in certain cancers, suggesting SMYD3 as a potential oncogene and attractive cancer drug target. Here we report the discovery of a novel SMYD3 inhibitor. We performed a thermal shift assay (TSA)-based high-throughput screening (HTS) with 410,000 compounds and identified a novel benzodiazepine-based SMYD3 inhibitor series. Crystal structures revealed that this series binds to the substrate binding site and occupies the hydrophobic lysine binding pocket via an unprecedented hydrogen bonding pattern. Biochemical assays showed substrate competitive behavior. Following optimization and extensive biophysical validation with surface plasmon resonance (SPR) analysis and isothermal titration calorimetry (ITC), we identified BAY-6035, which shows nanomolar potency and selectivity against kinases and other PKMTs. Furthermore, BAY-6035 specifically inhibits methylation of MAP3K2 by SMYD3 in a cellular mechanistic assay with an IC50 <100 nM. Moreover, we describe a congeneric negative control to BAY-6035. In summary, BAY-6035 is a novel selective and potent SMYD3 inhibitor probe that will foster the exploration of the biological role of SMYD3 in diseased and nondiseased tissues.

Are chief executive officers more likely to be first-borns?

We investigate the link between birth order and the career outcome of becoming Chief Executive Officer (CEO) of a company. CEOs are more likely to be the first-born, i.e., oldest, child of their family relative to what one would expect if birth order did not matter for career outcomes. Both male and female CEOs are more likely to be first-born. However, the first-born advantage seems to largely reflect the absence of an older brother, but not of an older sister. These results are more pronounced for family firms, where traditionally the oldest child is appointed to run the family business, but also hold for non-family firms.

Characterization of the Menin-MLL Interaction as Therapeutic Cancer Target.

Inhibiting the interaction of menin with the histone methyltransferase MLL1 (KMT2A) has recently emerged as a novel therapeutic strategy. Beneficial therapeutic effects have been postulated in leukemia, prostate, breast, liver and in synovial sarcoma models. In those indications, MLL1 recruitment by menin was described to critically regulate the expression of disease associated genes. However, most findings so far rely on single study reports. Here we independently evaluated the pathogenic functions of the menin-MLL interaction in a large set of different cancer models with a potent and selective probe inhibitor BAY-155. We characterized the inhibition of the menin-MLL interaction for anti-proliferation, gene transcription effects, and for efficacy in several in vivo xenografted tumor models. We found a specific therapeutic activity of BAY-155 primarily in AML/ALL models. In solid tumors, we observed anti-proliferative effects of BAY-155 in a surprisingly limited fraction of cell line models. These findings were further validated in vivo. Overall, our study using a novel, highly selective and potent inhibitor, shows that the menin-MLL interaction is not essential for the survival of most solid cancer models. We can confirm that disrupting the menin-MLL complex has a selective therapeutic benefit in MLL-fused leukemia. In solid cancers, effects are restricted to single models and more limited than previously claimed.

BET inhibition is an effective approach against KRAS-driven PDAC and NSCLC.

Effectively treating KRAS-driven tumors remains an unsolved challenge. The inhibition of downstream signaling effectors is a way of overcoming the issue of direct targeting of mutant KRAS, which has shown limited efficacy so far. Bromodomain and Extra-Terminal (BET) protein inhibition has displayed anti-tumor activity in a wide range of cancers, including KRAS-driven malignancies. Here, we preclinically evaluate the effect of BET inhibition making use of a new BET inhibitor, BAY 1238097, against Pancreatic Ductal Adenocarcinoma (PDAC) and Non-Small Cell Lung Cancer (NSCLC) models harboring RAS mutations both in vivo and in vitro. Our results demonstrate that BET inhibition displays significant therapeutic impact in genetic mouse models of KRAS-driven PDAC and NSCLC, reducing both tumor area and tumor grade. The same approach also causes a significant reduction in cell number of a panel of RAS-mutated human cancer cell lines (8 PDAC and 6 NSCLC). In this context, we demonstrate that while BET inhibition by BAY 1238097 decreases MYC expression in some cell lines, at least in PDAC cells its anti-tumorigenic effect is independent of MYC regulation. Together, these studies reinforce the use of BET inhibition and prompt the optimization of more efficient and less toxic BET inhibitors for the treatment of KRAS-driven malignancies, which are in urgent therapeutic need.

Preclinical evaluation of the BET bromodomain inhibitor BAY 1238097 for the treatment of lymphoma.

The epigenome is often deregulated in cancer and treatment with inhibitors of bromodomain and extra-terminal proteins, the readers of epigenetic acetylation marks, represents a novel therapeutic approach. Here, we have characterized the anti-tumour activity of the novel bromodomain and extra-terminal (BET) inhibitor BAY 1238097 in preclinical lymphoma models. BAY 1238097 showed anti-proliferative activity in a large panel of lymphoma-derived cell lines, with a median 50% inhibitory concentration between 70 and 208 nmol/l. The compound showed strong anti-tumour efficacy in vivo as a single agent in two diffuse large B cell lymphoma models. Gene expression profiling showed BAY 1238097 targeted the NFKB/TLR/JAK/STAT signalling pathways, MYC and E2F1-regulated genes, cell cycle regulation and chromatin structure. The gene expression profiling signatures also highly overlapped with the signatures obtained with other BET Bromodomain inhibitors and partially overlapped with HDAC-inhibitors, mTOR inhibitors and demethylating agents. Notably, BAY 1238097 presented in vitro synergism with EZH2, mTOR and BTK inhibitors. In conclusion, the BET inhibitor BAY 1238097 presented promising anti-lymphoma preclinical activity in vitro and in vivo, mediated by the interference with biological processes driving the lymphoma cells. Our data also indicate the use of combination schemes targeting EZH2, mTOR and BTK alongside BET bromodomains.

Benzoisoquinolinediones as Potent and Selective Inhibitors of BRPF2 and TAF1/TAF1L Bromodomains.

Bromodomains (BD) are readers of lysine acetylation marks present in numerous proteins associated with chromatin. Here we describe a dual inhibitor of the bromodomain and PHD finger (BRPF) family member BRPF2 and the TATA box binding protein-associated factors TAF1 and TAF1L. These proteins are found in large chromatin complexes and play important roles in transcription regulation. The substituted benzoisoquinolinedione series was identified by high-throughput screening, and subsequent structure-activity relationship optimization allowed generation of low nanomolar BRPF2 BD inhibitors with strong selectivity against BRPF1 and BRPF3 BDs. In addition, a strong inhibition of TAF1/TAF1L BD2 was measured for most derivatives. The best compound of the series was BAY-299, which is a very potent, dual inhibitor with an IC50 of 67 nM for BRPF2 BD, 8 nM for TAF1 BD2, and 106 nM for TAF1L BD2. Importantly, no activity was measured for BRD4 BDs. Furthermore, cellular activity was evidenced using a BRPF2- or TAF1-histone H3.3 or H4 interaction assay.

Targeting BET bromodomains for cancer treatment.

The bromodomain and extraterminal (BET) subfamily of bromodomain-containing proteins has emerged in the last few years as an exciting, novel target group. BRD4, the best studied BET protein, is implicated in a number of hematological and solid tumors. This is linked to its role in modulating transcription elongation of essential genes involved in cell cycle and apoptosis such as c-Myc and BCL2. Potent BET inhibitors with promising antitumor efficacy in a number of preclinical cancer models have been identified in recent years. This led to clinical studies focusing mostly on the treatment of leukemia and lymphoma, and first encouraging signs of efficacy have already been reported. Here we discuss the biology of BRD4, its known interaction partners and implication in different tumor types. Further, we summarize the current knowledge on BET bromodomain inhibitors.

Selective indole-based ECE inhibitors: synthesis and pharmacological evaluation.

Inhibition of the metalloprotease ECE-1 may be beneficial for the treatment of coronary heart disease, cancer, renal failure, and urological disorders. A novel class of indole-based ECE inhibitors was identified by high throughput screening. Optimization of the original screening lead structure 6 led to highly potent inhibitors such as 11, which bears a bisaryl amide moiety linked to the indole C2 position through an amide group. Docking of 11 into a model structure of ECE revealed a unique binding mode in which the Zn center of the enzyme is not directly addressed by the inhibitor, but key interactions are suggested for the central amide group. Testing of the lead compound 6 in hypertensive Dahl S rats resulted in a decrease in blood pressure after an initial period in which the blood pressure remained unchanged, most probably the result of ET-1 already present. Indole derivative 6 also displays a cardio-protective effect in a mouse model of acute myocardial infarction after oral administration. The more potent chloropyridine derivative 9 antagonizes big-ET-1-induced increase in blood pressure in rats at intravenous administration of 3 mg kg-1. All ECE inhibitors of the indole class showed high selectivity for ECE over related metalloproteases such as NEP and ACE. Therefore, these compounds might have further potential as drugs for the treatment of coronary heart diseases.

Novel, selective indole-based ECE inhibitors: lead optimization via solid-phase and classical synthesis.

A novel class of indole-based endothelin-converting enzyme (ECE) inhibitors was identified by high throughput screening. We report systematic optimization of this compound class by means of classical and solid-phase chemistry. Optimized compounds with a bisarylamide side chain at the 2-position of the indole skeleton exhibit low-nanomolar activity on ECE.

New class of bacterial phenylalanyl-tRNA synthetase inhibitors with high potency and broad-spectrum activity.

Phenylalanyl (Phe)-tRNA synthetase (Phe-RS) is an essential enzyme which catalyzes the transfer of phenylalanine to the Phe-specific transfer RNA (tRNA(Phe)), a key step in protein biosynthesis. Phenyl-thiazolylurea-sulfonamides were identified as a novel class of potent inhibitors of bacterial Phe-RS by high-throughput screening and chemical variation of the screening hit. The compounds inhibit Phe-RS of Escherichia coli, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus, with 50% inhibitory concentrations in the nanomolar range. Enzyme kinetic measurements demonstrated that the compounds bind competitively with respect to the natural substrate Phe. All derivatives are highly selective for the bacterial Phe-RS versus the corresponding mammalian cytoplasmic and human mitochondrial enzymes. Phenyl-thiazolylurea-sulfonamides displayed good in vitro activity against Staphylococcus, Streptococcus, Haemophilus, and Moraxella strains, reaching MICs below 1 micro g/ml. The antibacterial activity was partly antagonized by increasing concentrations of Phe in the culture broth in accordance with the competitive binding mode. Further evidence that inhibition of tRNA(Phe) charging is the antibacterial principle of this compound class was obtained by proteome analysis of Bacillus subtilis. Here, the phenyl-thiazolylurea-sulfonamides induced a protein pattern indicative of the stringent response. In addition, an E. coli strain carrying a relA mutation and defective in stringent response was more susceptible than its isogenic relA(+) parent strain. In vivo efficacy was investigated in a murine S. aureus sepsis model and a S. pneumoniae sepsis model in rats. Treatment with the phenyl-thiazolylurea-sulfonamides reduced the bacterial titer in various organs by up to 3 log units, supporting the potential value of Phe-RS as a target in antibacterial therapy.

Memory of Chirality in Electron Transfer Mediated Benzylic Umpolung Reactions of Arene-Cr(CO)3 Complexes.

As density functional calculations suggest, Cr(CO)3 -complexed benzylic radicals (such as 2) exhibit a significant degree of configurational stablility. This was exploited in an efficient method for the electron transfer mediated transformations of readily available 1-arylalkanol-Cr(CO)3 derivatives 1 to afford alkylated products 3 in good yields and with a high degree of stereochemical retention.