Lead Optimization of Small Molecule ENL YEATS Inhibitors to Enable In Vivo Studies: Discovery of TDI-11055.

Eleven-nineteen leukemia (ENL) is an epigenetic reader protein that drives oncogenic transcriptional programs in acute myeloid leukemia (AML). AML is one of the deadliest hematopoietic malignancies, with an overall 5-year survival rate of 27%. The epigenetic reader activity of ENL is mediated by its YEATS domain that binds to acetyl and crotonyl marks on histone tails and colocalizes with promoters of actively transcribed genes that are essential for leukemia. Prior to the discovery of TDI-11055, existing inhibitors of ENL YEATS showed in vitro potency, but had not shown efficacy in in vivo animal models. During the course of the medicinal chemistry campaign described here, we identified ENL YEATS inhibitor TDI-11055 that has an improved pharmacokinetic profile and is appropriate for in vivo evaluation of the ENL YEATS inhibition mechanism in AML.

Development of Potent Mcl-1 Inhibitors: Structural Investigations on Macrocycles Originating from a DNA-Encoded Chemical Library Screen.

Evasion of apoptosis is critical for the development and growth of tumors. The pro-survival protein myeloid cell leukemia 1 (Mcl-1) is an antiapoptotic member of the Bcl-2 family, associated with tumor aggressiveness, poor survival, and drug resistance. Development of Mcl-1 inhibitors implies blocking of protein-protein interactions, generally requiring a lengthy optimization process of large, complex molecules. Herein, we describe the use of DNA-encoded chemical library synthesis and screening to directly generate complex, yet conformationally privileged macrocyclic hits that serve as Mcl-1 inhibitors. By applying a conceptual combination of conformational analysis and structure-based design in combination with a robust synthetic platform allowing rapid analoging, we optimized in vitro potency of a lead series into the low nanomolar regime. Additionally, we demonstrate fine-tuning of the physicochemical properties of the macrocyclic compounds, resulting in the identification of lead candidates 57/59 with a balanced profile, which are suitable for future development toward therapeutic use.

Small-Molecule Inhibition of the Acyl-Lysine Reader ENL as a Strategy against Acute Myeloid Leukemia.

The chromatin reader eleven-nineteen leukemia (ENL) has been identified as a critical dependency in acute myeloid leukemia (AML), but its therapeutic potential remains unclear. We describe a potent and orally bioavailable small-molecule inhibitor of ENL, TDI-11055, which displaces ENL from chromatin by blocking its YEATS domain interaction with acylated histones. Cell lines and primary patient samples carrying MLL rearrangements or NPM1 mutations are responsive to TDI-11055. A CRISPR-Cas9-mediated mutagenesis screen uncovers an ENL mutation that confers resistance to TDI-11055, validating the compound's on-target activity. TDI-11055 treatment rapidly decreases chromatin occupancy of ENL-associated complexes and impairs transcription elongation, leading to suppression of key oncogenic gene expression programs and induction of differentiation. In vivo treatment with TDI-11055 blocks disease progression in cell line- and patient-derived xenograft models of MLL-rearranged and NPM1-mutated AML. Our results establish ENL displacement from chromatin as a promising epigenetic therapy for molecularly defined AML subsets and support the clinical translation of this approach. SIGNIFICANCE: AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.

Discovery of 2,4-1H-Imidazole Carboxamides as Potent and Selective TAK1 Inhibitors.

Herein we report the discovery of 2,4-1H-imidazole carboxamides as novel, biochemically potent, and kinome selective inhibitors of transforming growth factor ß-activated kinase 1 (TAK1). The target was subjected to a DNA-encoded chemical library (DECL) screen. After hit analysis a cluster of compounds was identified, which was based on a central pyrrole-2,4-1H-dicarboxamide scaffold, showing remarkable kinome selectivity. A scaffold-hop to the corresponding imidazole resulted in increased biochemical potency. Next, X-ray crystallography revealed a distinct binding mode compared to other TAK1 inhibitors. A benzylamide was found in a perpendicular orientation with respect to the core hinge-binding imidazole. Additionally, an unusual amide flip was observed in the kinase hinge region. Using structure-based drug design (SBDD), key substitutions at the pyrrolidine amide and the glycine resulted in a significant increase in biochemical potency.

Novel Autotaxin Inhibitor for the Treatment of Idiopathic Pulmonary Fibrosis: A Clinical Candidate Discovered Using DNA-Encoded Chemistry.

The activity of the secreted phosphodiesterase autotaxin produces the inflammatory signaling molecule LPA and has been associated with a number of human diseases including idiopathic pulmonary fibrosis (IPF). We screened a single DNA-encoded chemical library (DECL) of 225 million compounds and identified a series of potent inhibitors. Optimization of this series led to the discovery of compound 1 (X-165), a highly potent, selective, and bioavailable small molecule. Cocrystallization of compound 1 with human autotaxin demonstrated that it has a novel binding mode occupying both the hydrophobic pocket and a channel near the autotaxin active site. Compound 1 inhibited the production of LPA in human and mouse plasma at nanomolar levels and showed efficacy in a mouse model of human lung fibrosis. After successfully completing IND-enabling studies, compound 1 was approved by the FDA for a Phase I clinical trial. These results demonstrate that DECL hits can be readily optimized into clinical candidates.

The mRNA level of Charcot-Leyden crystal protein/galectin-10 is a marker for CRTH2 activation in human whole blood in vitro.

CRTH2 is one of the prostaglandin D2 receptors and plays a proinflammatory role in allergic diseases. Gene expression markers in whole blood induced by CRTH2 activation have not previously been reported. Using microarray analyses of 54 675 genes, we revealed modest gene expression changes in human whole blood stimulated in vitro by a selective CRTH2 agonist, DK-PGD2. Five genes were found to exhibit 1.5- to 2.6-fold changes in expression. The expression of Charcot-Leyden crystal protein/galectin-10 (CLC/Gal-10) in particular was consistently enhanced in human whole blood stimulated by DK-PGD2, as confirmed by quantitative real-time polymerase chain reaction analyses. DK-PGD(2)-induced increases in blood CLC/Gal-10 mRNA levels were largely attenuated by the CRTH2 antagonist CAY10471.Thus, the DK-PGD2-induced CLC/Gal-10 mRNA level can serve as a potential marker for monitoring pharmacodynamic effects of blood exposure to CRTH2 modulating agents.

Discovery of novel and potent leukotriene B4 receptor antagonists. Part 1.

The inhibition of LTB(4) binding to and activation of G-protein-coupled receptors BLT1 and BLT2 is the premise of a treatment for several inflammatory diseases. In a lead optimization effort starting with the leukotriene B(4) (LTB(4)) receptor antagonist (2), members of a series of 3,5-diarylphenyl ethers were found to be highly potent inhibitors of LTB(4) binding to BLT1 and BLT2 receptors, with varying levels of selectivity depending on the substitution. In addition, compounds 33 and 38 from this series have good in vitro ADME properties, good oral bioavailability, and efficacy after oral delivery in guinea pig LTB(4) and nonhuman primate allergen challenge models. Further profiling in a rat non-GLP toxicity experiment provided the rationale for differentiation and selection of one compound (33) for clinical development.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

Cellular and molecular characterization of ozone-induced pulmonary inflammation in the Cynomolgus monkey.

We investigated the cellular and molecular effects of ozone exposure in Cynomolgus monkeys. Thirty-six Cynomolgus monkeys were exposed to single or repeat ozone challenge. Pulmonary inflammation was assessed using bronchoalveolar lavage fluid (BAL) and histology. Gene expression profiling in lung and blood was performed. Ozone challenge evoked BAL cellular inflammation and increases in total protein, alkaline phosphatase and cytokines. Lung histology revealed cellular inflammation and epithelial necrosis. Gene expression profiling identified oxidative phosphorylation, immune response and cell adhesion pathways altered in response to ozone, with common and unique profiles in lung and blood. Lipocalin 2, CD177, the FK-506 and S100A8 binding proteins and ST-2 represent novel peripheral biomarkers of ozone toxicity. Repeat ozone challenge evoked reproducible inflammation but attenuated cell damage. These studies provide data on the molecular mechanisms and biomarker identification of ozone-evoked toxicity, and support the use of the Cynomolgus monkey as a model of human ozone challenge.

Pharmacokinetics, safety, and tolerability of R411, a dual alpha4beta1-alpha4beta7 integrin antagonist after oral administration at single and multiple once-daily ascending doses in healthy volunteers.

R411 is a dual alpha4beta1-alpha4beta7 integrin antagonist under development for the treatment of chronic asthma. The objective of this study was to investigate the pharmacokinetics and safety of R411 and its active metabolite, RO0270608, in humans. A 3-part phase I trial was conducted in 132 healthy volunteers: (1) 12 subjects received 200 mg R411 as a single oral dose or 100 mg RO0270608 as an intravenous infusion in a 1-sequence crossover design; (2) 7 groups of 10 subjects received 1 of 7 single oral doses of R411 (10-1200 mg) in a parallel, placebo-controlled, ascending adaptive dose design; and (3) 5 groups of 10 subjects each received repeated oral qd doses of R411 (50-900 mg) for up to 3 weeks in a parallel, placebo-controlled, ascending adaptive dose design. The absolute bioavailability of RO0270608 (mean +/- standard deviation) after oral administration of R411 was 27% +/- 4%, and the terminal half-life was 7.33 +/- 2.29 hours. After IV infusion of RO0270608, total clearance (mean +/- standard deviation) was 19.4 +/- 7.1 L/h, and the volume of distribution was 93.1 +/- 36.1 L. After single ascending oral doses of R411, area under the concentration-time curve from 0 to infinity of active metabolite RO0270608 increased proportionally from 150 to 1200 mg (P > .05). Following repeated administration, the oral clearance was independent of time. No drug accumulation was observed, and no safety concerns were revealed up to a dose of 900 mg after up to 3 weeks of treatment.

N-Cycloalkanoyl-L-phenylalanine derivatives as VCAM/VLA-4 antagonists.

A systematic structure-activity relationship investigation of the lead compound 1 resulted the identification of several N-[(substituted alkyl)cycloalkanoyl]-4-[((2,6-dichlorophenyl)carbonyl)amino]-L-phenylalanine derivatives as potent VCAM/VLA-4 antagonists. The data are consistent with a model of these compounds in which these alkanoylphenylalanines reside in a compact gauche (-) bioactive conformation.

N-Aroyl-L-phenylalanine derivatives as VCAM/VLA-4 antagonists.

A series of N-benzoyl-4-[(2,6-dichlorobenzoyl)amino]-L-phenylalanine derivatives was prepared in order to optimize the substitution on the N-benzoyl moiety for VCAM/VLA-4 antagonist activity. Disubstitution in the 2- and 6-positions is favored and a range of small alkyl and halogen are tolerated. A model of the bioactive conformation of these compounds is proposed.