Structure- and Property-Based Optimization of Efficient Pan-Bromodomain and Extra Terminal Inhibitors to Identify Oral and Intravenous Candidate I-BET787.

The bromodomain and extra terminal (BET) family of bromodomain-containing proteins are important epigenetic regulators that elicit their effect through binding histone tail N-acetyl lysine (KAc) post-translational modifications. Recognition of such markers has been implicated in a range of oncology and immune diseases and, as such, small-molecule inhibition of the BET family bromodomain-KAc protein-protein interaction has received significant interest as a therapeutic strategy, with several potential medicines under clinical evaluation. This work describes the structure- and property-based optimization of a ligand and lipophilic efficient pan-BET bromodomain inhibitor series to deliver candidate I-BET787 (70) that demonstrates efficacy in a mouse model of inflammation and suitable properties for both oral and intravenous (IV) administration. This focused two-phase explore-exploit medicinal chemistry effort delivered the candidate molecule in 3 months with less than 100 final compounds synthesized.

Identification and Optimization of a Ligand-Efficient Benzoazepinone Bromodomain and Extra Terminal (BET) Family Acetyl-Lysine Mimetic into the Oral Candidate Quality Molecule I-BET432.

The bromodomain and extra terminal (BET) family of proteins are an integral part of human epigenome regulation, the dysregulation of which is implicated in multiple oncology and inflammatory diseases. Disrupting the BET family bromodomain acetyl-lysine (KAc) histone protein-protein interaction with small-molecule KAc mimetics has proven to be a disease-relevant mechanism of action, and multiple molecules are currently undergoing oncology clinical trials. This work describes an efficiency analysis of published GSK pan-BET bromodomain inhibitors, which drove a strategic choice to focus on the identification of a ligand-efficient KAc mimetic with the hypothesis that lipophilic efficiency could be drastically improved during optimization. This focus drove the discovery of the highly ligand-efficient and structurally distinct benzoazepinone KAc mimetic. Following crystallography to identify suitable growth vectors, the benzoazepinone core was optimized through an explore-exploit structure-activity relationship (SAR) approach while carefully monitoring lipophilic efficiency to deliver I-BET432 (41) as an oral candidate quality molecule.

A discovery biotransformation strategy: combining in silico tools with high-resolution mass spectrometry and software-assisted data analysis for high-throughput metabolism.

Understanding compound metabolism in early drug discovery aids medicinal chemistry in designing molecules with improved safety and ADME properties. While advancements in metabolite prediction brings increased confidence, structural decisions require experimental data. In vitro metabolism studies using liquid chromatography and high-resolution mass spectrometry (LC-MS) are generally resource intensive and performed on very few compounds, limiting the chemical space that can be examined.Here, we describe a novel metabolism strategy increasing compound throughput using residual in vitro clearance samples conducted at drug concentrations of 0.5 µM. Analysis by robust ultra high-performance liquid chromatography separation and accurate-mass MS detection ensures major metabolites are identified from a single injection. In silico prediction (parent cLogD) tailors chromatographic conditions, with data-dependent tandem mass spectroscopy targeting predicted metabolites. Software-assisted data mining, structure elucidation and automatic reporting are used.Confidence in the globally aligned workflow is demonstrated with 16 marketed drugs. The approach is now implemented routinely across our laboratories. To date, the success rate for identification of at least one major metabolite is 85%. The utility of these data has been demonstrated across multiple projects, allowing earlier medicinal chemistry decisions to increase efficiency and impact of the design-make-test cycle thus improving the translatability of early in vitro metabolism data.

Design, Synthesis, and Evaluation of Lung-Retentive Prodrugs for Extending the Lung Tissue Retention of Inhaled Drugs.

A major limitation of pulmonary delivery is that drugs can exhibit suboptimal pharmacokinetic profiles resulting from rapid elimination from the pulmonary tissue. This can lead to systemic side effects and a short duration of action. A series of dibasic dipeptides attached to the poorly lung-retentive muscarinic M3 receptor antagonist piperidin-4-yl 2-hydroxy-2,2-diphenylacetate (1) through a pH-sensitive-linking group have been evaluated. Extensive optimization resulted in 1-(((R)-2-((S)-2,6-diaminohexanamido)-3,3-dimethylbutanoyl)oxy)ethyl 4-(2-hydroxy-2,2-diphenylacetoxy)piperidine-1-carboxylate (23), which combined very good in vitro stability and very high rat lung binding. Compound 23 progressed to pharmacokinetic studies in rats, where, at 24 h post dosing in the rat lung, the total lung concentration of 23 was 31.2 µM. In addition, high levels of liberated drug 1 were still detected locally, demonstrating the benefit of this novel prodrug approach for increasing the apparent pharmacokinetic half-life of drugs in the lungs following pulmonary dosing.

Carboxylesterase-1 Assisted Targeting of HDAC Inhibitors to Mononuclear Myeloid Cells in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Histone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD]. METHODS: CES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn's disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter. RESULTS: CES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis. CONCLUSIONS: We demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

Using a single, high mass resolution mass spectrometry platform to investigate ion suppression effects observed during tissue imaging.

RATIONALE: The signal intensity of a given molecule across a tissue section when measured using mass spectrometry imaging (MSI) is prone to changes caused by the molecular heterogeneity across the surface of the tissue. Here we propose a strategy to investigate these effects using electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) on a single high-resolution mass spectrometry (HRMS) platform. METHODS: A rat was administered with a single inhaled dose of a compound and sacrificed 1 h after dosing. Sections were prepared from the excised frozen lung and analysed using MALDI, liquid extraction surface analysis (LESA) nano-ESI-MS and nano-ESI liquid chromatography (LC)/MS. The ESI and MALDI ion sources were mounted either side of the ion transfer system of the same Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. RESULTS: MALDI MSI clearly demonstrated widespread distribution of the dosed molecule throughout the lung, with the exception of a non-lung section of tissue on the same sample surface. Comparison of the lipid signals across the sample indicated a change in signal between the lung and the adipose tissue present on the same section. Use of ESI and MALDI, with and without an internal standard, supported the evaluation of changes in the signal of the dosed molecule across the tissue section. CONCLUSIONS: The results demonstrate the successful application of a dual ion source HRMS system to the systematic evaluation of data from MALDI MSI, used to determine the distribution of an inhaled drug in the lung. The system discussed is of great utility in investigating the effects of ion suppression and evaluating the quantitative and qualitative nature of the MSI data.