Phase 1 and preclinical profiling of ESM-HDAC391, a myeloid-targeted histone deacetylase inhibitor, shows enhanced pharmacology and monocytopaenia.

AIMS: To improve the tolerability and therapeutic application of histone deacetylase inhibitors (HDACi), by application of an esterase-sensitive motif (ESM), to target pharmacological activity directly to mononuclear myeloid cells expressing the processing enzyme carboxylesterase-1 (CES1). METHODS: This first-in-human study comprised single and multiple ascending dose cohorts to determine safety and tolerability. Pharmacodynamic parameters included acetylation, cytokine inhibition and intracellular concentrations of processed acid metabolite in isolated monocytes. Mechanistic work was conducted in vitro and in a CES1/Es1elo mouse strain. RESULTS: ESM-HDAC391 showed transient systemic exposure (plasma half-life of 21-30 min) but selective retention of processed acid for at least 12 hours, resulting in robust targeted mechanistic engagement (increased acetylation in monocytes plus inhibition of ex vivo stimulated cytokine production). ESM-HDAC391 was well tolerated and clinical toxicities common to non-targeted HDACi were not observed. ESM-HDAC391 treatment was accompanied by the novel finding of a dose-dependent monocyte depletion that was transient and reversible and which plateaued at 0.06 × 109 monocytes/L after repeat dosing with 20 or 40 mg. Characterisation of monocyte depletion in transgenic mice (CES1/Es1elo ) suggested that colony stimulating factor 1 receptor loss on circulating cells contributed to ESM-HDAC-mediated depletion. Further mechanistic investigations using human monocytes in vitro demonstrated HDACi-mediated change in myeloid fate through modulation of colony stimulating factor 1 receptor and downstream effects on cell differentiation. CONCLUSION: These findings demonstrate selective targeting of monocytes in humans using the ESM approach and identify monocytopaenia as a novel outcome of ESM-HDACi treatment, with implications for potential benefit of these molecules in myeloid-driven diseases.

Discovery of a Novel Bromodomain and Extra Terminal Domain (BET) Protein Inhibitor, I-BET282E, Suitable for Clinical Progression.

The functions of the bromodomain and extra terminal (BET) family of proteins have been implicated in a wide range of diseases, particularly in the oncology and immuno-inflammatory areas, and several inhibitors are under investigation in the clinic. To mitigate the risk of attrition of these compounds due to structurally related toxicity findings, additional molecules from distinct chemical series were required. Here we describe the structure- and property-based optimization of the in vivo tool molecule I-BET151 toward I-BET282E, a molecule with properties suitable for progression into clinical studies.

IFN-γ Drives Human Monocyte Differentiation into Highly Proinflammatory Macrophages That Resemble a Phenotype Relevant to Psoriasis.

As key cells of the immune system, macrophages coordinate the activation and regulation of the immune response. Macrophages present a complex phenotype that can vary from homeostatic, proinflammatory, and profibrotic to anti-inflammatory phenotypes. The factors that drive the differentiation from monocyte to macrophage largely define the resultant phenotype, as has been shown by the differences found in M-CSF- and GM-CSF-derived macrophages. We explored alternative inflammatory mediators that could be used for in vitro differentiation of human monocytes into macrophages. IFN-γ is a potent inflammatory mediator produced by lymphocytes in disease and infections. We used IFN-γ to differentiate human monocytes into macrophages and characterized the cells at a functional and proteomic level. IFN-γ alone was sufficient to generate macrophages (IFN-γ Mϕ) that were phagocytic and responsive to polarization. We demonstrate that IFN-γ Mϕ are potent activators of T lymphocytes that produce IL-17 and IFN-γ. We identified potential markers (GBP-1, IP-10, IL-12p70, and IL-23) of IFN-γ Mϕ and demonstrate that these markers are enriched in the skin of patients with inflamed psoriasis. Collectively, we show that IFN-γ can drive human monocyte to macrophage differentiation, leading to bona fide macrophages with inflammatory characteristics.

Pharmacological validation of targets regulating CD14 during macrophage differentiation.

The signalling receptor for LPS, CD14, is a key marker of, and facilitator for, pro-inflammatory macrophage function. Pro-inflammatory macrophage differentiation remains a process facilitating a broad array of disease pathologies, and has recently emerged as a potential target against cytokine storm in COVID19. Here, we perform a whole-genome CRISPR screen to identify essential nodes regulating CD14 expression in myeloid cells, using the differentiation of THP-1 cells as a starting point. This strategy uncovers many known pathways required for CD14 expression and regulating macrophage differentiation while additionally providing a list of novel targets either promoting or limiting this process. To speed translation of these results, we have then taken the approach of independently validating hits from the screen using well-curated small molecules. In this manner, we identify pharmacologically tractable hits that can either increase CD14 expression on non-differentiated monocytes or prevent CD14 upregulation during macrophage differentiation. An inhibitor for one of these targets, MAP2K3, translates through to studies on primary human monocytes, where it prevents upregulation of CD14 following M-CSF induced differentiation, and pro-inflammatory cytokine production in response to LPS. Therefore, this screening cascade has rapidly identified pharmacologically tractable nodes regulating a critical disease-relevant process.

Structure-Based Design of a Bromodomain and Extraterminal Domain (BET) Inhibitor Selective for the N-Terminal Bromodomains That Retains an Anti-inflammatory and Antiproliferative Phenotype.

The bromodomain and extraterminal domain (BET) family of epigenetic regulators comprises four proteins (BRD2, BRD3, BRD4, BRDT), each containing tandem bromodomains. To date, small molecule inhibitors of these proteins typically bind all eight bromodomains of the family with similar affinity, resulting in a diverse range of biological effects. To enable further understanding of the broad phenotype characteristic of pan-BET inhibition, the development of inhibitors selective for individual, or sets of, bromodomains within the family is required. In this regard, we report the discovery of a potent probe molecule possessing up to 150-fold selectivity for the N-terminal bromodomains (BD1s) over the C-terminal bromodomains (BD2s) of the BETs. Guided by structural information, a specific amino acid difference between BD1 and BD2 domains was targeted for selective interaction with chemical functionality appended to the previously developed I-BET151 scaffold. Data presented herein demonstrate that selective inhibition of BD1 domains is sufficient to drive anti-inflammatory and antiproliferative effects.

Targeting Histone Deacetylases in Myeloid Cells Inhibits Their Maturation and Inflammatory Function With Limited Effects on Atherosclerosis.

Monocytes and macrophages are key drivers in the pathogenesis of inflammatory diseases. Epigenetic targets have been shown to control the transcriptional profile and phenotype of these cells. Since histone deacetylase protein inhibitors demonstrate profound anti-inflammatory activity, we wanted to test whether HDAC inhibition within monocytes and macrophages could be applied to suppress inflammation in vivo. ESM technology conjugates an esterase-sensitive motif (ESM) onto small molecules to allow targeting of cells that express carboxylesterase 1 (CES1), such as mononuclear myeloid cells. This study utilized an ESM-HDAC inhibitor to target monocytes and macrophages in mice in both an acute response model and an atherosclerosis model. We demonstrate that the molecule blocks the maturation of peritoneal macrophages and inhibits pro-inflammatory cytokine production in both models but to a lesser extent in the atherosclerosis model. Despite regulating the inflammatory response, ESM-HDAC528 did not significantly affect plaque size or phenotype, although histological classification of the plaques demonstrated a significant shift to a less severe phenotype. We hereby show that HDAC inhibition in myeloid cells impairs the maturation and activation of peritoneal macrophages but shows limited efficacy in a model of atherosclerosis.

Influence of discrete and continuous culture conditions on human mesenchymal stem cell lineage choice in RGD concentration gradient hydrogels.

Stem cells have shown lineage-specific differentiation when cultured on substrates possessing signaling groups derived from the native tissue. A distinct determinant in this process is the concentration of the signaling motif. While several groups have been working actively to determine the specific factors, concentrations, and mechanisms governing the differentiation process, many have been turning to combinatorial and gradient approaches in attempts to optimize the multiple chemical and physical parameters needed for the next advance. However, there has not been a direct comparison between the cellular behavior and differentiation of human mesenchymal stem cells cultured in gradient and discrete substrates, which quantitates the effect of differences caused by cell-produced, soluble factors due to design differences between the culture systems. In this study, the differentiation of human mesenchymal stem cells in continuous and discrete polyethylene glycol dimethacrylate (PEGDM) hydrogels containing an RGD concentration gradient from 0 to 14 mM were examined to study the effects of the different culture conditions on stem-cell behavior. Culture condition was found to affect every osteogenic (alkaline phosphatase, Runx 2, type 1 collagen, bone sailoprotein, and calcium content) and adipogenic marker (oil red and peroxisome proliferator-activated receptor gamma) examined regardless of RGD concentration. Only in the continuous gradient culture did RGD concentration affect human mesenchymal stem-cell lineage commitment with low RGD concentrations expressing higher osteogenic differentiation than high RGD concentrations. Conversely, high RGD concentrations expressed higher adipogenic differentiation than low RGD concentrations. Cytoskeletal actin organization was only affected by culture condition at low RGD concentrations, indicating that it played a limited role in the differences in lineage commitment observed. Therefore, the role of discrete versus gradient strategies in high-throughput experimentation needs to be considered when designing experiments as we show that the respective strategies alter cellular outcomes even though base scaffolds have similar material and chemical properties.

Maximizing phenotype constraint and extracellular matrix production in primary human chondrocytes using arginine-glycine-aspartate concentration gradient hydrogels.

New systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using the limited cell numbers obtained from primary human sources. Peptide functionalized hydrogels possessing continuous variations in physico-chemical properties are an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient of arginine-glycine-aspartic acid peptide (RGD) concentrations from 0mM to 10mM. The system is used to correlate primary human osteoarthritic chondrocyte proliferation, phenotype maintenance and extracellular matrix (ECM) production to the gradient hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher RGD concentrations, while regions with lower RGD concentrations maintained cell number and phenotype. Over three weeks of culture, hydrogel regions containing lower RGD concentrations experience an increase in ECM content compared to regions with higher RGD concentrations. Variations in actin amounts and vinculin organization were observed within the RGD concentration gradients that contribute to the differences in chondrogenic phenotype maintenance and ECM expression.