The epigenetic reader Brd4 is required for osteoblast differentiation.

Transcription networks and epigenetic mechanisms including DNA methylation, histone modifications, and noncoding RNAs control lineage commitment of multipotent mesenchymal progenitor cells. Proteins that read, write, and erase histone tail modifications curate and interpret the highly intricate histone code. Epigenetic reader proteins that recognize and bind histone marks provide a crucial link between histone modifications and their downstream biological effects. Here, we investigate the role of bromodomain-containing (BRD) proteins, which recognize acetylated histones, during osteogenic differentiation. Using RNA-sequencing (RNA-seq) analysis, we screened for BRD proteins (n = 40) that are robustly expressed in MC3T3 osteoblasts. We focused functional follow-up studies on Brd2 and Brd4 which are highly expressed in MC3T3 preosteoblasts and represent "bromodomain and extra terminal domain" (BET) proteins that are sensitive to pharmacological agents (BET inhibitors). We show that small interfering RNA depletion of Brd4 has stronger inhibitory effects on osteoblast differentiation than Brd2 loss as measured by osteoblast-related gene expression, extracellular matrix deposition, and alkaline phosphatase activity. Similar effects on osteoblast differentiation are seen with the BET inhibitor +JQ1, and this effect is reversible upon its removal indicating that this small molecule has no lasting effects on the differentiation capacity of MC3T3 cells. Mechanistically, we find that Brd4 binds at known Runx2 binding sites in promoters of bone-related genes. Collectively, these findings suggest that Brd4 is recruited to osteoblast-specific genes and may cooperate with bone-related transcription factors to promote osteoblast lineage commitment and maturation.

Lessons from a Minimal Genome: What Are the Essential Organizing Principles of a Cell Built from Scratch?

One of the primary challenges facing synthetic biology is reconstituting a living system from its component parts. A particularly difficult landmark is reconstituting a self-organizing system that can undergo autonomous chromosome compaction, segregation, and cell division. Here, we discuss how the syn3.0 minimal genome can inform us of the core self-organizing principles of a living cell and how these self-organizing processes can be built from the bottom up. The review underscores the importance of fundamental biology in rebuilding life from its molecular constituents.

Reactivity and mechanism of α-nucleophile scaffolds as catalytic organophosphate scavengers.

Despite their unique benefits imparted by their structure and reactivity, certain α-nucleophile molecules remain underexplored as chemical inactivators for the topical decontamination of reactive organophosphates (OPs). Here, we present a library of thirty α-nucleophile scaffolds, each designed with either a pyridinium aldoxime (PAM) or hydroxamic acid (HA) α-nucleophile core tethered to a polar or charged scaffold for optimized physicochemical properties and reactivity. These library compounds were screened for their abilities to catalyze the hydrolysis of a model OP, paraoxon (POX), in kinetic assays. These screening experiments led to the identification of multiple lead compounds with the ability to inactivate POX two- to four-times more rapidly than Dekon 139-the active ingredient currently used for skin decontamination of OPs. Our mechanistic studies, performed under variable pH and temperature conditions suggested that the differences in the reactivity and activation energy of these compounds are fundamentally attributable to the core nucleophilicity and pKa. Following their screening and mechanistic studies, select lead compounds were further evaluated and demonstrated greater efficacy than Dekon 139 in the topical decontamination of POX in an ex vivo porcine skin model. In addition to OP reactivity, several compounds in the PAM class displayed a dual mode of activity, as they retained the ability to reactivate POX-inhibited acetylcholine esterase (AChE). In summary, this report describes a rationale for the hydrophilic scaffold design of α-nucleophiles, and it offers advanced insights into their chemical reactivity, mechanism, and practical utility as OP decontaminants.

Hydrophilic scaffolds of oxime as the potent catalytic inactivator of reactive organophosphate.

Despite its efficacy as a skin decontaminant of reactive organophosphates (OP), Dekon 139-a potassium salt of 2,3-butanedione monooxime (DAM)-is associated with adverse events related to percutaneous absorption largely due to its small size and lipophilicity. In order to address this physicochemical issue, we synthesized and evaluated the activity of a focused library of 14 hydrophilic oxime compounds, each designed with either a DAM or monoisonitrosoacetone (MINA) oxime tethered to a polar or charged scaffold in order to optimize the size, hydrophilicity, and oxime acidity. High-throughput colorimetric assays were performed with paraoxon (POX) as a model OP to determine the kinetics of POX inactivation by these compounds under various pH and temperature conditions. This primary screening led to the identification of 6 lead compounds, predominantly in the MINA series, which displayed superb catalytic activity by reducing the POX half-life (t1/2) by 2-3 fold relative to Dekon 139. Our mechanistic studies show that POX inactivation by the oxime compounds occurred faster at a higher temperature and in a pH-dependent manner in which the negatively charged oximate species is ≥ 10-fold more effective than the neutral oxime species. Lastly, using one of the lead compounds, we demonstrated its promising efficacy for POX decontamination in porcine skin ex vivo, and showed its potent ability to protect acetylcholine esterase (AChE) through POX inactivation. In summary, we report the rational design and chemical biological validation of novel hydrophilic oximes which address an unmet need in therapeutic OP decontamination.

Oritavancin Retains a High Affinity for a Vancomycin-Resistant Cell-Wall Precursor via Its Bivalent Motifs of Interaction.

Despite its potent antibacterial activities against drug-resistant Gram-positive pathogens, oritavancin remains partially understood with respect to its primary mode of hydrogen bond interaction with a cell-wall peptide regarding the role of its lipophilic 4'-chlorobiphenyl moiety. Here we report a surface plasmon resonance (SPR) study performed in two cell-wall model surfaces, each prepared by immobilization with a vancomycin-susceptible Lys-d-Ala-d-Ala or vancomycin-resistant Lys-d-Ala-d-Lac peptide. Analysis of binding kinetics performed on the peptide surface showed that oritavancin bound ∼100-1000-fold more tightly than vancomycin on each model surface. Ligand competition experiments conducted by SPR and fluorescence spectroscopy provided evidence that such affinity enhancement can be attributed to its 4'-chlorobiphenyl moiety, possibly through a hydrophobic interaction that led to a gain of free energy with a contribution from enthalpy as suggested by a variable-temperature SPR experiment. On the basis of these findings, we propose a model for the bivalent motifs of interaction of oritavancin with cell-wall peptides, by which the drug molecule can retain a strong interaction even with the vancomycin-resistant peptide. In summary, this study advances our understanding of oritavancin and offers new insight into the significance of bivalent motifs in the design of glycopeptide antibiotics.