Antagonism of propofol anesthesia by alkyl-fluorobenzene derivatives.

Despite their frequent use across many clinical settings, general anesthetics are medications with lethal side effects and no reversal agents. A fluorinated analogue of propofol has previously been shown to antagonize propofol anesthesia in tadpoles and zebrafish, but little further investigation of this class of molecules as anesthetic antagonists has been conducted. A 13-member library of alkyl-fluorobenzene derivatives was tested in an established behavioral model of anesthesia in zebrafish at 5 days post fertilization. These compounds were examined for their ability to antagonize propofol and two volatile anesthetics, as well as their interaction with the anesthetic-binding model protein apoferritin. Two compounds provided significant antagonism of propofol, and when combined, were synergistic, suggesting more than one antagonist sensitive target site. These compounds did not antagonize the volatile anesthetics, indicating some selectivity amongst general anesthetics. For the compounds with the most antagonistic potency, similarities in structure and binding to apoferritin may be suggestive of competitive antagonism; however, this was not supported by a Schild analysis. This is consistent with multiple targets contributing to general anesthesia, but whether these are physiologic antagonists or are antagonists at only some subset of the many anesthetic potential targets remains unclear, and will require additional investigation.

Antagonism of Propofol Anesthesia by Alkyl-fluorobenzene Derivatives.

Despite their frequent use across many clinical settings, general anesthetics are medications with lethal side effects and no reversal agents. A fluorinated analogue of propofol has previously been shown to antagonize propofol anesthesia in tadpoles and zebrafish, but little further investigation of this class of molecules as anesthetic antagonists has been conducted. A 13-member library of alkyl-fluorobenzene derivatives was tested in an established behavioral model of anesthesia in zebrafish at 5 days post fertilization. These compounds were examined for their ability to antagonize propofol and two volatile anesthetics, as well as their binding to the anesthetic-binding model protein apoferritin. The two compounds demonstrating highest antagonistic potency were found to bind apoferritin in a manner similar to propofol. Selected compounds did not show antagonism of volatile anesthetics, indicating some selectivity of this antagonism. Similarities in structure and binding to apoferritin as well as a Schild analysis are suggestive of competitive antagonism, but like the anesthetics, the potential mechanism(s) of these antagonists will require further mechanistic investigation.

Synthesis and Characterization of a Diazirine-Based Photolabel of the Nonanesthetic Fropofol.

The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of ortho-azi-fropofol (AziFo) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.

Azi-medetomidine: Synthesis and Characterization of a Novel α2 Adrenergic Photoaffinity Ligand.

Agonists at the α2 adrenergic receptor produce sedation, increase focus, provide analgesia, and induce centrally mediated hypotension and bradycardia, yet neither their dynamic interactions with adrenergic receptors nor their modulation of neuronal circuit activity is completely understood. Photoaffinity ligands of α2 adrenergic agonists have the potential both to capture discrete moments of ligand-receptor interactions and to prolong naturalistic drug effects in discrete regions of tissue in vivo. We present here the synthesis and characterization of a novel α2 adrenergic agonist photolabel based on the imidazole medetomidine called azi-medetomidine. Azi-medetomidine shares protein association characteristics with its parent compound in experimental model systems and by molecular dynamics simulation of interactions with the α2A adrenergic receptor. Azi-medetomidine acts as an agonist at α2A adrenergic receptors, and produces hypnosis in Xenopus laevis tadpoles. Azi-medetomidine competes with the α2 agonist clonidine at α2A adrenergic receptors, which is potentiated by photolabeling, and azi-medetomidine labels moieties on the α2A adrenergic receptor as determined by mass spectrometry in a manner consistent with a simulated model. This novel α2 adrenergic agonist photolabel can serve as a powerful tool for in vitro and in vivo investigations of adrenergic signaling.

Peptide library approach to uncover phosphomimetic inhibitors of the BRCA1 C-terminal domain.

Many intracellular protein-protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein-protein interactions mediated by serine phosphorylation.

Replacing amino acids in translation: expanding chemical diversity with non-natural variants.

Here, we describe a strategy for synthesis of peptides with multiple unnatural amino acids (UAAs) using in vitro translation. Our method involves removing a natural amino acid and replacing it with an UAA variant in a reconstituted translation system. Whereas other systems require engineered components or chemical synthesis to charge UAAs onto tRNAs prior to translation, our strategy utilizes the wild-type machinery and charging occurs concomitant with translation. The design of the system allows for easy quantification of the UAA's incorporation efficiency and fidelity.

BRCA1-directed, enhanced and aberrant homologous recombination: mechanism and potential treatment strategies.

Despite intense studies, questions still remain regarding the molecular mechanisms leading to the development of hereditary breast and ovarian cancers. Research focused on elucidating the role of the breast cancer susceptibility gene 1 (BRCA1) in the DNA damage response may be of the most critical importance to understanding these processes. The BRCA1 protein has an N-terminal RING domain possessing E3 ubiquitinligase activity and a C-terminal BRCT domain involved in binding specific phosphoproteins. These domains are involved directly or indirectly in DNA double-strand break (DSB) repair. As the two terminal domains of BRCA1 represent two separate entities, understanding how these domains communicate and are functionally altered in regards to DSB repair is critical for understanding the development of BRCA1-related breast and ovarian cancers and for developing novel therapeutics. Herein, we review recent findings of how altered functions of these domains might lead to cancer through a mechanism of increased aberrant homologous recombination and possible implications for the development of BRCA1 inhibitors.

Delayed acquisition of neonatal reflexes in newborn primates receiving a thimerosal-containing hepatitis B vaccine: influence of gestational age and birth weight.

This study examined whether acquisition of neonatal reflexes in newborn rhesus macaques was influenced by receipt of a single neonatal dose of hepatitis B vaccine containing the preservative thimerosal (Th). Hepatitis B vaccine containing a weight-adjusted Th dose was administered to male macaques within 24 h of birth (n = 13). Unexposed animals received saline placebo (n = 4) or no injection (n = 3). Infants were tested daily for acquisition of nine survival, motor, and sensorimotor reflexes. In exposed animals there was a significant delay in the acquisition of root, snout, and suck reflexes, compared with unexposed animals. No neonatal responses were significantly delayed in unexposed animals. Gestational age (GA) and birth weight (BW) were not significantly correlated. Cox regression models were used to evaluate main effects and interactions of exposure with BW and GA as independent predictors and time-invariant covariates. Significant main effects remained for exposure on root and suck when controlling for GA and BW, such that exposed animals were relatively delayed in time-to-criterion. Interaction models indicated there were various interactions between exposure, GA, and BW and that inclusion of the relevant interaction terms significantly improved model fit. This, in turn, indicated that lower BW and/or lower GA exacerbated the adverse effects following vaccine exposure. This primate model provides a possible means of assessing adverse neurodevelopmental outcomes from neonatal Th-containing hepatitis B vaccine exposure, particularly in infants of lower GA or BW. The mechanisms underlying these effects and the requirements for Th requires further study.

WITHDRAWN: Delayed acquisition of neonatal reflexes in newborn primates receiving a thimerosal-containing Hepatitis B vaccine: Influence of gestational age and birth weight.

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