S-nitroso-L-cysteine stereoselectively blunts the adverse effects of morphine on breathing and arterial blood gas chemistry while promoting analgesia.

S-nitrosothiols exert multiple effects on neural processes in the central and peripheral nervous system. This study shows that intravenous infusion of S-nitroso-L-cysteine (SNO-L-CYS, 1 µmol/kg/min) in anesthetized male Sprague Dawley rats elicits (a) sustained increases in minute ventilation, via increases in frequency of breathing and tidal volume, (b) a decrease in Alveolar-arterial (A-a) gradient, thus improving alveolar gas-exchange, (c) concomitant changes in arterial blood-gas chemistry, such as an increase in pO2 and a decrease in pCO2, (d) a decrease in mean arterial blood pressure (MAP), and (e) an increase in tail-flick (TF) latency (antinociception). Infusion of S-nitroso-D-cysteine (SNO-D-CYS, 1 µmol/kg/min, IV), did not elicit similar responses, except for a sustained decrease in MAP equivalent to that elicited by SNO-L-CYS. A bolus injection of morphine (2 mg/kg, IV) in rats receiving an infusion of vehicle elicited (a) sustained decreases in frequency of breathing tidal volume, and therefore minute ventilation, (b) a sustained decrease in MAP, (c) sustained decreases in pH, pO2 and maximal sO2 with sustained increases in pCO2 and A-a gradient, and (d) a sustained increase in TF latency. In rats receiving SNO-L-CYS infusion, morphine elicited markedly smaller changes in minute ventilation, arterial blood gas chemistry, A-a gradient and MAP. In contrast, the antinociceptive effects of morphine were enhanced in rats receiving the infusion of SNO-L-CYS. The morphine-induced responses in rats receiving SNO-D-CYS infusion were similar to vehicle-infused rats. These data are the first to demonstrate that infusion of an S-nitrosothiol, such as SNO-L-CYS, can stereoselectively ameliorate the adverse effects of morphine on breathing and alveolar gas exchange while promoting antinociception.

S-Nitroso-L-Cysteine Stereoselectively Blunts the Deleterious Effects of Fentanyl on Breathing While Augmenting Antinociception in Freely-Moving Rats.

Endogenous and exogenously administered S-nitrosothiols modulate the activities of central and peripheral systems that control breathing. We have unpublished data showing that the deleterious effects of morphine on arterial blood-gas chemistry (i.e., pH, pCO2, pO2, and sO2) and Alveolar-arterial gradient (i.e., index of gas exchange) were markedly diminished in anesthetized Sprague Dawley rats that received a continuous intravenous infusion of the endogenous S-nitrosothiol, S-nitroso-L-cysteine. The present study extends these findings by showing that unanesthetized adult male Sprague Dawley rats receiving an intravenous infusion of S-nitroso-L-cysteine (100 or 200 nmol/kg/min) markedly diminished the ability of intravenous injections of the potent synthetic opioid, fentanyl (10, 25, and 50 µg/kg), to depress the frequency of breathing, tidal volume, and minute ventilation. Our study also found that the ability of intravenously injected fentanyl (10, 25, and 50 µg/kg) to disturb eupneic breathing, which was measured as a marked increase of the non-eupneic breathing index, was substantially reduced in unanesthetized rats receiving intravenous infusions of S-nitroso-L-cysteine (100 or 200 nmol/kg/min). In contrast, the deleterious effects of fentanyl (10, 25, and 50 µg/kg) on frequency of breathing, tidal volume, minute ventilation and non-eupneic breathing index were fully expressed in rats receiving continuous infusions (200 nmol/kg/min) of the parent amino acid, L-cysteine, or the D-isomer, namely, S-nitroso-D-cysteine. In addition, the antinociceptive actions of the above doses of fentanyl as monitored by the tail-flick latency assay, were enhanced by S-nitroso-L-cysteine, but not L-cysteine or S-nitroso-D-cysteine. Taken together, these findings add to existing knowledge that S-nitroso-L-cysteine stereoselectively modulates the detrimental effects of opioids on breathing, and opens the door for mechanistic studies designed to establish whether the pharmacological actions of S-nitroso-L-cysteine involve signaling processes that include 1) the activation of plasma membrane ion channels and receptors, 2) selective intracellular entry of S-nitroso-L-cysteine, and/or 3) S-nitrosylation events. Whether alterations in the bioavailability and bioactivity of endogenous S-nitroso-L-cysteine is a key factor in determining the potency/efficacy of fentanyl on breathing is an intriguing question.

D-cysteine ethyl ester and D-cystine dimethyl ester reverse the deleterious effects of morphine on arterial blood-gas chemistry and Alveolar-arterial gradient in anesthetized rats.

We determined whether intravenous injections of the membrane-permeable ventilatory stimulants, D-cysteine ethyl ester (ethyl (2 S)- 2-amino-3-sulfanylpropanoate) (D-CYSee) and D-cystine dimethyl ester (methyl (2 S)- 2-amino-3-[[(2 S)- 2-amino-3-methoxy-3-oxopropyl]disulfanyl] propanoate) (D-CYSdime), could overcome the deleterious actions of intravenous morphine on arterial blood pH, pCO2, pO2 and sO2, and Alveolar-arterial (A-a) gradient (i.e., the measure of exchange of gases in the lungs) in Sprague Dawley rats anesthetized with isoflurane. Injection of morphine (2 mg/kg, IV) caused pronounced reductions in pH, pO2 and sO2 accompanied by elevations in pCO2, all which are suggestive of diminished ventilation, and elevations in A-a gradient, which suggests a mismatch of ventilation-perfusion. Subsequent boluses of D-cysteine ethyl ester (2 ×100 µmol/kg, IV) or D-cystine dimethyl ester (2 ×50 µmol/kg, IV) rapidly reversed of the negative actions of morphine on pH, pCO2, pO2 and sO2, and A-a gradient. Similar injections of D-cysteine (2 ×100 µmol/kg, IV) were without effect, whereas injections of D-cystine (2 ×50 µmol/kg, IV) produced a modest reversal. Our data show that D-cysteine ethyl ester and D-cystine dimethyl ester readily overcome the deleterious effects of morphine on arterial blood gas (ABG) chemistry and A-a gradient by mechanisms that may depend upon their ability to rapidly enter cells. As a result of their known ability to enter the brain, lungs, muscles of the chest wall, and most likely the major peripheral chemoreceptors (i.e., carotid bodies), the effects of the thiolesters on changes in ABG chemistry and A-a gradient elicited by morphine likely involve central and peripheral mechanisms. We are employing target prediction methods to identify an array of in vitro and in vivo methods to test potential functional proteins by which D-CYSee and D-CYSdime modulate the effects of morphine on breathing.

D-Cystine di(m)ethyl ester reverses the deleterious effects of morphine on ventilation and arterial blood gas chemistry while promoting antinociception.

We have identified thiolesters that reverse the negative effects of opioids on breathing without compromising antinociception. Here we report the effects of D-cystine diethyl ester (D-cystine diEE) or D-cystine dimethyl ester (D-cystine diME) on morphine-induced changes in ventilation, arterial-blood gas chemistry, A-a gradient (index of gas-exchange in the lungs) and antinociception in freely moving rats. Injection of morphine (10 mg/kg, IV) elicited negative effects on breathing (e.g., depression of tidal volume, minute ventilation, peak inspiratory flow, and inspiratory drive). Subsequent injection of D-cystine diEE (500 µmol/kg, IV) elicited an immediate and sustained reversal of these effects of morphine. Injection of morphine (10 mg/kg, IV) also elicited pronounced decreases in arterial blood pH, pO2 and sO2 accompanied by pronounced increases in pCO2 (all indicative of a decrease in ventilatory drive) and A-a gradient (mismatch in ventilation-perfusion in the lungs). These effects of morphine were reversed in an immediate and sustained fashion by D-cystine diME (500 µmol/kg, IV). Finally, the duration of morphine (5 and 10 mg/kg, IV) antinociception was augmented by D-cystine diEE. D-cystine diEE and D-cystine diME may be clinically useful agents that can effectively reverse the negative effects of morphine on breathing and gas-exchange in the lungs while promoting antinociception. Our study suggests that the D-cystine thiolesters are able to differentially modulate the intracellular signaling cascades that mediate morphine-induced ventilatory depression as opposed to those that mediate morphine-induced antinociception and sedation.

Structures of SF3b1 reveal a dynamic Achilles heel of spliceosome assembly: Implications for cancer-associated abnormalities and drug discovery.

The pre-mRNA splicing factor SF3b1 exhibits recurrent mutations among hematologic malignancies and cancers, and consequently is a major therapeutic target of clinically-advanced spliceosome inhibitors. In this review, we highlight and rigorously analyze emerging views of SF3b1 conformational transitions, including the human SF3b particle either in isolation or bound to spliceosome inhibitors, and human or yeast spliceosome assemblies. Among spliceosome states characterized to date, an SF3b1 α-helical superhelix significantly closes to surround a U2 small nuclear RNA duplex with the pre-mRNA branch point sequence. The SF3b1 torus is locally unwound at an active site adenosine, whereas protein cofactors appear to stabilize overall closure in the spliceosome. Network analyses demonstrates that the natural SF3b1 dynamics mimic its conformational change in the spliceosome, raising the possibility of conformational selection underpinning spliceosome assembly. These dynamic SF3b1 conformations have consequences for gatekeeping of spliceosome assembly and therapeutic targeting of its cancer-associated dysfunction.

Molecular dynamics simulations reveal specific interactions of post-translational palmitoyl modifications with rhodopsin in membranes.

We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations-as well as their extensive fluctuations-suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin.

Concerted interconversion between ionic lock substates of the beta(2) adrenergic receptor revealed by microsecond timescale molecular dynamics.

The recently solved crystallographic structures for the A(2A) adenosine receptor and the beta(1) and beta(2) adrenergic receptors have shown important differences between members of the class-A G-protein-coupled receptors and their archetypal model, rhodopsin, such as the apparent breaking of the ionic lock that stabilizes the inactive structure. Here, we characterize a 1.02 mus all-atom simulation of an apo-beta(2) adrenergic receptor that is missing the third intracellular loop to better understand the inactive structure. Although we find that the structure is remarkably rigid, there is a rapid influx of water into the core of the protein, as well as a slight expansion of the molecule relative to the crystal structure. In contrast to the x-ray crystal structures, the ionic lock rapidly reforms, although we see an activation-precursor-like event wherein the ionic lock opens for approximately 200 ns, accompanied by movements in the transmembrane helices associated with activation. When the lock reforms, we see the structure return to its inactive conformation. We also find that the ionic lock exists in three states: closed (or locked), semi-open with a bridging water molecule, and open. The interconversion of these states involves the concerted motion of the entire protein. We characterize these states and the concerted motion underlying their interconversion. These findings may help elucidate the connection between key local events and the associated global structural changes during activation.

Role of solvent in determining conformational preferences of alanine dipeptide in water.

Evidence from a variety of spectroscopic probes indicates that (phi, psi) values corresponding to the left-handed polyproline II helix (P(II)) are preferred for short alanine-based peptides in water. On the basis of results from theoretical studies, it is believed that the observed preference is dictated by favorable peptide-solvent interactions, which are realized through formation of optimal hydrogen-bonding water bridges between peptide donor and acceptor groups. In the present study, we address this issue explicitly by analyzing the hydration structure and thermodynamics of 16 low-energy conformers of the alanine dipeptide (N-acetylalanine-N'-methylamide) in liquid water. Monte Carlo simulations in the canonical ensemble were performed under ambient conditions with all-atom OPLS parameters for the alanine dipeptide and the TIP5P model for water. We find that the number of hydrogen-bonded water molecules connecting the peptide group donor and acceptor atoms has no effect on the solvation thermodynamics. Instead, the latter are determined by the work done to fully hydrate the peptide. This work is minimal for conformations that are characterized by a minimal overlap of the primary hydration shells around the peptide donor and acceptor atoms. As a result, peptide-solvent interactions favor "compact" conformations that do not include P(II)-like geometries. Our main conclusion is that the experimentally observed preference for P(II) does not arise due to favorable direct interactions between the peptide and water molecules. Instead, the latter act to unmask underlying conformational preferences that are a consequence of minimizing intrapeptide steric conflicts.