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.

l-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.

Efavirenz is a second-generation nonnucleoside reverse transcriptase inhibitor (NNRTI) and a common component of clinically approved anti-AIDS regimens. NNRTIs are noncompetitive inhibitors that bind in a hydrophobic pocket in the p66 subunit of reverse transcriptase (RT) ∼10 Å from the polymerase active site. Hydrogen exchange mass spectrometry (HXMS) shows that efavirenz binding reduces molecular flexibility in multiple regions of RT heterodimer in addition to the NNRTI binding site. Of the 47 peptic fragments monitored by HXMS, 15 showed significantly altered H/D exchange rates in the presence of efavirenz. The slow cooperative unfolding of a β-sheet in the NNRTI binding pocket, which was previously observed in unliganded RT, is dramatically suppressed by efavirenz. HXMS also defines an extensive network of allosterically coupled sites, including four distinct regions of allosteric stabilization, and one region of allosteric destabilization. The effects of efavirenz binding extend > 60 Å from the NNRTI binding pocket. Allosteric changes to the structural dynamics propagate to the thumb and connection subdomains and RNase H domain of the p66 subunit as well as the thumb and palm subdomains of the p51 subunit. These allosteric regions may represent potential new drug targets.

There is an urgent need to develop novel compounds that prevent the deleterious effects of opioids such as fentanyl on minute ventilation while, if possible, preserving the analgesic actions of the opioids. We report that L-glutathione ethyl ester (GSHee) may be such a novel compound. In this study, we measured tail flick latency (TFL), arterial blood gas (ABG) chemistry, Alveolar-arterial gradient, and ventilatory parameters by whole body plethysmography to determine the responses elicited by bolus injections of fentanyl (75 μg/kg, IV) in male adult Sprague–Dawley rats that had received a bolus injection of GSHee (100 μmol/kg, IV) 15 min previously. GSHee given alone had minimal effects on TFL, ABG chemistry and A-a gradient whereas it elicited changes in some ventilatory parameters such as an increase in breathing frequency. In vehicle-treated rats, fentanyl elicited (1) an increase in TFL, (2) decreases in pH, pO2 and sO2 and increases in pCO2 (all indicative of ventilatory depression), (3) an increase in Alveolar-arterial gradient (indicative of a mismatch in ventilation-perfusion in the lungs), and (4) changes in ventilatory parameters such as a reduction in tidal volume, that were indicative of pronounced ventilatory depression. In GSHee-pretreated rats, fentanyl elicited a more prolonged analgesia, relatively minor changes in ABG chemistry and Alveolar-arterial gradient, and a substantially milder depression of ventilation. GSHee may represent an effective member of a novel class of thiolester drugs that are able to prevent the ventilatory depressant effects elicited by powerful opioids such as fentanyl and their deleterious effects on gas-exchange in the lungs without compromising opioid analgesia.

Crystal structures and simulations suggest that conformational changes are critical for the function of HIV-1 reverse transcriptase. The enzyme is an asymmetric heterodimer of two subunits, p66 and p51. The two subunits have the same N-terminal sequence, with the p51 subunit lacking the Cterminal RNase H domain. We used hydrogen exchange mass spectrometry to probe the structural dynamics of RT. H/D exchange revealed that the fingers and palm subdomains of both subunits form the stable core of the heterodimer. In the crystal structure, the tertiary fold of the p51 subunit is more compact than that of the polymerase domain of the p66 subunit, yet both subunits show similar flexibility. The p66 subunit contains both the polymerase and RNase H catalytic sites. H/D exchange indicated that the RNase H domain of p66 is highly flexible. The β-sheet β12-β13-β14 lies at the base of the thumb subdomain of p66, and contains highly conserved residues involved in template/ primer binding and NNRTI binding. Using the unique ability of hydrogen exchange mass spectrometry to resolve slowly interconverting species, we found that β-sheet β12-β13-β14 undergoes slow cooperative unfolding with a t 1/2 of 6.6 s. The H/D exchange results are discussed in relation to existing structural, simulation, and sequence information.Reverse transcriptase performs the first step in replication of HIV. 1 RT copies the singlestranded viral RNA genome into a double-stranded proviral DNA prior to insertion by integrase into a chromosome of the infected cell (1). RT has RNA-and DNA-dependent DNA polymerase activities and RNase H activity. The mature enzyme is a heterodimer of p66 and p51 subunits; p51 has the same N-terminal polymerase domain as p66, but lacks the C-terminal NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptRNase H domain. The enzyme has an asymmetric structure (2). The p66 subunit contains the enzyme active sites (3), whereas the p51 subunit appears to have a structural function. The p66 polymerase domain has a right-handed conformation like other polymerases with fingers (residues 1-84, 120-150), palm (residues 85-119, 151-243), and thumb (residues 244-322) subdomains plus a connection subdomain (residues 323-427). The fingers, palm, and thumb subdomains of p66 form the template/primer binding cleft with the polymerase active site residues (D110, D185, and D186) in the palm subdomain (4). Although the four subdomains of the polymerase domain have similar folds in p66 and p51, their relative orientations differ in the two subunits (Figure 1).Numerous crystal structures are available for wild-type and mutant HIV-1 RTs in the absence and presence of various substrates and inhibitors. The conformation of p51 subunit is essentially the same in all the structures, whereas the p66 polymerase domain adopts both open and closed positions of the fingers and thumb subdomains, suggesting that RT is quite flexible. The crystallographic B-factors of two unliganded wild-type RT structures (same space group) identify...