Characterization of S-linked conjugates of the endogenous tripeptide glutathione (gamma-glutamyl-cysteinylglycine, GSH) represents a valuable indirect approach for the identification of chemically reactive, electrophilic intermediates formed during the metabolism of both foreign compounds and endogenous substances. In most cases, GSH adducts generated in vitro or excreted in the bile of animals are detected by the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing survey scans based on characteristic fragmentations of this class of conjugates. However, a limitation of current LC-MS/MS approaches, which typically employ electrospray ionization with analysis of positive ions, is that no single survey scan exhibits broad utility in the detection of unknown GSH adducts, since different structural classes of conjugate (aromatic, benzylic, aliphatic, thioester, etc.) behave differently upon collision-induced dissociation (CID) of the respective [M + H]+ parent ions. In the present study, we evaluated MS/MS in the negative ion mode as an alternative approach and report herein that the spectra obtained by CID of the [M - H]- ions of a number of representative GSH adducts, as well as GSH itself, are dominated by fragments originating from the glutathionyl moiety of the tripeptide. In particular, the anion at m/z 272, corresponding nominally to deprotonated gamma-glutamyl-dehydroalanyl-glycine, was abundant in the negative ion spectra of free GSH and all GSH conjugates examined, suggesting that scanning for precursors of this ion may provide a generally applicable technique for the detection of adducts of unknown structure. The utility of this novel detection strategy was demonstrated in a series of in vitro and in vivo experiments where compounds known to undergo metabolic activation were examined for their propensity to form conjugates with GSH. In all cases, scanning for precursors of m/z 272 in the negative ion mode revealed the presence of the expected adducts and in some instances revealed additional conjugates that had not been reported previously. Positive ion MS/MS, on the other hand, was more useful than the corresponding negative ion scans in providing information on the molecular structure of GSH conjugates.
Conformational restrictions in the form of [i, i + 4] lactam bridges were sequentially incorporated into the shortest fragment of hPTH with recognized efficacy in the OVX rat model of osteoporosis, hPTH-(1-31)NH 2 (1). Cyclo(Lys 18 -Asp 22 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 22 ,Leu 27 ]hPTH(1-31)NH 2 (2) is a potent agonist of the PTH/PTHrP receptor located on the surface of ROS 17/2.8 cells as measured by its ability to stimulate adenylyl cyclase activity (EC 50 ) 0.29 nM). A second analogue, which constrains the entire C-terminal receptor binding domain, bicyclo(Lys 18 -Asp 22 ,Lys 26 -Asp 30 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 22 ,Leu 27 ] hPTH(1-31)NH 2 ( 6), is also shown to be a potent agonist (EC 50 ) 0.13 nM), thus providing further evidence for an extended helix as the relevant bioactive conformation in this region of the hormone. Adjacent lactam bridges were incorporated into the analogue bicyclo(Lys 13 -Asp 17 ,Lys 18 -Asp 22 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 17,22 ,Leu 27 ]hPTH(1-31)NH 2 (7) to evaluate the receptor's tolerance to conformational restriction in the midregion of the peptide. In fact, peptide 7 is also a highly potent agonist (EC 50 ) 0.43 nM) in the cAMP-based assay, which suggests that at least one bioactive form of the hormone requires a helical conformation extending from residue 13 to residue 22. Incorporation of all three lactam bridges afforded the most conformationally constrained PTH peptide agonist yet reported, tricyclo(Lys 13 -Asp 17 ,Lys 18 -Asp 22 ,Lys 26 -Asp 30 )[Ala 1 ,Nle 8 ,Lys 18 ,Asp 17,22 ,Leu 27 ]hPTH(1-31)NH 2 (9). Peptide 9 (EC 50 ) 0.14 nM) forces residues 13-30 into an extended helical conformation and is a more potent PTH receptor agonist than the parent linear hPTH(1-31)NH 2 (1, EC 50 ) 4.7 nM). Comparative circular dichroism studies indicate that peptide 9 is highly helical even in the absence of TFE, indicating that residues 1-12 are also likely to be helical in the bioactive conformation. Taken together, these results provide strong structural evidence that hPTH binds to its receptor in an extended helical conformation.
Current pain therapeutics suffer from undesirable psychotropic and sedative side effects, as well as abuse potential. Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels expressed in nerves of the spinal dorsal horn, where their activation is believed to reduce transmission of painful stimuli. Herein, we describe the identification and hit-to-lead optimization of a novel class of tricyclic sulfonamides as allosteric GlyR potentiators. Initial optimization of high-throughput screening (HTS) hit 1 led to the identification of 3, which demonstrated ex vivo potentiation of glycine-activated current in mouse dorsal horn neurons from spinal cord slices. Further improvement of potency and pharmacokinetics produced in vivo proof-of-concept tool molecule 20 (AM-1488), which reversed tactile allodynia in a mouse spared-nerve injury (SNI) model. Additional structural optimization provided highly potent potentiator 32 (AM-3607), which was cocrystallized with human GlyRα3 to afford the first described potentiator-bound X-ray cocrystal structure within this class of ligand-gated ion channels (LGICs).
Total scattering pair distribution function (TSPDF) analysis of synchrotron X-ray diffraction data has been used to study the structural characteristics of amorphous lactose and its subsequent recrystallization on aging. This shows that the recrystallization kinetics vary substantially between spray dried, lyophilized, and melt quenched samples aged at 40 °C/75% relative humidity (RH), although all samples consistently form the stable α monohydrate when crystallization does occur. Using TSPDF it was possible to quantify the amount of amorphous and crystalline phases present, as well as to extract other structural information such as crystallite size, as a function of time during aging from the different starting materials. We also were able to determine a correlation between a higher degree of local molecular ordering in the amorphous phase with decreased stability against recrystallization. This study shows the rich information that may be obtained from a TSPDF analysis of recrystallization from the amorphous state in organic systems.
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