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•We found new crystalline forms of bromhexine hydrochloride and determined their crystal structures.•Br-K edge X-ray fine structure spectroscopy can be used to identify the crystal ...form of bromhexine.•X-ray fine structure spectroscopy can be used to identify the crystalline form of active pharmaceutical ingredients in commercial tablets packaged within PTP sheets.
Different crystal forms of active pharmaceutical ingredients (APIs) may display variations in physicochemical properties. During the drug development process, the definitive purpose is to maintain homogeneous quality in a single crystalline form. Hence, it is important to evaluate and understand the properties of each crystal form of APIs in pharmaceutics. In this study, forms 0, Ⅰ, Ⅱ, III of bromhexine hydrochloride, and form S of bromhexine were characterized by the commonly used methods X-ray powder diffraction, thermogravimetry–differential thermal analysis, and single crystal structure X-ray diffraction. Additionally, X-ray absorption fine structure spectroscopy (XAFS), a seldom used method in the pharmaceutics discipline was also applied to explore the chemical environment of bromine atoms in forms 0, Ⅰ, Ⅱ and S as well as chloride ions in forms 0 to Ⅱ. The XAFS spectra of each form were different from each of the other forms which indicated the chemical environment around target elements in the crystal polymorphs were distinct. Then, we measured the commercial bromhexine hydrochloride tablets with XAFS measurement and found that XAFS could distinguish the crystal form in the tablets. Hence, we demonstrated that XAFS measurements would be applicable as one of the methods for the direct detection of APIs in the tablets.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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Structural biology of the core autophagy machinery Suzuki, Hironori; Osawa, Takuo; Fujioka, Yuko ...
Current opinion in structural biology,
April 2017, 2017-Apr, 2017-04-00, 20170401, Volume:
43
Journal Article
Peer reviewed
Open access
•Atg13 facilitates autophagy initiation complex formation and supramolecular assembly.•Autophagy-specific phosphatidylinositol 3-kinase complex has a V-shaped architecture.•Atg9 vesicles, with the ...help of the autophagy initiation complexes, form membrane source of autophagosomes.
In autophagy, which is an intracellular degradation system that is conserved among eukaryotes, degradation targets are sequestered through the de novo synthesis of a double-membrane organelle, the autophagosome, which delivers them to the lysosomes for degradation. The core autophagy machinery comprising 18 autophagy-related (Atg) proteins in yeast plays an essential role in autophagosome formation; however, the molecular role of each Atg factor and the mechanism of autophagosome formation remain elusive. Recent years have seen remarkable progress in structural biological studies on the core autophagy machinery, opening new avenues for autophagy research. This review summarizes recent advances in structural biological and mechanistic studies on the core autophagy machinery and discusses the molecular mechanisms of autophagosome formation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 ...subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.
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•An essential autophagy factor Atg13 has a large intrinsically disordered region (IDR)•The IDR of Atg13 contains two distinct regions binding intermolecularly to Atg17•Tethering two Atg17 molecules by Atg13 leads to supramolecular Atg1 complex assembly•The Atg13-mediated supramolecular assembly is responsible for autophagy initiation
Yamamoto et al. uncover insights into early steps of yeast autophagosome assembly, where autophagy-related (Atg) proteins localize to a perivacuolar site to organize the pre-autophagosomal structure (PAS). Atg13, via an intrinsically disordered region, provides a flexible linkage between two Atg17 molecules to mediate multimeric Atg1 complex self-assembly for PAS organization.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy analyses were used to evaluate the crystals of the active pharmaceutical ingredients, eletriptan hydrobromide, ...dextromethorphan hydrobromide and scopolamine hydrobromide salts and the solid dispersion of eletriptan hydrobromide. The crystals and the solid dispersion of the active pharmaceutical ingredient (API) salts could be discriminated based on the shape of the XANES spectra. The differences in the shape of XANES spectra was ascribable to the differences in the interatomic interactions of the bromine ions based on the crystal structures. Ratio of the eletriptan hydrobromide α-form crystal in mixed powders of α-form and monohydrate crystals could be quantified by the linear-combination fitting using their XANES spectra. These results indicated that the XANES spectroscopy are a potent method for evaluating the APIs of pharmaceutical formulations even at the higher energy region around the bromine K-edge of 13470 eV.
Polymorphic crystals of ambroxol, forms I and II, and form A ambroxol hydrochloride crystals were characterized with bromine K-edge X-ray absorption near-edge structure (XANES) spectroscopy and ...single-crystal X-ray structure analysis. The XANES spectra had unique shapes depending on the crystal forms. Refined single-crystal structures revealed different interatomic interactions around bromine atoms, such as C–H…Br and N–H…Br hydrogen bonds, Br…O halogen bonds, and N–H…π interactions. Differences in these weak interactions could affect the electronic states of the bromines, resulting in differences in the XANES spectra. The results demonstrated that weak non-conventional interatomic interactions could alter the shape of XANES spectra. Hence, the spectra could be used for evaluating polymorphs of active pharmaceutical ingredients.
X-ray absorption near-edge structure (XANES) spectroscopy is a new method for the characterization of active pharmaceutical ingredients. XANES spectra show unique features depending on the electronic ...states of the X-ray absorbing elements and provide information about the chemical environment that affects the electronic states. In this study, six bisphosphonate hydrate crystals were used to investigate, for the first time, how the phosphorus K-edge XANES spectra are affected by the interatomic interactions and charged states of phosphonate moieties. Phosphorus K-edge XANES spectra showed several differences among the bisphosphonates. In particular, the chlorine atoms covalently bonded near the phosphonate and the number of electric charges of the phosphonate moieties seemed to have large effects on peak shape in XANES spectra. Unique shapes of the XANES spectra demonstrated that differences in interactions at the oxygen atoms of the phosphonate moieties could change the shapes of the XANES spectrum peaks to the extent that each material was distinguished based on the spectra. Since slight differences in interatomic interactions and charged states lead to variations in the spectra, XANES spectroscopy could be widely applied as the fingerprint method to evaluate active pharmaceutical ingredients.
Chlorine K-edge X-ray absorption near-edge spectroscopy (XANES) measurements were performed to characterize the crystal polymorphs of identical active pharmaceutical ingredients (APIs) containing ...chloride atoms and their amorphous solid dispersions (ASDs). Indomethacin (IMC), of which three crystal forms (α, β, and γ) have been reported, was used as a model API. The shape of XANES spectra was unique to each IMC crystal. The analysis of the crystal structures of IMC revealed that chlorine atoms of the IMCα form had unique intermolecular interactions and halogen bonds with oxygen atoms, while those of the IMCγ form do not have any notable interactions. This result showed that XANES measurements can detect weak interatomic interactions. The shapes of the ASD spectra were clearly different from those of the crystals, suggesting that the environment around the Cl atom of IMC was different from that of the crystals. A thermal stress test was then performed to study the transformation from the amorphous form to the crystalline form of IMC in the ASD. The powder X-ray diffraction (PXRD) patterns indicated that amorphous IMC transformed into crystals during the thermal stress test. In accordance with the PXRD results, the XANES spectra also transformed from ASD to crystalline form. These results indicate that the IMC transformation could be monitored by XANES measurement. Our findings led us to conclude that XANES measurement is a novel approach for the evaluation of crystal polymorphs of APIs and the crystalline state of APIs in ASDs.
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IJS, KILJ, NUK, PNG, UL, UM
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Amorphous forms of disodium etidronate were prepared by three manufacturing methods, heat drying, freeze drying, and anti-solvent precipitation, and the effects of these methods on ...the physical properties of disodium etidronate amorphous forms were evaluated for the first time. Variable temperature X-ray powder diffraction and thermal analyses revealed that these amorphous forms had different physical properties such as glass transition point, water desorption, and crystallization temperatures. These differences can be explained by the molecular mobility and water content in amorphous forms. The differences in the structural characteristics related to the differences in these physical properties could not be detected clearly by the spectroscopic methods like Raman spectroscopy and X-ray absorption near-edge spectroscopy. Dynamic vapor sorption analyses demonstrated that all amorphous forms were hydrated to form I, a tetrahydrated form, at above 50% relative humidity, and the transition to form I was irreversible. These amorphous forms require strict humidity control to avoid crystallization. Among the three amorphous forms of disodium etidronate, the amorphous form prepared by heat drying was the most suitable for manufacturing the solid formulation, considering the low water content and low molecular mobility.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The crystal forms of active pharmaceutical ingredients must be precisely determined to properly regulate their physical properties. In this study, chlorine K-edge X-ray absorption near-edge structure ...(XANES) measurements were performed on four different crystals of the gastroprokinetic drug mosapride, which contains a covalently bound chlorine atom. The XANES spectrum of the mosapride propylene glycol solvate crystal (Mosa-PG) showed unique features compared to those of the other three crystals despite the identical chemical structures of the chlorine atoms. Single-crystal structural analyses revealed unique interatomic interactions in the Mosa-PG crystal, viz., the close van der Waals contact between the chlorine and hydrogen atoms and the hydrogen bond scheme of the amino moiety that forms a close intramolecular interaction with the chlorine atom. These results showed that chlorine K-edge XANES is sensitive to weak interatomic interactions, suggesting that XANES spectroscopy may be widely applicable to studying drug polymorphs.
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IJS, KILJ, NUK, PNG, UL, UM
Atg101 is an essential component of the autophagy-initiating ULK complex in higher eukaryotes, but it is absent from the functionally equivalent Atg1 complex in budding yeast. Here, we report the ...crystal structure of the fission yeast Atg101-Atg13 complex. Atg101 has a Hop1, Rev7 and Mad2 (HORMA) architecture similar to that of Atg13. Mad2 HORMA has two distinct conformations (O-Mad2 and C-Mad2), and, intriguingly, Atg101 resembles O-Mad2 rather than the C-Mad2-like Atg13. Atg13 HORMA from higher eukaryotes possesses an inherently unstable fold, which is stabilized by Atg101 via interactions analogous to those between O-Mad2 and C-Mad2. Mutational studies revealed that Atg101 is responsible for recruiting downstream factors to the autophagosome-formation site in mammals via a newly identified WF finger. These data define the molecular functions of Atg101, providing a basis for elucidating the molecular mechanisms of mammalian autophagy initiation by the ULK complex.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SBMB, UILJ, UKNU, UL, UM, UPUK
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