Niemann-Pick C1 (NPC1) is a lysosomal membrane protein that exports cholesterol derived from receptor-mediated uptake of LDL, and it also mediates cellular entry of Ebola virus. Cholesterol export is ...inhibited by nanomolar concentrations of U18666A, a cationic sterol. To identify the target of U18666A, we synthesized U-X, a U18666A derivative with a benzophenone that permits ultraviolet-induced crosslinking. When added to CHO cells, U-X crosslinked to NPC1. Crosslinking was blocked by U18666A derivatives that block cholesterol export, but not derivatives lacking blocking activity. Crosslinking was prevented by point mutation in the sterol-sensing domain (SSD) of NPC1, but not by point mutation in the N-terminal domain (NTD). These data suggest that the SSD contains a U18666A-inhibitable site required for cholesterol export distinct from the cholesterol-binding site in the NTD. Inasmuch as inhibition of Ebola requires 100-fold higher concentrations of U18666A, the high affinity U16888A-binding site is likely not required for virus entry.
Cancer-specific inhibitors that reflect the unique metabolic needs of cancer cells are rare. Here we describe Gboxin, a small molecule that specifically inhibits the growth of primary mouse and human ...glioblastoma cells but not that of mouse embryonic fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises oxygen consumption in glioblastoma cells. Gboxin relies on its positive charge to associate with mitochondrial oxidative phosphorylation complexes in a manner that is dependent on the proton gradient of the inner mitochondrial membrane, and it inhibits the activity of F
F
ATP synthase. Gboxin-resistant cells require a functional mitochondrial permeability transition pore that regulates pH and thus impedes the accumulation of Gboxin in the mitochondrial matrix. Administration of a metabolically stable Gboxin analogue inhibits glioblastoma allografts and patient-derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin, and shows that the increased proton gradient and pH in cancer cell mitochondria is a mode of action that can be targeted in the development of antitumour reagents.
Niemann-Pick C1 (NPC1), a lysosomal protein of 13 transmembrane helices (TMs) and three lumenal domains, exports low-density-lipoprotein (LDL)-derived cholesterol from lysosomes. TMs 3-7 of NPC1 ...comprise the Sterol-Sensing Domain (SSD). Previous studies suggest that mutation of the NPC1-SSD or the addition of the anti-fungal drug itraconazole abolishes NPC1 activity in cells. However, the itraconazole binding site and the mechanism of NPC1-mediated cholesterol transport remain unknown. Here, we report a cryo-EM structure of human NPC1 bound to itraconazole, which reveals how this binding site in the center of NPC1 blocks a putative lumenal tunnel linked to the SSD. Functional assays confirm that blocking this tunnel abolishes NPC1-mediated cholesterol egress. Intriguingly, the palmitate anchor of Hedgehog occupies a similar site in the homologous tunnel of Patched, suggesting a conserved mechanism for sterol transport in this family of proteins and establishing a central function of their SSDs.
Niemann–Pick C1 (NPC1), amembrane protein of lysosomes, is required for the export of cholesterol derived from receptor-mediated endocytosis of LDL. Lysosomal cholesterol export is reportedly ...inhibited by itraconazole, a triazole that is used as an antifungal drug Xu et al. (2010) Proc Natl Acad Sci USA 107:4764–4769. Here we show that posaconazole, another triazole, also blocks cholesterol export from lysosomes. We prepared P-X, a photoactivatable cross-linking derivative of posaconazole. P-X cross-linked to NPC1 when added to intact cells. Cross-linking was inhibited by itraconazole but not by ketoconazole, an imidazole that does not block cholesterol export. Cross-linking of P-X was also blocked by U18666A, a compound that has been shown to bind to NPC1 and inhibit cholesterol export. P-X also cross-linked to purified NPC1 that was incorporated into lipid bilayer nanodiscs. In this in vitro system, cross-linking of P-X was inhibited by itraconazole, but not by U18666A. P-X cross-linking was not prevented by deletion of the N-terminal domain of NPC1, which contains the initial binding site for cholesterol. In contrast, P-X cross-linking was reduced when NPC1 contained a point mutation (P691S) in its putative sterol-sensing domain. We hypothesize that the sterol-sensing domain has a binding site that can accommodate structurally different ligands.
Biomimetic synthesis describes the field of organic chemistry that aims to emulate the natural, biosynthetic processes toward natural products. As well as providing insight into how molecules are ...formed in nature, the benefits of this approach to total synthesis are numerous and extend beyond the gains typical of traditional synthesis. For example, using biosynthetic proposals to design a synthetic route can highlight alternative methods to the desired target. The pursuit of biomimetic syntheses also promotes the development of new reactions to prove or disprove a biosynthetic proposal or to unravel mechanistic implications of a proposed biosynthesis and can lead to the identification of new natural products. Here we look at some recent compelling examples and examine how biomimetic synthesis has led to the discovery of new procedures and principles that would not have been found by other approaches.
Summary
The sleep disorder narcolepsy, a hypocretin deficiency disorder thought to be due to degeneration of hypothalamic hypocretin/orexin neurons, is currently treated symptomatically. We evaluated ...the efficacy of two small molecule hypocretin/orexin receptor‐2 (HCRTR2) agonists in narcoleptic male orexin/tTA; TetO‐DTA mice. TAK‐925 (1–10 mg/kg, s.c.) and ARN‐776 (1–10 mg/kg, i.p.) were injected 15 min before dark onset in a repeated measures design. EEG, EMG, subcutaneous temperature (Tsc) and activity were recorded by telemetry; recordings for the first 6 h of the dark period were scored for sleep/wake and cataplexy. At all doses tested, TAK‐925 and ARN‐776 caused continuous wakefulness and eliminated sleep for the first hour. Both TAK‐925 and ARN‐776 caused dose‐related delays in NREM sleep onset. All doses of TAK‐925 and all but the lowest dose of ARN‐776 eliminated cataplexy during the first hour after treatment; the anti‐cataplectic effect of TAK‐925 persisted into the second hour for the highest dose. TAK‐925 and ARN‐776 also reduced the cumulative amount of cataplexy during the 6 h post‐dosing period. The acute increase in wakefulness produced by both HCRTR2 agonists was characterised by increased spectral power in the gamma EEG band. Although neither compound provoked a NREM sleep rebound, both compounds affected NREM EEG during the second hour post‐dosing. TAK‐925 and ARN‐776 also increased gross motor activity, running wheel activity, and Tsc, suggesting that the wake‐promoting and sleep‐suppressing activities of these compounds could be a consequence of hyperactivity. Nonetheless, the anti‐cataplectic activity of TAK‐925 and ARN‐776 is encouraging for the development of HCRTR2 agonists.
Platinum(II) and an unusual cationic gold(I) complex were identified as mild catalysts for the room temperature cycloisomerization or tandem hydroalkoxylation/acetal formation of unactivated internal ...alkynols. Under the appropriate conditions, 5-endo, 5-exo, 6-endo, and 6-exo cycloisomerization modes are all available.
The isolation, characterization, and total synthesis of the macrocyclic polyene mangrolide D is reported. A 16-step total synthesis relies on robust Suzuki and ring-closing metathesis reactions, and ...an iron-catalyzed hydroazidation of an exomethylene substituted tetrahydropyran as a key step for the synthesis of the appended 4-epi-vancosamine sugar. Although mangrolide D did not display antibiotic activity, this work should prove enabling toward the synthesis of the antitubercular tiacumicins which display a virtually identical macrocyclic backbone.
Efficient approaches that enable the synthesis of analogs of natural product antibiotics are needed to keep up with the emergence of multiply-resistant strains of pathogenic organisms. One promising ...candidate in this area is fidaxomicin, which boasts impressive in vitro anti-tubercular activity but has poor systemic bioavailability. We designed a flexible synthetic route to this target to enable the exploration of new chemical space and the future development of analogs with superior pharmacokinetics. We developed a robust approach to each of the key macrocyclic and sugar fragments, their union via stereoselective glycosylation, and a convergent late-stage macrolide formation with fully glycosylated fragments. Although we were able to demonstrate that the final Suzuki cross-coupling and ring-closing metathesis steps enabled macrocycle formation in the presence of the northern resorcylic rhamnoside and southern novioside sugars, these final steps were hampered by poor yields and the formation of the unwanted Z-macrocycle as the major stereoisomer.
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•Efficient stereoselective synthesis of the key polyketide fragments of Fidaxomicin.•Efficient stereoselective synthesis of Fidaxomicin’s resorcylic rhamnoside and novioside sugar fragments.•Efficient and selective glycosylation of the polyketide fragments with the sugar fragments.•Krische hydrogenative C–C coupling towards Fidaxomicin’s branched dienoic acid fragment.•2,3-Wittig rearrangement tactic to the polyketide stereodiad embedded within Fidaxomicin’s southern polyketide fragment.
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