Spoxazomicin D and Oxachelin C, Potent Neuroprotective Carboxamides from the Appalachian Coal Fire-Associated Isolate Streptomyces sp. RM-14-6

Abstract

The isolation and structure elucidation of six new bacterial metabolites [spoxazomicin D (2), oxachelins B and C (4, 5), and carboxamides 6–8] and 11 previously reported bacterial metabolites (1, 3, 9–12a, and 14–18) from Streptomyces sp. RM-14-6 is reported. Structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry data analysis, along with direct comparison to synthetic standards for 2, 11, and 12a,b. Complete 2D NMR assignments for the known metabolites lenoremycin (9) and lenoremycin sodium salt (10) were also provided for the first time. Comparative analysis also provided the basis for structural revision of several previously reported putative aziridine-containing compounds [exemplified by madurastatins A1, B1, C1 (also known as MBJ-0034), and MBJ-0035] as phenol-dihydrooxazoles. Bioactivity analysis [including antibacterial, antifungal, cancer cell line cytotoxicity, unfolded protein response (UPR) modulation, and EtOH damage neuroprotection] revealed 2 and 5 as potent neuroprotectives and lenoremycin (9) and its sodium salt (10) as potent UPR modulators, highlighting new functions for phenol-oxazolines/salicylates and polyether pharmacophores.

Identification of Neuroprotective Spoxazomicin and Oxachelin Glycosides via Chemoenzymatic Glycosyl-Scanning

“…Compound 4a was also found to decrease EtOH-induced increases in propidium iodide uptake. Post hoc analyses demonstrated this reduction in propidium iodide by 4a was observed at 0.10 and 1.0 μ M (Figure 4F; ~10 3 less potent than the parent 4 61 ). In EtOH-naïve medium, coexposure to compound 4a did reveal significant increases in prodidium iodide uptake at 0.01 and 0.10 μ M. In contrast, glycosylation had no apparent effect on antibacterial, antifungal, or anticancer activity, as both the parental NPs and all corresponding glycosides were inactive at the concentrations tested (≤60–120 μ M antibacterial/fungal; ≤20 μ M anticancer).…”

“…Putative substrates evaluated included 17 recently characterized metabolites from Streptomyces sp. RM-14-6 ( 3 – 7 and 9; 10 – 20 ; Figures 2 and S1), 61 five of which [spoxazomicin C ( 3 ), spoxazomicin D ( 4 ), N -salicyloyl-2-aminopropane-1,3-diol ( 6 ) (2 R )- N -salicyloyl-2-aminopropan-1-ol ( 7 ), and o -hydroxybenzamide ( 9 ); Figure 2] were identified as putative substrates (Figure 3A and B). Subsequent hit validation confirmed the results from the colorimetric screen with turnovers ranging from 6% to 68% based on HPLC integration, where, in some cases (e.g., 3 ), the multiple products observed were attributed to putative variant glycoside regioisomers as previously reported for OleD-catalyzed reactions.…”

“…29–39 While the fundamental mechanism(s) of neuroprotection remain to be determined, this study presents important structure–activity relationships that may support future target identification (e.g., via incorporation of sugars bearing reactive handles for affinity pull-down studies) and/or, given the impact of glucose conjugation on improving blood–brain barrier activity transport, 64–68 may also enhance biodistribution in the context of advancing leads to treat neurological deficits resulting from alcohol abuse. 61,69–71 …”

“…RM-14-6. 61 This analysis revealed the permissive engineered OleD Loki to catalyze the glycosylation of spoxazomicin C [an antitrypanosomal NP first reported from Streptosporangium oxazolinicum K07-0460(T)] 62 and oxachelin (a metal chelator first reported from Streptomyces sp. GW9/1258), 63 providing the first reported corresponding glycosides of naturally occurring phenol/oxazolines.…”

“…Cumulatively, this study further extends the demonstrated substrate scope of OleD Loki and, given the impact of glucose conjugation on improving blood–brain barrier transport, 64–68 may also offer a convenient strategy to modulate the properties of novel NPs to treat neurological deficits resulting from alcohol abuse. 61,69–71 …”