Thiophosphate Analogs of Coenzyme A and Its Precursors-Synthesis, Stability, and Biomimetic Potential

Abstract

Coenzyme A (CoA) is ubiquitous and essential for key cellular processes in any living organism. Primary degradation of CoA occurs by enzyme-mediated pyrophosphate hydrolysis intracellularly and extracellularly to form adenosine 3',5'-diphosphate and 4'-phosphopantetheine (PPanSH). The latter can be recycled for intracellular synthesis of CoA. Impairments in the CoA biosynthetic pathway are linked to a severe form of neurodegeneration with brain iron accumulation for which no disease-modifying therapy is available. Currently, exogenous administration of PPanSH is examined as a therapeutic intervention. Here, we describe biosynthetic access to thiophosphate analogs of PPanSH, 3'-dephospho-CoA, and CoA. The stabilizing effect of thiophosphate modifications toward degradation by extracellular and peroxisomal enzymes was studied in vitro. Experiments in a CoA-deficient cell model suggest a biomimetic potential of the PPanSH thiophosphate analog P_SPanSH (C1). According to our findings, the administration of P_SPanSH may provide an alternative approach to support intracellular CoA-dependent pathways.

Keywords: CoA; CoA biosynthesis; CoA deficiency; CoA degradation; HoPan; NBIA; Nudt7; PKAN; PPanSH; phosphorothioate.

Figure 1

Synthesis of thiophosphate analogs of PPanSH, dpCoA, and CoA starting from pantetheine. (A,B) Reaction scheme for CoA and CoA thiophosphate analogs. The phosphorylation catalyzed by E. coli pantothenate kinase (PanK) yields PPanSH or a thiophosphate analog of PPanSH. The AMP transfer catalyzed by E. coli phosphopantetheine adenylyltransferase (PPAT) yields dpCoA or thiophosphate analogs of dpCoA. The phosphorylation catalyzed by E. coli dpCoA kinase (DPCK) yields CoA or thiophosphate analogs of CoA. (B) Colored letters in parentheses show the type of atom (oxygen or sulfur) in position X, Y, or Z of the respective compound shown in (A). Path descriptions display whether ATP or a thiophosphate analog of ATP was used to produce the respective compound. Black lines show the canonical path. Green lines show the paths to accessible novel products. Red lines show the paths to inaccessible products (n.a.: not accessible). (C) Approximate retention times of compounds during purification by reversed-phase high-performance liquid chromatography. (D) ³¹P nuclear magnetic resonance (NMR) spectra of purified molecules and commercially obtained CoA. Obtained amounts of C6b were too low to record a ³¹P NMR spectrum (ppm: parts per million).

Figure 2

Stability of thiophosphate analogs of PPanSH, dpCoA, and CoA. (A) Overview of potential degradation pathways of C1. PanSH: pantetheine. (B) Stability of C1 in fetal bovine serum (FBS). The sample was monitored by ³¹P NMR spectroscopy. The signal of C1 was integrated and normalized to the phosphate peak arising from FBS. (C) Overview of pyrophosphate hydrolysis of dpCoA and CoA as well as thiophosphate analogs thereof. (D) Quantification of the conversion of dpCoA and C2a to PPanSH or C3a and C3b to C1 after incubation in FBS. (E) Quantification of the conversion of CoA and C4 to PPanSH after incubation in FBS. (F) Quantification of the conversion of dpCoA and C2a to PPanSH or C3a and C3b to C1 after incubation with human Nudt7. (D–F) Thiol compounds are quantified after conjugation to the fluorescence dye 7-diethylamino-3-(4-maleimidophenyl)-4-methyl-coumarin (CPM) and subsequent RP-HPLC separation. The peak heights of CPM conjugates were measured at 465 nm emission, and the product-to-substrate ratio was calculated. Error bars represent the standard deviation (n = 3).

Figure 3

Supplementation of cell cultures with C1. (A) Experiment design overview. HEK 293T cells were seeded on day 1 and treated with calcium hopantenate (HoPan), PPanSH, C1, or a combination on day 2. Viable cells were quantified by a colorimetric cell proliferation assay on day 5. (B) Percentage of viable cells after growth in the presence of varying concentrations of PPanSH or C1 relative to untreated cells. (C) Percentage of viable cells after growth in the presence of 500 µM HoPan, 500 µM HoPan, and 25 µM PPanSH, or 500 µM HoPan and 25 µM C1 relative to untreated cells (control). The statistical analysis was carried out by two-sample t-test (n = 6), and double asterisks (**) represent a p-value < 0.01. (B,C) Error bars represent the standard deviation (n = 6). (D) Simplified scheme for the intracellular fate of C1. Red arrows indicate non-conversion. The green arrow indicates conversion. Black arrows are still hypothetical. PanSH: pantetheine.