The chemical basis of serine palmitoyltransferase inhibition by myriocin.

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.

The relationship between plasma albumin, alkaline phosphatase and pyridoxal phosphate concentrations in plasma and red cells: Implications for assessing vitamin B6 status.

BACKGROUND: Plasma concentrations of most vitamins decrease as part of the systemic inflammatory response (SIR). Thus low plasma values do not necessarily indicate deficiency. Vitamin B6 status is usually assessed by measurement of pyridoxal phosphate (PLP) in plasma, although vitamin concentrations in blood cells tend to be better markers of cellular stores. In health, plasma PLP appears to be determined primarily by intake, its binding to albumin, and its hydrolysis by alkaline phosphatase (ALP). OBJECTIVE: To examine, using in vitro studies, the effect of albumin concentration and ALP activity on PLP concentration in plasma and red blood cells of healthy subjects (HS) and critically ill patients (CI). DESIGN: Heparin and EDTA (ALP inhibited) whole blood samples from HS (n = 8) and CI (n = 26) were incubated with PLP. Concentration of PLP in plasma and red cells was measured. Albumin and ALP levels were determined in plasma. RESULTS: In PLP incubated heparin samples, there was a strong direct relationship between albumin in the concentration range 10-44 g/L and increase in plasma PLP concentration (rs = 0.93, P < 0.001) and an inverse relationship with increase in red cell PLP concentration (rs = -0.90, P < 0.001). In contrast, ALP activity was inversely associated with increase in plasma PLP concentration (rs = -0.42; P = 0.013) and directly associated with red cell PLP concentration (rs = 0.49; P = 0.003). CONCLUSIONS: Plasma albumin concentration and to a lesser extent ALP activity influences PLP concentration in plasma and red cells. In conditions associated with low albumin (e.g. SIR) or altered ALP activity, red cell PLP measurements are more likely to be reliable than plasma measurements in differentiating true from apparent vitamin B6 deficiency and to guide vitamin B6 supplementation.

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous measurement of voriconazole, posaconazole and itraconazole.

Background Azole-based antifungals are the first-line therapy for some of the most common mycoses and are now also being used prophylactically to protect immunocompromised patients. However, due to variability in both their metabolism and bioavailability, therapeutic drug monitoring is essential to avoid toxicity but still gain maximum efficacy. Methods Following protein precipitation of serum with acetonitrile, 20 µL of extract was injected onto a 2.1 × 50 mm Waters Atlantis dC18 3 µm column. Detection was via a Waters Quattro Premier XE tandem mass spectrometer operating in ESI-positive mode. Multiple reaction monitoring (MRM) detected two product ions for each compound and one for each isotopically labelled internal standard. Ion suppression, linearity, stability, matrix effects, recovery, imprecision, lower limits of measuring interval and detection were all assessed. Results Optimal chromatographic separation was achieved using gradient elution over 8 minutes. Voriconazole, posaconazole and itraconazole eluted at 1.71, 2.73 and 3.41 min, respectively. The lower limits of measuring interval for all three compounds was 0.1 mg/L. The assay was linear to 10 mg/L for voriconazole (R2 = 0.995) and 5 mg/L for posaconazole (R2 = 0.990) and itraconazole (R2 = 0.991). The assay was both highly accurate and precise with % bias of voriconazole, posaconazole and itraconazole, respectively, when compared with previous NEQAS samples. The intra-assay precision (CV%) was 1.6%, 2.5% and 1.9% for voriconazole, posaconazole and itraconazole, respectively, across the linear range. Conclusion A simple and robust method has been validated for azole antifungal therapeutic drug monitoring. This new assay will result in a greatly improved sample turnaround time and will therefore vastly increase the clinical utility of azole antifungal drug monitoring.

Reconstitution of the pyridoxal 5'-phosphate (PLP) dependent enzyme serine palmitoyltransferase (SPT) with pyridoxal reveals a crucial role for the phosphate during catalysis.

The pyridoxal 5'-phosphate (PLP)-dependent enzyme serine palmitoyltransferase (SPT) is required for de novo sphingolipid biosynthesis. A previous study revealed a novel and unexpected interaction between the hydroxyl group of the l-serine substrate and the 5'-phosphate group of PLP. By using pyridoxal (PL), the dephosphorylated analogue of vitamin B6, we show here that this interaction is important for substrate specificity and optimal catalytic efficiency.