1-O-Octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-GS-441524 (V2043). Evaluation of Oral V2043 in a Mouse Model of SARS-CoV-2 Infection and Synthesis and Antiviral Evaluation of Additional Phospholipid Esters with Enhanced Anti-SARS-CoV-2 Activity

Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease caused by COVID-19. An orally bioavailable RDV analog may facilitate earlier treatment of non-hospitalized COVID-19 patients. Here we describe the synthesis and evaluation of alkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. Oral treatment of SARS-CoV-2-infected BALB/c mice with 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (60 mg/kg orally, once daily for 5 days starting 12h after infection) reduced lung viral load by 1.5 log10 units versus vehicle at day 2 and to below the limit of detection at day 5. Structure/activity evaluation of additional analogs that have hydrophobic ethers at the sn-2 of glycerol revealed improved in vitro antiviral activity by introduction of a 3-fluoro-4-methoxy-substituted benzyl or a 3- or 4-cyano-substituted benzyl. Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections

Subcellular and submitochondrial localization of the biosynthesis of cardiolipin and related phospholipids in rat liver

1. 1. Intact mitochondria, inner and outer mitochondrial membranes and microsomes were isolated from rat liver and their purity determined with specific marker enzymes. 2. 2. Diphosphatidylglycerol synthesis was found exclusively in the inner mitochondrial membrane. Phosphatidylglycerol was predominantly synthesized in theinner membrane although a small contribution was noted in the outer membrane. CDP and dCDP diglyceride synthesis occurred primarily in the microsomal fraction. 3. 3. Diphosphatidylglycerol synthesis from phosphatidylglycerol was shown to require CDP diglyceride or dCDP diglyceride and a divalent cation (either Mg24, Mn2+ or Co2+). The reaction was strongly inhibited by nonionic detergents such as Triton X-10

The mechanism of cardiolipin biosynthesis in liver mitochondria

1. 1. When added exogenously, both CDP- and dCDP-diglyceride supported diphosphatidylglycerol synthesis from phosphatidyl[1'-14C] glycerol in intact mitochondria. The maximum rate observed for CDP-diglyceride was about 2-fold greater than for dCDP-diglyceride. At optimal concentrations of CDP-diglyceride a 30-fold stimulation of diphosphatidylglycerol synthesis was observed. 2. 2. [14C] Glycerol was not formed in significant amounts during mitochondrial conversion of phosphatidyl[1'-14C] glycerol to diphosphatidylglycerol. 3. 3. In mitochondria, [2-3H]phosphatidyl[1'-14C] glycerol was converted in the presence of unlabeled CDP-diglyceride to a diphosphatidylglycerol having a nearly identical 3H/14C ratio. The above evidence confirms that diphosphatidylglycerol is formed from phosphatidylglycerol and CDP-diglyceride in mitochondria. 4. 4. In contrast, in preparations of membranes from Escherichia coli K12, the diphosphatidylglycerol formed at low concentrations of CDP-diglyceride had a 3H/14C ratio of 2.0 relative to its precursor [2-3H]phosphatidyl[1'-14C] glycerol. Furthermore, significant amounts of [14C] glycerol were isolated after incubation of the E. coli membranes with phosphatidyl[1'-14C]glycerol. These findings indicate that in E. coli diphosphatidylglycerol is formed from two molecules of phosphatidylglycerol. However, evidence is presented which suggests the operation of the CDP-diglyceride pathway in E. coli at higher concentrations of CDP-diglyceride

Biosynthesis of cardiolipin in liver mitochondria

Diphosphatidylglycerol (cardiolipin) biosynthesis from CDP-diglyceride and sn-glycerol-3-phosphate has been demonstrated in a preparation of mitochondria from rat liver in a reaction requiring Mg²⁺. The identity of the diphosphatidylglycerol was established by thin-layer chromatography and mild alkaline hydrolysis. Diphosphatidylglycerol formation from [³H]phosphatidylglycerol was shown to occur only in the presence of CDP-diglyceride. The rate of diphosphatidylglycerol formation appears to be quite slow in mitochondria and may explain why it was not previously observed. Evidence is presented which eliminates the possibility of a bacterial contribution to the biosynthetic process observed

Some studies on the metabolism of phospholipids in plasma membranes from rat liver

1. 1. Rat liver plasma membranes were isolated by rate-isopycnic zonal centrifugation. A method is described for the size 14 zonal rotor. The isolated membranes had an isopycnic banding density of 41.2% sucrose (w/w). On the basis of studies with eleven marker enzymes and electron microscopy, at least 90% of the fraction consisted of plasma membranes. The electron micrographs showed a predominantly vesicular appearance with few junctional complexes. 2. 2. Plasma membranes exhibited virtually no CDP-choline: 1, 2-diacyl-sn-glycerol cholinephosphotransferase activity and thus appear incapable of significant in vitro synthesis of phosphatidylcholine by the cytidine nucleotide pathway. 3. 3. Phosphatidylglycerol was the major product of sn-glycerol-3-phosphate esterification by plasma membranes in the presence of CDP-diglyceride at pH 7.5. The product was identified by thin-layer chromatography using pure phosphatidylglycerol as a reference compound, and by paper chromatography of the products obtained by hydrolysis with phospholipase D. 4. 4. Incubation of plasma membranes with labeled exogenous phospholipids in the presence of Ca2+ resulted in the formation of fatty acids and monoacylphosphoglycerides. Analyses of the reaction products indicated that the phospholipase A mainly attacks the 2-position of the phosphoglyceride molecule. Plasma membrane phospholipase A also acted on labeled endogenous phosphatidylethanolamine in the presence of Ca2+. 5. 5. A partial characterization of the phospholipase indicated that it required Ca2+ for optimal activity, was insensitive to N-ethylmaleimide and deoxycholate, and was relatively heat stable. Its pH optimum was 8.0. The following was the preferred order of hydrolysis: phosphatidylethanolamine > phosphatidylglycerol > > > phosphatidylcholine. 6. 6. Lysophospholipase was not detected in rat liver plasma membranes under conditions in which maximal activity of this enzyme was observed in rat liver 100 000 × g supernatant

The synthesis and utilization of dCDP-diglyceride by a mitochondrial fraction from rat liver

1. 1. Mitochondrial preparations from rat liver incorporate added dCTP into a compound that is readily extracted with chloroform. This compound was identified as dCDP-diglyceride by co-chromatography with authentic dCDP-diglyceride on silica gels with three different solvent systems and by the absence of an effect of borate on its RF value. 2. 2. The apparent Km for dCTP in dCDP-diglyceride synthesis by intact mitochondria was 4 · 10−5 M; the vmax was 1.1 · 10−2 nmoles/min per mg protein. Synthesis was inhibited by rCTP, whereas the incorporation of the rCDP moiety of rCTP into rCDP-diglyceride was inhibited by dCTP. 3. 3. dCDP-Diglyceride substituted for rCDP-diglyceride in the synthesis of phosphatidylglycerol from sn-glycero-3-phosphate by these mitochondrial preparations. The apparent Km's were 2 · 10−5 M for dCDP-diglyceride and 7 · 10−5 M for rCDP-diglyceride. The vmax was 0.42 · 10−2 nmole/min per mg protein with dCDP-diglyceride and 2.5 · 10−2 nmole/min per mg protein with rCDP-diglyceride as substrate

Phospholipid transfer activities in morris hepatomas and the specific contribution of the phosphatidylcholine exchange protein

Phospholipid transfer activities for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were measured in three hepatomas of increasing growth rate and degree of dedifferentiation, the hepatomas 7787, 9633 and 7777, and compared to the activities found in normal and host liver. A 2–3-fold increase was found in the phosphatidylcholine and phosphatidyinositol transfer activities in the fast-growing 7777 hepatoma, while these activities were moderately or not increased in the 7787 and 9633 hepatomas. Phosphatidylethanolamine transfer was found to be extremely low in all three hepatomas. The possible significance of these findings with respect to the altered phospholipid content and composition of the hepatoma membranes is discussed. The contribution of the phosphatidylcholine specific exchange protein to the total phosphatidylcholine transfer activity was determined in normal and host liver and in the hepatomas 7777 and 9633 with the aid of a phosphatidylcholine exchange protein specific antiserum. To this end a new procedure for the purification of the phosphatidylcholine exchange protein from rat liver was developed which leads to a final purification factor of 5300 and a high overall yield of 17%. In addition, this protein was chemically and immunologically characterized and its properties were compared to those of the bovine phosphatidylcholine exchange protein purified in our laboratory previously