Metabolism of GS-7340, a novel phenyl monophosphoramidate intracellular prodrug of PMPA, in blood

PMPA, an acyclic nucleoside phosphonate analog, is a potent inhibitor of HIV. In the cells, PMPA is efficiently phosphorylated by intracellular kinases to produce PMPApp, the pharmacologically active metabolite. Despite its demonstrated antiviral potency, PMPA has limited cell permeability presumably resulting from the presence of two negative charges on the phosphonyl group. To enhance intracellular concentrations of PMPA, we developed a prodrug, selectively metabolized inside cells. GS-7340 (9-[(R)-2-[[[[(S)-1-(isopropoxycarbonyl)ethyl] amino] phenoxy-phosphinyl]-methoxy] propyl] adenine) is a prodrug which is orally bioavailable in dogs as the intact prodrug and has demonstrated anti-HIV activity in cell culture of over 1000-fold greater than that of PMPA. The metabolism of PMPA in peripheral blood mononuclear cells (PBMC), red blood cells (RBC) and plasma was examined following exposure of whole blood to PMPA or GS-7340 at concentrations similar to ones observed systemically following oral administration in dogs. Following 1 hour incubation with whole blood, GS-7340 was stable in plasma, produced high levels of PMPA and its phosphorylated metabolites in PBMC but not in RBC. No intact prodrug was present in PBMC. The only other species present in PBMC was monoalaninyl PMPA. The levels of PMPA and the phosphorylated metabolites were over 20 times greater than those after incubation with PMPA. The dog and human blood data were similar. The intracellular levels of PMPA and PMPApp were roughly proportional to GS-7340 over a 10-fold concentration range indicating a lack of saturability of uptake and phosphorylation. Since PMPApp is the species responsible for antiviral activity of PMPA, the high intracellular levels of PMPApp should be an important indicator of greater clinical efficacy of GS-7340.     

METABOLISM OF GS-7340, A NOVEL PHENYL MONOPHOSPHORAMIDATE INTRACELLULAR PRODRUG OF PMPA,IN BLOOD

PMPA, an acyclic nucleoside phosphonate analog, is a potent inhibitor of HIV. In the cells, PMPA is efficiently phosphorylated by intracellular kinases to produce PMPApp, the pharmacologically active metabolite. Despite its demonstrated antiviral potency, PMPA has limited cell permeability presumably resulting from the presence of two negative charges on the phosphonyl group. To enhance intracellular concentrations of PMPA, we developed a prodrug, selectively metabolized inside cells. GS-7340 (9-[R)-2-[[[[S)-1-(isopropoxycarbonyl)ethyl] amino] phenoxy-phosphinyl]-methoxy] propyl] adenine) is a prodrug which is orally bioavailable in dogs as the intact prodrug and has demonstrated anti-HIV activity in cell culture of over 1000-fold greater than that of PMPA. The metabolism of PMPA in peripheral blood mononuclear cells (PBMC), red blood cells (RBC) and plasma was examined following exposure of whole blood to PMPA or GS-7340 at concentrations similar to ones observed systemically following oral administration in dogs. Following 1 hour incubation with whole blood, GS-7340 was stable in plasma, produced high levels of PMPA and its phosphorylated metabolites in PBMC but not in RBC. No intact prodrug was present in PBMC. The only other species present in PBMC was monoalaninyl PMPA. The levels of PMPA and the phosphorylated metabolites were over 20 times greater than those after incubation with PMPA. The dog and human blood data were similar. The intracellular levels of PMPA and PMPApp were roughly proportional to GS-7340 over a 10-fold concentration range indicating a lack of saturability of uptake and phosphorylation. Since PMPApp is the species responsible for antiviral activity of PMPA, the high intracellular levels of PMPApp should be an important indicator of greater clinical efficacy of GS-7340.     

High-performance liquid chromatographic determination of GS4071, a potent inhibitor of influenza neuraminidase, in plasma by precolumn fluorescence derivatization with naphthalenedialdehyde

GS4071 is a potent inhibitor of influenza neuraminidase. A precolumn fluorescence derivatization HPLC method is described for the analysis of GS4071 in rat plasma. Plasma samples were subjected to solid-phase extraction on C₁₈ extraction columns. After extraction, GS4071 was derivatized with naphthalenedialdehyde in the presence of potassium cyanide to produce highly fluorescent cyano[f]benzoisoindole derivatives. Derivatized samples were stable for >24 h at 4°C. The samples were analyzed by an isocratic HPLC method using fluorescence detection at 420 nm excitation and 470 nm emission wavelength. The method was validated and applied to the analysis of plasma samples from pre-clinical pharmacokinetic studies in rats. The limit of detection for GS4071 was 20 ng/ml. For five replicate samples at 50, 400, and 1000 ng/ml, the within-day precision values were 16.9, 9.4 and 4.5%, respectively, and the between-day precision values were 16.9, 7.9, and 2.1%, respectively. The method was linear from 25 to 1600 ng/ml and the total recovery was >68% over this concentration range.     

A cold-active esterase with a substrate preference for vinyl esters from a psychrotroph, Acinetobacter sp. strain no. 6: gene cloning, purification, and characterization

It has recently been shown that fatty acid vinyl esters serve as effective acylating agents for the synthesis of esters by enzymatic transesterification in high yields. To enhance the usefulness of this system at low temperatures, we have searched for the gene coding for a cold-active lipolytic enzyme with a substrate preference for fatty acid vinyl esters and obtained it from the genomic library of Acinetobacter sp. strain no. 6, a psychrotroph isolated from Siberian soil. The gene (termed aelh, 777 bp) encoded a protein of 258 amino acids, and sequence analysis revealed that the enzyme shows a high sequence similarity to β-ketoadipate enol-lactone hydrolase involved in the β-ketoadipate pathway for the bacterial catabolism of benzoic acid. The aelh gene was expressed in the E. coli C600 cells under the control of lac promoter and the expression product was purified to homogeneity and characterized. It was a monomeric esterase preferentially catalyzing the hydrolysis of enol esters, such as fatty acid vinyl esters with a short-chain acyl group. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, a specific inhibitor for serine hydrolases. The enzyme could also catalyze transesterification, for example, between vinyl propionate and propanol yielding propyl propionate at 4 °C. These results indicate the usefulness of an esterase (termed AELH) for the enzymatic synthesis of esters by transesterification using vinyl esters as an acyl donor.     

Nucleotide sequence of the Streptococcus mutans gtfD gene encoding the glucosyltransferase-S enzyme

The nucleotide sequence of the Streptococcus mutans GS-5 gtfD gene coding for the glucosyltransferase which synthesizes water-soluble glucan (GTF-S) has been determined. The complete gene contains 4293 base pairs and the unprocessed protein is composed of 1430 amino acids with a molecular mass of 159814 Da. The amino terminus of the unprocessed protein resembles the signal sequences of other extracellular proteins secreted by S. mutans and that of the GTF-I secreted by Streptococcus downei. In addition, the GTF-S protein exhibits high amino acid similarity with the strain GS-5 enzymes responsible for insoluble glucan synthesis (GTF-I, GTF-SI) previously isolated and sequenced in this laboratory. These results indicate that all three gtf genes evolved from a common ancestral gene.     

The Spleen Tyrosine Kinase Inhibitor,Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

Tyrosine kinase inhibitors (TKIs) are important in managing lymphoid malignancies by targeting B-cell receptor signaling pathways. Entospletinib (GS-9973) is an oral, selective inhibitor of spleen tyrosine kinase (Syk), currently in the phase II clinical trials for the treatment of chronic lymphocytic leukemia. Syk is abundantly present in the cells of hematopoietic lineage that mediates cell proliferation, differentiation, and adhesion. In this current study, we evaluated the efficacy of GS-9973 to overcome multidrug resistance (MDR) due to the overexpression of the ABCG2 transporter in the non-small cell lung cancer (NSCLC) cell line, NCI-H460/MX20. In vitro, 3 μM of GS-9973 reversed the drug resistance of NCI-H460/MX20 cell line to mitoxantrone or doxorubicin. GS-9973, at 3 μM reverses ABCG2-mediated MDR by blocking ABCG2 efflux activity and downregulating ABCG2 expression at the protein level but did not alter the ABCG2 mRNA expression and subcellular localization of the ABCG2 protein compared to drug-resistant cells incubated with the vehicle. GS-9973 produced a moderate concentration-dependent increase in the ATPase activity of ABCG2 (EC₅₀ = 0.42 µM) and molecular docking data indicated that GS-9973 had a high affinity (-10.226 kcal/mol) for the substrate-binding site of ABCG2. Finally, HPLC analysis proved that the intracellular concentration of GS-9973 is not significantly different in both parental and resistant cell lines. In conclusion, our study suggests that in vitro, GS-9973 in combination with certain anticancer drugs, represent a strategy to overcome ABCG2-mediated MDR cancers.     

Binding Adaptation of GS-441524 Diversifies Macro Domains and Downregulates SARS-CoV-2 de-MARylation Capacity

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD?GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.     

Development of a phosphatase-resistant, L-tyrosine derived LPA1/LPA3 dual antagonist

Lysophosphatidic acid (LPA) is a bioactive compound that has gained attention due to its role in neoplastic diseases. Our group has developed a potent dual LPA1/LPA3 receptor antagonist, VPC51098 (LPA1 IC(50) = 84 nM, LPA1 IC(50) = 48 nM) that contained a labile phosphate head group. This lability has impaired our evaluation of our scaffold of LPA receptor antagonists in vivo. We wished to replace the phosphate with a potentially more stable head group while retaining potency at both LPA1 and LPA3 to facilitate future in vivo studies. We tested in vitro potency of all head groups including α-methylene, α-fluoromethylene, α-hydroxymethylene; vinyl phosphonates; α-fluoro vinyl phosphonates. The most potent compound was found to be a low micromolar inhibitor VPC51299 that contained a vinyl phosphonate and possessed a half-life of approximately 90 min in rats when dosed intravenously. Herein, we describe the synthesis and initial biological evaluation of these compounds.     

Effect of service number on estrus duration in dairy goats

The object of this research was to study the effect of sterile service number on estrus duration in dairy goats. Twenty-four Nubian goats (20 nulliparous and 4 multiparous) were randomly assigned to 1 of 4 treatment groups (n = 6 animals per group). The following Groups were formed: no service (GS-0); 1 service (GS-1); 2 services (GS-2); 3 services (GS-3). Estrus was synchronized by using fluorogestone acetate intravaginal pessaries (40 mg) over a 12-d period plus 400 IU im pregnant mare serum gonadotropin (PMSG) at pessary removal. Estrus was detected by using a vasectomized buck at 6-h intervals over 5 d after pessary removal (at 0600, 1200, 1800 and 2400 h). In the GS-0 group the teaser was outfitted with an apron and was permitted to mount. In the GS-1, GS-2 and GS-3 groups, the teaser was permitted to mount and service 1, 2 and 3 times, respectively, within the first 12 h after initiation of estrus. The duration of estrus for the 4 groups (GS-0, GS-1, GS-2 and GS-3) was (mean +/- SD) 41.0 +/- 5.9, 24.0 +/- 5.4, 22.0 +/- 4.9 and 22.0 +/- 7.2 h, respectively. These results show differences between the serviced groups and the nonserviced group (P<0.01), but they fail to show differences among the serviced groups (P>0.05). It is concluded that sterile service shortens estrus duration and that service number (1, 2 or 3) does not affect estrus duration.     

Pharmacological evaluation of GS-389, a novel tetrahydroisoquinoline analog related to higenamine, on vascular smooth muscle.

The effects of GS-389, a novel tetrahydroisoquinoline analog, on isolated rat and mouse thoracic aorta rings, were investigated. Both GS-389 and papaverine induced endothelium-independent, concentration-dependent relaxations of the rat and mouse aortae precontracted with phenylephrine (PE). The GS-389-induced inhibition of the contractile response to PE was noncompetitive. The initial phasic contraction to PE elicited in Ca(2+)-free media was also attenuated by pretreatment with GS-389, indicating that GS-389 may interfere with the release of intracellular Ca2+ and/or the effects of intracellular Ca2+ release. GS-389 potentiated the vasodilatory effects of isoproterenol and sodium nitroprusside in rat and mouse aortae. GS-389 significantly increased cGMP levels in the rat aorta and inhibited cGMP phosphodiesterase from the rabbit brain. Methylene blue, but not propranolol, inhibited the vasodilatory effect of GS-389. These results suggest that the vasorelaxant effect of GS-389 may be due, at least in part, to inhibition of cGMP metabolism.     

Quantitation of peptide hormone in single cultured secretory neurons of the crab,Cardisoma carnifex

The content of crustacean hyperglycemic hormone (CHH) in single cultured neurons of the crab Cardisoma carnifex was determined by a sensitive enzyme-linked immunosorbent assay (ELISA), using purified CHH (1–50 pg) of the crab Carcinus maenas as standard. The somata were dissociated from the group of 150 peptidergic neurons that form the X-organ—sinus gland neuroendocrine system. As previously reported, the neurons show immediate regenerative outgrowth in defined culture conditions, and develop, generally, into one of two morphological types: cells that produce broad, lamelliform growth cones (veils), and others that are characterized by branching of neurites. In this study, all but one of 64 veiling cells taken after various times in culture up to 12 days contained CHH. They could be readily categorized as having high (>33 pg; mean 86±5, S.E., n=47) or low (33pg; mean 22±2.5; n=17) Carcinus CHH equivalents. Thus, CHH is associated with neurons showing veiling outgrowth, but veiling neurons with low CHH form a distinct, but not morphologically distinguishable group. They may contain an isoform of CHH with limited cross-reactivity. In 24 branching neurons assayed, Carcinus CHH equivalents averaged 7.2±2 pg. This figure includes 14 neurons in which CHH was undetectable, and one that had 40 pg of Carcinus CHH equivalents. There was no significant change of the hormone content in cells of either type during 6 days of culturing.     

Effects of neonatal treatment with phytoestrogens, genistein and daidzein, on sex difference in female rat brain function: estrous cycle and lordosis

It is well known that neonatal exposure to estrogen induces masculinization or defeminization of the brain. In this study, the effects of neonatal treatment with two kinds of soybean isoflavone aglycone, genistein (GS) and daidzein (DZ), on the estrous cycle and lordosis behavior were investigated. Female rats were injected subcutaneously with 1 mg GS, 1 mg DZ, 100 microg estradiol (E2), or oil daily for 5 days from birth. As a result, vaginal opening was advanced in GS- or E2-treated females. A vaginal smear check indicated that oil- or DZ-treated females showed a constant 4- or 5-day estrous cycle, whereas GS- or E2-treated rats showed a persistent or prolonged estrus. Ovariectomy was performed in all females at 60 days of age. The ovaries in the GS- or E2-treated groups were smaller than those in the oil- and DZ-treated groups and contained no corpora lutea. In the DZ group, although corpora lutea were seen, ovaries were smaller than that of control females. Behavioral tests were carried out after implantation of E2-tubes. All of the oil- or DZ-treated females showed lordosis with a high lordosis quotient (LQ). On the other hand, as male rats, LQs were extremely low in the E2-treated group, when compared to the oil-treated group. In the GS-treated group, the mean LQ was lower than that in the oil-treated group, but higher than those in the E2-treated female or male groups. These results suggest that genistein acts as an estrogen in the sexual differentiation of the brain and causes defeminization of the brain in regulating lordosis and the estrous cycle in rats. In addition, neonatal daidzein also has some influence on ovarian function.     

Mutations in Multiple Domains of Gag Drive the Emergence of In Vitro Resistance to the Phosphonate-Containing HIV-1 Protease Inhibitor GS-8374

GS-8374 is a potent HIV protease inhibitor (PI) with a unique diethyl-phosphonate moiety. Due to a balanced contribution of enthalpic and entropic components to its interaction with the protease (PR) active site, the compound retains activity against HIV mutants with high-level multi-PI resistance. We report here the in vitro selection and characterization of HIV variants resistant to GS-8374. While highly resistant viruses with multiple mutations in PR were isolated in the presence of control PIs, an HIV variant displaying moderate (14-fold) resistance to GS-8374 was generated only after prolonged passaging for >300 days. The isolate showed low-level cross-resistance to darunavir, atazanavir, lopinavir, and saquinavir, but not other PIs, and contained a single R41K mutation in PR combined with multiple genotypic changes in the Gag matrix, capsid, nucleocapsid, and SP2 domains. Mutations also occurred in the transframe peptide and p6* domain of the Gag-Pol polyprotein. Analysis of recombinant HIV variants indicated that mutations in Gag, but not the R41K in PR, conferred reduced susceptibility to GS-8374. The Gag mutations acted in concert, since they did not affect susceptibility when introduced individually. Analysis of viral particles revealed that the mutations rendered Gag more susceptible to PR-mediated cleavage in the presence of GS-8374. In summary, the emergence of resistance to GS-8374 involved a combination of substrate mutations without typical resistance mutations in PR. These substrate changes were distributed throughout Gag and acted in an additive manner. Thus, they are classified as primary resistance mutations indicating a unique mechanism and pathway of resistance development for GS-8374.