Production of Four Flavonoid C-Glucosides in Escherichia coli.

Flavonoid C-glucosides, which are found in several plant families, are characterized by several biological properties, including antioxidant, anticancer, anti-inflammatory, neuroprotective, hepatoprotective, cardioprotective, antibacterial, antihyperalgesic, antiviral, and antinociceptive activities. The biosynthetic pathway of flavonoid C-glucosides in plants has been elucidated. In the present study, a pathway was introduced to Escherichia coli to synthesize four flavonoid C-glucosides, namely, isovitexin, vitexin, kaempferol 6-C-glucoside, and kaempferol 8-C-glucoside. A five- or six-step metabolic pathway for synthesizing flavonoid aglycones from tyrosine was constructed and two regioselective flavonoid C-glycosyltransferases from Wasabia japonica (WjGT1) and Trollius chinensis (TcCGT) were used. Additionally, the best shikimate gene module construct was selected to maximize the titer of each C-glucoside flavonoid. Isovitexin (30.2 mg/L), vitexin (93.9 mg/L), kaempferol 6-C-glucoside (14.4 mg/L), and kaempferol 8-C-glucoside (38.6 mg/L) were synthesized using these approaches. The flavonoid C-glucosides synthesized in this study provide a basis for investigating and unraveling their novel biological properties.

In Vitro Hepatic Uptake in Human and Monkey Hepatocytes in the Presence and Absence of Serum Protein and Its In Vitro to In Vivo Extrapolation.

It is well documented that human hepatic clearance based on in vitro metabolism or transporter assays systematically resulted in underprediction; therefore, large empirical scalars are often needed in either static or physiologically based pharmacokinetic (PBPK) models to accurately predict human pharmacokinetics (PK). In our current investigation, we assessed hepatic uptake in hepatocyte suspension in Krebs-Henseleit buffer in the presence and absence of serum. The results showed that the unbound intrinsic active clearance (CLu,int,active) values obtained by normalizing the unbound fraction in the buffer containing 10% serum were generally higher than the CLu,int,active obtained directly from protein free buffer, suggesting "protein-facilitated" uptake. The differences of CLu,int,active in the buffer with and without protein ranged from 1- to 925-fold and negatively correlated to the unbound serum binding of organic anion transporting polypeptide substrates. When using the uptake values obtained from buffer containing serum versus serum-free buffer, the median of scaling factors (SFs) for CLu,int,active reduced from 24.2-4.6 to 22.7-7.1 for human and monkey, respectively, demonstrating the improvement of in vitro to in vivo extrapolation in a PBPK model. Furthermore, values of CLu,int,active were significantly higher in monkey hepatocytes than that in human, and the species differences appeared to be compound dependent. Scaling up in vitro uptake values derived in assays containing species-specific serum can compensate for the species-specific variabilities when using cynomolgus monkey as a probe animal model. Incorporating SFs calibrated in monkey and together with scaled in vitro data can be a reliable approach for the prospective human PK prediction in early drug discovery. SIGNIFICANCE STATEMENT: We investigated the protein effect on hepatic uptake in human and monkey hepatocytes and improved the in vitro to in vivo extrapolation using parameters obtained from the incubation in the present of serum protein. In addition, significantly higher active uptake clearances were observed in monkey hepatocytes than in human, and the species differences appeared to be compound dependent. The physiologically based pharmacokinetic model that incorporates scaling factors calibrated in monkey and together with scaled in vitro human data can be a reliable approach for the prospective human pharmacokinetics prediction.

Efficacy of Oral Tenofovir Alafenamide/Emtricitabine Combination or Single-Agent Tenofovir Alafenamide Against Vaginal Simian Human Immunodeficiency Virus Infection in Macaques.

BACKGROUND: Tenofovir alafenamide (TAF)-based regimens are being evaluated for pre-exposure prophylaxis (PrEP). We used a macaque model of repeated exposures to simian human immunodeficiency virus (SHIV) to investigate whether TAF alone or the combination of TAF and emtricitabine (FTC) can prevent vaginal infection. METHODS: Pigtail macaques were exposed vaginally to SHIV162p3 once a week for up to 15 weeks. Animals received clinical doses of FTC/TAF (n = 6) or TAF (n = 9) orally 24 hours before and 2 hours after each weekly virus exposure. Infection was compared with 21 untreated controls. RESULTS: Five of the 6 animals in the FTC/TAF and 4 of the 9 animals in the TAF alone group were protected against infection (P = .001 and P = .049, respectively). The calculated efficacy of FTC/TAF and TAF was 91% (95% confidence interval [CI], 34.9%-98.8%) and 57.8% (95% CI, -8.7% to 83.6%), respectively. Infection in FTC/TAF but not TAF-treated macaques was delayed relative to controls (P = .005 and P = .114). Median tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells (PBMCs) were similar among infected and uninfected macaques receiving TAF PrEP (351 and 143 fmols/106 cells, respectively; P = .921). CONCLUSIONS: Emtricitabine/TAF provided a level of protection against vaginal challenge similar to FTC/TFV disoproxil fumarate combination in the macaque model. Our results support the clinical evaluation of FTC/TAF for PrEP in women.

Nucleotide Prodrug Containing a Nonproteinogenic Amino Acid To Improve Oral Delivery of a Hepatitis C Virus Treatment.

Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2'-C-methyl-1'-cyano-7-deaza-adenosine C-nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the (S) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620.

Sofosbuvir and Ribavirin Liver Pharmacokinetics in Patients Infected with Hepatitis C Virus.

Sofosbuvir and ribavirin exert their anti-hepatitis C virus (anti-HCV) activity following metabolic activation in the liver. However, intrahepatic concentrations of the pharmacologically active nucleotide metabolites in humans are poorly characterized due to the inaccessibility of tissue and technical challenges with measuring nucleotide levels. A clinical study assessing the efficacy of sofosbuvir and ribavirin administered prior to liver transplantation to prevent HCV recurrence provided a unique opportunity to quantify nucleotide concentrations in human liver. We analyzed nucleotides using high-performance liquid chromatography coupled to tandem mass spectrometry in liver tissue from 30 HCV-infected patients with hepatocellular carcinoma who were administered sofosbuvir (400 mg/day) and ribavirin (1,000 to 1,200 mg/day) for 3 to 52 weeks prior to liver transplantation. Median total hepatic metabolite concentrations (the sum of nucleoside and mono-, di-, and triphosphates) were 77.1 µM for sofosbuvir and 361 µM for ribavirin in patients on therapy at the time of transplantation. Ribavirin and sofosbuvir efficiently loaded the liver, with total hepatic metabolite concentrations exceeding maximal levels in plasma by approximately 30-fold. Ribavirin metabolite levels suggest that its monophosphate is in great excess of its inhibition constant for IMP dehydrogenase and that its triphosphate is approaching the binding constant for incorporation by the HCV NS5B RNA-dependent RNA polymerase. In accordance with the potent antiviral activity of sofosbuvir, these results demonstrate that the liver triphosphate levels achieved following sofosbuvir administration greatly exceed the inhibition constant for HCV NS5B. In conclusion, this study expands the quantitative understanding of the pharmacology of sofosbuvir and ribavirin by establishing efficient hepatic delivery in the clinic. (This study has been registered at ClinicalTrials.gov under identifier NCT01559844.).

Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.

Emerging viral infections are difficult to control because heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. Severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) successively emerged, causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. We show that a nucleotide prodrug, GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems, including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat CoVs, prepandemic bat CoVs, and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory function. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and, possibly most importantly, emerging CoV of the future.

Discovery of a 2'-fluoro-2'-C-methyl C-nucleotide HCV polymerase inhibitor and a phosphoramidate prodrug with favorable properties.

A series of 2'-fluorinated C-nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2'-fluoro-2'-C-methyl adenosine C-nucleoside (15) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant (53) with favorable pharmacokinetic properties including efficient liver delivery in animals.

Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys.

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

Implications of efficient hepatic delivery by tenofovir alafenamide (GS-7340) for hepatitis B virus therapy.

Tenofovir alafenamide (TAF) is a prodrug of tenofovir (TFV) currently in clinical evaluation for treatment for HIV and hepatitis B virus (HBV) infections. Since the target tissue for HBV is the liver, the hepatic delivery and metabolism of TAF in primary human hepatocytes in vitro and in dogs in vivo were evaluated here. Incubation of primary human hepatocytes with TAF resulted in high levels of the pharmacologically active metabolite tenofovir diphosphate (TFV-DP), which persisted in the cell with a half-life of >24 h. In addition to passive permeability, studies of transfected cell lines suggest that the hepatic uptake of TAF is also facilitated by the organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3, respectively). In order to inhibit HBV reverse transcriptase, TAF must be converted to the pharmacologically active form, TFV-DP. While cathepsin A is known to be the major enzyme hydrolyzing TAF in cells targeted by HIV, including lymphocytes and macrophages, TAF was primarily hydrolyzed by carboxylesterase 1 (CES1) in primary human hepatocytes, with cathepsin A making a small contribution. Following oral administration of TAF to dogs for 7 days, TAF was rapidly absorbed. The appearance of the major metabolite TFV in plasma was accompanied by a rapid decline in circulating TAF. Consistent with the in vitro data, high and persistent levels of TFV-DP were observed in dog livers. Notably, higher liver TFV-DP levels were observed after administration of TAF than those given TDF. These results support the clinical testing of once-daily low-dose TAF for the treatment of HBV infection.

Sensitivity of mitochondrial transcription and resistance of RNA polymerase II dependent nuclear transcription to antiviral ribonucleosides.

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

Metabolic activation of the anti-hepatitis C virus nucleotide prodrug PSI-352938.

PSI-352938 is a novel cyclic phosphate prodrug of ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-methylguanosine-5'-monophosphate with potent anti-HCV activity. In order to inhibit the NS5B RNA-dependent RNA polymerase, PSI-352938 must be metabolized to the active triphosphate form, PSI-352666. During in vitro incubations with PSI-352938, significantly larger amounts of PSI-352666 were formed in primary hepatocytes than in clone A hepatitis C virus (HCV) replicon cells. Metabolism and biochemical assays were performed to define the molecular mechanism of PSI-352938 activation. The first step, removal of the isopropyl group on the 3',5'-cyclic phosphate moiety, was found to be cytochrome P450 (CYP) 3A4 dependent, with other CYP isoforms unable to catalyze the reaction. The second step, opening of the cyclic phosphate ring, was catalyzed by phosphodiesterases (PDEs) 2A1, 5A, 9A, and 11A4, all known to be expressed in the liver. The role of these enzymes in the activation of PSI-352938 was confirmed in primary human hepatocytes, where prodrug activation was reduced by inhibitors of CYP3A4 and PDEs. The third step, removal of the O(6)-ethyl group on the nucleobase, was shown to be catalyzed by adenosine deaminase-like protein 1. The resulting monophosphate was consecutively phosphorylated to the diphosphate and to the triphosphate PSI-352666 by guanylate kinase 1 and nucleoside diphosphate kinase, respectively. In addition, formation of nucleoside metabolites was observed in primary hepatocytes, and ecto-5'-nucleotidase was able to dephosphorylate the monophosphate metabolites. Since CYP3A4 is highly expressed in the liver, the CYP3A4-dependent metabolism of PSI-352938 makes it an effective liver-targeted prodrug, in part accounting for the potent antiviral activity observed clinically.