Discovery and development of oseltamivir at Gilead Sciences.

John Martin's untimely death in March 2021 was a huge loss for us personally, Gilead Sciences, the company he built over 30 years and the scientific community concerned with antiviral therapies. We wish to honor John's legacy by retelling the discovery and history of Tamiflu and his contributions to it. Without his vision, persistence, and keen eye for opportunities, Tamiflu would not exist and Gilead's path would not have been the same. His strategic thinking around the first oral flu drug is still quite relevant today, when we are still in the SARS-CoV-2 pandemic. John explained it simply in an interview with the Science History Institute in May 2020: "…most of my colleagues, we travel with Tamiflu when we go internationally, because it works for treatment and prevention, and hopefully, there will be a solution like that, eventually, for the Covid virus in addition to vaccines. Most people will get a flu vaccine every year, but there is still disease, we need a pill for treatment and prevention.".

Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses.

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1'-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3-14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [ Nature 2016 , 531 , 381 - 385 ]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.

Discovery of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerases.

Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.

Structure-activity relationships of diamine inhibitors of cytochrome P450 (CYP) 3A as novel pharmacoenhancers. Part II: P2/P3 region and discovery of cobicistat (GS-9350).

The HIV protease inhibitor (PI) ritonavir (RTV) has been widely used as a pharmacoenhancer for other PIs, which are substrates of cytochrome P450 3A (CYP3A). However the potent anti-HIV activity of ritonavir may limit its use as a pharmacoenhancer with other classes of anti-HIV agents. Ritonavir is also associated with limitations such as poor physicochemical properties. To address these issues a series of compounds with replacements at the P2 and/or P3 region was designed and evaluated as novel CYP3A inhibitors. Through these efforts, a potent and selective inhibitor of CYP3A, GS-9350 (cobicistat) with improved physiochemical properties was discovered.

In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile.

GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (K(i) = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4(+) T cells (50% effective concentration [EC(50)] = 3.4 to 11.5 nM), and macrophages (EC(50) = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC(50)s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients.

Synthesis and anti-HIV activity of cyclic pyrimidine phosphonomethoxy nucleosides and their prodrugs: a comparison of phosphonates and corresponding nucleosides.

Cyclic phosphonomethoxy pyrimidine nucleosides that are bioisosteres of the monophosphate metabolites of HIV reverse transcriptase (RT) inhibitors AZT, d4T, and ddC have been synthesized. The RT inhibitory activities of the phosphonates were reduced for both dideoxy (dd) and dideoxydidehydro (d4) analogs compared to the nucleosides. Bis-isopropyloxymethylcarbonyl (BisPOC) prodrugs were prepared on selected compounds and provided > 150-fold improvements in antiviral activity.

Synthesis, anti-HIV activity, and resistance profile of thymidine phosphonomethoxy nucleosides and their bis-isopropyloxymethylcarbonyl (bisPOC) prodrugs.

Phosphonomethoxy nucleoside analogs of the thymine containing nucleoside reverse transcriptase inhibitors (NRTIs), 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), and 2',3'-dideoxythymidine (ddT), were synthesized. The anti-HIV activity against wild-type and several major nucleoside-resistant strains of HIV-1 was evaluated together with the inhibition of wild-type HIV reverse transcriptase (RT). Phosphonomethoxy analog of d4T, 8 (d4TP), demonstrated antiviral activity with an EC(50) value of 26 microM, whereas, phosphonomethoxy analogs of ddT, 7 (ddTP), and AZT, 6 (AZTP), were both inactive at concentrations up to 200 microM. Bis-isopropyloxymethylcarbonyl (bisPOC) prodrugs improved the anti-HIV activity of 7 and 8 by >150-fold and 29-fold, respectively, allowing for antiviral resistance to be determined. The K65R RT mutant virus was more resistant to the bisPOC prodrugs of 7 and 8 than bisPOC PMPA (tenofovir DF) 1. However, bisPOC prodrug of 7 demonstrated superior resistance toward the RT virus containing multiple thymidine analog mutations (6TAMs) indicating that new phosphonate nucleoside analogs may be suitable for targeting clinically relevant nucleoside resistant HIV-1 strains.

Cathepsin A is the major hydrolase catalyzing the intracellular hydrolysis of the antiretroviral nucleotide phosphonoamidate prodrugs GS-7340 and GS-9131.

GS-7340 and GS-9131 {9-[(R)-2-[[(S)-[[(S)-1-(isopropoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-propyl]adenine and 9-(R)-4'-(R)-[[[(S)-1-[(ethoxycarbonyl)ethyl]amino]phenoxyphosphinyl]methoxy]-2'-fluoro-1'-furanyladenine, respectively} are novel alkylalaninyl phenyl ester prodrugs of tenofovir {9-R-[(2-phosphonomethoxy)propyl]adenine} (TFV) and a cyclic nucleotide analog, GS-9148 (phosphonomethoxy-2'-fluoro-2', 3'-dideoxydidehydroadenosine), respectively. Both prodrugs exhibit potent antiretroviral activity against both wild-type and drug-resistant human immunodeficiency virus type 1 strains and excellent in vivo pharmacokinetic properties. In this study, the main enzymatic activity responsible for the initial step in the intracellular activation of GS-7340 and GS-9131 was isolated from human peripheral blood mononuclear cells and identified as lysosomal carboxypeptidase A (cathepsin A [CatA]; EC 3.4.16.5). Biochemical properties of the purified hydrolase (native complex and catalytic subunit molecular masses of 100 and 29 kDa, respectively; isoelectric point [pI] of 5.5) matched those of CatA. Recombinant CatA and the isolated prodrug hydrolase displayed identical susceptibilities to inhibitors and identical substrate preferences towards a panel of tenofovir phosphonoamidate prodrugs. Incubation of both enzymes with 14C-labeled GS-7340 or [3H]difluorophosphonate resulted in the covalent labeling of identical 29-kDa catalytic subunits. Finally, following a 4-h incubation with GS-7340 and GS-9131, the intracellular concentrations of prodrug metabolites detected in CatA-negative fibroblasts were approximately 7.5- and 3-fold lower, respectively, than those detected in normal control fibroblasts. Collectively, these data demonstrate the key role of CatA in the intracellular activation of nucleotide phosphonoamidate prodrugs and open new possibilities for further improvement of this important class of antiviral prodrugs.

Suppression of HIV-1 protease inhibitor resistance by phosphonate-mediated solvent anchoring.

The introduction of human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) markedly improved the clinical outcome and control of HIV-1 infection. However, cross-resistance among PIs due to a wide spectrum of mutations in viral protease is a major factor limiting their broader clinical use. Here we report on the suppression of PI resistance using a covalent attachment of a phosphonic acid motif to a peptidomimetic inhibitor scaffold. The resulting phosphonate analogs maintain high binding affinity to HIV-1 protease, potent antiretroviral activity, and unlike the parent molecules, display no loss of potency against a panel of clinically important PI-resistant HIV-1 strains. As shown by crystallographic analysis, the phosphonate moiety is highly exposed to solvent with no discernable interactions with any of the enzyme active site or surface residues. We term this effect "solvent anchoring" and demonstrate that it is driven by a favorable change in the inhibitor binding entropy upon the interaction with mutant enzymes. This type of thermodynamic behavior, which was not found with the parent scaffold fully buried in the enzyme active site, is a result of the increased degeneracy of inhibitor binding states, allowing effective molecular adaptation to the expanded cavity volume of mutant proteases. This strategy, which is applicable to various PI scaffolds, should facilitate the design of novel PIs and potentially other antiviral therapeutics.

Small molecules VP-14637 and JNJ-2408068 inhibit respiratory syncytial virus fusion by similar mechanisms.

Here we present data on the mechanism of action of VP-14637 and JNJ-2408068 (formerly R-170591), two small-molecule inhibitors of respiratory syncytial virus (RSV). Both inhibitors exhibited potent antiviral activity with 50% effective concentrations (EC50s) of 1.4 and 2.1 nM, respectively. A similar inhibitory effect was observed in a RSV-mediated cell fusion assay (EC50=5.4 and 0.9 nM, respectively). Several drug-resistant RSV variants were selected in vitro in the presence of each compound. All selected viruses exhibited significant cross-resistance to both inhibitors and contained various single amino acid substitutions in two distinct regions of the viral F protein, the heptad repeat 2 (HR2; mutations D486N, E487D, and F488Y), and the intervening domain between HR1 and HR2 (mutation K399I and T400A). Studies using [3H]VP-14637 revealed a specific binding of the compound to RSV-infected cells that was efficiently inhibited by JNJ-2408068 (50% inhibitory concentration=2.9 nM) but not by the HR2-derived peptide T-118. Further analysis using a transient T7 vaccinia expression system indicated that RSV F protein is sufficient for this interaction. F proteins containing either the VP-14637 or JNJ-2408068 resistance mutations exhibited greatly reduced binding of [3H]VP-14637. Molecular modeling analysis suggests that both molecules may bind into a small hydrophobic cavity in the inner core of F protein, interacting simultaneously with both the HR1 and HR2 domains. Altogether, these data indicate that VP-14637 and JNJ-2408068 interfere with RSV fusion through a mechanism involving a similar interaction with the F protein.

Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissue.

An isopropylalaninyl monoamidate phenyl monoester prodrug of tenofovir (GS 7340) was prepared, and its in vitro antiviral activity, metabolism, and pharmacokinetics in dogs were determined. The 50% effective concentration (EC(50)) of GS 7340 against human immunodeficiency virus type 1 in MT-2 cells was 0.005 microM compared to an EC(50) of 5 microM for the parent drug, tenofovir. The (L)-alaninyl analog (GS 7340) was >1,000-fold more active than the (D)-alaninyl analog. GS 7340 has a half-life of 90 min in human plasma at 37 degrees C and a half-life of 28.3 min in an MT-2 cell extract at 37 degrees C. The antiviral activity (>10 x the EC(50)) and the metabolic stability in MT-2 cell extracts (>35 x) and plasma (>2.5 x) were also sensitive to the stereochemistry at the phosphorus. After a single oral dose of GS 7340 (10 mg-eq/kg tenofovir) to male beagle dogs, the plasma bioavailability of tenofovir compared to an intravenous dose of tenofovir was 17%. The total intracellular concentration of all tenofovir species in isolated peripheral blood mononuclear cells at 24 h was 63 microg-eq/ml compared to 0.2 microg-eq/ml in plasma. A radiolabeled distribution study with dogs resulted in an increased distribution of tenofovir to tissues of lymphatic origin compared to the commercially available prodrug tenofovir DF (Viread).

Inhibition of respiratory syncytial virus fusion by the small molecule VP-14637 via specific interactions with F protein.

Human respiratory syncytial virus (RSV) is a major cause of respiratory tract infections worldwide. Several novel small-molecule inhibitors of RSV have been identified, but they are still in preclinical or early clinical evaluation. One such inhibitor is a recently discovered triphenol-based molecule, VP-14637 (ViroPharma). Initial experiments suggested that VP-14637 acted early and might be an RSV fusion inhibitor. Here we present studies demonstrating that VP-14637 does not block RSV adsorption but inhibits RSV-induced cell-cell fusion and binds specifically to RSV-infected cells with an affinity corresponding to its inhibitory potency. VP-14637 is capable of specifically interacting with the RSV fusion protein expressed by a T7 vaccinia virus system. RSV variants resistant to VP-14637 were selected; they had mutations localized to two distinct regions of the RSV F protein, heptad repeat 2 (HR2) and the intervening domain between heptad repeat 1 (HR1) and HR2. No mutations arose in HR1, suggesting a mechanism other than direct disruption of the heptad repeat interaction. The F proteins containing the resistance mutations exhibited greatly reduced binding of VP-14637. Despite segregating with the membrane fraction following incubation with intact RSV-infected cells, the compound did not bind to membranes isolated from RSV-infected cells. In addition, binding of VP-14637 was substantially compromised at temperatures of < or =22 degrees C. Therefore, we propose that VP-14637 inhibits RSV through a novel mechanism involving an interaction between the compound and a transient conformation of the RSV F protein.