Discovery of ANA975: an oral prodrug of the TLR-7 agonist isatoribine.

ANA975, a 5-amino-3-beta -D-ribofuranosyl-3H-thiazolo[4,5-d]pyrimidin-2-one derivative, was synthesized in the search of an oral prodrug of isatoribine, a small molecule toll-like receptor 7 (TLR-7) agonist. Several strategies were studied to enable the kilogram-scale synthesis of ANA975. Three general total syntheses are described. In the phase I clinical study of ANA975 against hepatitis C virus (HCV), conversion to isatoribine in plasma was rapid and effective, delivering levels of isatoribine that have been shown to be clinically relevant.

Syntheses of novel myxopyronin B analogs as potential inhibitors of bacterial RNA polymerase.

Based upon observations from our initial findings, additional myxopyronin B analogs have been prepared and tested for in vitro inhibitory activity against DNA-dependent RNA polymerase and antibacterial activity against Escherichia coli and Staphylococcus aureus.

Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery.

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

Masked oral prodrugs of toll-like receptor 7 agonists: a new approach for the treatment of infectious disease.

Toll-like receptor (TLR)7 is a pattern-recognition receptor that activates the innate immune response. Stimulation of TLR7 induces type I interferons, pro-inflammatory cytokines, the upregulation of co-stimulatory molecules and leads to the development of an adaptive immune response. Small-molecule TLR7 agonists with broad-spectrum antiviral activities in animal models have been identified. Such compounds have been examined clinically for a number of different infectious disease indications, leading to marketing approval of one of these agents, imiquimod, for the topical treatment of external genital and perianal papillomavirus infections. In contrast with topical and intravenous routes of administration, compounds delivered orally have exhibited poor tolerability at desired doses, with substantial adverse events associated with gastrointestinal toxicity. However, dosing with masked oral prodrugs of TLR7 agonists, such as ANA-975, limits the adverse events associated with the activation of responsive gastrointestinal immune tissue. As a consequence, the treatment of systemic diseases, such as chronic hepatitis C, can now be explored with orally administered TLR7 agonists.

Isatoribine, an agonist of TLR7, reduces plasma virus concentration in chronic hepatitis C infection.

Immune-based therapy is the mainstay treatment for chronic hepatitis C virus (HCV) infection but causes multiple side effects and achieves durable viral clearance in only approximately 50% of patients. Most new investigational anti-HCV compounds are direct-acting antivirals for which durability of response and risk of viral mutations and resistance are not yet known. Therefore, continuing discovery and development of new immune-based treatments is desirable. Toll-like receptors (TLRs) are pathogen recognition receptors that initiate the innate immune response. The responsiveness of HCV or other ongoing chronic systemic infections to treatment with a selective TLR agonist has not been reported. Isatoribine is a selective agonist of TLR7. In a proof-of-concept study, we found that once-daily 7-day treatment with intravenous isatoribine 800 mg caused a significant (P = .001) reduction of plasma HCV RNA (mean, -0.76; range, -2.85 to +0.21 log(10) units) in otherwise untreated patients (n = 12) who were chronically infected with HCV. Viral load reduction occurred in patients infected with genotype 1 as well as non-genotype 1 HCV. The reduction of viral load was correlated with induction of markers of a heightened immune antiviral state, including 2'-, 5'- oligoadenylate synthetase levels in whole blood. This treatment was well tolerated, with a low frequency of mild to moderate adverse events. In conclusion, systemic administration of the selective TLR7 agonist isatoribine resulted in dose-dependent changes in immunologic biomarkers and a statistically significant antiviral effect with relatively few and mild side effects.

Structure-based characterization and optimization of novel hydrophobic binding interactions in a series of pyrrolidine influenza neuraminidase inhibitors.

The structure-activity relationship (SAR) of a novel hydrophobic binding interaction within a subsite of the influenza neuraminidase (NA) active site was characterized and optimized for a series of trisubstituted pyrrolidine inhibitors modified at the 4-position. Previously, potent inhibitors have targeted this subsite with hydrophilic substituents such as amines and guanidines. Inhibitor-bound crystal structures revealed that hydrophobic substituents with sp(2) hybridization could achieve optimal interactions by virtue of a low-energy binding conformation and favorable pi-stacking interactions with the residue Glu119. From a lead methyl ester, investigation of five-membered heteroaromatic substituents at C-4 produced a 3-pyrazolyl analogue that improved activity by making a targeted hydrogen bond with Trp178. The SAR of substituted vinyl substituents at C-4 produced a Z-propenyl analogue with improved activity over the lead methyl ester. The C-1 ethyl ester prodrugs of the substituted C-4 vinyl analogues gave compounds with excellent oral bioavailability (F > 60%) when dosed in rat.

A diketopiperazine dimer from a marine-derived isolate of Aspergillus niger.

The new diketopiperazine dimer 1, as well as the known compounds TMC-256A1 (2), TMC-256C1 (3), and demethylkotanin (4), were isolated from a culture of Aspergillus niger. The gross structure of 1 was determined by 2D NMR studies and comparison with literature data, and the absolute stereochemistry was elucidated by chiral HPLC analysis of the hydrolysis products.

In vitro selection and characterization of influenza A (A/N9) virus variants resistant to a novel neuraminidase inhibitor, A-315675.

With the recent introduction of neuraminidase (NA) inhibitors into clinical practice for the treatment of influenza virus infections, considerable attention has been focused on the potential for resistance development and cross-resistance between different agents from this class. A-315675 is a novel influenza virus NA inhibitor that has potent enzyme activity and is highly active in cell culture against a variety of strains of influenza A and B viruses. To further assess the therapeutic potential of this compound, in vitro resistance studies have been conducted and a comparative assessment has been made relative to oseltamivir carboxylate. The development of viral resistance to A-315675 was studied by in vitro serial passage of influenza A/N9 virus strains grown in MDCK cells in the presence of increasing concentrations of A-315675. Parallel passaging experiments were conducted with oseltamivir carboxylate, the active form of a currently marketed oral agent for the treatment of influenza virus infections. Passage experiments with A-315675 identified a variant at passage 8 that was 60-fold less susceptible to the compound. Sequencing of the viral population identified an E119D mutation in the NA gene, but no mutations were observed in the hemagglutinin (HA) gene. However, by passage 10 (2.56 microM A-315675), two mutations (R233K, S339P) in the HA gene appeared in addition to the E119D mutation in the NA gene, resulting in a 310-fold-lower susceptibility to A-315675. Further passaging at higher drug concentrations had no effect on the generation of further NA or HA mutations (20.5 microM A-315675). This P15 virus displayed 355-fold-lower susceptibility to A-315675 and >175-fold-lower susceptibility to zanamivir than did wild-type virus, but it retained a high degree of susceptibility to oseltamivir carboxylate. By comparison, virus variants recovered from passaging against oseltamivir carboxylate (passage 14) harbored an E119V mutation and displayed a 6,000-fold-lower susceptibility to oseltamivir carboxylate and a 175-fold-lower susceptibility to zanamivir than did wild-type virus. Interestingly, this mutant still retained susceptibility to A-315675 (42-fold loss). This suggests that cross-resistance between A-315675- and oseltamivir carboxylate-selected variants in vitro is minimal.

In vitro characterization of A-315675, a highly potent inhibitor of A and B strain influenza virus neuraminidases and influenza virus replication.

A-315675 is a novel, pyrrolidine-based compound that was evaluated in this study for its ability to inhibit A and B strain influenza virus neuraminidases in enzyme assays and influenza virus replication in cell culture. A-315675 effectively inhibited influenza A N1, N2, and N9 and B strain neuraminidases with inhibitor constant (K(i)) values between 0.024 and 0.31 nM. These values were comparable to or lower than the K(i) values measured for oseltamivir carboxylate (GS4071), zanamivir, and BCX-1812, except for the N1 enzymes that were found to be the most sensitive to BCX-1812. The time-dependent inhibition of neuraminidase catalytic activity observed with A-315675 is likely due to its very low rate of dissociation from the active site of neuraminidase. The half times for dissociation of A-315675 from B/Memphis/3/89 and A/Tokyo/3/67 (H3N2) influenza virus neuraminidases of 10 to 12 h are significantly slower than the half times measured for oseltamivir carboxylate (33 to 60 min). A-315675 inhibited the replication of several laboratory strains of influenza virus in cell culture with potencies that were comparable or superior to those for oseltamivir carboxylate and BCX-1812, except for the A/H1N1 viruses that were found to be two- to fourfold more susceptible to BCX-1812. A-315675 and oseltamivir carboxylate exhibited comparable potencies against a panel of A/H1N1 and A/H3N2 influenza virus clinical isolates, but A-315675 was found to be significantly more potent than oseltamivir carboxylate against the B strain isolates. The favorable in vitro results relative to other clinically effective agents provide strong support for the further investigation of A-315675 as a potential therapy for influenza virus infections.