Discovery of the pan-genotypic hepatitis C virus NS3/4A protease inhibitor voxilaprevir (GS-9857): A component of Vosevi®

Treatment of hepatitis C virus (HCV) infection has been historically challenging due the high viral genetic complexity wherein there are eight distinct genotypes and at least 86 viral subtypes. While HCV NS3/4A protease inhibitors are an established treatment option for genotype 1 infection, limited coverage of genotypes 2 and/or 3 combined with serum alanine transaminase (ALT) elevations for some compounds has limited the broad utility of this therapeutic class. Our discovery efforts were focused on identifying an NS3/4A protease inhibitor with pan-genotypic antiviral activity, improved coverage of resistance associated substitutions, and a decreased risk of hepatotoxicity. Towards this goal, distinct interactions with the conserved catalytic triad of the NS3/4A protease were identified that improved genotype 3 antiviral activity. We further discovered that protein adduct formation strongly correlated with clinical ALT elevation for this therapeutic class. Improving metabolic stability and decreasing protein adduct formation through structural modifications ultimately resulted in voxilaprevir. Voxilaprevir, in combination with sofosbuvir and velpatasvir, has demonstrated pan-genotypic antiviral clinical activity. Furthermore, hepatotoxicity was not observed in Phase 3 clinical trials with voxilaprevir, consistent with our design strategy. Vosevi^® (sofosbuvir, velpatasvir, and voxilaprevir) is now an approved pan-genotypic treatment option for the most difficult-to-cure individuals who have previously failed direct acting antiviral therapy.     

Discovery of O6-benzyl glaziovianin A,a potent cytotoxic substance and a potent inhibitor of α,β-tubulin polymerization

We have discovered O⁶-benzyl glaziovianin A, which showed stronger inhibition of microtubule polymerization (IC₅₀ = 2.1 μM) than known α,β-tubulin inhibitors, such as colchicine and glaziovianin A. Also, we performed competition binding experiments of O⁶-benzyl glaziovianin A and revealed that O⁶-benzyl glaziovianin A binds to the colchicine binding site with high affinity. It is interesting that glaziovianin A derivatives change their mode of action in benzylation at the O⁶ (α,β-tubulin inhibitor) or O⁷ (γ-tubulin-specific inhibitor) position.     

GDC-0449—A potent inhibitor of the hedgehog pathway

SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5 mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC.     

The introduction of P4 substituted 1-methylcyclohexyl groups into Boceprevir®: A change in direction in the search for a second generation HCV NS3 protease inhibitor

In the search for a second generation HCV protease inhibitor, molecular modeling studies of the X-ray crystal structure of Boceprevir® 1 bound to the NS3 protein suggest that expansion into the S4 pocket could provide additional hydrophobic Van der Waals interactions. Effective replacement of the P4 tert-butyl with a cyclohexylmethyl ligand led to inhibitor 2 with improved enzyme and replicon activities. Subsequent modeling and SAR studies led to the pyridine 38 and sulfone analogues 52 and 53 with vastly improved PK parameters in monkeys, forming a new foundation for further exploration.     

Discovery of potent α-glucosidase inhibitor flavonols: Insights into mechanism of action through inhibition kinetics and docking simulations

Beside other pharmaceutical benefits, flavonoids are known for their potent α-glucosidase inhibition. In the present study, we investigated α-glucosidase inhibitory effects of structurally related 11 flavonols, among which quercetin-3-O-(3″-O-galloyl)-β-galactopyranoside (8) and quercetin 3-O-(6″-O-galloyl)-β-glucopyranoside (9) showed significant inhibition compared to the positive control, acarbose, with IC₅₀ values of 0.97 ± 0.02 and 1.35 ± 0.06 µM, respectively. It was found that while sugar substitution to C3-OH of C ring reduced the α-glucosidase inhibitory effect, galloyl substitution to these sugar units increased it. An enzyme kinetics analysis revealed that 7 was competitive, whereas 1, 2, 8, and 9 were uncompetitive inhibitors. In the light of these findings, we performed molecular docking studies to predict their inhibition mechanisms at atomic level.     

Discovery of ONO-8590580: A novel,potent and selective GABAA α5 negative allosteric modulator for the treatment of cognitive disorders

The identification and SAR development of a series of negative allosteric modulators of the GABA_A α5 receptor is described. This novel series of compounds was optimised to provide analogues with high GABA_A α5 binding affinity, high α5 negative allosteric modulatory activity, good functional subtype selectivity and low microsomal turnover, culminating in identification of ONO-8590580.     

Discovery of potent and selective 5-azaindazole inhibitors of leucine-rich repeat kinase 2 (LRRK2) – Part 1

Parkinson’s disease is a relatively common neurological disorder with incidence increasing with age. Present treatments merely alleviate the symptoms and do not alter the course of the disease, thus identification of disease modifying therapies represents a significant unmet medical need. Mutations in the LRRK2 gene are risk-factors for developing PD and it has been hypothesized that the increased kinase activity of certain LRRK2 mutants are responsible for the damage of the dopaminergic neurons, thus LRRK2 inhibitors offer the potential to target an underlying cause of the disease. In this communication, we describe hit-to-lead medicinal chemistry program on a novel series of 5-azaindazoles. Compound 1, obtained from high-throughput screening was optimized to a highly potent, selective series of molecules with promising DMPK properties. Introduction of heterocycles at the 3-position were found to significantly increase the potency and kinase selectivity, whilst changes to the 4-chlorobenzyl group improved the physicochemical properties. Our series was licensed to a major pharmaceutical company for further development.     

Tissue pharmacokinetics and pharmacodynamics of AmBisome® (L-AmBis) in uninfected and infected animals and their effects on dosing regimens

Abstract

By selecting a unique combination of lipids and amphotericin B, the liposome composition for AmBisome® (L-AmBis) has been optimized resulting in a formulation that is minimally toxic, targets to fungal cell walls, and distributes into and remains for days to weeks in various host tissues at drug levels above the MIC for many fungi. Procedures have been standardized to ensure that large scale production of the drug retains the drug’s low toxicity profile, favorable pharmacokinetics and antifungal efficacy. Tissue accumulation and clearance with single or multiple intravenous administration is similar in uninfected and infected animal species, with tissue accumulation being dose-dependent and the liver and spleen retaining the most drug. The efficacy in animals appears to be correlated with drug tissue levels although the amount needed in a given organ varies depending upon the type of infection. The long-term tissue retention of bioactive L-AmBis in different organs suggests that for some indications, prophylactic and intermittent drug dosing would be efficacious reducing the cost and possible toxic side-effects. In addition, preliminary preclinical studies using non-intravenous routes of delivery, such as aerosolized L-AmBis, catheter lock therapy, and intravitreal administration, suggest that alternative routes could possibly provide additional therapeutic applications for this antifungal drug.     

Identification of a novel and potent inhibitor of phospholipase A(2) in a medicinal plant: crystal structure at 1.93Å and Surface Plasmon Resonance analysis of phospholipase A(2) complexed with berberine

Crystal of Russell Viper venom phospholipase A(2) complexed with an isoquinoline alkaloid, berberine from a herbaceous plant Cardiospermum halicacabum, was prepared and its structure was solved by X-ray crystallography. The crystal diffracted up to 1.93? and the structure solution clearly located the position of berberine in the active site of the enzyme. Two hydrogen bonds, one direct and the other water mediated, were formed between berberine and the enzyme. Gly 30 and His 48 made these two hydrogen bonds. Additionally, the hydrophobic surface of berberine made a number of hydrophobic contacts with side chains of neighboring amino acids. Surface Plasmon Resonance studies revealed strong binding affinity between berberine and phospholipase A(2). Enzyme inhibition studies proved that berberine is a competitive inhibitor of phospholipase A(2). It was inferred that the isoquinoline alkaloid, berberine, is a potent natural inhibitor of phospholipaseA(2).     

Discovery of a potent and highly selective transforming growth factor β receptor-associated kinase 1 (TAK1) inhibitor by structure based drug design (SBDD)

A novel thienopyrimidinone analog was discovered as a potent and highly selective TAK1 inhibitor using the SBDD approach. TAK1 plays a key role in inflammatory and immune signaling, so TAK1 is considered to be an attractive molecular target for the treatment of human diseases (inflammatory disease, cancer, etc.). After the hit compound had been obtained, our modifications successfully increased TAK1 inhibitory activity and solubility, but metabolic stability was still unsatisfactory. To improve metabolic stability, we conducted metabolic identification. Although the obtained metabolite was fortunately a potent TAK1 inhibitor, its kinase selectivity was low. Subsequently, to achieve high kinase selectivity, we used SBDD to follow two strategies: one targeting unique amino acid residues in TAK1, especially the combination of Ser111 and Asn114; the other decreasing the interaction with Tyr106 at the hinge position in TAK1. As expected, our designed compound showed an excellent kinase selectivity profile in both an in-house and a commercially available panel assay of over 420 kinases and also retained its potent TAK1 inhibitory activity (TAK1 IC₅₀ = 11 nM).     

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (R_P/S_P). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (k_on = 174 M^?1 s^?1) was ca. 8-fold more potent than the d-isomer (k_on = 21.5 M^?1 s^?1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be S_P. The S_P-isomers inhibited human GGT (k_on = 21.5–174 M^?1 s^?1), while the R_P-isomers were inactive even at concentrations of 0.1 mM.     

Aminoparthenolides as novel anti-leukemic agents: Discovery of the NF-κB inhibitor,DMAPT (LC-1)

A series of aminoparthenolide analogs (6–37) were synthesized and evaluated for their anti-leukemic activity. Eight compounds exhibited good anti-leukemic activity with LD₅₀’s in the low μM range (1.5–3.0 μM). Compounds 16, 24 and 30 were the most potent compounds in the series, causing greater than 90% cell death at 10 μM concentration against primary AML cells in culture, with LD₅₀ values of 1.7, 1.8 and 1.6 μM.     

Spasmolytic and tonic effect of Iberogast® (STW 5) in intestinal smooth muscle

STW 5 (Iberogast^®) is a fixed combination of nine medicinal plant extracts effective in the treatment of functional dyspepsia and irritable bowel syndrome. The effects of STW 5, a combination of Iberis amara fresh plant extract, and other eight plant extracts as well as single extract components including extracts from Menthae piperitae folium, Matricariae flos and Liquiritiae radix, were assayed in guinea pig ileum with or without stimulation with acetylcholine or histamine, in order to find a possible effect on the contractility of intestinal smooth muscle.STW 5 decreased acetylcholine- and histamine-induced contraction of guinea pig ileum. This was also true for extracts of Menthae piperitae folium, Matricariae flos and L. radix. Extract from I. amara, however, showed no spasmolytic action; in contrary, it increased the basal resting tone and contraction of atonic ileal segments. This was also true when STW 5 was employed.A spasmolytic action of STW 5 could also be observed in duodenum, jejunum and colon.These data are the first to show not only the spasmolytic effects of STW 5 and its component extracts in intestinal muscle but also the tonicising effects of STW 5 through its component Iberis amara extract in relaxed intestinal muscle. Thus, pharmacological evidence suggests a dual-action principle and may explain, at least in part, the clinically observed therapeutic efficacy of STW 5 (Iberogast^®) in both hypotonic and spastic dysmotility symptoms of functional dyspepsia and irritable bowel syndrome.     

Measurement of homonuclear three-bond J(HNHα) coupling constants in unlabeled peptides complexed with labeled proteins: Application to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of hepatitis C virus (HCV)

A new isotope-filtered experiment has been designed to measure homonuclear three-bond J(H^NH) coupling constants of unlabeled peptides complexed with labeled proteins. The new experiment is based on the 3D HNHA pulse scheme, and belongs to the `quantitative J-correlation' type. It has been applied to a decapeptide inhibitor bound to the proteinase domain of the NS3 protein of human hepatitis C virus (HCV).     

Ganoderol B: A potent α-glucosidase inhibitor isolated from the fruiting body of Ganoderma lucidum

α-Glucosidase inhibitor has considerable potential as a diabetes mellitus type 2 drug because it prevents the digestion of carbohydrates. The search for the constituents reducing α-glucosidase activity led to the finding of active compounds in the fruiting body of Ganoderma lucidum. The CHCl₃ extract of the fruiting body of G. lucidum was found to show inhibitory activity on α-glucosidase in vitro. The neutral fraction, with an IC₅₀ of 88.7 μg/ml, had stronger inhibition than a positive control, acarbose, with an IC₅₀ of 336.7 μg/ml (521.5 μM). The neutral fraction was subjected to silica gel column chromatography and repeated p-HPLC to provide an active compound, (3β,24E)-lanosta-7,9(11),24-trien-3,26-diol (ganoderol B). It was found to have high α-glucosidase inhibition, with an IC₅₀ of 48.5 μg/ml (119.8 μM).     

5,5′- and 6,6′-Dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of HCV NS5B polymerase

The discovery of 5,5′- and 6,6′-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC₅₀ <0.10 μM). In vitro DMPK data for selected compounds as well as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.     

Histochemical Demonstration of the Inhibitory Effect of Nuvan® and Neguvon® on Cholinesterase Activity in Pseudodactylogyrus anguillae (Monogenea)

The nervous system of Pseudodactylogyrus anguillae, a gill parasite from European eel (Anguilla anguilla) is demonstrated histochemically using the acetylthiocholine iodide method. It was shown that the staining of the nervous system was reduced or eliminated due to the inhibitory effect of Nuvan® and Neguvon®.     

Synthesis of 5α-cholestan-6-one derivatives and their inhibitory activities of NO production in activated microglia: Discovery of a novel neuroinflammation inhibitor

Glial activation-mediated neuroinflammation plays a pivotal role in the process of several neuroinflammatory diseases including stroke, Alzheimer’s diseases, Parkinson’s diseases, multiple sclerosis and ischemia. Inhibition of microglial activation may ameliorate neuronal degeneration under the inflammatory conditions. In the present study, a number of 5α-cholestan-6-one derivatives were prepared and the anti-inflammatory effects of these compounds were evaluated in LPS-stimulated BV-2 microglia cells. Those derivatives were synthesized from readily available hyodeoxycholic acid (1). Among the tested compounds, several analogs (16–18, 25, 35, 38) exhibited potent inhibitory activities on nitric oxide production with no or weak cell toxicity. Compound 16 also significantly suppressed the expression of TNF-α, interleukin (IL)-1β, cyclooxygenase (COX-2) as well as inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 microglia cells. In addition, compound 16 markedly reduced infarction volume in a focal ischemic mice model.     

Integrating thin film microfluidics in developing a concise synthesis of DGJNAc: A potent inhibitor of α-N-acetylgalctosaminidases

A simple synthesis, which utilizes a thin film microfluidic reactor for a problematic step, of a potent inhibitor of α-N-acetylhexosaminidases, DGJNAc, has been developed.