Evaluation of Cytologic Sample Preparations for Compatibility With Nucleic Acid Analysis.

Objectives: In this study, the influence of several key elements of the cytologic sample workflow on DNA and RNA content was evaluated.

Methods: The A549 cell line, patient-derived organoids, and pleural effusions were used to investigate the effect of (1) several collection media and delayed time to processing; (2) cytology specimens; (3) cytologic staining; and (4) formalin-fixed, paraffin-embedded (FFPE) cell block processing on nucleic acid quality and quantity as determined by fragment analyzer, Qubit analysis (Thermo Fisher Scientific), and quantitative polymerase chain reaction-based analysis on the Idylla platform (Biocartis).

Results: Alcohol-based collection media (CytoRich Red [Thermo Fisher Scientific] and EtOH95%) displayed high DNA and RNA preservation capacity, while phosphate-buffered saline and, to a lesser extent, formalin were associated with high RNA quality.

Discovery and early development of TMC647055, a non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Structure-based macrocyclization of a 6-carboxylic acid indole chemotype has yielded potent and selective finger-loop inhibitors of the hepatitis C virus (HCV) NS5B polymerase. Lead optimization in conjunction with in vivo evaluation in rats identified several compounds showing (i) nanomolar potency in HCV replicon cells, (ii) limited toxicity and off-target activities, and (iii) encouraging preclinical pharmacokinetic profiles characterized by high liver distribution. This effort culminated in the identification of TMC647055 (10a), a nonzwitterionic 17-membered-ring macrocycle characterized by high affinity, long polymerase residence time, and broad genotypic coverage.

TMC647055, a potent nonnucleoside hepatitis C virus NS5B polymerase inhibitor with cross-genotypic coverage.

Hepatitis C virus (HCV) infection is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. There remains an unmet medical need for efficacious and safe direct antivirals with complementary modes of action for combination in treatment regimens to deliver a high cure rate with a short duration of treatment for HCV patients. Here we report the in vitro inhibitory activity, mode of action, binding kinetics, and resistance profile of TMC647055, a novel and potent nonnucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase.

Finger loop inhibitors of the HCV NS5b polymerase. Part II. Optimization of tetracyclic indole-based macrocycle leading to the discovery of TMC647055.

Optimization of a novel series of macrocyclic indole-based inhibitors of the HCV NS5b polymerase targeting the finger loop domain led to the discovery of lead compounds exhibiting improved potency in cellular assays and superior pharmacokinetic profile. Further lead optimization performed on the most promising unsaturated-bridged subseries provided the clinical candidate 27-cyclohexyl-12,13,16,17-tetrahydro-22-methoxy-11,17-dimethyl-10,10-dioxide-2,19-methano-3,7:4,1-dimetheno-1H,11H-14,10,2,9,11,17-benzoxathiatetraazacyclo docosine-8,18(9H,15H)-dione, TMC647055 (compound 18a). This non-zwitterionic 17-membered ring macrocycle combines nanomolar cellular potency (EC(50) of 82 nM) with minimal associated cell toxicity (CC(50)>20 μM) and promising pharmacokinetic profiles in rats and dogs.

Finger-loop inhibitors of the HCV NS5b polymerase. Part 1: Discovery and optimization of novel 1,6- and 2,6-macrocyclic indole series.

Novel conformationaly constrained 1,6- and 2,6-macrocyclic HCV NS5b polymerase inhibitors, in which either the nitrogen or the phenyl ring in the C2 position of the central indole core is tethered to an acylsulfamide acid bioisostere, have been designed and tested for their anti-HCV potency. This transformational route toward non-zwitterionic finger loop-directed inhibitors led to the discovery of derivatives with improved cell potency and pharmacokinetic profile.

Antiviral activity and mode of action of TMC647078, a novel nucleoside inhibitor of the hepatitis C virus NS5B polymerase.

Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times.

In vitro resistance profile of the hepatitis C virus NS3/4A protease inhibitor TMC435.

TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent.

TMC125 displays a high genetic barrier to the development of resistance: evidence from in vitro selection experiments.

TMC125 is a potent new investigational nonnucleoside reverse transcriptase inhibitor (NNRTI) that is active against human immunodeficiency virus type 1 (HIV-1) with resistance to currently licensed NNRTIs. Sequential passage experiments with both wild-type virus and NNRTI-resistant virus were performed to identify mutations selected by TMC125 in vitro. In addition to "classic" selection experiments at a low multiplicity of infection (MOI) with increasing concentrations of inhibitors, experiments at a high MOI with fixed concentrations of inhibitors were performed to ensure a standardized comparison between TMC125 and current NNRTIs.