Increasing extracellular protein concentration reduces intracellular antiretroviral drug concentration and antiviral effect.

The objective of this study was to develop an in vitro pharmacodynamic (PD) system to test the impact of protein binding on antiretroviral (ARV) drug effect and intracellular ARV distribution. CD4(+) T cells were isolated from peripheral blood mononuclear cells (PBMCs) and exposed to varying and physiologically relevant concentrations of human serum albumin (HSA) and the ARV drugs efavirenz (EFV), raltegravir (RAL), etravirine (ETR), and enfuvirtide (ENF). The effect of varying extracellular protein concentration on the intracellular distribution of EFV, RAL, and ETR was assessed using ultraperformance liquid chromatography tandem mass spectrometry. HIV infectivity was assessed using an HIV-1 reporter virus expressing an Env-green fluorescent protein (GFP) and quantified using flow cytometry. Increasing extracellular HSA concentration was associated with increased relative infectivity for all drugs tested as well as decreased intracellular concentrations for EFV, RAL, and ETR. Median-effect plots indicate linearity between log10 antiviral effect (fraction of virus affected divided by fraction unaffected) and log10 intracellular drug concentration. The median [interquartile range (IQR)] slope (m) of the median-effect plots was 2.97 (2.26-5.85) for EFV, 3.52 (3.11-3.74) for ETR, and 2.39 (2.15-3.74) for RAL. The intracellular ARV concentrations associated with half-maximal antiviral effect (IC50) of EFV, ETR, and RAL were 1.2 (0.51-5.39), 39.06 (30.10-51.76), and 4.67 (3.91-5.02) ng/ml, respectively. This study demonstrates a significant reduction in cell penetration and antiviral effect of highly bound ARVs due to increasing extracellular concentration of HSA. This study is therefore the first to demonstrate experimentally how protein binding impacts intracellular distribution and the efficacy of ARVs.

Ler interdomain linker is essential for anti-silencing activity in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) expresses a type III secretion system (T3SS) required for pathogenesis. Regulation of the genes encoding the T3SS is complex; two major regulators control transcription, the silencer H-NS, and the related H-NS-like protein Ler. Our laboratory is interested in understanding the molecular differences that distinguish the anti-silencer Ler from H-NS, and how Ler differentially regulates EPEC virulence genes. Here, we demonstrate that mutated Ler proteins either containing H-NS alpha-helices 1 and 2, missing from Ler, or truncated for the 11 aa C-terminal extension compared with the related H-NS protein, did not appreciably alter Ler function. In contrast, mutating the proline at position 92 of Ler, in the conserved C-terminal DNA binding motif, eliminated Ler activity. Inserting 11 H-NS-specific amino acids, 11 alanines or 6 alanines into the Ler linker severely impaired the ability of Ler to increase LEE5 transcription. To extend our analysis, we constructed six chimeric proteins containing the N terminus, linker region or C terminus of Ler in different combinations with the complementary domains of H-NS, and monitored their in vivo activities. Replacing the Ler linker domain with that of H-NS, or replacing the Ler C-terminal, DNA binding domain with that of H-NS eliminated the ability of Ler to increase transcription at the LEE5 promoter. Thus, the linker and C-terminal domains of Ler and H-NS are not functionally equivalent. Conversely, replacing the H-NS linker region with that of Ler caused increased transcription at LEE5 in a strain deleted for hns. In summary, the interdomain linker specific to Ler is necessary for anti-silencing activity in EPEC.