Recombinant approach for the production of HIV fusion inhibitor Enfuvirtide using Escherichia coli.

The use of antiretroviral drugs is gaining importance in the recent past for the treatment of human immunodeficiency virus infection. Enfuvirtide (T20) is one of the fusion inhibitors, inhibits the fusion between the virus and healthy target CD4 cells. The treatment with T20 involves very high therapeutic dose. In addition to its high dose, production of T20 by synthetic methods is expensive and cumbersome. We report an alternative recombinant approach for the production of the T20 peptide through a novel short fusion-tag expression system. This expression system consists of the hydrophobic region of growth hormone (GH) as the fusion tag, a factor Xa cleavage site upstream to the T20. The fusion protein was expressed in Escherichia coli as inclusion bodies. We also report here, a simple and an efficient down-stream strategy for the purification of recombinant T20 peptide (rT20). Our study is the first to demonstrate a novel approach using GH fusion tag, ensured the peptide stability, for the production of rT20 which yields more than 250mg/L with 98% purity. The biological activity of the rT20 is comparable to its synthetic counterpart. Thus, this novel approach could be an alternate method of choice for production of therapeutically important small peptides.

Artificial ion channel biosensor in human immunodeficiency virus gp41 drug sensing.

An ion channel biosensor is described for label-free detection of inhibitors which bind to the coiled coil domain of human immunodeficiency virus type 1 gp41. gp41 is the viral transmembrane glycoprotein responsible for fusion between HIV-1 and host cells. The N-terminal coiled coil domain binds three antiparallel C-heptad repeat peptides in the six helix bundle structure of trimeric gp41 that forms during fusion. Compounds able to prevent six-helix bundle formation by binding to the gp41 coiled coil could inhibit fusion and have important therapeutic potential. We have immobilized on gold a positively charged metallopeptide mimic of a section of the gp41 coiled coil containing a hydrophobic pocket suitable for small molecule binding. We demonstrate that the resulting sensor is able to transmit a current in the presence of negatively charged redox marker ions, therefore acting as an artificial ion channel. The electrochemical signal, measured by cyclic voltammetry, was modulated by specific analyte binding to the coiled coil. Nanomolar quantities of peptides and small molecules that bind in the hydrophobic pocket could be selectively detected, providing a method for label-free detection of binding to gp41.

Optimized sample preparation for MALDI mass spectrometry analysis of protected synthetic peptides.

The recent development and commercialization of Fuzeon (enfuvirtide) demonstrated that a convergent strategy comprised of both solid- and solution-phase synthetic methodologies presents a viable route for peptide manufacturing on a multi-ton scale. In this strategy, the target sequence is prepared by stepwise solid-phase synthesis of protected peptide fragments, which are then coupled together in the solution-phase to give the full-length sequence. These synthetic methodologies pose a unique challenge for mass spectrometry (MS), as protected peptide intermediates are often marked by poor solubility, structural lability, and low ionization potential. Matrix-assisted laser desorption/ionization (MALDI) MS is uniquely suited to such analytes; however, generalized protocols for MALDI analysis of protected peptides have yet to be demonstrated. Herein, we report an operationally simple sample preparation method for MALDI analysis of protected peptides, which greatly facilitates the collection and interpretation of MS data. In this method, the difficulty in MS analysis of protected peptides has been greatly diminished by use of dithranol as a matrix and CsCl as an additive, giving rise to intentionally-formed Cs(+) adducts. With greatly reduced fragmentation, better crystalline morphology, and easier data interpretation, we anticipate that these findings will find utility in peptide process development and manufacturing settings for reaction monitoring, troubleshooting, and quality control.

Rapid and automated fluorescence-linked immunosorbent assay for high-throughput screening of HIV-1 fusion inhibitors targeting gp41.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays an important role in the virus entry. During the process of fusion between the viral and target cell membranes, the N- and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.

Development and automation of a 384-well cell fusion assay to identify inhibitors of CCR5/CD4-mediated HIV virus entry.

This article describes the automation of an in vitro cell-based fusion assay for the identification of novel inhibitors of receptor mediated HIV-1 entry. The assay utilises two stable cell lines: one expressing CD4, CCR5 and an LTR-promoter/beta-galactosidase reporter construct, and the other expressing gp160 and tat. Accumulation of beta-galactosidase can only occur following fusion of these two cell lines via the gp160 and receptor mediators, as this event facilitates the transfer of the tat transcription factor between the two cell types. Although similar cell fusion systems have been described previously, they have not met the requirements for HTS due to complexity, throughput and reagent cost. The assay described in this article provides significant advantage, as (a) no transfection/infection events are required prior to the assay, reducing the potential for variability, (b) cells are mixed in solution, enhancing fusion efficiency compared to adherent cells, (c) miniaturization to low volume enables screening in 384-well plates; and (d) online cell dispensing facilitates automated screening. This assay has been employed to screen approximately 650,000 compounds in a singleton format. The data demonstrate that the assay is robust, with a Z' consistently above 0.6, which compares favourably with less complex biochemical assays.

Clinical pharmacology of antiretroviral therapy.

Antiretroviral therapy represents by all means a new branch of anti-infective chemotherapy, and in order to describe the mode of action of antiretrovirals, a series of inferences from anti-bacterial chemotherapy were made. The currently available antiretroviral agents can be classified as time-dependent drugs, and therefore the key pharmacokinetic parameter adopted in their clinical-pharmacological assessment is the concentration at the end of the dosing interval (Ctrough). By focusing on this parameter, the application of Therapeutic Drug Monitoring (TDM) allows for the successful individual tailoring of the drug dosage in some clinical circumstances, such as treatment of drug-resistant infections, drug-drug interactions and side effects. While this procedure has now been sufficiently standardized for protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs), no clinical applications are yet recognized for nucleoside/nucleotide reverse transcriptase inhibitors (N/NtRTIs) and fusion inhibitors. The main unfavourable peculiarity of HIV infection, such as the need for lifelong treatment, is one of the reasons why increasing attention is being paid to pharmacological aspects of antiretroviral therapy. Issues like treatment potency, maintenance over time of the immunovirological benefit and long-term side effects require intensive pharmacological investigation in order to obtain the information on which basing the most convenient strategy to be adopted for the therapeutical management of this condition.

Cloning and characterization of functional subtype A HIV-1 envelope variants transmitted through breastfeeding.

Previous studies of HIV-1 variants transmitted from mother-to-infant have focused primarily on computational analyses of partial envelope gene sequences, rather than analyses of functional envelope variants. There are very few examples of well-characterized functional envelope clones from mother-infant pairs, especially from envelope variants representing the most prevalent subtypes worldwide. To address this, we amplified the envelope variants present in 4 mother-infant transmission pairs, all of whom were infected with subtype A and three of whom presumably transmitted HIV-1 during the breastfeeding period. Functional envelope clones were constructed, either encoding full-length envelope sequences from the mother and baby or by making chimeric envelope clones in a common backbone sequence. The infant envelope sequences were genetically homogeneous compared to the maternal viruses, and pseudoviruses bearing these envelopes all used CCR5 as a coreceptor. The infant viruses were generally resistant to neutralization by maternal antibodies present near the time of transmission. There were no notable differences in sensitivity of the mother and infant envelope variants to neutralization by heterologous plasma or monoclonal antibodies 2G12 and b12, or to inhibition by sCD4, PSC-RANTES or TAK779. This collection of viral envelopes, which can be used for making pseudotyped viruses, may be useful for examining the efficacy of interventions to block mother-infant transmission, including sera from vaccine candidates, purified antibodies under consideration for passive immunization and viral entry inhibitors.