Semisynthetic analogues of PSC-RANTES, a potent anti-HIV protein

Department of Structural Biology and Bioinformatics, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.
Bioconjugate Chemistry (Impact Factor: 4.51). 03/2008; 19(2):480-9. DOI: 10.1021/bc7003044
Source: PubMed


New HIV prevention methods are needed, and among those currently being explored are "microbicides", substances applied topically to prevent HIV acquisition during sexual intercourse. The chemokine analogue PSC-RANTES (N(alpha)(n-nonanoyl)-des-Ser(1)-[ L-thioprolyl(2), L-cyclohexylglycyl(3)]-RANTES(4-68)) is a highly potent HIV entry inhibitor which has shown promising efficacy in its initial evaluation as a candidate microbicide. However, a way must be found to produce the molecule by cheaper means than total chemical synthesis. Since the only noncoded structures are located at the N-terminus, a possible solution would be to produce a protein fragment representing all but the N-terminal region using low-cost recombinant production methods and then to attach, site specifically, a short synthetic fragment containing the noncoded N-terminal structures. Here, we describe the evaluation of a range of different conjugation chemistries in order to identify those with potential for development as economical routes to production of a PSC-RANTES analogue with antiviral activity as close as possible to that of the parent protein. The strategies tested involved linkage through oxime, hydrazone/hydrazide, and Psi[CH2-NH] bonds, as well as through a peptide bond obtained either by a thiazolidine rearrangement or by direct alpha-amino acylation of a protein fragment in which 4 of the 5 lysine residues of the native sequence were replaced by arginine (the fifth lysine is essential for activity). Where conjugation involved replacement of one or more residues with a linker moiety, the point in the main chain at which the linker was introduced was varied. The resulting panel of 22 PSC-RANTES analogues was evaluated for anti-HIV activity in an entry inhibition assay. The [Arg (25,45,56,57)] PSC-RANTES analogue has comparable potency to PSC-RANTES, and one of the oxime linked analogues, 4L-57, has potency only 5-fold lower, with scope for improvement. Both represent promising leads for development as microbicide compounds that could be produced at low cost via semisynthesis.

7 Reads
  • Source
    • "Human serum albumin (HSA) was a gift from GeneProt (Geneva, Switzerland). Rantes 10–68 was chemically synthesized using Boc chemistry solid phase peptide synthesis and native chemical ligation as previously described [21]. CYAKYAKL, CPYAKYAKL, GCYAKYAKL and TYAKYAKL were chemically synthesized as previously described [19]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The detection of low abundance proteins in complex biological samples is still a challenge in proteomics. To circumvent this obstacle a number of strategies involving the targeting of subsets of proteins or peptides were developed. The following work describes a new approach to simplify peptide mixtures by enrichment of N-terminal cysteinyl peptides (and to some extent N-terminal threonine peptides). The strategy is based on the use of an isolation method, so-called covalent capture (CC), which relies on the formation of a covalent bond between an N-terminal free cysteine or N-terminal free threonine and an aldehyde fixed on a solid support. The CC is highly selective. It permits extensive washes of the resin for the elimination of non-specific moieties before the release of the captured peptides. The application of the CC to proteomics was evaluated on tryptic peptides of standard proteins and test protein mixtures. The procedure demonstrated a significant reduction in sample complexity, while allowing the identification of N-terminal cysteinyl peptides hidden in the non-fractionated samples. This new strategy provides an efficient tool to existing proteomics approaches to reduce sample complexity and potentially identify less abundance proteins.
    Journal of Proteomics 12/2008; 71(6):647-61. DOI:10.1016/j.jprot.2008.11.009 · 3.89 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: There is a pressing need for microbicides—vaginally applied drug delivery systems that create a pharmacological barrier to HIV—to prevent the male-tofemale sexual transmission of HIV. Numerous antiretroviral agents with a wide range of mechanisms of inhibiting HIV are under development. Delivery systems are required that complement the antiviral agents through maximizing their safety, efficacy and user adherence within cost constraints such that they are affordable in resource poor nations with high HIV prevalence. To this end, three drug delivery systems were engineered for microbicide application in this thesis. The first delivery system was designed to promote uniform distribution and retention of the antivirals in the vaginal lumen, and provide semen triggered delivery into semen—the carrier of HIV in male-to-female sexual transmission. The delivery system consisted of a temperature and pH sensitive gel composed of a terpolymer of N-isopropylacrylamide, acrylic acid and butyl methacrylate. The neutralization of acidic pH in the vagina upon exposure to semen was used as the trigger to release the active agents from the terpolymer gel. The thermosensitive design employed was such that the delivery vehicle is applied as a liquid at room temperature to allow uniform coating, and gels postapplication to promote retention. In vitro characterizations under simulated physiological conditions confirmed that the designed system was liquid at room temperature, gels as the temperature is increased from room to body temperature, and provides burst release of active agents upon exposure to semen fluid simulant. The second and third systems were intravaginal rings (IVRs) for sustained delivery of dapivirine, a potent inhibitor of HIV replication after the virus has entered the host cells. A sustained delivery of replication inhibitors is pursued to allow enough time for antiviral drugs to diffuse into the vaginal epithelium and ensure that inhibitory concentrations are established before the viral attack. Monolithic IVRs were fabricated from biomedical grade polyurethanes and degradable polyurethanes synthesized with hydrolytically labile ester groups in the polymer backbone. The ability of the utilized polyurethane matrix to provide a zero-order delivery of dapivirine enabled a monolithic design that can be manufactured using a cost-effective melt extrusion procedure. The inexpensive IVR design loaded with a potent drug and with the high user adherence associated with IVRs offer a promising solution for an effective microbicide.
  • [Show abstract] [Hide abstract]
    ABSTRACT: To highlight promising areas of research and preview future generations of microbicides, this review will focus on reports that described new cellular or viral targets, drug substances, or strategies that are specifically intended for topical microbicides. Those reports that dealt with the design, discovery, and synthesis of anti-HIV agents for use in oral or parenteral formulations, while important for the microbicide field, are beyond the scope of this review. Drug substances intended for topical microbicides are becoming increasingly target specific and, structurally, more complex. New production methods might reduce the cost of microbicides that contain these complex molecules. Genetically engineered probiotic vaginal bacteria express an even wider range of antiviral compounds, perhaps resulting in uninterrupted, coitally independent protection. Combination microbicides that contain two or more drug substances frequently act synergistically. The discovery of new cellular targets such as syndecan-3 might lead to more effective microbicides. Future generations of microbicides will likely contain one or more complex or highly specific drug substances, resulting in safer and more effective products. Since compliance issues continue to confound HIV and herpes simplex virus trials, efforts to bring practical, coitally independent microbicides to developing countries will become a top priority.
    Current opinion in HIV and AIDS 10/2008; 3(5):548-53. DOI:10.1097/COH.0b013e32830ab9dd · 4.68 Impact Factor
Show more