title: Charge modification of plasma and milk proteins results in antiviral active compounds.
abstract: Previous studies have shown that acylated plasma and milk proteins with increased negative charge, derived from various animal and human sources, are potent anti-HIV compounds. The antiviral effects seemed to correlate positively with the number of negative charges introduced into the various polypeptides: proteins with a high content of basic amino acids in which all of the available epsilonNH2 groups were anionized yielded the most potent anti-HIV compounds. It remained unclear however whether the total net negative charge of the various derivatized proteins, or rather the charge density on the protein backbone, is essential for the observed anti-HIV activity. Earlier studies have shown that acylated albumins preferentially block the process of HIV/cell fusion through binding to the HIV envelope proteins gp120 and gp41 as well as to the cell surface of the HIV target cells. Some of these polyanionic proteins have been shown to interfere also with the gp120-CD4 mediated virus/cell binding. The relative contribution of these effects to the anti-HIV activity may depend both on the total negative charge introduced as well as the hydrophobicity of the acylating reagent added to the particular proteins. In this study we show that the higher the charge density of the derivatized proteins, the more potent their HIV replication inhibiting effects are. In contrast, the addition of positive charge to the studied plasma and milk proteins through amination resulted in a reduced anti-HIV activity but a clearly increased anti-HCMV activity, with IC50 values in the low micromolar concentration range. Interestingly, native lactoferrin (Lf) was antivirally active against both HIV and HCMV. Acylation or amination of Lf increased the anti-HIV and anti-HCMV activity, respectively. The N-terminal portion of Lf appeared essential for its anti-HCMV effect: N-terminal deletion variants of human Lf were less active against HCMV. Circular dichroism of the modified proteins showed that the secondary structure of the tested proteins was only moderately influenced by acylation and/or covalent attachment of drugs, making these (derivatized) proteins useful candidates as antiviral agents and/or intrinsically active drug carriers. The relatively simple chemical derivatization as well as the abundant sources of blood plasma and milk proteins provides attractive opportunities for the preparation of potent and relatively cheap antiviral agents for systemic or local applications.
authors.affiliation: Groningen University Institute for Drug Exploration, Department of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, The Netherlands. swart.nl@yamanouchi-eu.com
authors.forename: P J
authors.initials: PJ
authors.lastname: Swart
authors.forename: M C
authors.initials: MC
authors.lastname: Harmsen
authors.forename: M E
authors.initials: ME
authors.lastname: Kuipers
authors.forename: A A
authors.initials: AA
authors.lastname: Van Dijk
authors.forename: B W
authors.initials: BW
authors.lastname: Van Der Strate
authors.forename: P H
authors.initials: PH
authors.lastname: Van Berkel
authors.forename: J H
authors.initials: JH
authors.lastname: Nuijens
authors.forename: C
authors.initials: C
authors.lastname: Smit
authors.forename: M
authors.initials: M
authors.lastname: Witvrouw
authors.forename: E
authors.initials: E
authors.lastname: De Clercq
authors.forename: M P
authors.initials: MP
authors.lastname: de Béthune
authors.forename: R
authors.initials: R
authors.lastname: Pauwels
authors.forename: D K
authors.initials: DK
authors.lastname: Meijer
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D001798:Blood Proteins / Q000737:chemistry*; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003587:Cytomegalovirus / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects; D006801:Humans; D008826:Microbial Sensitivity Tests; D008894:Milk Proteins / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D000998:Antiviral Agents; D001798:Blood Proteins; D008894:Milk Proteins