title: The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins.
abstract: The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.
authors: Department of Pharmacokinetics and Drug Delivery, University Center for Pharmacy, Groningen University Institute for Drug Exploration (GUIDE), Ant. Deusinglaan 1, 9713 AV, Groningen, The Netherlands. l.beljaars@farm.rug.nl
authors: Leonie
authors: L
authors: Beljaars
authors: René
authors: R
authors: Floris
authors: Ben
authors: B
authors: Berkhout
authors: Catharina
authors: C
authors: Smit
authors: Dirk K F
authors: DK
authors: Meijer
authors: Grietje
authors: G
authors: Molema
mesh_terms: D000156:Aconitic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D019380:Anti-HIV Agents / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects*; D006493:Heparin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D023421:Models, Animal; D011487:Protein Conformation; D051381:Rats; D017208:Rats, Wistar; D012709:Serum Albumin / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D000075462:Serum Albumin, Human; D013386:Succinates; D019802:Succinic Acid / Q000737:chemistry / Q000494:pharmacology*; D014018:Tissue Distribution
chemical_list: D019380:Anti-HIV Agents; D000925:Anticoagulants; D015699:HIV Envelope Protein gp120; C459130:Hep-HSA; D012709:Serum Albumin; C459126:Suc-HSA; D013386:Succinates; C108785:polyaconitylated human serum albumin; D006493:Heparin; D000156:Aconitic Acid; D019802:Succinic Acid; D000075462:Serum Albumin, Human