Probing the Interaction of Dengue Virus Envelope Protein with Heparin: Assessment of Glycosaminoglycan-Derived Inhibitors Rory M. Marks, † Hong Lu, † Renuka Sundaresan, † Toshihiko Toida, ‡ Atsushi Suzuki, ‡ Toshio Imanari, ‡ Marı ´a J. Herna ´ iz, § and Robert J. Linhardt* ,§ Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109, Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, Japan, and Division of Medicinal and Natural Products Chemistry, Departments of Chemistry and Chemical and Biochemical Engineering, University of Iowa, Iowa City, Iowa 52242 Received September 20, 2000 A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an under- standing of the structural requirements for polyanion binding to dengue virus envelope protein has been established. Introduction Dengue virus is a mosquito-transmitted flavivirus that causes a febrile disease in humans. 1 More than 2.5 billion people in over 100 countries are at risk of infection, and there are at least 20 million infections per year. 2 There is no treatment for dengue infection, and no vaccine is available. New approaches for the control of dengue infection are urgently needed. Studies of dengue virus pathogenesis have been hampered by the lack of suitable animal models, and there is little understanding of the pathophysiology of infection. 1 Studies of other pathogenic viruses have revealed that the binding of viral ectodomain molecules to specific receptors on target cells is a critical factor in pathoge- nicity since it determines cell and tissue tropism. 3 We previously showed that dengue virus envelope protein utilizes a highly sulfated form of the glycosaminoglycan (GAG) heparan sulfate as a receptor. 4 Five distinct cell types, derived from five different organs and five dif- ferent species 4 (and unpublished), utilize heparan sul- fate as a receptor; thus, it is likely that a single cell- surface GAG represents a conserved physiologically relevant receptor for dengue virus. Although many microorganisms interact with glycosaminoglycans, the dengue virus interaction is unusual for its high affinity and its specificity for an highly sulfated form of heparan sulfate. 4 Since a heparan sulfate GAG is a putative receptor for dengue virus envelope protein, soluble GAGs and other highly charged polyanions could be effective inhibitors of viral infectivity. In support of this hypoth- esis, we found that heparin, heparin-derived oligosac- charides, and the polysulfonated urea Suramin, inhib- ited dengue virus envelope protein binding and viral infectivity in vitro. 4 We are building on these data to systematically generate and test related chemical struc- tures for antiviral activity and potential use as thera- peutics. We developed a 96-well-based competition assay for testing the ability of soluble compounds to inhibit the binding of dengue virus envelope protein to im- mobilized heparin. We used this assay, in conjunction with surface plasmon resonance spectroscopy (SPR), to investigate the interaction of heparin-like molecules with dengue virus envelope protein and report the relationship of molecular size, level of sulfation, and other structural properties to inhibitory activity. Materials and Methods Chemicals. Chondroitin sulfate (molecular weight average (MWav) 15 000), from bovine tracheal cartilage, and dermatan sulfate (MWav, 30 000) from porcine skin were a kind gift from Shin-Nippon Yakugyo Co. (Tokyo, Japan). Hyaluronan (MWav, 100 000) from Streptococcus zooepidemicus was purchased from Kibun Food Chemipha Co. (Tokyo, Japan). Heparin (MW av, 16 000) and heparan sulfate (MWav, 14 800) from porcine intestinal mucosa were purchased from Celsus (Cin- cinnati, OH). Suramin was from Sigma (St. Louis, MO). Sulfated lactobionic acid was a generous gift from Professor Jawed Fareed (Loyola University Medical Center, Maywood, IL). Sulfated -cyclodextrin was from American Maize (Ham- mond, IN). Sucrose octasulfate was from Toronto Research Chemicals (Toronto, Canada). Hyaluronidase from Streptomy- ces hyalurolyticus (lyase, E. C.4.2.2.1) was purchased from Seikagaku Kogyo Co., Tokyo, Japan. Sephadex G-50 (super- fine) and Hi-Trap desalting columns were purchased from Pharmacia Biotech. Dialysis tubing (MWCO 500) was pur- * Correspondence to Dr. Robert J. Linhardt, PHAR S328, University of Iowa, Iowa City, Iowa 52242. Phone: (319) 335-8834. Fax: (319) 335-6634. E-mail: robert-linhardt@uiowa.edu. † University of Michigan. ‡ Chiba University. § University of Iowa. 2178 J. Med. Chem. 2001, 44, 2178-2187 10.1021/jm000412i CCC: $20.00 © 2001 American Chemical Society Published on Web 05/09/2001