A Potent Trivalent Sialic Acid Inhibitor of Adenovirus Type 37 Infection of Human Corneal Cells**

Viruses of the Adenoviridae family are widespread in society and are associated with a wide variety of clinical symptoms in humans, including respiratory, gastrointestinal, and ocular diseases. Epidemic keratoconjunctivitis (EKC) is a severe ocular infection and is caused by the highly contagious adenoviruses Ad8, Ad19, and Ad37. Besides keratitis and conjunctivitis, other common symptoms of EKC are pain, lacrimation, red and swollen eyes, as well as decreased vision that may last for months or even years. No antiviral drugs are currently available for the treatment of EKC or any other infection caused by adenoviruses. The initial event leading to EKC is binding of the viruses to glycans that contain sialic acid moieties on epithelial cells in the cornea or conjunctiva through trimeric fiber structures extending from the viral particles. The receptor-binding domain, the fiber knob, is located at the C terminus of each fiber and contains three separate pockets that each can accommodate one sialic acid residue. Ad37 was recently shown to bind to cell-surface glycoproteins carrying a glycan structure similar to the GD1a ganglioside. The GD1a glycan is a branched hexasaccharide with a terminal sialic acid residue on each of its two arms. Structural studies showed that the two sialic acid moieties dock into two of three sialic acid binding sites in the trimeric knob of the Ad37 fiber protein. Most likely, multiple fiber proteins simultaneously engage several host-cell epitopes containing terminal sialic acids; internalization and subsequent infection follow. If these sialic acid–protein interactions can be blocked, for example, by a multivalent sialic acid conjugate, infection might be prevented. To date, several carbohydrate-based or glycomimetic drugs have reached the market; however, the development of additional therapies is still hampered by challenges such as poor absorption and/or rapid elimination. The topical administration of sialic acid conjugates directly to the eye, that is, the site of infection, circumvents many of the pharmacokinetic hurdles and has the potential to prevent or even cure EKC. In the search for new antiviral substances against Ad37, we synthesized and evaluated multivalent human serum albumin (HSA) conjugates of both 3’-sialyllactose and sialic acid as adenoviral inhibitors. These conjugates efficiently inhibited Ad37 cell attachment and the subsequent infection of human corneal epithelial (HCE) cells. Both types of conjugates were equally efficient as Ad37 inhibitors. From the crystal structure of the fiber-knob protein as a complex with sialyllactose, it was evident that the sialic acid acetamide group is positioned in a relatively large hydrophobic pocket. To improve the potency of the more advantageous multivalent sialic acid conjugates, we used structure-based design and synthesized a library of ten Nacyl-modified sialic acid derivatives with the overall goal of improving hydrophobic interactions and thus affinity and efficacy. Unfortunately, none of the designed conjugates were as potent as the original sialic acid–HSA conjugate, although X-ray crystallography revealed that the modified saccharides interacted with the fiber-knob protein as expected. On the basis of the structural features of the interaction of the GD1a glycan with the Ad37 knob, and with our previous results 9] in mind, we then designed and synthesized sialic acid containing compounds by using small non-protein scaffolds. The crystal structure of the fiber-knob protein shows that the three known sialic acid binding sites are separated by distances of about 10 . We therefore considered the design of a compact and rigid scaffold decorated with three correctly positioned sialic acids as too complex. Instead we selected the three small and flexible scaffolds tris(2aminoethyl)amine (2), 2-(aminomethyl)-2-methyl-1,3-propanediamine (3), and 2,2-diaminomethyl-1,3-propanediamine (4) for conjugation with the sialic acid derivative 1 through [*] Dr. S. Spjut, Dr. W. Qian, Prof. M. Elofsson Department of Chemistry Ume Centre for Microbial Research (UCMR) and Laboratory for Molecular Infection Medicine Sweden (MIMS) Ume University, 90187 Ume (Sweden) E-mail: [email protected]