Rational design of potent sialidase-based inhibitors of influenza virus replication

Abstract

Two potent inhibitors based on the crystal structure of influenza virus sialidase have been designed. These compounds are effective inhibitors not only of the enzyme, but also of the virus in cell culture and in animal models. The results provide an example of the power of rational, computer-assisted drug design, as well as indicating significant progress in the development of a new therapeutic or prophylactic treatment for influenza infection.

Influenza neuraminidase

“…In the 1970’s a trifluoroacetyl derivative of DANA was found to be a potent inhibitor of NA in vitro and of virus replication in tissue culture, but it failed to protect animal models, apparently due to difficulty in crossing cell membranes coupled with rapid metabolism 1 . By examining how sialic acid sits in the active site, it was predicted and demonstrated that addition of a 4-guanidino group to DANA would improve its binding, and this compound (zanamivir) is now marketed as Relenza 65 . Scientists at Gilead took a more a priori approach, using the crystal structure of the active site to find a backbone that was easier to synthesize than sialic acid and that had better bioavailability, and the result was oseltamivir and its ethyl ester pro-drug marketed as Tamiflu.…”

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

“…Therefore, we propose that influenza NA follows a double-displacement mechanism similar to the classical retaining glycosidase mechanism 16 . The covalently linked 3ax-fluoro-Neu5Ac moiety adopts a chair conformation, whereas a-Neu5Ac and Neu5Ac2en adopt boat and half-chair conformations 10 , respectively, when bound to influenza NA (Fig.3b). In our N2-covalent complex, the unique C-3 fluorine of the 3ax-fluoro-Neu5Ac moiety is 3.51-3.55 Å away from the closest neighbouring residue Arg118.…”

“…Two more recently developed NA inhibitors, laninamivir and peramivir, are approved for use in Japan and still under phase III clinical trials in the United States. All of these current NA inhibitors were designed based upon the structure of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en), a putative NA transition-state analogue 8,[10][11][12] . Insight into the NA catalytic mechanism and the proposed oxocarbenium ion transition-state intermediate were crucial for the design of Neu5Ac2en analogues with potent NA inhibitory activity 13,14 .…”

“…However, numerous studies still indicate that the influenza NA may possibly function in a unique manner. For example, 4-guanidinoNeu5Ac2en (zanamivir), 4-amino-Neu5Ac2en and even Neu5Ac2en all exhibit selectivity for influenza NA over NA from other species 10 ; a computational study by Thomas et al 23 concluded that a direct hydroxylation mechanism with no covalent influenza NA-sialosyl intermediate is actually the energetically preferred pathway; moreover, some bacterial NAs that possess the same canonical protein fold and conserved active-site tyrosine still produce completely different products 16,24 . Taken together, the above studies clearly illustrate that the influenza virus NA catalytic mechanism may be quite unique.…”

Protein Structure from X-Ray Diffraction

“…The SBDD methodology requires an iterative procedure in which compounds are designed, synthesised and crystal structures of the protein-drug complexes are then determined to test the modelling predictions. Successful examples of this approach include the currently used cocktail of HIV protease inhibitors against AIDS [6], thymidylate synthase inhibitors against cancer [7] and neuraminidase inhibitors against influenza [8]. In the case of thymidylate synthase inhibitors, more than 100 enzyme-inhibitor complex structures were solved [9].…”