Novel α- and β-Amino Acid Inhibitors of Influenza Virus Neuraminidase

Abstract

In an effort to discover novel, noncarbohydrate inhibitors of influenza virus neuraminidase we hypothesized that compounds which contain positively charged amino groups in an appropriate position to interact with the Asp 152 or Tyr 406 side chains might be bound tightly by the enzyme. Testing of 300 ␣-and ␤-amino acids led to the discovery of two novel neuraminidase inhibitors, a phenylglycine and a pyrrolidine, which exhibited K i values in the 50 M range versus influenza virus A/N2/Tokyo/3/67 neuraminidase but which exhibited weaker activity against influenza virus B/Memphis/3/89 neuraminidase. Limited optimization of the pyrrolidine series resulted in a compound which was about 24-fold more potent than 2-deoxy-2,3-dehydro-N-acetylneuraminic acid in an anti-influenza cell culture assay using A/N2/Victoria/3/75 virus. X-ray structural studies of A/N9 neuraminidase-inhibitor complexes revealed that both classes of inhibitors induced the Glu 278 side chain to undergo a small conformational change, but these compounds did not show time-dependent inhibition. Crystallography also established that the ␣-amino group of the phenylglycine formed hydrogen bonds to the Asp 152 carboxylate as expected. Likewise, the ␤-amino group of the pyrrolidine forms an interaction with the Tyr 406 hydroxyl group and represents the first compound known to make an interaction with this absolutely conserved residue. Phenylglycine and pyrrolidine analogs in which the ␣-or ␤-amino groups were replaced with hydroxyl groups were 365-and 2,600-fold weaker inhibitors, respectively. These results underscore the importance of the amino group interactions with the Asp 152 and Tyr 406 side chains and have implications for anti-influenza drug design.The catalytic power of enzymes arises from their ability to bind the altered substrate in the transition state much more tightly than the substrate in its unaltered, ground state form (28,29). The magnitude of this binding affinity discrimination can be estimated from a comparison of the rate constants for the enzymatic and nonenzymatic reactions (29) and appears to range between 10 8 -and 10 17 -fold for most enzymes (7, 30). Any stable compound whose chemical structure resembles that of the transition state should capture some fraction of this binding affinity advantage, resulting in a potent and specific inhibitor of the target enzyme. Compounds believed to mimic the structure of the transition state or of an intermediate in the reaction pathway have been described for well over 100 different enzymes (29, 31).Influenza neuraminidase is one enzyme for which a putative transition state analog has been described (10). This enzyme is present on the viral surface and functions to cleave terminal ␣-ketosidically linked N-acetylneuraminic acid residues from glycoproteins, glycolipids, and oligosaccharides in a reaction which is essential for effective replication of the influenza virus (20). Enzyme kinetic isotope effect studies (5) have established that the neuraminidase hydrolytic reaction proceeds throug...

In Vitro Characterization of A-315675, a Highly Potent Inhibitor of A and B Strain Influenza Virus Neuraminidases and Influenza Virus Replication

“…ues reported in Table 1. Indeed, the potency of A-315675 against the B/Memphis/3/89 neuraminidase represents a 10 7 -fold improvement in binding affinity relative to the original pyrrolidine screening hit that started our influenza neuraminidase inhibitor program (16). Although influenza neuraminidase inhibitor potencies are often reported as IC 50 s (2, 3, 22), we have elected to measure potencies as K i values.…”

“…The cytotoxicities of the test compounds were studied by exposing MDCK cells growing in log phase to test compounds for 4 days. The cytopathogenic effects were then quantitated using a slight modification of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye metabolism procedure described previously (16).…”

Phenotypic Drug Susceptibility Assay for Influenza Virus Neuraminidase Inhibitors

“…The search for better and more useful NA inhibitors is under way. The pyrrolidinebased compound A-315675, the pyrazinecarboximide-based compound T-750, and the cyclopentane peramivir (RWJ-270201) are under study for their activities against influenza type A and B viruses (1,12,26,27,38). Peramivir has in vitro and in vivo activities equal to or greater than those of zanamivir and oseltamivir carboxylate against a number of influenza type A and B viruses (2,4,5,10,18,(46)(47)(48)(49).…”

Combining docking, scoring and molecular field analyses to probe influenza neuraminidase–ligand interactions

“…A set of 121 diverse neuraminidase inhibitors belonging to the following chemical classes: 5,6-dihydro-4H-pyranes, cyclohexenes, benzoic acid derivatives, pyridines and pyrrolidines [14,18,[71][72][73][74][75] (Fig. 1 and Table A (in supplementary data)) were used for modeling.…”