Serendipitous discovery of a potent influenza virus a neuraminidase inhibitor.

We have previously reported a potent neuraminidase inhibitor that comprises a carbocyclic analogue of zanamivir in which the hydrophilic glycerol side chain is replaced by the hydrophobic 3-pentyloxy group of oseltamivir. This hybrid inhibitor showed excellent inhibitory properties in the neuraminidase inhibition assay (Ki =0.46 nM; Ki (zanamivir) =0.16 nM) and in the viral replication inhibition assay in cell culture at 10(-8) M. As part of this lead optimization, we now report a novel spirolactam that shows comparable inhibitory activity in the cell culture assay to that of our lead compound at 10(-7) M. The compound was discovered serendipitously during the attempted synthesis of the isothiourea derivative of the original candidate. The X-ray crystal structure of the spirolactam in complex with the N8 subtype neuraminidase offers insight into the mode of inhibition.

Structural basis for a class of nanomolar influenza A neuraminidase inhibitors.

The influenza virus neuraminidase (NA) is essential for the virus life cycle. The rise of resistance mutations against current antiviral therapies has increased the need for the development of novel inhibitors. Recent efforts have targeted a cavity adjacent to the catalytic site (the 150-cavity) in addition to the primary catalytic subsite in order to increase specificity and reduce the likelihood of resistance. This study details structural and in vitro analyses of a class of inhibitors that bind uniquely in both subsites. Crystal structures of three inhibitors show occupation of the 150-cavity in two distinct and novel binding modes. We believe these are the first nanomolar inhibitors of NA to be characterized in this way. Furthermore, we show that one inhibitor, binding within the catalytic site, offers reduced susceptibility to known resistance mutations via increased flexibility of a pendant pentyloxy group and the ability to pivot about a strong hydrogen-bonding network.

Exploitation of the catalytic site and 150 cavity for design of influenza A neuraminidase inhibitors.

We report here the exploitation of the 150 cavity in the active site of influenza A viral neuraminidases for the design of novel C-6 triazole-containing Tamiflu derivatives. A general and convenient synthetic route was developed by utilizing a highly substituted cyclic Baylis-Hillman acetate as an active precursor for azide substitution via suprafacial allylic azide [3,3]-sigmatropic rearrangement. Virus replication inhibitory assays in vitro of these triazole derivatives containing either an amino or guanidino function indicated that the guanidinium compound showed the higher efficacy against a strain with N2 subtype at a concentration of 2 × 10(-5) M but did not inhibit replication of a strain with N1 subtype even at a concentration of 10(-4) M. In order to probe the nature of the enzyme-inhibitor interactions, molecular dynamics simulations were performed on complexes of these compounds with different neuraminidase enzymes. The results indicated that the candidate inhibitors occupy both the 150 cavity and catalytic site but with alternating occupancy.

Mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity.

Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here on a new class of specific, mechanism-based anti-influenza drugs that function through the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and we confirm this mode of action with structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad-spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates.

Replication inhibition activity of carbocycles related to oseltamivir on influenza A virus in vitro.

We have recently demonstrated that newly synthesized oseltamivir derivatives that contain a substituted triazole ring at the C-5 amino group interact with the 150 cavity found specifically in the group-1 neuraminidase (NA) subtypes of influenza A virus. These compounds exhibited in vitro inhibition activity of a group-1 NA enzyme incorporated in virus-like particles (VLPs). In the current study, we tested these nine triazole-containing carbocycles as well as an amino- and a guanidino-substituted derivative in virus replication inhibitory assays in vitro. None of the triazole-containing carbocycles significantly inhibited influenza A virus replication in MDCK cells with either a virus strain containing a group-1 or a group-2 subtype NA. In contrast, the amino- and guanidino-substituted derivatives clearly inhibited the cytopathic effect or spread of virus infection detected by immunostaining in MDCK monolayers as well as progeny virus release; these compounds were also reported to have shown the highest inhibition of group-1 NA in the context of VLPs. These results, together with the structures of these compounds, suggest that hydrogen-bonding interactions between the polar amino or guanidino functions and complementary groups in the neuraminidase active site (e.g. Asp151, Glu 119) may be essential for strong inhibition of the neuraminidase enzyme and, in turn, the inhibition of influenza A virus replication.