Protein-Structure Assisted Optimization of 4,5-Dihydroxypyrimidine-6-Carboxamide Inhibitors of Influenza Virus Endonuclease

Abstract

Influenza is a serious hazard to human health that causes hundreds of thousands of deaths annually. Though vaccines and current therapeutics can blunt some of the perilous impact of this viral infection, new treatments are needed due to the constantly evolving nature of this virus. Recently, our growing understanding of an essential influenza viral protein, PA, has led to the development of focused libraries of new small molecules that specifically target the active site of the PA influenza endonuclease, which we report here. Our overarching approach has been to proactively develop lead inhibitors that are less likely to rapidly develop clinical resistance by optimizing inhibitors that retain activity against induced resistant mutants. Here, we report details behind the discovery of new potent inhibitors of wild type and resistant mutant endonucleases along with their high-resolution co-crystal structure-activity relationships. These results add to our understanding of nuclease protein targets and potentially serve as starting points for a new therapeutic approach to the treatment of influenza.

Figure 1

Compounds targeting the two-metal nuclease active site, including the FDA approved HIV integrase inhibitor raltegravir (Isentress), along with the first influenza lead compound in this class, L-742,001 (1), and several recently developed experimental influenza endonuclease inhibitors (2 , 3 , 4 , and 5 ). The general structure 6 shows the structure of the active inhibitors reported in this publication.

Figure 2

Tables A–D. Fluorescent polarization structure-activity relationships of inhibitors for wild type PA organized by small molecule 2D structural type. Table A. Initial diamides. Table B. 3,5-Dichloroisonicotinyl and N-methyl derivatives. Table C. Linker heteroatom exploration. Table D. R₂ optimization. The fluorescent polarization assay conditions have previously been reported^,.

Figure 3

The wild type PA_N high affinity bound conformations of inhibitors from high-resolution co-crystal structures. (A) The PA_N inhibitor co-crystal structures of 7b (left panel, which shows the cis (44%) and trans (56%) bound conformations) and 7a (right panel, which shows the trans bound conformation). (B) The bound conformation of 8f (left panel) in the PA_N active site and the bound conformation of 8e (right panel) in the wild type PA_N construct showing potential hydrogen-bond interactions with crystallographic water molecules, active-site side chains, and a main chain interaction. Both structures feature H-bond donation from Lys34 to the pyrrolidine amide carbonyl (3.4 Å and 3.3 Å, respectively), and numerous interactions near the two-metal site, which are common to all DHPC inhibitors. In 8e, note the main chain (Leu106) H-bond (3.1 Å) to one sulfone oxygen and the H-bond (3.0 Å) donation from Tyr24 the other sulfone oxygen. Mn²⁺⁺ ions are shown as violet spheres and Mg²⁺⁺ as green spheres. See Supporting Figure S1b for electron density maps for examples of these and other relevant structures.

Figure 4

Examples of some aryl linker bound conformations with wild type PA. (A) A depiction of the co-crystal structure showing two binding modes of 10e with comparable binding energies in the wild type PA_N; showing active-site side-chains Tyr111 on the left and Tyr24 on the right. (B) Top Panel: Overlay of the bound conformation of conformationally constrained phenethylamine analogs 10i (tan) and 10j (cyan). Bottom Panel: Overlay of 10j (cyan) with 10k (purple).

Figure 5

The bound conformations (cis = 49%, inverted = 51%) of 10e in complex with the E119D mutant of PA_N construct. The cis mutant conformation includes a chloro carbonyl intramolecular interaction that is not present in the wild type structure with the same inhibitor.

Figure 6

A comparison of bound high affinity wild type versus E119D inhibitor conformations. Left Panel: The co-crystal structure of 9k (tan) overlaid with the conformation 9b (cyan) in the wild type PA_N construct. Right Panel: The conformation of 9b (cyan) overlaid with the conformation of 9k (tan) in the E119D mutant. Note that the amide bond with the 2-Cl phenyl group is cis in both the wild type and mutant structures.