Fragments vs Dengue virus polymerase

Dengue fever, evocatively called “breakbone fever” for the severe pain it can inflict, is caused by a mosquito-borne virus that infects hundreds of millions of people each year. There are no approved antiviral treatments. Two papers from researchers at the Novartis Institute for Tropical Diseases and the University of Texas Galveston provide some promising early leads.

The first, in J. Biol. Chem., by Christian Noble, Pei-Yong Shi, and collaborators, describes a crystallographic screen of 1408 fragments against Dengue virus RNA-dependent RNA polymerase (DENV RdRp), which is highly conserved among the four serotypes of Dengue virus. Crystals were soaked in pools of eight fragments, with each present at only 0.625 mM, ten to one hundred times lower than other recent crystallographic screens. Perhaps because of this low concentration, only a single hit was identified – compound JF-31-MG46. The crystal structure revealed that the molecule binds in the “palm subdomain” of the protein, which is analogous to a druggable site on the hepatitis C virus protein.

Surface plasmon resonance (SPR) showed that this fragment had a dissociation constant of 0.21 mM against RdRp from serotype 3 and 0.61 mM against RdRp from serotype 4, suggesting weak but real binding. Isothermal titration calorimetry (ITC) was not successful, perhaps because of compound solubility, but replacing the terminal phenyl group with a thiophene led to more potent compounds which could be characterized both by SPR and ITC. The compounds were also active in an enzymatic assay, with IC₅₀ values comparable to their affinities.

The second paper, by Fumiaki Yokokawa and collaborators and published in J. Med. Chem., describes the optimization of these fragments. Fragment growing was performed to try to displace a bound water molecule, resulting in the low micromolar compound 17. Compounds that contain carboxylic acids often have low cell permeability, so several bioiosteres were tested to try to replace this moiety, and compound 23 showed increased affinity. However, this compound was still quite polar, showed poor permeability, and no cell activity. Adding a lipophilic substituent and decreasing the acidity led to compound 27, with nanomolar affinity and enzymatic inhibition of all four Dengue virus serotypes. Importantly, this compound also showed low micromolar activity against all four serotypes in cell assays.

The J. Med. Chem. paper notes that a high-throughput screen against RdRp had been plagued with false positives. One validated low micromolar hit was optimized to nanomolar potency, but this was very lipophilic and displayed no cell activity. It is interesting that the fragment-derived leads initially displayed no cell activity for the opposite reason: they were too polar. This is a useful reminder that physicochemical properties matter. The successful optimization of the fragment-derived series suggests that it can be easier to make leads more lipophilic than less.