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.
1 comment:
BSL-2+ makes this more accessible than other recent tropical diseases.
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