Don't get your kinases in a twist

New drugs that block kinase enzymes irreversibly could be used in cancer therapy as well as in studying how members of this class of enzymes, also known as the phosphotransferases, function. The kinases play a role in cancer, diabetes and metabolism, epilepsy, inflammation, immunity and infection, hypertension and cardiovascular disease, neurodegenerative diseases, the central nervous system, autism, vision, cognition, osteoporosis, allergic response, circadian rhythms, reproduction and development.

Kinases are among the most important drug discovery targets, particularly in cancer therapy. There are well over 500 kinases in the human body, which help transmit signals and control a vast array of complex cellular processes. The enormous diversity of these enzymes and their specific roles make them an important target for drug discovery. Indeed, finding novel inhibitors is one of the most fruitful areas of research.

A team in France has now found lead compounds based on resorcylic acid lactone. This structure has been investigated previously, but they have carried out "molecular editing" to find diverse derivatives, including two fluoroenones with improved properties compared with the parent compound. The compounds are not only useful drug leads but might also be used to study the kinases themselves, and lead to a better understanding of these ubiquitous enzymes.

So far, most small-molecule inhibitors are heterocycles reminiscent of the adenosine scaffold, which target the nucleotide binding site of the kinase enzyme, the researchers explain. Resorcylic acid lactones (RAL) bearing a suitably positioned cis-enone, such as hypothemycin, LL-Z1640-2, L-783277, radicicol A, however, have been shown to irreversibly inhibit select kinases and so represent what the team describes as "a unique pharmacophore", which is effective in vivo. Previous researchers used a bioinformatics analysis to identify 46 individual kinase enzymes that could potentially be targeted by this family of compounds.

Now, Rajamalleswaramma Jogireddy, Sofia Barluenga, and Nicolas Winssinger of the Institute of Supramolecular Science and Engineering at the University of Strasbourg, France, have focused their efforts on expanding the diversity of this natural pharmacophore. In work funded by grants from France's National Research Agency (ANR) and Conectus they have identified several modifications and substitutions in these small molecules, particularly at the benzylic carbon by an oxygen atom which can make the compounds more accessible to synthetic organic chemists.

The team points out that these compounds react with the cysteine residue in the kinase nucleotide binding site through a Michael addition, which means that controlling the electronic properties of the enone group within the molecule should boost their inhibitory properties. Their previous work showed that substitution at the beta position of the enone with a methyl group completely suppresses activity, a point reported at the same time by another team. However, they then reasoned that a fluorine substituent at the alpha position might have the opposite effect without changing the shape of the molecule overall.

The team has now termed these small, but significant changes, "molecular editing" (after Samuel Danishefsky et al. of Columbia University and the Memorial Sloan-Kettering Cancer Center, New York) and suggest that the approach could be used to improve the molecules and avoid isomerisation side reactions in their synthesis.

The team tested their "edits" three times on standard kinase enzyme assays and analysed the raw data by first converting to percent receptor autophosphorylation relative to stimulated controls. The so-called IC50 values were determined on GraphPad Prism 5.01 software with the bottom constrained to 0 and the top to 100 using a nonlinear regression curve fit with variable hill slope. IC50 is the half maximal inhibitory concentration which measures effectiveness of a compound in inhibiting biological or biochemical function.