Fragments vs Trypanosoma cruzi spermidine synthase, allosterically

Chagas disease, caused by Trypanosoma cruzi, is spread throughout Central and South America by a nasty blood-sucking insect. A couple drugs are approved to treat it, but they can cause severe nausea and peripheral neuropathy, so there is room for improvement. In a recent paper in Acta Cryst D., Yasushi Amano and colleagues at Astellas Pharma describe their efforts against the T. cruzi spermidine synthase (TcSpdSyn).

TcSpdSyn transfers an aminopropyl moiety from the cofactor decarboxylated S-adenosylmethionine (dcSAM) to the evocatively-named putrescine (1,4-diaminobutane) as one step in the synthesis of an essential antioxidant. Small amines can bind in the putrescine-binding pocket and inhibit the enzyme with low micomolar activity, so the researchers decided to find other fragments that could bind in this pocket. They screened in the presence of dcSAM, using surface-plasmon resonance (SPR), with each fragment present at 0.25 mM, as well as in thermal shift assays, with each fragment present at 2 mM. Although nothing is reported about library size or hit rate, hits from either assay were taken into crystallography, resulting in six structures described in detail and deposited in the Protein Data Bank (pdb).

Two fragments were found that bind in the putrescine-binding pocket, and in both cases the enzyme shows some conformational changes to accommodate the fragments. Although these two fragments have only modest potency (IC₅₀ = 0.18-0.48 mM), they do have satisfying ligand efficiencies, and are good starting points for structure-based design.

Unexpectedly, the other four fragments bound not in the putrescine-binding pocket but at an interface between two proteins of TcSpdSyn, which forms a homodimer. One of these fragments, an isothiazolinone, showed mid-nanomolar activity in a functional assay. Readers may recall a paper we pilloried earlier this year which also reported an isothiazolinone as a screening hit. In that case, the researchers failed to recognize that this PAINS compound has ample precedent for reacting with thiols. Happily, in the current paper the researchers are not only aware of this, they actually see covalent bond formation between the fragment and a cysteine residue in the crystal structure. Interestingly though, the fragment reacts with only a single cysteine residue at the dimer interface, despite the presence of six other cysteine residues in the protein.

The researchers carefully analyzed this structure and found that binding of the fragment disrupts the putrescine-binding pocket; in other words, the fragment is an allosteric inhibitor. Moreover, the other three fragments that bind at the dimer interface also appear to act allosterically, and one of them is a single digit micromolar inhibitor.

This is a nice example of how even PAINS compounds can be useful if they are well-characterized and not hyped. Moreover, the structures suggest new approaches for tackling a target for a neglected tropical disease, either covalently or more conventionally.