Structural basis of malaria parasite phenylalanine tRNA-synthetase inhibition by bicyclic azetidines

Abstract

The inhibition of Plasmodium cytosolic phenylalanine tRNA-synthetase (cFRS) by a novel series of bicyclic azetidines has shown the potential to prevent malaria transmission, provide prophylaxis, and offer single-dose cure in animal models of malaria. To date, however, the molecular basis of Plasmodium cFRS inhibition by bicyclic azetidines has remained unknown. Here, we present structural and biochemical evidence that bicyclic azetidines are competitive inhibitors of L-Phe, one of three substrates required for the cFRS-catalyzed aminoacylation reaction that underpins protein synthesis in the parasite. Critically, our co-crystal structure of a PvcFRS-BRD1389 complex shows that the bicyclic azetidine ligand binds to two distinct sub-sites within the PvcFRS catalytic site. The ligand occupies the L-Phe site along with an auxiliary cavity and traverses past the ATP binding site. Given that BRD1389 recognition residues are conserved amongst apicomplexan FRSs, this work lays a structural framework for the development of drugs against both Plasmodium and related apicomplexans.

Structural analyses of the malaria parasite aminoacyl‐tRNA synthetases provide new avenues for antimalarial drug discovery

“…The Pf FRS genes code for three proteins that are localised to the subcellular compartments in the malaria parasite 14,46 . This FRS enzyme is unique as it has α‐ and β‐subunits, exists as a heterodimer and further dimerizes into a hetero‐tetrameric (αβ) 2 assembly 47,48 . The FRS (αβ) 2 heterotetramer binds to two molecules of tRNA Phe .…”

“…The Pf FRS α‐subunit is ~500 residue longer than the bacterial one and the β‐subunit is ~200 residues shorter than the bacterial counterpart. The α‐ and β‐subunit interface is occupied by a Mg 2+ that may be critical for enzyme activity and stabilization of the complex 47 . The plasmodial species contain two copies of tRNA Phe each for the nucleus and apicoplast.…”

“…The (αβ) 2 heterodimer arrangement of the Pv FRS cyto consists of eight domains 47 . Among these eight domains, two are present in the α‐subunit (α1‐catalytic and α2) and six are within the β‐subunit (β1, β3‐β4 (constitute the editing site), β5, β6 (catalytic‐like) and β7) 47 . The α‐subunit is associated with β‐subunit via two‐subdomains of the β‐subunit, namely B1 and B2’ (Figure 5a).…”

“…Bicyclic azetidine based small molecules were identified from a library of ~100,000 compounds (stereo‐chemically and structurally diverse compounds) synthesized by Broad Institute through Diversity Oriented Synthesis (DOS) 49–51 . The potent bicyclic azetidine BRD1389 compound is bound to the α‐subunit of the Pv FRS cyto enzyme 47 . These bicyclic azetidine compounds exhibited killing activity against the different parasitic stages of Pf , that is, liver, blood and the transmission stages 49,50 .…”

“…Further, in vivo studies showed low propensity for the mutations against BRD compounds, which specifically inhibit Pf cFRS enzyme activity in nanomolar range 49 . BRD1389 shows high selectivity for Pf cFRS with an IC50 of 12 nM versus an IC 50 of 1,200 nM for Hs cFRS 47,49 . Subsequently, BRD1389 was enzymatically characterized as a competitive inhibitor for L‐Phe, but noncompetitive inhibition was observed against ATP 47 .…”