Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase

Abstract

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure activity relationship and the selectivity mechanism.

Fig. 1.. Structures of OSM-S-106, OSM-S-106 derivatives and adenosine 5’-sulfamate.

(A) OSM-S-106. (B) OSM-E-32. (C) OSM-S-137. (D) OSM-S-488. (E) OSM-LO-80. (F) OSM-LO-81. (G) OSM-LO-87. (H) OSM-LO-88. (I) AMS. Structural differences between OSM-S-106 and derivatives (B-H) are highlighted in red.

Fig. 2.. Identification of the P. falciparum target of OSM-S-106

(A) P. falciparum cultures (Cam3.II-rev; trophozoite stage; 30–35 h p.i.) were exposed to OSM-S-106 for 6 h. Protein translation was assessed in the last two hours of the incubation, via the incorporation of OPP. Aliquots of inhibitor-exposed cultures were washed and returned to cultures, and viability was estimated at the trophozoite stage of the next cycle. IC₅₀ (Translation) = 0.51 μM, IC₅₀ (Viability) = 4.7 μM. Error bars correspond to SEM of three independent experiments. (B) Trophozoite stage Cam3.II_rev parasites (30–35 h p.i.) were incubated with 0.05% DMSO (Mock), 50 nM borrelidin (BOR) or 2.5 μM OSM-S-106 or 2.5 μM OSM-S-137 for 3 h. Western blots of lysates were probed for phosphorylated eIF2α with PfBiP as a loading control. Additional blots are presented in Supplementary Fig. 1C. (C) Schematic showing aaRS-catalysed attack of a nucleoside sulfamate on an activated amino acid to form an amino acid adduct. (D) Structure of Asn-OSM-S-106. (E) P. falciparum-infected RBCs were treated with 10 μM OSM-S-106 for 3 h. Extracts were subjected to LCMS. The extracted ion chromatograms of the Asn-OSM-S-106 adduct generated by P. falciparum (upper panel) and the synthetic conjugate at m/z 421.0753 (lower panel). The inset shows MS analysis of the parasite-generated Asn-OSM-S-106 adduct. (F) Sensitivity to OSM-S-106 exposure (72-h) for a cloned wildtype line (Dd2) and a CRISPR-edited clone harbouring PfAsnRS_R487S. Data represent 5 replicates and error bars correspond to SD. See Supplementary Table 6 for data values. (G,H) Sensitivity to OSM-S-106 exposure (72-h) for aptamer-regulatable PfAsnRS (G) and PfNT4 (H) lines upon addition of aTc (closed circles) and with the target expression reduced (open circles), with data normalized to a no drug control. Data represent the mean of three replicates and error bars correspond to SD. See Supplementary Table 7 for data values.

Fig. 3.. OSM-S-106 hijacks PfAsnRS enzyme activity but is less effective against PfAsnRS_R487 and HsAsnRS.

(A) ATP consumption by wildtype PfAsnRS, PfAsnRS_R487S and full-length HsAsnRS in the presence and absence of E. coli tRNA. ATP is consumed during the formation (and release) of AMP-Asn in the initial phase of the aminoacylation reaction. Reactions were incubated at 37°C for 1 h. PfAsnRS and PfAsnRS_R487S: 0.5 μM; HsAsnRS: 0.2 μM. Data represent the average of at least three independent assays and error bars correspond to SD. (B) Effects of increasing concentrations of OSM-S-106 on ATP consumption at 37°C, over a period of 2.5 h, by wildtype PfAsnRS and PfAsnRS_R487S in the presence or absence of E. coli tRNA. Enzyme concentration = 0.5 μM. IC₅₀ values: Plus E. coli tRNA = 7.3 μM for PfAsnRS and 26.3 μM for PfAsnRS_R487S; minus E. coli tRNA > 500 μM. Data represent the average of at least three independent assays. Error bars correspond to SEM. (C) Effects of increasing concentrations of OSM-S-106 and OSM-LO-80 on ATP consumption by PfAsnRS and HsAsnRS. Reactions were incubated at 37°C for 1 h with 0.05 μM PfAsnRS or 0.2 μM HsAsnRS in the presence of E. coli tRNA. IC₅₀ values for OSM-S-106: 3.6 μM for PfAsnRS and >100 μM for HsAsnRS. IC₅₀ values for OSM-LO-80: >100 μM for PfAsnRS and HsAsnRS. Data are the average of at least three independent experiments. Error bars represent SEM. (D) Effects of AMS on ATP consumption by PfAsnRS and HsAsnRS. Reactions were incubated at 37°C for 1 h with increasing concentrations of AMS and 0.05 μM PfAsnRS or 0.2 μM HsAsnRS. IC₅₀ values: 3.7 nM for PfAsnRS; 25 μM for HsAsnRS. Data represent the average of three independent experiments and error bars correspond to SEM. (E,F) Effects of synthetic Asn-OSM-S-106 on ATP consumption by PfAsnRS and HsAsnRS (E) and PfAsnRS and PfAsnRS_R487S (F). Reactions were incubated at 37°C for 1 or 2.5 h with increasing concentrations of Asn-OSM-S-106, and 0.5 μM of enzymes without tRNA. IC₅₀ values: 2.6 / 3.3 μM for PfAsnRS; 12 μM for HsAsnRS, 1.9 μM for PfAsnRS_R487S. Data points represent the average of at least three independent experiments and error bars correspond to SEM. The component concentrations for all above reactions are ATP (10 μM), asparagine (200 μM), pyrophosphatase (1 unit/mL) and E. coli tRNA (2.5 mg/mL), if present.

Fig. 4.. Structures of the Asn-AMP/CDHsAsnRS and OSM-S-106/CDHsAsnRS complexes.

(A) Structure of the CDHsAsnRS dimer in complex with Asn-AMP. The bound Asn-AMP is circled (dotted red lines) and the two chains of the dimer are coloured differently. (B) Key inhibitor contact residues in the Asn-AMP/CDHsAsnRS complex. Hydrogen bonds are indicated by yellow dashed lines. (C) Key inhibitor contact residues in the Asn-OSM-S-106/CDHsAsnRS complex. Hydrogen bonds are indicated by yellow dashed lines. Abbreviations: 2PN – imidodiphosphoric acid, GOL – glycerol.

Fig. 5.. A model of the PfAsnRS-Asn-tRNA complex and compound docking reveal mechanisms for differential compound activity.

(A) AlphaFold-Multimer model of the PfAsnRS dimer. Each chain of the dimer, and the long, disordered PfAsnRS-specific insert are depicted. (B) Model of the PfAsnRS-Asn-tRNA complex, generated by overlay of the PfAsnRS model with the E. coli AspRS/Asp-tRNA complex (PDB ID 1C0A, ). The position of the bound ligand is highlighted with a dotted red line. Residue R487 (arrowed) lies close to the tRNA binding site. (C-G) Representative in silico docks of compounds to the PfAsnRS/Asn-tRNA model for (C) AMP, (D) AMS, (E) OSM-S-106 (see also Supplementary Fig. 11C), (F) OSM-E-32, and (G) OSM-S-488. Two orientations of each docked compound are shown to illustrate alignment of the reactive groups with the tRNA-Asn carbonyl carbon. The binding poses of AMP, AMS and OSM-S-106 are similar to the corresponding parts of our experimentally determined structures of the Asn-AMP/CDHsAsnRS, Asn-AMS/CDHsAsnRS, and Asn-OSM-S-106/CDHsAsnRS complexes. The AMS sulfamate and the OSM-S-106 sulfonamide are in a suitable position to attack the carbonyl carbon of Asn-tRNA.

Scheme 1.. Aminothieno pyrimidine sulfamate synthesis protocol.