Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

Abstract

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 microg x l(-1) to >400 microg x l(-1) by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale 'piggyback' inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 microM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 microM.

Figure 1. Purification of recombinant PfNMT

Recombinant PfNMT was enriched in three stages from E. coli soluble lysate, prepared following protein expression in a 50 litre fermenter. After pooling of fractions, products showing NMT activity from each purification stage were separated by SDS/PAGE as shown. Lane 1, capture stage using Ni-Sepharose IMAC; lane 2, intermediate purification on Sephacryl S-100 size-exclusion column; lane 3, final stage using ReSourceS cation-exchange (pooled active NMT fractions shown). M, molecular mass markers. In lane 3, the band migrating at approx. 50 kDa (+) was identified as PfNMT by peptide mass fingerprinting. The major impurity (*) was characterized by peptide mass fingerprinting and is most likely to be an E. coli Cap-DNA recognition protein (gi:2098303), consistent with the observed molecular mass of 24 kDa.

Figure 2. Development of SPA for NMT activity

(A) The SPA was conducted with 500 nM PfARFlong substrate, 500 nM myristoyl CoA and 100 ng of PfNMT at 37 °C. Sampling was conducted at five different time points (0, 40, 90, 150 and 240 min) to monitor reaction progression. Four different samples were used in a typical reaction timecourse (▼), a reaction with no peptide (○), no NMT (●) and AlaARF peptide replacing the peptide substrate (□). (B) The concentration of NMT was titrated by serial dilution in otherwise identical reactions incubated for 30 min using 125 nM ARFlong peptide and 125 nM myristoyl-CoA, in a final volume of 100 μl. Enzyme concentration, as a percentage of the highest concentration used, is plotted against c.p.m. for reactions using CaNMT (△), PfNMT (■) and HsNMT1 (○). A close approximation to a linear fit demonstrates that enzyme concentrations can be selected so that the amount of enzyme is rate-limiting under the experimental conditions selected. a.u., arbitrary units. (C) An IC₅₀ value for inhibitor UK-362091 with CaNMT was determined using the SPA. Inhibitor concentrations were varied from 1 μM to 42 pM in otherwise identical reactions (125 nM PfARFlong peptide, 125 nM myristoyl-CoA and 3 ng CaNMT). A plot of c.p.m., after 30 min incubation at 37 °C, against inhibitor concentration allows a four-parameter fit to derive the point at which the signal is half maximal, the IC₅₀. Values are means±S.D. for three experiments. The IC₅₀ value obtained is 11.6 nM (see inset), consistent with a previous evaluation of 19 nM (Pfizer, personal communication).

Figure 3. Structures of PfNMT inhibitors based around a benzothiazole core structure

Structures of the seven compounds which demonstrated the highest levels of PfNMT inhibition are shown together with the parent compound UK-370485. All compounds contain a benzothiazole core. Compounds 1–3 are identical, apart from the C-6 substitution on the benzothiazole ring. Compounds 4–7 are more diverse in structure but all contain a cyclohexyl linker with 1R,3S stereochemistry (as compared with 1S,4S in compounds 1–3) and a dimethylamide as the C-6 benzothiazole substituent.

Figure 4. Inhibitors of PfNMT in vitro reduce P. falciparum parasitaemia in culture

Each inhibitor was applied to synchronized mid-trophozoite erythrocytic stages of P. falciparum (27 h) at 100 μM and 10 μM and the culture was allowed to grow until controls had reached a similar stage in the following cycle (approx. 48 h later). The total parasitaemia of each sample was evaluated by microscopic assessment of Giesma-stained blood smears. Values are means±S.D. for three experiments. All experiments were initiated at a parasitaemia of approx. 1%. RBC, red blood cell.