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. 2019 Jan 11;5(1):111-122.
doi: 10.1021/acsinfecdis.8b00226. Epub 2018 Nov 12.

Pharmacological Validation of N-Myristoyltransferase as a Drug Target in Leishmania donovani

Victoriano Corpas-Lopez  1 , Sonia Moniz  1 , Michael Thomas  1 , Richard J Wall  1 , Leah S Torrie  1 , Dorothea Zander-Dinse  2 , Michele Tinti  1 , Stephen Brand  1 , Laste Stojanovski  1 , Sujatha Manthri  1 , Irene Hallyburton  1 , Fabio Zuccotto  1 , Paul G Wyatt  1 , Manu De Rycker  1 , David Horn  1 , Michael A J Ferguson  1 , Joachim Clos  2 , Kevin D Read  1 , Alan H Fairlamb  1 , Ian H Gilbert  1 , Susan Wyllie  1
Affiliations

Pharmacological Validation of N-Myristoyltransferase as a Drug Target in Leishmania donovani

Victoriano Corpas-Lopez et al. ACS Infect Dis. .

Abstract

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and L. infantum, is responsible for ∼30 000 deaths annually. Available treatments are inadequate, and there is a pressing need for new therapeutics. N-Myristoyltransferase (NMT) remains one of the few genetically validated drug targets in these parasites. Here, we sought to pharmacologically validate this enzyme in Leishmania. A focused set of 1600 pyrazolyl sulfonamide compounds was screened against L. major NMT in a robust high-throughput biochemical assay. Several potent inhibitors were identified with marginal selectivity over the human enzyme. There was little correlation between the enzyme potency of these inhibitors and their cellular activity against L. donovani axenic amastigotes, and this discrepancy could be due to poor cellular uptake due to the basicity of these compounds. Thus, a series of analogues were synthesized with less basic centers. Although most of these compounds continued to suffer from relatively poor antileishmanial activity, our most potent inhibitor of LmNMT (DDD100097, K i of 0.34 nM) showed modest activity against L. donovani intracellular amastigotes (EC50 of 2.4 μM) and maintained a modest therapeutic window over the human enzyme. Two unbiased approaches, namely, screening against our cosmid-based overexpression library and thermal proteome profiling (TPP), confirm that DDD100097 (compound 2) acts on-target within parasites. Oral dosing with compound 2 resulted in a 52% reduction in parasite burden in our mouse model of VL. Thus, NMT is now a pharmacologically validated target in Leishmania. The challenge in finding drug candidates remains to identify alternative strategies to address the drop-off in activity between enzyme inhibition and in vitro activity while maintaining sufficient selectivity over the human enzyme, both issues that continue to plague studies in this area.

Keywords: Leishmania; N-myristoyltransferase; drug discovery; target validation; thermal proteome profiling (TPP).

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Chemical structures.
Figure 2
Figure 2
LmNMT assay development. (A) Linearity of the assay with respect to enzyme concentration (data shown as mean CPM ± SD, n = 3). (B) CAP5.5 Km determination (all data points shown, n = 2). (C) Myristoyl CoA Km determination (all data points shown, n = 3). (D) Assay linearity with respect to time under the final assay conditions of 5 nM LmNMT, 0.5 μM CAP5.5, and 125 nM myristoyl CoA (data shown as mean CPM ± SD, n = 3).
Figure 3
Figure 3
Enzymatic and cellular efficacy of LdNMT inhibitors. (A) Correlation of pIC50 values for a focused set of compounds against LmNMT and HsNMT1 in enzymatic assays. Solid line represents equipotent activity while the dashed line represents a linear regression of the data (r2 = 0.856). (B) Correlation of potencies for a focused set of compounds against LmNMT (pIC50) in enzymatic assays versus L. donovani axenic amastigotes (pEC50) cell-based assays. Solid line represents equipotent activity while the dashed line represents a linear regression of the data (r2 = 0.358).
Figure 4
Figure 4
Target deconvolution utilizing a cosmid library approach. (A) Schematic representing the cosmid library workflow. (B) Cumulative growth of cosmid library population treated with compound 2 (80 nM, closed circles) and untreated (open circles). (C) Genome-wide map indicating cosmid library hits from screening of compound 2. A single primary hit was identified, indicated in blue. (D) Focus on primary “hit” on chromosome 32. NMT (LdBPK_320080), green bar; other genes, black bars. The blue/pink and black/green peaks indicate independent cosmid inserts in different orientations. (E) Dose response curves of compound 2 against promastigotes constitutively overexpressing NMT (clone 1, black circles; clone 2, gray circles) and WT promastigotes (open circles). Data are the mean ± SD of three technical replicates and are representative of two independent experiments.
Figure 5
Figure 5
Target deconvolution utilizing TPP. (A) Schematic representing our TPP workflow. TMT: tandem mass tags. (B) Venn diagram of proteins identified as candidate targets of compound 2 from duplicate experiments (biological replicates). UniProt identifiers are used to represent individual proteins. (C) Melting curves for L. donovani NMT following incubation with 0.4 μM compound 2 (red) or vehicle (0.1% DMSO, blue). Data from two technical replicates (circles and squares) are shown, and the mean shift in melting temperature (ΔTm) for NMT was 3.3 °C. Data from an independent duplicate experiment is presented in Supplementary Figure 2 and Supplementary Table 4.
Figure 6
Figure 6
Efficacy of compound 2 in a mouse model of VL. Efficacy of compound 2 was assessed at 50 mg kg–1 b.i.d. for 5 days, alongside vehicle (orally administered b.i.d. for 5 days), miltefosine (orally administered 30 mg kg–1 q.d. for 5 days), and sodium stibogluconate (administered by subcutaneous injection at 15 mg kg–1 q.d. for 5 days). Each arm was carried out with 5 mice. Mean reduction in parasite burden expressed as Leishman Donovan Units (LDU), equivalent to the number of amastigotes per 500 nucleated cells multiplied by the organ weight in milligrams. Treatment with miltefosine resulted in an 88% reduction in liver parasite burden, while dosing with both compound 2 and sodium stibogluconate resulted in 52% reductions. Unpaired t tests confirmed the reduction in parasitaemia evident in all three groups of treated mice is significant, compared to untreated control animals, with P = 0.0021 (*), P < 0.0001 (***), and P = 0.0009 (**) for sodium stibogluconate, miltefosine, and compound 2, respectively.
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