Structure-Guided Identification of Resistance Breaking Antimalarial N‑Myristoyltransferase Inhibitors

Abstract

Summary The attachment of myristate to the N-terminal glycine of certain proteins is largely a co-translational modification catalyzed by N-myristoyltransferase (NMT), and involved in protein membrane-localization. Pathogen NMT is a validated therapeutic target in numerous infectious diseases including malaria. In Plasmodium falciparum , NMT substrates are important in essential processes including parasite gliding motility and host cell invasion. Here, we generated parasites resistant to a particular NMT inhibitor series and show that resistance in an in vitro parasite growth assay is mediated by a single amino acid substitution in the NMT substrate-binding pocket. The basis of resistance was validated and analyzed with a structure-guided approach using crystallography, in combination with enzyme activity, stability, and surface plasmon resonance assays, allowing identification of another inhibitor series unaffected by this substitution. We suggest that resistance studies incorporated early in the drug development process help selection of drug combinations to impede rapid evolution of parasite resistance.

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

“…Arg586 of HsUBA1 was previously suggested as a residue that could mediate specificity for the development of inhibitors of HsUBA1 over other E1 enzymes present in humans [19], and its replacement with the shorter Asn639 side chain in PfUBA1 suggests that inhibitors with extensions in the distal direction may specifically attack PfUBA1. The evolution of parasite resistance to such inhibitors is also an important concern that should be address early in the drug discovery process [25]. A previous study showed that upon MLN7243 treatment HsUBA1 Ala580, which has been designated as the gatekeeper residue, is replaced with a serine [54], thereby providing drug resistance.…”

“…The cells were then fixed in 2% paraformaldehyde, 0.2% glutaraldehyde and parasite DNA stained for 10 min with 2 µg/ml Hoechst 33342 dye. Cells were examined using a BD LSRFortessa X-20 flow cytometer and an argon laser tuned for UV (355 nm), with fluorescence at 465nm; Parasites with one nucleus can be clearly distinguished from those with 2 or more nuclei and were sorted by DNA content [25].…”

“…To validate the on-target specificity of antimalarial compounds it is now possible to use genetic approaches. Application of CRISPR-Cas9 technology to modify the parasite genome, allows genes to be deleted using an inducible Cre-recombinase system [22][23][24], or single codon changes conferring drug resistance to be introduced [25].…”

Global profiling of myristoylation inToxoplasma gondiireveals key roles for lipidation in CDPK1 and MIC7 function

“…In light of this, we predict that NMT inhibition would exert severe pleiotropic effects on the Toxoplasma lytic cycle. Although potent and selective TgNMT inhibitors are yet to be reported, extensive work in other protozoan parasites (Schlott et al, 2019;Wright et al, 2014;Wright et al, 2016;Wright et al, 2015) demonstrates that selective NMT inhibition could provide an attractive strategy to combat infection.…”

“…It is catalysed by N-myristoyl transferase (NMT), which is conserved in Toxoplasma and has been reported to be a prominent drug target in fungal (Devadas et al, 1995;Nagarajan et al, 1997), Trypanosome (Frearson et al, 2010;Wright et al, 2016) and Leishmania infections (Hutton et al, 2014;Wright et al, 2015). In Plasmodium falciparum (the causative agent of malaria), inhibition of NMT leads to severe pleiotropic consequences affecting parasite development (Schlott et al, 2019;Wright et al, 2014), highlighting the importance of myristoylation for pathogen survival and progression.…”