NMT (malaria)

n-myristoyl transferase (NMT) mode of action

acts co-translationally at ribosome, coordinated with methionine aminopeptidase (MetAP)

takes off initial Met, leaving a glycine at the end of the polypeptide

NMT1/2 and Myr-CoA myristoylate the nascent protein

NMT characteristics

established mechanism and recombinant enz available

multiple substrates of diverse function

essential for parasite viability (no gene knockouts)

druggable target in fungi (not broad spectrum)

found in most eukaryotes

how NMT inhibitors were discovered, developed and characterised

screening against large chemical compounds libraries of drug-like molecules

looking for hit inhibitors of NMT or parasite killing

use medicinal chem to improve potency and selectivity of hits

refine molecular characteristics for drug development

cell-based screen

screen compounds against killing parasite

target-based screen

target molecules/enzymes and find inhibitors

know how it works, but there is a risk of not actually killing the parasite down the line

NMT reaction mechanism

enzyme binds myristoyl coA, then the protein substrate to form a ternary complex

reaction takes place, CoA is released first, then the protein substrate which is now myristoylated

P. vivax NMT substrate recognition sequence

sparsely conserved, expected to vary between species

H2n-Gly-X-X-X-X-Ser-Lys

Piggybacking approach to NMT-targeted anti-malarials using Benzofuran

Benzofuran has modest potency against PfNMT

PfNMT IC50 = 50uM (good start, is 20x more selective than HsNMT)

want to drive up the potency (lower IC50 and Ki)

IC50

concentration of inhibitor needed to halve the rate of an enzyme-catalysed reaction under specific conditions

HTS approach for NMT inhibitors

used Scintillation assay with radioactive myristoyl-CoA joined to biotin-containing peptide (enz transfers radioactivity onto peptide), the proposed inhibitor and PfNMT

room temp, 80 mins, add streptavidin-coated SPA beads which capture biotin

measure light emitted

hit compounds were narrowed down based on activity and selectivity

crystal structures show PfNMT inhibitors bind in peptide binding site

how was it shown that NMT inhibitors act on-target in cells

ratio of EC50/Ki50 of compounds shows extent to which enz is inhibited to extent to which parasite is killed

shows the parasite is killed because the enzyme is inhibited

if cannot be concluded, use YnMyr to measure if enz is inhibited inside a parasite cell

YnMyr

a tool compound for exploring protein myristoylation in blood stage malaria parasites

like myristic acid with an alkyne group that cells convert into YnMyr-CoA - substrate mimic for NMT

allows selective attachment of alkyne into NMT substrate proteins - metabolic tagging

chemoselective labelling of alkyne-tagged proteins

azide of azido-TAMRA-biotin is used to selectively label alkynes in cell lysates

biotin used for enrichment on streptavidin-coated beads

TAMRA as a fluorescent reporter for protein visualisation in gels

how to define the myristome

trypsin digestion and LC-MS-MS analysis

an assay for in vivo myristoylation

quantification of NMT inhibition in live parasites

as conc of inhibitor increases, fluorescence decreases (not tagged as they are inhibited)

similar ratio of killing to tagging

how is parasite killing correlated with inhibition of in-vivo tagging

inhibition of tagging correlates well with parasite killing

inhibitors are acting on-target

NMT is chemically validated as a drug target in P.falciparum

NMT mechanism of killing

relative to controls, they

reduce number of parasite nuclei per RBC

block parasite egress from the RBC

reduce parasite burden in a mouse malaria model

human rhinovirus (HRV)

common cold

picornavirus family

very diverse, rapidly replicating and evolving - no vaccines or viral inhibitors

RNA genome - translated into polyprotein which processed by viral proteases to form a capsid precursor and non-structural proteins

role of protein myristoylation in the human rhinovirus infection

NMT myristoylated HRV capsid precursor

proteases break this up and they assemble into a promoter

this assembles into 5x promoter pentamer, then 12x pentamer

this and the RNA genome gives an infectious virion

using IMP-72 (NMT inhibitor) as a base for inhibiting HRV replication

fragment merging of IMP-72 with IMP-358 (synergism) increased potency 20,000-fold

IMP-1088

inhibits HRV replication by blocking virion assembly

further refined to improve ADME and is used as an inhibitor against cancer cells (MYX1715) as an ADC