Identification of the Binding Site of Apical Membrane Antigen 1 (AMA1) Inhibitors Using a Paramagnetic Probe

Abstract

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small‐molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin‐labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)‐based NMR screening, the 1H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE‐derived distance constraints can be used to identify binding sites and guide further hit optimisation.

A Computational Journey across Nitroxide Radicals: From Structure to Spectroscopic Properties and Beyond

“…Nitroxides are one of the few families of stable organic free radicals and this feature, together with the remarkable sensitivity of their structure and spectroscopic properties to environmental effects has stimulated their widespread use as spin labels and spin probes in both biological and material chemistry [ 1 , 2 , 3 ]. The interest for this class of radicals has seen very recently a remarkable increase especially in connection with the analysis of dynamical and environmental effects by state-of-the-art computational approaches and with protein crystallography refinement [ 4 , 5 , 6 ]. Moreover, while the EPR (electronic paramagnetic resonance) spectra of a huge number of molecular systems including the NO moiety have been recorded and interpreted by means of quantum chemical (QC) computations [ 7 , 8 , 9 , 10 ], the situation is different concerning accurate molecular structures and, especially, vibrational spectra.…”

“…As a matter of fact, the EPR studies of spin labelled proteins can be connected with crystallographic experiments allowing the determination of a static atomistic description of the whole molecular model through structure refinement procedures [ 6 ]. The refinement requires additional a priori information, supplied in the form of chemical restraints, to compensate for the lack of high-resolution data and/or other experimental issues.…”

Computational and experimental studies of the breathing motion of a protein loop: implications in PfAMA1-PfRON2 late-stage binding event

“…An overall understanding of this process remained difficult to address, as all AMA1-RON2 complex crystal structures available in the Protein Data Bank show missing electron densities (Lys351-Ala387) reiterating the intrinsic disorderness of the DII loop (7). Although a loop-closed crystal structure has been solved recently [PDB:6N87, (8)], it is still far from clear how the DII loop interacts with specific parts of the Pf RON2 2021-2059 peptide or how the interim dynamics of the loop contributes to the overall stability of the complex. In the present work, we have addressed the questions mentioned above using atomistic molecular dynamics (MD) simulations (both unbiased and umbrella sampled, summing up to approximately 1.8 microsecond) coupled with biophysical experiments with systematic alanine substitution on the terminal helix of the Pf RON2 2021-2059 peptide.…”

Investigating the Characteristics and Evolution of Apical Membrane Antigen 1 (AMA1) of Plasmodium sp. using Phylogenetic Approach in Searching for Drug Candidate

“…Page 66 IC-BIOLIS [10]. The polymorphic feature of AMA-1 leads to a challenge in the process of vaccine design [8], [11].…”