Rapid Optimization of a Peptide Inhibitor of Malaria Parasite Invasion by Comprehensive N-Methyl Scanning

Abstract

Apical membrane antigen 1 (AMA1) of the malaria parasite Plasmodium falciparum has been implicated in the invasion of host erythrocytes and is an important vaccine candidate. We have previously described a 20-residue peptide, R1, that binds to AMA1 and subsequently blocks parasite invasion. Because this peptide appears to target a site critical for AMA1 function, it represents an important lead compound for anti-malarial drug development. However, the effectiveness of this peptide inhibitor was limited to a subset of parasite isolates, indicating a requirement for broader strain specificity. Furthermore, a barrier to the utility of any peptide as a potential therapeutic is its susceptibility to rapid proteolytic degradation. In this study, we sought to improve the proteolytic stability and AMA1 binding properties of the R1 peptide by systematic methylation of backbone amides (N-methylation). The inclusion of a single N-methyl group in the R1 peptide backbone dramatically increased AMA1 affinity, bioactivity, and proteolytic stability without introducing global structural alterations. In addition, N-methylation of multiple R1 residues further improved these properties. Therefore, we have shown that modifications to a biologically active peptide can dramatically enhance activity. This approach could be applied to many lead peptides or peptide therapeutics to simultaneously optimize a number of parameters.

Synthesis of Chemically Modified Bioactive Peptides: Recent Advances, Challenges and Developments for Medicinal Chemistry

“…Naturally occurring peptides containing N-methyl amino acids, including a variety of cyclic peptides isolated from marine organisms, show a range of biological activities [110]. Similarly to previously mentioned derivatizations, N-methylation results in pharmacokinetically improved analogs, enhancing proteolytic stability [111] and membrane permeability [112]. The introduction of a methyl group at a backbone amide brings about several effects Figure 13.…”

The cysteine-rich regions of Plasmodium falciparum RON2 bind with host erythrocyte and AMA1 during merozoite invasion

“…Purified preimmune mice serum antibodies were used as control merozoite invasion. The anti-AMA1 antibodies inhibit merozoite invasion, and an AMA1-binding R1 peptide block critical invasion steps after reorientation of the merozoite and apical end attachment (Harris et al 2005(Harris et al , 2009Treeck et al 2009). Recent discovery of rhoptry neck protein complex consisting of RON-2, RON-4, and RON-5 helped to understand the functional role of AMA1 during invasion and process of MJ formation.…”

Surface plasmon resonance for vaccine design and efficacy studies: recent applications and future trends

“…A previously identified AMA1-binding peptide was examined to improve proteolytic stability by N-methylation of backbone amides. The augmentation in affinity and retained activity was confirmed by SPR-based ana lysis, demonstrating affinity improvement for the 3D7, W2mef and HB3 strains, in addition to preventing invasion [89].…”

“…This rationally designed vaccine antigen can potentially elicit an antibody response preventing MSP-1 42 processing and hence may have a beneficial effect in suppressing blood-stage parasitemia [88]. The apical membrane antigen (AMA)1 is an integral membrane protein common to all Plasmodium species that stabilizes binding of the invasive form of the parasite into hepatocytes (expressed in both pre-erythroctic and erythrocytic stages) [89]. The AMA1 ectoplasmic domain is significant for malarial vaccine development due to its critical role in the lifecycle of the parasite and elicitation of protection in animal models [89,90].…”

“…No substantial parasite growth inhibition was observed, which was possibly due to the fact that the level of inhibition was below the observable threshold for the ex vivo growth inhibition assay. However, the importance of characterization of dominant epitopes for vaccine candidate design is highlighted [90] and the requirement to target critical functionally conserved regions of AMA1 is apparent [89]. The novel antigen receptor from sharks (IgNAR), a single-domain antibody (sdAb), was employed to examine its binding to AMA1 using a Biacore T100 instrument and to investigate the crystal structure of IgNAR in complex with AMA1 [91].…”