Inhibition of a malaria host-pathogen interaction by a computationally designed inhibitor

Abstract

Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria-causing parasite putting 2.5 billion people at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood-stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. A unique characteristic of P. vivax is that it depends on the Duffy antigen/receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease-resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria.

Keywords: Plasmodium vivax; protease selections; protein design; protein scaffolds; three-helical bundles.

FIGURE 1

(a) Binding mode of Duffy antigen/receptor for chemokines (DARC) with Duffy binding protein (DBP‐II) when membrane embedded. DARC is based on the AlphaFold homology model AF‐Q16570‐F1 (Uniprot). (b) Models of designed receptor mimicries and their interference of DBP binding and dimerization. (c) Histogram illustrating which topology permutation can be successfully made after an initial time limited design trajectory. (d) Overview of the design workflow for the new helical scaffold proteins.

FIGURE 2

Summary of initial yeast surface display screen. (a) Scheme selecting of folded designed proteins using yeast surface display combined with protease digestions. (b) Data of selection of the second round of selection at the three different protease conditions. (c) Comparison between stable and unstable designs for the designed helical bundles.

FIGURE 3

Site‐saturation mutagenesis (SSM) library results of the lead candidate Dbb16. (a) Heat maps of enrichment values at 250 nM DBP‐II and 1 μM Duffy binding protein region II (DBP‐II). Red indicates improvement of binding. (b) Model of Dbb16 colored by Shannon entropy computed from the enrichments; blue colors represent more conserved residues. (c) Model of bound Dbb16 turned by 180°.

FIGURE 4

Library selections and biochemical characterization of Dbb16. (a) Fluorescent‐activated cell sorting (FACS) rounds for affinity maturation of Dbb16. The concentration of biotinylated Duffy binding protein region II (bDBP‐II) was steadily decreased. Round 2 started by first incubating the displaying yeast cells with 0.25 μM trypsin and 0.05 μM chymotrypsin before washing and incubating with 50 nM bDBP‐II. (b) Sequences of identified clones after three and four rounds of sorting. (c) Size‐exclusion chromatography (SEC) profiles monitored at 280 nm absorption wavelength of soluble identified variants after expression in Escherichia coli. (d) Far‐ultraviolet CD spectra of variants; 25°C (blue), 95°C (orange), and at 25°C after heating it up to 95°C (green). (e) Biolayer interferometry of variants binding to immobilized bDBP‐II.

FIGURE 5

In vitro inhibition and proposed mode of action. (a) Inhibition of Duffy binding protein region II (DBP‐II) binding to reticulocytes. (b) Model of the inhibitor (pink) bound to one unit of DBP‐II (green) which avoids binding to the host receptor Duffy antigen/receptor for chemokines (DARC) (helical interface fragment in grey); the inhibitor would clash with the second DBP‐II domain (yellow) and thereby would prevent their dimerization.