Proposed model for MMV291 interference in profilin-mediated filamentous actin polymerisation.

(A) Treadmilling model of profilin’s role in sequestering G-actin and stimulating the exchange of ADP for ATP before delivering the subunits to the barbed end of the growing filament. Here, formin initiates the polymerisation process to form F-actin. Hydrolysis of the G-actin-ATP occurs at this end to produce G-actin-ADP and inorganic phosphate (Pi), to stabilise the filament. The slow release of Pi at the pointed end induces filament instability and proteins such as ADF1 bind to G-actin-ADP to aid in the release of the subunits, thereby severing the filaments. (B) A potential mechanism for MMV291’s inhibitory activity could be through the stabilisation of the G-actin/profilin dimer therefore inhibiting the formation of F-actin and preventing the generation of force required for invasion. ADF1, actin depolymerising factor 1; F-actin, filamentous actin; G-actin, globular actin.</p

Source data for graphs in this paper.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.</div

MMV291 pretreatment of uninfected RBCs does not inhibit merozoite invasion.

Uninfected RBCs were pretreated with invasion inhibitory compounds (blue); 10 μM MMV291, 100 μg/mL heparin, 2 μM cytochalasin D (CytD), or 0.0025% glutaraldehyde (Glut) for 30 minutes at 37°C, after which the cells were washed (W/O) to remove the inhibitors. Purified merozoites were then allowed to invade the pretreated RBCs. In parallel, merozoites were added to untreated RBCs in the presence of these inhibitors (red). After incubation for 30 minutes at 37°C, the compounds were washed out and parasites allowed to grow for 24 hours. Successful invasion was assessed by measuring the bioluminescence levels of trophozoite-stage parasites expressing a nanoluciferase reporter, and invasion rate was normalised to the DMSO vehicle control. This demonstrated that unlike the fixative glutaraldehyde, pretreatment with MMV291 did not reduce merozoite invasion of RBCs, producing a similar profile to the invasion inhibitory molecules, heparin and CytD. Error bars represent the standard deviation of 2 biological replicates with statistical analyses performed in GraphPad Prism using a one-way ANOVA with pretreated RBCs compared to glutaraldehyde (blue) and merozoite treatment compared to heparin (red). **** indicates P P > 0.05). Source data can be found in S1 Data. (TIF)</p

MMV291 does not affect actin filaments in HeLa cells.

HeLa cells labelled with SiR-Actin imaged by lattice light-sheet microscopy upon stimulation with DMSO control (A), 5 μM Latrunculin B (B), 200 nM Cytochalasin D (CytD) (C), 2.5 μM MMV291 (D), 5 μM MMV291 (E), 10 μM MMV291 (F), and 20 μM MMV291 (G). Images represent a 100 × 100 μm subregion of a larger 250 × 250 μm field of view. Images are presented as maximum intensity projections with the contrast scaled between 100–400 counts. The images show the same region of cells imaged across multiple time points. Time is presented as HH:MM, and the scale bar represents 20 μm. (TIF)</p

Introduction of the SNPs in <i>profilin</i> and <i>actin-1</i> into 3D7 parasites mediates resistance to MMV291.

(A) i Strategy to create the donor plasmid to introduce PFN(N154Y), PFN(K124N), and ACT1(M356L) SNPs into 3D7 parasites. Homology regions (HRs) were designed to the 5′ flank (HR1) and 3′ flank (HR2) whereby HR1 was made up of the endogenous genes’ sequence (HR1A) and recodonised fragments (HR1B), encompassing the resistant mutation alleles. A synthetic guide RNA (gRNA) was designed for either profilin or actin-1 to direct Cas9 to the cleavage site and induce double crossover homologous recombination. WR99210 was used to select for integrated parasites via the human hydrofolate reductase (hDHFR). ii Integration into the profilin or actin-1 locus was validated whereby a 5′ UTR primer (i/v) was used in combination with a primer located in the glmS region (k). B) i Integrated parasites were tested in a 72-hour LDH growth assay, which revealed the resistant mutations conferred resistance against MMV291 and confirmed the profilin and actin-1 proteins as involved in the MoA of the compound. Growth has been normalised to that of parasites grown in 0.1% DMSO, and error bars indicate the standard deviation of 3 biological replicates. Source data can be found in S1 Data. ii EC50 values derived from nonlinear regression curves in GraphPad Prism with 95% confidence intervals shown in brackets. und = undefined.</p

MMV291 series show limited activity against <i>T</i>. <i>gondii</i> invasion.

Nanoluciferase expressing parasites were liberated from their host cell and incubated with the MMV291 analogues before being added back to fibroblasts and allowed to invade for 1 hour before compounds were washed out. After a 24-hour incubation, cells were then lysed and the relative light units was quantified to correlate with T. gondii invasion rate. This showed MMV291 analogues S-W936 (A), R-W936 (B), S-MMV291 (C), and R-MMV291 (D) had some inhibitory activity against invasion at high concentrations. DMSO was included to the same amount as the highest concentration of analogue to account for DMSO-related effects (30% reduction in invasion). Error bars indicate the standard deviation from 2 biological repeats. Source data can be found in S1 Data. (TIF)</p

Resistant genomes pooled variant summary.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.</div

MMV291 analogues interfere with actin polymerisation in the presence of profilin in vitro.

PfACT1 (4 μM) under polymerizing conditions was quantified in the supernatant and pellet fractions in the presence of the MMV291 analogues (25 μM) or DMSO and upon addition of PfPFN (16 μM). (A) In the absence of PfPFN, 80 ± 4% of PfACT1 sedimented to the pellet fraction with the vehicle DMSO treatment. S-W936 decreased the amount of actin in the pellet to 68 ± 7%, while the remaining compounds had no significant effects on actin sedimentation. (B) Upon addition of PfPFN, actin sedimentation decreased to 21 ± 1% with DMSO treatment. All MMV291 analogues, S-MMV291, R-MMV291, S-W936, R-W936, S-W414, and S-W827, decreased the amount of actin in the pellet further to 11 ± 1%, 15 ± 2%, 8 ± 2%, 10 ± 4%, 9 ± 4%, and 5 ± 2%, respectively. Results are plotted as mean ± standard deviation of the relative amounts of actin in the pellet fraction. The data are based on at least 3 independent assays each performed in triplicate. Statistical significances were determined using an unpaired two-tailed t test, where ** P ≤ 0.01 and *** P ≤ 0.001, and **** ≤ 0.0001. No bar indicates not significant. Source data can be found in S1 Data.</p

A model of the comparison between mutation locations in human and <i>P</i>. <i>falciparum</i>.

An X-ray structure human profilin (gold) (PDB: 2PBD) and a homology model of human actin (green) (created by SWISS-MODEL [106] using O. cuniculus actin (PDB: 2PBD) [56] aligned with P. falciparum profilin (pink) (PDB: 2JKG) [36] and actin (blue) (PDB: 6I4E) [42] showing the similarity of the heterodimeric complex. The positions of the MMV291 P. falciparum mutations and the associated human amino acids are shown for comparison. (TIF)</p

Model of <i>P</i>. <i>falciparum</i> profilin and actin-1 with known actin binders.

P. falciparum profilin (pink) (PDB: 2JKG) ([36]; structure 16: 1638) and actin-1 (blue) (ATP, magenta) (PDB: 6I4E) [42] heterodimeric complex showing regions of the proteins where actin inhibitors are known to bind relative to the MMV291 P. falciparum mutations. The actin inhibitors aligned to P. falciparum actin-1 and shown are Bistramide A (blue) (aligned from O. cuniculus actin, PDB: 2FXU) [101], Cytochalasin D (grey) (aligned from D. melanogaster actin, PDB: 3EKU) [102], Jasplakinolide (yellow) (aligned from P. falciparum F-actin, PDB: 5OGW) [48], Latrunculin B (gold) and Pectenotoxin-2 (green) (aligned from O. cuniculus actin, PDB: 2Q0U) [103], Phalloidin (maroon) (aligned from G. gallus F-actin, PDB: 7BTI) [104], and Reidispongiolide A (orange) (aligned from O. cuniculus actin PDB: 2ASM) [105]. (TIF)</p