Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics

Abstract 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

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

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

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

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, 5 μM Latrunculin B, 200 nM CytD, 2.5 μM MMV291, 10 μM MMV291, and 20 μM MMV291 over a time course of 3 hours. Movies represent maximum intensity projections with the contrast scaled between 100–400 counts. Time is presented as HH:MM, and the scale bar represents 20 μm. (MP4)</p

In vitro resistance to MMV291.

Viable parasites recovered after 3 rounds of drug cycling were tested against a titration of MMV291 in a 72-hour lactate dehydrogenase (LDH) growth assay. Parasite growth was normalised to parasite’s grown in 0.1% DMSO, which indicated 3 resistant populations were obtained (B, C, and D) with an 8- to 14-fold increase in EC50 compared to 3D7. Data points represent the average of 3 technical replicates. C.I indicates 95% confidence intervals for EC50 values, which were derived from nonlinear regression curves in GraphPad Prism. N.D = not determined. Source data can be found in S1 Data. (TIF)</p

MMV291 has no effect on sporozoite motility or invasion.

(A) Sporozoites expressing GFP were used to measure motility via the quantification of fluorescent trails. This revealed that similarly to DMSO, MMV291 had no effect on sporozoite motility in P. berghei (i) or P. falciparum (ii), while cytochalasin D (CytD) significantly reduced motility. 3+ indicates 3 or more trails observed. (B) In vitro human liver cells were incubated with a titration of MMV291 in the presence of 20,000 sporozoites expressing a luciferase protein. After 52 hours, cells were lysed and luciferase activity was measured to correlate with sporozoite invasion rate. In contrast with CytD (10 μM) treatment, MMV291 did not reduce invasion rate of sporozoites at concentrations tested. Error bars represent the standard deviation across 3 biological replicates each comprised of 3 technical replicates. Statistical analysis performed via a chi-squared (A) and unpaired t test (B) using GraphPad Prism. * P P P S1 Data. (TIF)</p

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