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

Published: April 13, 2023With 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.Madeline G. Dans, Henni Piirainen, William Nguyen, Sachin Khurana, Somya Mehra, Zahra Razook, Niall D. Geoghegan, Aurelie T. Dawson, Sujaan Das, Molly Parkyn Schneider, Thorey K. Jonsdottir, Mikha Gabriela, Maria R. Gancheva, Christopher J. Tonkin, Vanessa Mollard, Christopher Dean Goodman, Geoffrey I. McFadden, Danny W. Wilson, Kelly L. Rogers, Alyssa E. Barry, Brendan S. Crabb, Tania F. de Koning-Ward, Brad E. Sleebs, Inari Kursula, Paul R. Gilso

Solubilization of Itraconazole by Surfactants and Phospholipid-Surfactant Mixtures: Interplay of Amphiphile Structure, pH and Electrostatic Interactions

Although surfactants are frequently used in enabling formulations of poorly water-soluble drugs, the link between their structure and drug solubilization capacity is still unclear. We studied the solubilization of the “brick-dust” molecule itraconazole by 16 surfactants and 3 phospholipid:surfactant mixtures. NMR spectroscopy was used to study in more details the drug-surfactant interactions. Very high solubility of itraconazole (up to 3.6 g/L) was measured in anionic surfactant micelles at pH = 3, due to electrostatic attraction between the oppositely charged (at this pH) drug and surfactant molecules. 1H NMR spectroscopy showed that itraconazole is ionized at two sites (2+ charge) at these conditions: in the phenoxy-linked piperazine nitrogen and in the dioxolane-linked triazole ring. The increase of amphiphile hydrophobic chain length had a markedly different effect, depending on the amphiphile type: the solubilization capacity of single-chain surfactants increased, whereas a decrease was observed for double-chained surfactants (phosphatidylglycerols). The excellent correlation between the chain melting temperatures of phosphatidylglycerols and itraconazole solubilization illustrated the importance of hydrophobic chain mobility. This study provides rules for selection of itraconazole solubilizers among classical single-chain surfactants and phospholipids. The basic physics underpinning the described effects suggests that these rules should be transferrable to other “brick-dust” molecules

Albendazole solution formulation via vesicle-to-micelle transition of phospholipid-surfactant aggregates

<p><b>Objective:</b> To reveal the physicochemical mechanisms governing the solubilization of albendazole in surfactant and phospholipid-surfactant solutions and, on this basis, to formulate clinically relevant dose of albendazole in solution suitable for parenteral delivery.</p> <p><b>Significance:</b> (1) A new drug delivery system for parenteral delivery of albendazole is proposed, offering high drug solubility and low toxicity of the materials used; (2) New insights on the role of surface curvature on albendazole solubilization in surfactant and surfactant-phospholipid aggregates are provided.</p> <p><b>Methods:</b> The effect of 17 surfactants and 6 surfactant-phospholipid mixtures on albendazole solubility was studied. The size of the colloidal aggregates was determined by light-scattering. The dilution stability of the proposed formulation was assessed by experiments with model human serum.</p> <p><b>Results:</b> Anionic surfactants increased very strongly drug solubility at pH = 3 (up to 4 mg/mL) due to strong electrostatic attraction between the oppositely charged (at this pH) drug and surfactant molecules. This effect was observed with all anionic surfactants studied, including sodium dodecyl sulfate, double chain sodium dioctylsulfosuccinate (AOT), and the bile salt sodium taurodeoxycholate. The phospholipid-surfactant mixture of 40% sodium dipalmitoyl-phosphatidylglycerol +60% AOT provided highest albendazole solubilization (4.4 mg/mL), smallest colloidal aggregate size (11 nm) and was stable to dilution with model human serum at (and above) 1:12 ratio.</p> <p><b>Conclusions:</b> A new albendazole delivery system with high drug load and low toxicity of the materials used was developed. The high solubility of albendazole was explained with vesicle-to-micelle transition due to the larger interfacial curvature preferred for albendazole solubilization locus.</p

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

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

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

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