Using Dental Pulp Stem Cells for Stroke Therapy

Stroke is a leading cause of permanent disability world-wide, but aside from rehabilitation, there is currently no clinically-proven pharmaceutical or biological agent to improve neurological disability. Cell-based therapies using stem cells, such as dental pulp stem cells, are a promising alternative for treatment of neurological diseases, including stroke. The ischaemic environment in stroke affects multiple cell populations, thus stem cells, which act through cellular and molecular mechanisms, are promising candidates. The most common stem cell population studied in the neurological setting has been mesenchymal stem cells due to their accessibility. However, it is believed that neural stem cells, the resident stem cell of the adult brain, would be most appropriate for brain repair. Using reprogramming strategies, alternative sources of neural stem and progenitor cells have been explored. We postulate that a cell of closer origin to the neural lineage would be a promising candidate for reprogramming and modification towards a neural stem or progenitor cell. One such candidate population is dental pulp stem cells, which reside in the root canal of teeth. This review will focus on the neural potential of dental pulp stem cells and their investigations in the stroke setting to date, and include an overview on the use of different sources of neural stem cells in preclinical studies and clinical trials of stroke

Structure activity refinement of phenylsulfonyl piperazines as antimalarials that block erythrocytic invasion

The emerging resistance to combination therapies comprised of artemisinin derivatives has driven a need to identify new antimalarials with novel mechanisms of action. Central to the survival and proliferation of the malaria parasite is the invasion of red blood cells by Plasmodium merozoites, providing an attractive target for novel therapeutics. A screen of the Medicines for Malaria Venture Pathogen Box employing transgenic P. falciparum parasites expressing the nanoluciferase bioluminescent reporter identified the phenylsulfonyl piperazine class as a specific inhibitor of erythrocyte invasion. Here, we describe the optimization and further characterization of the phenylsulfonyl piperazine class. During the optimization process we defined the functionality required for P. falciparum asexual stage activity and determined the alpha-carbonyl S-methyl isomer was important for antimalarial potency. The optimized compounds also possessed comparable activity against multidrug resistant strains of P. falciparum and displayed weak activity against sexual stage gametocytes. We determined that the optimized compounds blocked erythrocyte invasion consistent with the asexual activity observed and therefore the phenylsulfonyl piperazine analogues described could serve as useful tools for studying Plasmodium erythrocyte invasion.William Nguyena, Madeline G. Dansc, Anna Ngoa, Maria R. Ganchevaf, Ornella Romeof, Sandra Duffy ... et al

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

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