Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

Discovering New Transmission-Blocking Antimalarial Compounds: Challenges and Opportunities.

The ability to target human-mosquito parasite transmission challenges global malaria elimination. However, it is not obvious what a transmission-blocking drug will look like; should it target only parasite transmission stages; be combined with a partner drug killing the pathogenic asexual stages; or kill both the sexual and asexual blood stages, preferably displaying polypharmacology? The development of transmission-blocking antimalarials requires objective analyses of the current strategies. Here, pertinent issues and questions regarding the target candidate profile of a transmission-blocking compound, and its role in malaria elimination strategies, are highlighted and novel perspectives proposed. The essential role of a test cascade that integrates screening and validation strategies to identify next-generation transmission-blocking antimalarials is emphasised.

Localization and interactions of Plasmodium falciparum SWIB/MDM2 homologues.

BACKGROUND: Malaria remains a global health problem and the majority of deaths are caused by Plasmodium falciparum parasites. Due to the rapid emergence of drug-resistant strains, novel avenues of research on the biology of the parasite are needed. The massive proliferation of asexual, intra-erythrocytic parasites every 48 h could kill the human host prior to transmission of slow-developing gametocytes to the mosquito vector. A self-induced P. falciparum programmed cell death mechanism has been hypothesized to maintain this balance between the parasite and its two hosts, but molecular participants of the cell death pathway in P. falciparum have not been characterized. Proteins with SWIB/MDM2 domains play a key role in metazoan programmed cell death and this study provides the first evaluation of two parasite SWIB/MDM2 homologues, PF3D7_0518200 (PfMDM2) and PF3D7_0611400 (PfSWIB). METHODS: The function of these proteins was assessed by predicting their structural topology with the aid of bioinformatics and determining their location within live transgenic parasites, expressing green fluorescent protein-tagged PfMDM2 and PfSWIB under normal and elevated temperatures, which mimic fever and which are known to induce a programmed cell death response. Additionally, P. falciparum phage display library technology was used to identify binding partners for the two parasite SWIB/MDM2 domains. RESULTS: Structural features of the SWIB/MDM2 domains of PfMDM2 and PfSWIB, suggested that they are chromatin remodelling factors. The N-terminal signal sequence of PfMDM2 directed the protein to the mitochondrion under both normal and heat stress conditions. Plasmodium falciparum phage display library technology revealed that the C-terminal SWIB/MDM2 domain of PfMDM2 interacted with a conserved protein containing a LisH domain. PfSWIB localized to the cytoplasm under normal growth conditions, while approximately 10% of the heat-stressed trophozoite-stage parasites presented a rapid but short-lived nuclear localization pattern. Two PfSWIB binding partners, a putative Aurora-related kinase and a member of the inner membrane complex, were identified. CONCLUSION: These novel data provide insight into the function of two parasite SWIB/MDM2 homologues and suggest that PfMDM2 plays a role within the mitochondrion and that PfSWIB is involved in a stage-specific, heat-stress, response pathway.

A Novel Pyrazolopyridine with in Vivo Activity in Plasmodium berghei- and Plasmodium falciparum-Infected Mouse Models from Structure-Activity Relationship Studies around the Core of Recently Identified Antimalarial Imidazopyridazines.

Toward improving pharmacokinetics, in vivo efficacy, and selectivity over hERG, structure-activity relationship studies around the central core of antimalarial imidazopyridazines were conducted. This study led to the identification of potent pyrazolopyridines, which showed good in vivo efficacy and pharmacokinetics profiles. The lead compounds also proved to be very potent in the parasite liver and gametocyte stages, which makes them of high interest.

Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells.

The malaria parasite Plasmodium falciparum invades human erythrocytes through multiple pathways utilizing several ligand-receptor interactions. These interactions are broadly classified in two groups according to their dependency on sialic acid residues. Here, we focus on the sialic acid-dependent pathway by using purified glycophorins and red blood cells (RBCs) to screen a cDNA phage display library derived from P. falciparum FCR3 strain, a sialic acid-dependent strain. This screen identified several parasite proteins including the erythrocyte-binding ligand-1, EBL-1. The phage cDNA insert encoded the 69-amino acid peptide, termed F2i, which is located within the F2 region of the DBL domain, designated here as D2, of EBL-1. Recombinant D2 and F2i polypeptides bound to purified glycophorins and RBCs, and the F2i peptide was found to interfere with binding of D2 domain to its receptor. Both D2 and F2i polypeptides bound to trypsin-treated but not neuraminidase or chymotrypsin-treated erythrocytes, consistent with known glycophorin B resistance to trypsin, and neither the D2 nor F2i polypeptide bound to glycophorin B-deficient erythrocytes. Importantly, purified D2 and F2i polypeptides partially inhibited merozoite reinvasion in human erythrocytes. Our results show that the host erythrocyte receptor glycophorin B directly interacts with the DBL domain of parasite EBL-1, and the core binding site is contained within the 69 amino acid F2i region (residues 601-669) of the DBL domain. Together, these findings suggest that a recombinant F2i peptide with stabilized structure could provide a protective function at blood stage infection and represents a valuable addition to a multi-subunit vaccine against malaria.

Phage display: a useful tool for malaria research?

Defining the molecular intricacies of malaria pathogenesis is a vital area of medical and scientific research. Sophisticated methods have been developed to identify and characterise host-parasite interactions that are important in infection. Phage display involves the combinatorial display of proteins or peptides on the surface of bacteriophage. The technology provides an invaluable tool for screening diverse libraries for polypeptides that have a high affinity for a given target. Phage display in malaria research has proven successful, not only in mapping the protein-protein interactions that are important in Plasmodium biology, but also in the identification of molecules that might be exploited in the design of therapeutic agents or vaccines.

Idiopathic myelofibrosis without dacryocytes.

Idiopathic myelofibrosis (IMF) typically presents with marrow fibrosis, splenomegaly, progressive anemia, and a leukoerythroblastic blood smear with dacryocytes. We present a patient with IMF who did not have dacryocytes.

Myosin-like sequences in the malaria parasite Plasmodium falciparum bind human erythrocyte membrane protein 4.1.

BACKGROUND AND OBJECTIVES: Plasmodium falciparum malaria is one of the most lethal infectious diseases afflicting humanity. During development within the erythrocyte, P. falciparum induces significant modifications to the structure and function of the human erythrocyte membrane. This study focused on the identification of new protein-protein interactions between host and parasite. DESIGN AND METHODS: A novel application of in vitro display technology was used: P. falciparum phage display expression libraries were screened against purified human erythrocyte protein 4.1. DNA sequencing and bioinformatic analyses were used to identify parasite proteins that bind protein 4.1. RESULTS: P. falciparum proteins displaying strong binding specificity toward protein 4.1 included five hypothetical proteins, erythrocyte binding antigen-175, erythrocyte binding ligand-1 like protein and a putative serine/threonine kinase. A common binding motif displaying homology to muscle myosin and neurofilament sequences was also identified in four of the eight proteins. INTERPRETATION AND CONCLUSIONS: These proteins are potentially involved in the invasion and/or release, as well as the growth and survival of malaria parasites during development with the red blood cell. The characterization of novel protein interactions between P. falciparum and erythrocyte membrane protein 4.1 will lead to a better understanding of malaria pathogenesis and parasite biology.

Real-time quantitative RT-PCR for human telomere elongation reverse transcriptase in chronic myeloid leukemia.

Telomeres cap chromosome ends and are pivotal for DNA stability. Deregulation of the telomere stabilising enzyme telomerase in malignancy has implications in diagnosis, prognosis and therapeutics of cancer. Quantification of the expression of the telomerase catalytic subunit, hTERT, using the LightCycler TeloTAGGG hTERT Quantification kit is not optimal for analysis of chronic myeloid leukemia (CML) samples. The internal control, porphobilinogen deaminase (PBGD) is amplified in a separate tube to hTERT and has an unstable genomic localisation of 11q23. Our laboratory thus developed a real-time reverse transcriptase polymerase chain reaction which co-amplifies hTERT and either mitochondrial single-stranded DNA binding protein 1 (ssBP1) or ubiquitin C (UBC).