Summary
Invasion of hepatocytes by sporozoites is essential for Plasmodium to initiate infection of the mammalian host. The parasite's subsequent intracellular differentiation in the liver is the ...first developmental step of its mammalian cycle. Despite their biological significance, surprisingly little is known of the signalling pathways required for sporozoite invasion. We report that sporozoite invasion of hepatocytes requires signalling through two second‐messengers – cGMP mediated by the parasite's cGMP‐dependent protein kinase (PKG), and Ca2+, mediated by the parasite's calcium‐dependent protein kinase 4 (CDPK4). Sporozoites expressing a mutated form of Plasmodium berghei PKG or carrying a deletion of the CDPK4 gene are defective in invasion of hepatocytes. Using specific and potent inhibitors of Plasmodium PKG and CDPK4, we demonstrate that PKG and CDPK4 are required for sporozoite motility, and that PKG regulates the secretion of TRAP, an adhesin that is essential for motility. Chemical inhibition of PKG decreases parasite egress from hepatocytes by inhibiting either the formation or release of merosomes. In contrast, genetic inhibition of CDPK4 does not significantly decrease the number of merosomes. By revealing the requirement for PKG and CDPK4 in Plasmodium sporozoite invasion, our work enables a better understanding of kinase pathways that act in different Plasmodium stages.
Malaria infection begins with the injection of Plasmodium sporozoites into humans by a feeding mosquito. We demonstrate that two parasite kinases, PKG and CDPK4 are crucial for sporozoite motility and consequently their entry into hepatocytes. Our results have implications for understanding the role of second messenger molecules, cGMP and Ca2+ in regulating the exo‐erythrocytic cycle of Plasmodium.
Abstract
Background
We evaluated the efficacy, pharmacokinetics (PK), and safety of clofazimine (CFZ) in patients living with human immunodeficiency virus (HIV) with cryptosporidiosis.
Methods
We ...performed a randomized, double-blind, placebo-controlled study. Primary outcomes in part A were reduction in Cryptosporidium shedding, safety, and PK. Primary analysis was according to protocol (ATP). Part B of the study compared CFZ PK in matched individuals living with HIV without cryptosporidiosis.
Results
Twenty part A and 10 part B participants completed the study ATP. Almost all part A participants had high viral loads and low CD4 counts, consistent with failure of antiretroviral (ARV) therapy. At study entry, the part A CFZ group had higher Cryptosporidium shedding, total stool weight, and more diarrheal episodes compared with the placebo group. Over the inpatient period, compared with those who received placebo, the CFZ group Cryptosporidium shedding increased by 2.17 log2 Cryptosporidium per gram stool (95% upper confidence limit, 3.82), total stool weight decreased by 45.3 g (P = .37), and number of diarrheal episodes increased by 2.32 (P = .87). The most frequent solicited adverse effects were diarrhea, abdominal pain, and malaise. One placebo and 3 CFZ participants died during the study. Plasma levels of CFZ in participants with cryptosporidiosis were 2-fold lower than in part B controls.
Conclusions
Our findings do not support the efficacy of CFZ for the treatment of cryptosporidiosis in a severely immunocompromised HIV population. However, this trial demonstrates a pathway to assess the therapeutic potential of drugs for cryptosporidiosis treatment. Screening persons living with HIV for diarrhea, and especially Cryptosporidium infection, may identify those failing ARV therapy.
Clinical Trials Registration
NCT03341767.
We evaluated clofazimine for treatment of adults living with human immunodeficiency virus and cryptosporidiosis. Clofazimine was well tolerated but did not reduce Cryptosporidium excretion or diarrhea compared with adults treated with placebo. This trial forms a blueprint for future cryptosporidiosis therapeutic trials.
The NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium established to apply structural genomics approaches to potential drug targets from NIAID priority ...organisms for biodefense and emerging and re-emerging diseases. The mission of the SSGCID is to determine approximately 400 protein structures over the next five years. In order to maximize biomedical impact, ligand-based drug-lead discovery campaigns will be pursued for a small number of high-impact targets. Here we review the center's target selection processes, which include pro-active engagement of the infectious disease research and drug therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. This is followed by a brief overview of the SSGCID structure determination pipeline and ligand screening methodology. Finally, specifics of our resources available to the scientific community are presented. Physical materials and data produced by SSGCID will be made available to the scientific community, with the aim that they will provide essential groundwork benefiting future research and drug discovery.
This is a brief review of 42 cytokines and interleukins that are involved in inflammatory and immune responses. The cytokines are listed in tables organized as hematopoietic growth factors, ...interferons, lymphokines, monokines, chemokines, and other cytokines. Information on each cytokine includes the most commonly used abbreviations, the former or alternative names and abbreviations of the cytokines, the cells that form the major sources of production of the cytokines, the major biologic actions of the cytokines, and references to recent reviews or primary literature. Minor biologic actions and minor cellular sources of the cytokines may not be listed. This review should be useful as a quick reference guide to the cytokines and interleukins.
The TDR Targets Database (http://tdrtargets.org) has been designed and developed as an online resource to facilitate the rapid identification and prioritization of molecular targets for drug ...development, focusing on pathogens responsible for neglected human diseases. The database integrates pathogen specific genomic information with functional data (e.g. expression, phylogeny, essentiality) for genes collected from various sources, including literature curation. This information can be browsed and queried using an extensive web interface with functionalities for combining, saving, exporting and sharing the query results. Target genes can be ranked and prioritized using numerical weights assigned to the criteria used for querying. In this report we describe recent updates to the TDR Targets database, including the addition of new genomes (specifically helminths), and integration of chemical structure, property and bioactivity information for biological ligands, drugs and inhibitors and cheminformatic tools for querying and visualizing these chemical data. These changes greatly facilitate exploration of linkages (both known and predicted) between genes and small molecules, yielding insight into whether particular proteins may be druggable, effectively allowing the navigation of chemical space in a genomics context.
In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate ...dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD+ interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N 6-(1-naphthalenemethyl)-2‘-deoxy-2‘-(3-methoxybenzamido)adenosine (6e), N 6-(substituted-naphthalenemethyl)-2‘-deoxy-2‘-(substituted-benzamido)adenosine analogues were investigated. N 6-(1-Naphthalenemethyl)-2‘-deoxy-2‘-(3,5-dimethoxybenzamido)adenosine (6m), N 6-1-(3-hydroxynaphthalene)methyl-2‘-deoxy-2‘-(3,5-dimethoxybenzamido)adenosine (7m), N 6-1-(3-methoxynaphthalene)methyl-2‘-deoxy-2‘-(3,5-dimethoxybenzamido)adenosine (9m), N 6-(2-naphthalenemethyl)-2‘-deoxy-2‘-(3-methoxybenzamido)adenosine (11e), and N 6-(2-naphthalenemethyl)-2‘-deoxy-2‘-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC50's of these compounds were in the range 2−12 μM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure−activity relationships of this class of compounds, a library of 240 N 6-(substituted)-2‘-deoxy-2‘-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N6 and C2‘ substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5‘-amido substituents and found that 2‘,5‘-dideoxy-2‘-(3,5-dimethoxybenzamido)-5‘-(diphenylacetamido)adenosine (49) displays an IC50 of 60−100 μM against the three parasite GAPDH's.
By means of a differential screening technique, a 92-kDa antigen, designated Tp92, was identified from Treponema pallidum subspecies pallidum. This protein is similar in sequence to the protective ...surface antigens D15 from Haemophilus influenzae and Oma87 from Pasteurella multocida. Amino acid sequence analyses revealed a cleavable N-terminal signal sequence and predicted the outer membrane location for Tp92. In support of this, antiserum raised against recombinant Tp92 promotes opsonization and phagocytosis of T. pallidum by rabbit macrophages, and anti-Tp92 reactivity is absent from washed treponemal preparations presumed to be lacking outer membranes. The Tp92 amino acid sequence is 95.5%–100% conserved among 11 strains representing 4 pathogenic treponemes, and immunization with recombinant Tp92 partially protected rabbits from subsequent T. pallidum challenge. These results demonstrate that Tp92 is an invariant, immunoprotective antigen that may be present on the surface of T. pallidum and may represent a potential vaccine candidate for syphilis.
New therapeutics to combat malaria are desperately needed. Here we show that the enzyme protein farnesyltransferase (PFT) from the malaria parasite Plasmodium falciparum (P. falciparum) is an ideal ...drug target. PFT inhibitors (PFTIs) are well tolerated in man, but are highly cytotoxic to P. falciparum. Because of their anticancer properties, PFTIs comprise a highly developed class of compounds. PFTIs are ideal for the rapid development of antimalarials, allowing “piggy-backing” on previously garnered information. Low nanomolar concentrations of tetrahydroquinoline (THQ)-based PFTIs inhibit P. falciparum PFT and are cytotoxic to cultured parasites. Biochemical studies suggest inhibition of parasite PFT as the mode of THQ cytotoxicity. Studies with malaria-infected mice show that THQ PFTIs dramatically reduce parasitemia and lead to parasite eradication in the majority of animals. These studies validate P. falciparum PFT as a target for the development of antimalarials and describe a potent new class of THQ PFTIs with antimalaria activity.
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