12 research outputs found
Paratransgenic Control of Vector Borne Diseases
Conventional methodologies to control vector borne diseases with chemical pesticides are often associated with environmental toxicity, adverse effects on human health and the emergence of insect resistance. In the paratransgenic strategy, symbiotic or commensal microbes of host insects are transformed to express gene products that interfere with pathogen transmission. These genetically altered microbes are re-introduced back to the insect where expression of the engineered molecules decreases the host's ability to transmit the pathogen. We have successfully utilized this strategy to reduce carriage rates of Trypanosoma cruzi, the causative agent of Chagas disease, in the triatomine bug, Rhodnius prolixus, and are currently developing this methodology to control the transmission of Leishmania donovani by the sand fly Phlebotomus argentipes. Several effector molecules, including antimicrobial peptides and highly specific single chain antibodies, are currently being explored for their anti-parasite activities in these two systems. In preparation for eventual field use, we are actively engaged in risk assessment studies addressing the issue of horizontal gene transfer from the modified bacteria to environmental microbes
Paratransgenic Control of Vector Borne Diseases
<p>Conventional methodologies to control vector borne diseases with chemical pesticides are often associated with environmental toxicity, adverse effects on human health and the emergence of insect resistance. In the paratransgenic strategy, symbiotic or commensal microbes of host insects are transformed to express gene products that interfere with pathogen transmission. These genetically altered microbes are re-introduced back to the insect where expression of the engineered molecules decreases the host's ability to transmit the pathogen. We have successfully utilized this strategy to reduce carriage rates of <i>Trypanosoma cruzi,</i> the causative agent of Chagas disease, in the triatomine bug, <i>Rhodnius prolixus</i>, and are currently developing this methodology to control the transmission of <i>Leishmania donovani</i> by the sand fly <i>Phlebotomus argentipes</i>. Several effector molecules, including antimicrobial peptides and highly specific single chain antibodies, are currently being explored for their anti-parasite activities in these two systems. In preparation for eventual field use, we are actively engaged in risk assessment studies addressing the issue of horizontal gene transfer from the modified bacteria to environmental microbes.</p
The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites
Biochemical Screening of Five Protein Kinases from <i>Plasmodium falciparum</i> against 14,000 Cell-Active Compounds
<div><p>In 2010 the identities of thousands of anti-<i>Plasmodium</i> compounds were released publicly to facilitate malaria drug development. Understanding these compoundsā mechanisms of actionāi.e., the specific molecular targets by which they kill the parasiteāwould further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Childrenās Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of <i>Plasmodium falciparum</i> calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC<sub>50</sub> < 1 Ī¼M) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple <i>Plasmodium</i> kinase targets without harming human cells is challenging but feasible.</p></div
Assessment of compound promiscuity with human kinases.
<p>Kinobeads were incubated with K562 cell extract either in the presence of vehicle (DMSO) or TCAMS compound, respectively (20 Ī¼M-0.03 Ī¼M). Protein kinases captured by the beads (140ā150 kinases per experiment) were quantified following tryptic digestion, isobaric peptide tagging, and LC-MS/MS analysis. Kinases were identified as potential targets by virtue of their reduced capture in the presence of excess TCAMS compounds. Apparent dissociation constants (K<sub>d</sub>ās) were calculated from the extent to which capture of each kinase was reduced at each compound concentration. K<sub>d</sub> values from duplicate experiments generally agreed with each other quite well (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.s002" target="_blank">S2 Fig</a>). Colored bands indicate kinase-ligand complexes with apparent pK<sub>d</sub>ās of ā„6, with darker shades denoting higher pK<sub>d</sub>ās. Kinases that did not have an apparent pK<sub>d</sub> of ā„6 for any of the compounds are not represented; only names of every other targeted kinase are shown due to space limitations. These results are summarized numerically in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.t003" target="_blank">Table 3</a>.</p
A comparison of different CDPK inhibitorsā cytotoxicity to human cells.
<p>Inhibition of HepG2 cell growth at compound concentrations of 10 Ī¼M is shown for CDPK4 inhibitors in scaffolds D and G (top) and for CDPK1 inhibitors in scaffolds F and H (bottom).</p
Summary of kinobead competition assays (results reflect two independent experiments).
<p>Summary of kinobead competition assays (results reflect two independent experiments).</p
Human cytotoxicity of inhibitors of 1, 2, or 3 of the <i>P</i>. <i>falciparum</i> kinases studied.
<p>Inhibition of HepG2 cell growth at compound concentrations of 10 Ī¼M were previously reported by Gamo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.ref003" target="_blank">3</a>].</p
Clustering of <i>P</i>. <i>falciparum</i> protein kinase hits into chemical scaffolds.
<p>Inhibition of HepG2 cell growth at compound concentrations of 10 Ī¼M were previously reported by Gamo et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149996#pone.0149996.ref003" target="_blank">3</a>]. For some scaffolds, target counts exceed the number of hits because some compounds hit more than one target.</p