High-throughput mechanistic screening of non-equilibrium inhibitors by a fully automated data analysis pipeline in early drug-discovery.

Recent efforts for increasing the success in drug discovery focus on an early, massive, and routine mechanistic and/or kinetic characterization of drug-target engagement as part of a design-make-test-analyze strategy. From an experimental perspective, many mechanistic assays can be translated into a scalable format on automation platforms and thereby enable routine characterization of hundreds or thousands of compounds. However, now the limiting factor to achieve such in-depth characterization at high-throughput becomes the quality-driven data analysis, the sheer scale of which outweighs the time available to the scientific staff of most labs.

Multifaceted C-X-C Chemokine Receptor 4 (CXCR4) Inhibition Interferes with Anti-Vascular Endothelial Growth Factor Therapy-Induced Glioma Dissemination.

Resistance to antiangiogenic therapy in glioblastoma (GBM) patients may involve hypoxia-induced expression of C-X-C motif chemokine receptor 4 (CXCR4) on invading tumor cells, macrophage/microglial cells (MGCs), and glioma stem cells (GSCs). We determined whether antagonizing CXCR4 with POL5551 disrupts anti-vascular endothelial growth factor (VEGF) therapy-induced glioma growth and dissemination. Mice bearing orthotopic CT-2A or GL261 gliomas received POL5551 and/or anti-VEGF antibody B20-4.

Identification and characterization of novel small molecules as potent inhibitors of the plasmodial calcium-dependent protein kinase 1.

Malaria remains a major killer in many parts of the world. Recently, there has been an increase in the role of public-private partnerships inciting academic and industrial scientists to merge their expertise in drug-target validation and in the early stage of drug discovery to identify potential new medicines. There is a need to identify and characterize new molecules showing high efficacy, low toxicity with low propensity to induce resistance in the parasite.

A soluble pyrophosphatase, a key enzyme for polyphosphate metabolism in Leishmania.

We report the functional characterization in Leishmania amazonensis of a soluble pyrophosphatase (LaVSP1) that localizes in acidocalcisomes, a vesicular acidic compartment. LaVSP1 is preferentially expressed in metacyclic forms. Experiments with dominant negative mutants show the requirement of LaVSP1 functional expression for metacyclogenesis and virulence in mice.

Trypanosomatid flagellum biogenesis: ARL-3A is involved in several species.

Overexpression in Leishmania amazonensis promastigotes of the GTPase-deficient small G protein LdARL-3A-Q70L specifically provokes the loss of the flagella without affecting cell viability and body size. However, motility is lost and, remarkably, cells do not survive in the insect vector Lutzomyia longipalpis gut, leading to interruption of parasite transmission. We report here that overexpression of the same protein in Leishmania major, Leishmania donovani, and Crithidia fasciculata also led to significant alterations of the flagella.

A pyrophosphatase regulating polyphosphate metabolism in acidocalcisomes is essential for Trypanosoma brucei virulence in mice.

We report the functional characterization of a soluble pyrophosphatase (TbVSP1), which localizes to acidocalcisomes, a vesicular acidic compartment of Trypanosoma brucei. Depending on the pH and the cofactors Mg(2+) or Zn(2+), both present in the compartment, the enzyme hydrolyzes either inorganic pyrophosphate (PP(i)) (k(cat) = 385 s(-1)) or tripolyP (polyP(3)) and polyphosphate (polyP) of 28 residues (polyP(28)) with k(cat) values of 52 and 3.5 s(-1), respectively.

A vacuolar-type H+-pyrophosphatase governs maintenance of functional acidocalcisomes and growth of the insect and mammalian forms of Trypanosoma brucei.

Vacuolar proton pyrophosphatases (V-H(+)-PPases) are electrogenic proton pumps found in many organisms of considerable industrial, environmental, and clinical importance. V-H(+)-PPases of several parasites were shown to be associated with acidic vacuoles named acidocalcisomes, which contain polyphosphate and calcium. In this work we functionally characterized a Trypanosoma brucei V-H(+)-PPase gene by using double-stranded RNA interference methodology to produce inducible V-H(+)-PPase-deficient strains of procyclic and bloodstream forms (PFiVP1 and BFiVP1).