ATH434, a promising iron-targeting compound for treating iron regulation disorders.

Cytotoxic accumulation of loosely bound mitochondrial Fe2+ is a hallmark of Friedreich's Ataxia (FA), a rare and fatal neuromuscular disorder with limited therapeutic options. There are no clinically approved medications targeting excess Fe2+ associated with FA or the neurological disorders Parkinson's disease and Multiple System Atrophy. Traditional iron-chelating drugs clinically approved for systemic iron overload that target ferritin-stored Fe3+ for urinary excretion demonstrated limited efficacy in FA and exacerbated ataxia. Poor treatment outcomes reflect inadequate binding to excess toxic Fe2+ or exceptionally high affinities (i.e. ≤10-31) for non-pathologic Fe3+ that disrupts intrinsic iron homeostasis. To understand previous treatment failures and identify beneficial factors for Fe2+-targeted therapeutics, we compared traditional Fe3+ chelators deferiprone (DFP) and deferasirox (DFX) with additional iron-binding compounds including ATH434, DMOG, and IOX3. ATH434 and DFX had moderate Fe2+ binding affinities (Kd's of 1-4  µM), similar to endogenous iron chaperones, while the remaining had weaker divalent metal interactions. These compounds had low/moderate affinities for Fe3+(0.46-9.59 µM) relative to DFX and DFP. While all compounds coordinated iron using molecular oxygen and/or nitrogen ligands, thermodynamic analyses suggest ATH434 completes Fe2+ coordination using H2O. ATH434 significantly stabilized bound Fe2+ from ligand-induced autooxidation, reducing reactive oxygen species (ROS) production, whereas DFP and DFX promoted production. The comparable affinity of ATH434 for Fe2+ and Fe3+ position it to sequester excess Fe2+ and facilitate drug-to-protein iron metal exchange, mimicking natural endogenous iron binding proteins, at a reduced risk of autooxidation-induced ROS generation or perturbation of cellular iron stores.

Factors governing attachment of Rhizobium leguminosarum to legume roots at acid, neutral, and alkaline pHs.

Rhizobial attachment to host legume roots is the first physical interaction of bacteria and plants in symbiotic nitrogen fixation. The pH-dependent primary attachment of Rhizobium leguminosarum biovar viciae 3841 to Pisum sativum (pea) roots was investigated by genome-wide insertion sequencing, luminescence-based attachment assays, and proteomic analysis. Under acid, neutral, or alkaline pH, a total of 115 genes are needed for primary attachment under one or more environmental pH, with 22 genes required for all. These include components of cell surfaces and membranes, together with enzymes that construct and modify them. Mechanisms of dealing with stress also play a part; however, exact requirements vary depending on environmental pH. RNASeq showed that knocking out the two transcriptional regulators required for attachment causes massive changes in the bacterial cell surface. Approximately half of the 54 proteins required for attachment at pH 7.0 have a role in the later stages of nodule formation. We found no evidence for a single rhicadhesin responsible for alkaline attachment, although sonicated cell surface fractions inhibited root attachment at alkaline pH. Our results demonstrate the complexity of primary root attachment and illustrate the diversity of mechanisms involved. IMPORTANCE: The first step by which bacteria interact with plant roots is by attachment. In this study, we use a combination of insertion sequencing and biochemical analysis to determine how bacteria attach to pea roots and how this is influenced by pH. We identify several key adhesins, which are molecules that enable bacteria to stick to roots. This includes a novel filamentous hemagglutinin which is needed at all pHs for attachment. Overall, 115 proteins are required for attachment at one or more pHs.

Rhizobium determinants of rhizosphere persistence and root colonization.

Bacterial persistence in the rhizosphere and colonization of root niches are critical for the establishment of many beneficial plant-bacteria interactions including those between Rhizobium leguminosarum and its host legumes. Despite this, most studies on R. leguminosarum have focused on its symbiotic lifestyle as an endosymbiont in root nodules. Here, we use random barcode transposon sequencing to assay gene contributions of R. leguminosarum during competitive growth in the rhizosphere and colonization of various plant species. This facilitated the identification of 189 genes commonly required for growth in diverse plant rhizospheres, mutation of 111 of which also affected subsequent root colonization (rhizosphere progressive), and a further 119 genes necessary for colonization. Common determinants reveal a need to synthesize essential compounds (amino acids, ribonucleotides, and cofactors), adapt metabolic function, respond to external stimuli, and withstand various stresses (such as changes in osmolarity). Additionally, chemotaxis and flagella-mediated motility are prerequisites for root colonization. Many genes showed plant-specific dependencies highlighting significant adaptation to different plant species. This work provides a greater understanding of factors promoting rhizosphere fitness and root colonization in plant-beneficial bacteria, facilitating their exploitation for agricultural benefit.

Targeting metals rescues the phenotype in an animal model of tauopathy.

Tauopathies are characterized by the pathological accumulation of the microtubule associated protein tau within the brain. We demonstrate here that a copper/zinc chaperone (PBT2, Prana Biotechnology) has rapid and profound effects in the rTg(tauP301L)4510 mouse model of tauopathy. This was evidenced by significantly improved cognition, a preservation of neurons, a decrease in tau aggregates and a decrease in other forms of "pathological" tau (including phosphorylated tau and sarkosyl-insoluble tau). Our data demonstrate that one of the primary mechanisms of action of PBT2 in this model may be driven by an interaction on the pathways responsible for the dephosphorylation of tau. Specifically, PBT2 increased protein levels of both the structural and catalytic subunits of protein phosphatase 2A (PP2A), decreased levels of the methyl esterase (PME1) that dampens PP2A activity, and increased levels of the prolyl isomerase (Pin1) that stimulates the dephosphorylation activity of PP2A. None of these effects were observed when the metal binding site of PBT2 was blocked. This highlights the potential utility of targeting metal ions as a novel therapeutic strategy for diseases in which tau pathology is a feature, which includes conditions such as frontotemporal dementia and Alzheimer's disease.

The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease.

Elevated iron in the SNpc may play a key role in Parkinson's disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.

Synthesis of chiral building blocks for use in drug discovery.

In the past decade there has been a significant growth in the sales of pharmaceutical drugs worldwide, but more importantly there has been a dramatic growth in the sales of single enantiomer drugs. The pharmaceutical industry has a rising demand for chiral intermediates and research reagents because of the continuing imperative to improve drug efficacy. This in turn impacts on researchers involved in preclinical discovery work. Besides traditional chiral pool and resolution of racemates as sources of chiral building blocks, many new synthetic methods including a great variety of catalytic reactions have been developed which facilitate the production of complex chiral drug candidates for clinical trials. The most ambitious technique is to synthesise homochiral compounds from non-chiral starting materials using chiral metal catalysts and related chemistry. Examples of the synthesis of chiral building blocks from achiral materials utilizing asymmetric hydrogenation and asymmetric epoxidation are presented.

Chiral building blocks: enantioselective syntheses of benzyloxymethyl phenyl propionic acids.

The synthesis of (2S)-2-benzyloxymethyl-3-(2-fluoro-4-methoxyphenyl)- propionic acid, (2S)-2-benzyloxymethyl-3-(2-fluoro-4-methylphenyl)propionic acid and (2S)-2-benzyl-oxymethyl-3-(2,4-dimethylphenyl)propionic acid has been achieved by TiCl4 mediated alkylation of the corresponding (4R)-4-benzyl-3-[3-(2-fluoro-4-methoxyphenyl-, 2-fluoro-4-methylphenyl-, 2,4- dimethylphenyl-)propionyl]-2-oxazolidinones, followed by hydrolysis of the chiral auxiliary. The stereochemistry of the alkylation reaction was confirmed by an X-ray crystal structure of (4R)-4-benzyl-3-[(2S)-2-benzyloxymethyl-3-(2- fluoro-4-methylphenyl)propionyl]-2-oxazolidinone.