Tower 1978

1978 yearbook of Westbrook College in Portland, Maine.https://dune.une.edu/wchc_yearbooks/1007/thumbnail.jp

Impact of Substrate Protonation and Tautomerization States on Interactions with the Active Site of Arginase I

Human arginase is a binuclear manganese metalloenzyme that participates in the urea cycle. Arginase catalyzes the hydrolysis of l-arginine into l-ornithine and urea and is linked to several disorders such as asthma and cancer. Currently, the protonation and tautomerization state of the substrate when bound to the active site, which contains two manganese ions, is not known. Knowledge of the charge-dependent behavior of arginine in the arginase I environment would be of utility toward understanding the catalytic mechanism and designing inhibitors of this enzyme. The arginine^+/0 species, including all possible neutral tautomers, were modeled using an aminoimidazole analog as template. All-atom molecular dynamics simulations were then performed on each of the charged and neutral species. In addition, a hydroxide ion was included in selected simulations to test its importance. Results show that the positively charged state of arginine is stable in the active site of arginase I, with that stabilization facilitated by the presence of hydroxide. Glu277 is indicated to play a role in stabilizing arginine in the active site and facilitating its ability to assume a catalytically competent conformation in the presence of hydroxide. The reported interactions and modeled arginine-bound arginase I structures can be used as a tool for structure-based inhibitor design, as experimental data on the structure of the substrate–enzyme complex is lacking

FX2149 improved the brain penetration and inhibition of LRRK2 GTP binding and kinase activities.

<p>FX2149 (10 mg/kg) and 68 (10 and 20 mg/kg) were injected intraperitoneally into G2019S-LRRK2 BAC transgenic mice at 6–12 weeks of age for 1 hour. There were 6 mice in each experimental group. The brain homogenates were used to detect LRRK2 GTP-binding and kinase activities. A and B, LRRK2 GTP-binding assays. C and D, LRRK2 phosphorylation assays using anti-phospho-LRRK2 antibodies. E and F, FX2149 reduced G2019S-LRRK2-induced 4E-BP phosphorylation determined by anti-phospho-4E-BP western blot analysis. Ntg: non-transgenic mouse. *<i>p</i> < 0.05 by ANOVA compared with G2019S-LRRK2 transgenic mice treated with vehicle.</p

FX2149 inhibits LRRK2 GTP binding activity.

<p>WT or mutant LRRK2 was pulled down from lysates of transfected HEK293T cells using GTP-agarose in the absence or presence of FX2149 at 1 and 10 nM concentrations. The resulting precipitates were subjected to western blot analysis using anti-Flag antibodies. A and C. Representative blots from GTP binding assays. B and D. Quantification of A and C. K1347A-LRRK2, non GTP binding genetic control. All experiments were repeated three times with similar results. *<i>p</i><0.05 by ANOVA, <i>vs</i> vehicle control.</p

FX2149 reduced LPS-induced microglia activation and LRRK2-upregulation.

<p>G2019S-LRRK2 BAC transgenic mice (6–12 weeks) were injected with LPS (5 μg) and FX2149 (10 mg/kg) as described in the methods section. Serial coronal sections through the substantia nigra were subjected to immunohistochemistry analysis. A. Representative immunofluorescent images with anti-isolectin (green) and anti-LRRK2 (red) staining. B. Quantification of immunofluorescence of A by unbiased stereology. *<i>p</i> < 0.05 by ANOVA compared with vehicle group. ^#<i>p</i> < 0.05 by ANOVA compared with LPS treated group. C. Representative immunostaining with anti-phospho-LRRK2-S935 and anti-isolectin B4 (marker for microglia) antibodies by DAB detection.</p

A. Chemical structures of 68 and FX2149; B. Synthesis of FX2149.

<p>A. Chemical structures of 68 and FX2149; B. Synthesis of FX2149.</p

Small Molecule Antivirulents Targeting the Iron-Regulated Heme Oxygenase (HemO) of <i>P. aeruginosa</i>

Bacteria require iron for survival and virulence and employ several mechanisms including utilization of the host heme containing proteins. The final step in releasing iron is the oxidative cleavage of heme by HemO. A recent computer aided drug design (CADD) study identified several inhibitors of the bacterial HemOs. Herein we report the near complete HN, N, CO, Cα, and Cβ chemical shift assignment of the <i>P. aeruginosa</i> HemO in the absence and presence of inhibitors (<i>E</i>)-3-(4-(phenylamino)­phenylcarbamoyl)­acrylic acid (<b>3</b>) and (<i>E</i>)-<i>N</i>′-(4-(dimethylamino)­benzylidene) diazenecarboximidhydrazide (<b>5</b>). The NMR data confirm that the inhibitors bind within the heme pocket of HemO consistent with in silico molecular dynamic simulations. Both inhibitors and the phenoxy derivative of <b>3</b> have activity against <i>P. aeruginosa</i> clinical isolates. Furthermore, <b>5</b> showed antimicrobial activity in the in vivo C. elegans curing assay. Thus, targeting virulence mechanisms required within the host is a viable antimicrobial strategy for the development of novel antivirulants

FX2149 attenuated G2019S-LRRK2-induced neuronal degeneration in SH-SY5Y cells.

<p>SH-SY5Y cells were co-transfected with GFP and various pcDNA3.1-LRRK2 plasmids at a 1:15 ratio as described in the method section. After 4-h transfection, cells were treated with FX2149 for 48 hours. A. Cell viability was measured by counting the healthy viable GFP positive cells that contained at least one smooth extension (neurite) that was twice the length of the cell body. *<i>p</i>< 0.05 by ANOVA compared to wild type LRRK2. ^#<i>p</i> < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle. B. TUNEL assays. The experiments were repeated three times. *<i>p</i> < 0.05 by ANOVA compared to vector control. ^#<i>p</i> < 0.05 by ANOVA compared to G2019S-LRRK2 treated with vehicle.</p

Crystallographic and Computational Insights into Isoform-Selective Dynamics in Nitric Oxide Synthase

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors

Iminoguanidines as Allosteric Inhibitors of the Iron-Regulated Heme Oxygenase (HemO) of <i>Pseudomonas aeruginosa</i>

New therapeutic targets are required to combat multidrug resistant infections, such as the iron-regulated heme oxygenase (HemO) of <i>Pseudomonas aeruginosa</i>, due to links between iron and virulence and dependence on heme as an iron source during infection. Herein we report the synthesis and activity of a series of iminoguanidine-based inhibitors of HemO. Compound <b>23</b> showed a binding affinity of 5.7 μM and an MIC₅₀ of 52.3 μg/mL against <i>P. aeruginosa</i> PAO1. An in cellulo activity assay was developed by coupling HemO activity to a biliverdin-IXα-dependent infrared fluorescent protein, in which compound <b>23</b> showed an EC₅₀ of 11.3 μM. The compounds showed increased activity against clinical isolates of <i>P. aeruginosa</i>, further confirming the target pathway. This class of inhibitors acts by binding to an allosteric site; the novel binding site is proposed in silico and supported by saturation transfer difference (STD) NMR as well as by hydrogen exchange mass spectrometry (HXMS)