Synthesis of a Small Molecule Nitrosocysteine Inhibitor to Reduce the Activity of Caspase-1

Caspase-1 is an enzyme that is overactive in autoimmune and autoinflammatory diseases cleaving pro-interleukin-1β to the cytokine interleukin-1β (IL-1β), which leads to inflammatory symptoms. The inhibition of caspase-1 will cause a decrease in the concentration of interleukin-1β (IL-1β), resulting in the reduction of inflammatory symptoms. Recent research has revealed that the appending of a nitric oxide (NO) or nitroxyl (HNO) donating group to non-steroidal anti-inflammatory drugs (NSAIDs) reduced, or avoided, the side effects caused by currently available treatments. A small molecule based on a known caspase-1 inhibitor with a nitrosocysteine appended on was synthesized to look at the effect of NO donation on caspase-1

The Susceptibility of Trypanosomatid Pathogens to PI3/mTOR Kinase Inhibitors Affords a New Opportunity for Drug Repurposing

In our study we describe the potency of established phosphoinositide-3-kinase (PI3K) and mammalian Target of Rapamycin (mTOR) kinase inhibitors against three trypanosomatid parasites: Trypanosoma brucei, T. cruzi, and Leishmania sp., which are the causative agents for African sleeping sickness, Chagas disease, and leishmaniases, respectively. We noted that these parasites and humans express similar kinase enzymes. Since these similar human targets have been pursued by the drug industry for many years in the discovery of cellular growth and proliferation inhibitors, compounds developed as human anti-cancer agents should also have effect on inhibiting growth and proliferation of the parasites. With that in mind, we selected eight established PI3K and mTOR inhibitors for profiling against these pathogens. Among these inhibitors is an advanced clinical candidate against cancer, NVP-BEZ235, which we demonstrate to be a highly potent trypanocide in parasite cultures, and in a mouse model of T. brucei infection. Additionally, we describe observations of these inhibitors' effects on parasite growth and other cellular characteristics

Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn2+ binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn2+ was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn2+) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn2+. A stable binary complex, [Zn(BPA)]2+, which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes

Phenotypic observations of parasites upon dosage with NVP-BEZ235.

<p>(A) NIH-3T3 host cells were incubated with <i>T. cruzi</i> trypomastigotes for 2 h before addition of NVP-BEZ235 (350 nM). Cells were incubated for 4 days during which time the parasites differentiate and replicate as amastigotes. At that time cells were fixed and stained with an anti-<i>T. cruzi</i> antiserum (green) and DAPI to visualize DNA (blue). The upper panel shows control cells with intact amastigotes, and the lower panel shows debris of parasite proteins throughout the host cell cytoplasm. (B,C) Fluorescence-activated cell sorter (FACS) analysis of cell size (Forward Scatter, FSC) and DNA content after drug treatment. (B) Bloodstream form culture of <i>T. b. brucei</i> was subjected to different drugs, indicated to the side, and analyzed by FACS for cell size and DNA content stained by propidium iodide. Cell cultures were incubated during 16 h with PI-103 (1 µM), WYE-354 (2 µM), Pp242 (2 µM) and NVP-BEZ235 (0.1 µM), represented with dark lines, and with DMSO as control population, represented as shaded area. (C) Treatment of <i>Leishmania major</i> promastigotes. Dark lines are WT parasites examined when in logarithmic growth phase; shaded areas are parasites grown in the presence of the indicated concentration of drug. Cell size (FSC) and DNA content (PI staining) were determined as indicated in Panel B and/or as described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001297#s2" target="_blank">Methods</a>. The subpanels show data for PI-103 (4 µM, ∼EC₉₀); WYE-354 (25 µM, ∼EC₆₀); NVP-BEZ235 (0.5 µM, EC₉₀) and Pp242 (dashed lines 12.5 µM/∼EC₉₀, shaded area 25 µM/∼EC₉₀).</p

Summary of potency data of mTOR/PI3K inhibitors against trypanosomatid cultures.

a<p> <i>promastigotes, average of three replicates;</i></p>b<p> <i>axenic amastigotes, average of three replicates;</i></p>c<p> <i>trypomastigotes, average of three replicates, within ±10.2%;</i></p>d<p> <i>bloodstream form, average of three replicates.</i></p>*<p> <i>p<0.05 for L. major vs. L. donovani promastigotes;</i></p>#<p> <i>p<0.05 for L. major promastigotes vs. L. donovani promastigotes or amastigotes.</i></p><p>Effective concentration (EC₅₀) values are shown in micromolar concentrations except as noted.</p