Autophagy-Related Proteins Are Required for Degradation of Peroxisomes in Arabidopsis Hypocotyls during Seedling Growth

Plant peroxisomes play a pivotal role during postgerminative growth by breaking down fatty acids to provide fixed carbons for seedlings before the onset of photosynthesis. The enzyme composition of peroxisomes changes during the transition of the seedling from a heterotrophic to an autotrophic state; however, the mechanisms for the degradation of obsolete peroxisomal proteins remain elusive. One candidate mechanism is autophagy, a bulk degradation pathway targeting cytoplasmic constituents to the lytic vacuole. We present evidence supporting the autophagy of peroxisomes in Arabidopsis thaliana hypocotyls during seedling growth. Mutants defective in autophagy appeared to accumulate excess peroxisomes in hypocotyl cells. When degradation in the vacuole was pharmacologically compromised, both autophagic bodies and peroxisomal markers were detected in the wild-type vacuole but not in that of the autophagy-incompetent mutants. On the basis of the genetic and cell biological data we obtained, we propose that autophagy is important for the maintenance of peroxisome number and cell remodeling in Arabidopsis hypocotyls.     

Structural hybridization of pyrrolidine-based T-type calcium channel inhibitors and exploration of their analgesic effects in a neuropathic pain model

Highly effective and safe drugs for the treatment of neuropathic pain are urgently required and it was shown that blocking T-type calcium channels can be a promising strategy for drug development for neuropathic pain. We have developed pyrrolidine-based T-type calcium channel inhibitors by structural hybridization and subsequent assessment of in vitro activities against Ca_v3.1 and Ca_v3.2 channels. Profiling of in vitro ADME properties of compounds was also carried out. The representative compound 17h showed comparable in vivo efficacy to gabapentin in the SNL model, which indicates T-type calcium channel inhibitors can be developed as effective therapeutics for neuropathic pain.     

Rapid and accurate structure-based therapeutic peptide design using GPU accelerated thermodynamic integration

Peptide-based therapeutics are an alternative to small molecule drugs as they offer superior specificity, lower toxicity, and easy synthesis. Here we present an approach that leverages the dramatic performance increase afforded by the recent arrival of GPU accelerated thermodynamic integration (TI). GPU TI facilitates very fast, highly accurate binding affinity optimization of peptides against therapeutic targets. We benchmarked TI predictions using published peptide binding optimization studies. Prediction of mutations involving charged side-chains was found to be less accurate than for non-charged, and use of a more complex 3-step TI protocol was found to boost accuracy in these cases. Using the 3-step protocol for non-charged side-chains either had no effect or was detrimental. We use the benchmarked pipeline to optimize a peptide binding to our recently discovered cancer target: EME1. TI calculations predict beneficial mutations using both canonical and non-canonical amino acids. We validate these predictions using fluorescence polarization and confirm that binding affinity is increased. We further demonstrate that this increase translates to a significant reduction in pancreatic cancer cell viability.     

Effect of a therapeutic lifestyle change diet on immune functions of moderately hypercholesterolemic humans

Hypercholesterolemia is a risk factor for coronary heart disease (CHD) and also could contribute to impaired immune response. The National Cholesterol Education Program Expert Panel recommends a therapeutic lifestyle change (TLC) diet to reduce the risk for CHD. We investigated the effects of changing from a high-fat Western diet to a low-fat diet in accordance with a TLC diet on immune functions of older adults with hypercholesterolemia to determine whether improving the lipid profile via dietary intervention would have beneficial effects on immune functions. In a double-blind study, 18 subjects consumed both a Western diet (38% fat) and a TLC diet (28% fat) for 32 days in a randomized order. Measures of cellular immune responses, including delayed-type hypersensitivity (DTH) response, in vitro lymphocyte proliferation, and interleukin (IL)-2 production, and production of proinflammatory mediators, including tumor necrosis factor-alpha, IL-6, IL-1beta, and prostaglandin E2, were determined. DTH response and lymphocyte proliferative response increased significantly (29% and 27%, respectively) after consumption of a TLC diet. Our results indicate that consumption of a TLC diet enhances T cell-mediated immune functions in older adults with elevated cholesterol level. This might be a clinically important benefit, considering the decline of T cell-mediated immune functions with aging and evidence of impaired immune function associated with hypercholesterolemia.     

Classification of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>l. japonica nipponbare) immunophilins (FKBPs,CYPs) and expression patterns under water stress

Background  

FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses.     

Design, synthesis, and biological evaluation of 1,3-dioxoisoindoline-5-carboxamide derivatives as T-type calcium channel blockers

A small molecule library of 1,3-dioxoisoindoline-5-carboxamides 4 was designed based on the pharmacophore model, synthesized and biologically evaluated as potential T-type calcium channel blockers. The most active compounds 4d and 4n show T-type calcium channel blocking activity with IC50 values of 0.93 and 0.96 microM, respectively.     

3D pharmacophore based virtual screening of T-type calcium channel blockers

Virtual screening of the commercial databases was done by using a three dimensional pharmacophore previously developed for T-type calcium channel blockers using CATALYSTtrade mark program. Biological evaluation of 25 selected virtual hits resulted in the discovery of a highly potent compound VH04 with IC(50) value of 0.10 microM, eight times as potent as the known selective T-type calcium channel blocker, mibefradil. Search for similar compounds yielded several hits with micro-molar IC(50) values and high T-type calcium channel selectivity. Based on the structure of the virtual hits, small molecule libraries with novel scaffolds were designed, synthesis and biological evaluation of which are currently in progress. This result shows a successful example of ligand based drug discovery of potent T-type calcium channel blockers.     

Synthesis of cinnamoyl ketoamides as hybrid structures of antioxidants and calpain inhibitors

The excessive calpain activation causes serious cellular damage or even cell death in neurological disorders such as stroke and Alzheimer’s disease. Oxidative stress has also been implicated in the initiation or progression of neurodegenerative diseases. In the present studies, a series of cinnamoyl ketoamides 4a-4j were synthesized as hybrid structures of antioxidants and calpain inhibitors. Cinnamoyl ketoamides, possessing an alkyl chain at the α-position, showed potent μ-calpain inhibitory activities indicating that the cinnamoyl skeleton can be regarded as an acyclic variant of calpain inhibitory chromone carboxamide 2. Among synthesized, compound 4e was the most potent inhibitor of μ-calpain (IC₅₀ = 0.13 μM) and also exhibited strong antioxidant activities in DPPH and superoxide anion radical scavenging and lipid peroxidation inhibition assay systems.