Department of Pharmaceutical Sciences, UCI

Neuronal nitric oxide synthase (nNOS) is a promising target for addressing various neurological disorders and melanoma. Our discovery of a series of truncated pyridinylbenzylamines has yielded potent, selective, and membrane permeable inhibitors of human neuronal nitric oxide synthase. By implementing an efficient synthetic procedure using the Suzuki-Miyaura cross-coupling reaction, we were able to rapidly identify a potent inhibitor. This new inhibitor (18, 6-(2,3-difluoro-5-((methylamino)methyl)phenyl)-4-methylpyridin-2-amine dihydrochloride) exhibits excellent potency, with K_i values of 30 nM for human nNOS and 40 nM for rat nNOS. It also demonstrates high isoform selectivity, showing an 821-fold preference for human nNOS over human endothelial NOS (eNOS) and a 75-fold selectivity over human inducible NOS (iNOS). Additionally, inhibitor 18 displays high permeability (P_e = 10.7 × 10^-6 cm s^-1) in an artificial membrane permeability assay. The crystal structures of several NOS-inhibitor complexes provide valuable structural insights into the potency and selectivity of this series of novel inhibitors. A particularly notable finding is the unexpected role of a Cl^- anion bound to heNOS, which contributes to the high isoform selectivity of these inhibitors and explains why heNOS binds Cl^-, while hnNOS does not. This unique Cl^- binding site could be important in future inhibitor design, opening new avenues for the development of more selective NOS inhibitors. Additionally, the presented crystal structures reveal the key factors required to maintain both high potency and selectivity in the simplified inhibitors discussed in this study. Abbreviations: NO, nitric oxide; nNOS, neuronal nitric oxide synthase; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; rnNOS, rat neuronal nitric oxide synthase; hnNOS, human neuronal nitric oxide synthase; hiNOS, human inducible nitric oxide synthase; heNOS, human endothelial nitric oxide synthase; l-Arg, l-arginine; NADPH, reduced nicotinamide adenine dinucleotide phosphate; CaM, calmodulin; H₄B, (6R)-5,6,7,8-tetrahydrobiopterin; FAD, flavin adenine dinucleotide; FMN, Flavin mononucleotide, BBB, blood-brain barrier; CNS, central nervous system; PAMPA, parallel artificial membrane permeability assay; P-gp, P-glycoprotein; ER, efflux ratio; P_e, effective permeability; P_app, apparent permeability; Caco-2, cancer coli-2; TLC, thin layer chromatography; TBAF, tetra-n-butylammonium fluoride; TFA, trifluoroacetic acid.

Oxidoreductase enzymes are widely used biocatalysts due to their high enantioselectivity and broad substrate compatibility in useful transformations. Many oxidoreductases require nicotinamide cofactors (i.e., NAD(P)H). To replace this costly natural cofactor, synthetic nicotinamide cofactor biomimetics (NCBs) offer different shapes, binding affinities, and reducing potentials that exceed the capabilities of wild-type NAD(P)H. However, the ill-defined structure-activity relationships (SARs) of various NCBs slow rationally guided innovation, such as customized reducing potentials. Here, we dissect two essential elements of NCB design, holding the nicotinamide invariant. First, the linker length between the nicotinamide and an unconjugated aromatic ring uncovered unexpected benefits to redox activity for two or three carbon linkers. Second, substitution on this unconjugated aryl group (Ring 2) might not be expected to affect activity. However, SAR trends demonstrate substantial benefits to reductive potential conferred by electron-donating functionalities on Ring 2. Furthermore, catalysis by two enzymes demonstrates enzyme-dependent tolerance or sensitivity to the NCB structures. Density functional theory (DFT) and computational modeling provide a theoretical framework to understand and build upon these observations. Ring 2 reaches up to the nicotinamide to stabilize its positive charge after oxidation through π-π stacking and charge transfer. Thus, the systematic examination of NCB's stability, electrochemical redox potentials, and kinetics uncovers trends for the improved design of NCBs.

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The gut microbiota confers host protection against pathogen colonization early after infection. Several mechanisms underlying the protection have been described, but the contributions of nutrient competition versus direct inhibition are controversial. Using an ex vivo model of Salmonella growth in the mouse cecum with its indigenous microbes, we find that nutrient limitation and typical inhibitory factors alone cannot prevent pathogen growth. However, the addition of certain amino acids markedly reverses the microbiota's ability to suppress pathogen growth. Enhanced Salmonella colonization after antibiotic treatment is ablated by exclusion of dietary protein, which requires the presence of the microbiota. Thus, dietary protein and amino acids are important regulators of colonization resistance.

High shear spinning top (ST) typhoon-like fluid flow in a rapidly rotating inclined tube within a vortex fluidic device (VFD) approaches homochirality throughout the liquid with toroids of bundled single-walled carbon nanotubes (SWCNTs) twisted into stable chiral lemniscates (in the shape of Figure 8s), predominantly as the R-or S-structures, for the tube rotating clockwise (CW) or counterclockwise (CCW). However, this is impacted by the Earth's magnetic field (B_E). Theory predicts 1-20 MPa pressure for their formation, with their absolute chirality determined from scanning electron microscopy (SEM) and atomic force microscopy (AFM) images. Thus, the resultant lemniscate structures establish the absolute chirality of the inner and outer components of the ST flow. These chiral flows and lemniscates can be flipped to the opposite chirality by changing the orientation of the tube relative to the inclination angle of B_E, by moving the geographical location. Special conditions prevail where the tangential angle of the outer and inner flow of the ST becomes periodically aligned with B_E, which respectively dramatically reduce the formation of toroids (and thus lemniscates) and formation of lemniscates from the toroids formed by the double-helical (DH) flow generated by side wall Coriolis forces and Faraday waves.

Polymerases are among the most powerful tools in the molecular biology toolbox; however, access to large quantities of chemically modified nucleoside triphosphates for diverse applications remains hindered by the need for purification by high-performance liquid chromatography (HPLC). Here, we describe a scalable approach to modified nucleoside triphosphates that proceeds through a P(III)-P(V) mixed anhydride intermediate obtained from the coupling of a P(III) nucleoside phosphoramidite and a P(V) pyrene pyrophosphate reagent. The synthetic strategy allows the coupling, oxidation, and deprotection steps to proceed as stepwise transformations in a single one-pot reaction. The fully protected nucleoside triphosphates are purified by silica gel chromatography and converted to their desired compounds on scales exceeding those achievable by conventional strategies. The power of this approach is demonstrated through the synthesis of several natural and modified nucleoside triphosphates using protocols that are efficient and straightforward to perform.

Many strategies have been implemented to enrich an RNA population for a selectable function, but demarcation of the optimal functional motifs or minimal structures within longer libraries remains a lengthy and tedious process. To overcome this problem, we have developed a technique that isolates minimal active segments from complex heterogeneous pools of RNAs. This method allows for truncations to occur at both 5' and 3' ends of functional domains and introduces independent primer-binding sequences, thereby removing sequence and structure bias introduced by constant-sequence regions. We show examples of minimization for genomic and synthetic aptamers and demonstrate that the method can directly reveal an active RNA assembled from multiple strands, facilitating the development of heterodimeric structures used in cellular sensors. This approach provides a pipeline to experimentally define the boundaries of active domains and accelerate the discovery of functional RNAs.

Spermidine is a naturally occurring polyamine present in all cells and is necessary for viability in eukaryotic cells. The cellular levels of spermidine decline as an organism ages, and its supplementation has been found to extend lifespan in yeast, worms, flies, mice, and human cultured cells. The lifespan extending effect of spermidine is thought to be due to its ability to induce autophagy, a turnover of cellular components. Mitochondrial dysfunction is believed to be a major driver of the aging process. We asked whether spermidine could rescue mitochondrial dysfunction using the yeast Saccharomyces cerevisiae lacking mtDNA (ρ0 cells) as a model. Not only was spermidine unable to rescue survival in ρ0 cells, but it appeared to exhibit toxicity resulting in a shortened lifespan. This toxicity appears to not be due to the loss of mitochondrial respiration, elevated oxidative stress, or depleted ATP. Spermidine toxicity could be recapitulated by the genetic or pharmacological inactivation of mitochondrial complex III. It can also be prevented by the impairment of autophagy, through the inactivation of ATG8, or by impairment of mitochondrial complex II through the inactivation of SDH2. Spermidine toxicity in ρ0 cells was present in yeast strains BY4741 and W303, but not D273-10B, demonstrating genetic variance in the phenotype. Thus, caution may be suggested regarding the use of spermidine to alleviate aging in humans. Depending on the genotype of the individual, spermidine could potentially harm the very individuals it is intended to help.

Nitric oxide synthase (NOS) is a pivotal enzyme that regulates various physiological processes, and the dysregulation of neuronal NOS (nNOS) is implicated in neurodegenerative diseases. In our efforts to leverage existing X-ray crystallography data to develop novel aminoquinoline-pyridine hybrid inhibitors and evaluate their inhibitory activities and interactions with NOS isoforms, we identified compounds 8 and 9 as potent human nNOS inhibitors (K _i = 38 and 22 nM, respectively). Notably, compound 8 displayed an unprecedented binding mode, displacing the essential cofactor tetrahydrobiopterin (H₄B). Furthermore, compound 9 exhibited excellent selectivity, with a 900-fold preference for human nNOS over human eNOS, making it one of the most potent and selective aminoquinoline-based nNOS inhibitors reported to date. Herein we present our inhibitor design rationale, the synthesis, and the biochemical/physical evaluation of binding along with X-ray crystallographic studies with three NOS isoforms, providing detailed insights into the observed potency and selectivity of these inhibitors.