Design and synthesis of thiophene dihydroisoquinolines as novel BACE1 inhibitors.

Utilizing a structure based design approach, combined with extensive medicinal chemistry execution, highly selective, potent and novel BACE1 inhibitor 8 (BACE1 Alpha assay IC50=8nM) was made from a weak µM potency hit in an extremely efficient way. The detailed SAR and general design approaches will be discussed.

Structure-based design of novel dihydroisoquinoline BACE-1 inhibitors that do not engage the catalytic aspartates.

The structure-activity relationship of a series of dihydroisoquinoline BACE-1 inhibitors is described. Application of structure-based design to screening hit 1 yielded sub-micromolar inhibitors. Replacement of the carboxylic acid of 1 was guided by X-ray crystallography, which allowed the replacement of a key water-mediated hydrogen bond. This work culminated in compounds such as 31, which possess good BACE-1 potency, excellent permeability and a low P-gp efflux ratio.

Nuclear resonance vibrational spectroscopy (NRVS) of rubredoxin and MoFe protein crystals.

We have applied 57Fe nuclear resonance vibrational spectroscopy (NRVS) for the first time to study the dynamics of Fe centers in Fe-S protein crystals, including oxidized wild type rubredoxin crystals from Pyrococcus furiosus, and the MoFe protein of nitrogenase from Azotobacter vinelandii. Thanks to the NRVS selection rule, selectively probed vibrational modes have been observed in both oriented rubredoxin and MoFe protein crystals. The NRVS work was complemented by extended X-ray absorption fine structure spectroscopy (EXAFS) measurements on oxidized wild type rubredoxin crystals from Pyrococcus furiosus. The EXAFS spectra revealed the Fe-S bond length difference in oxidized Pf Rd protein, which is qualitatively consistent with the X-ray crystal structure.

Design and synthesis of hydroxyethylamine (HEA) BACE-1 inhibitors: structure-activity relationship of the aryl region.

The structure-activity relationship of the prime region of hydroxyethylamine BACE inhibitors is described. Variation in the aryl linker region with 5- and 6-membered heterocycles provided compounds such as 33 with improved permeability and reduced P-gp liability compared to benzyl amine analog 1.

Improving the permeability of the hydroxyethylamine BACE-1 inhibitors: structure-activity relationship of P2' substituents.

Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2' position of the inhibitor with more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic analog 1 but with much higher observed passive permeability in MDCK cellular assay.

Regulation of nif gene expression and the energetics of N2 fixation over the diel cycle in a hot spring microbial mat.

Nitrogen fixation, a prokaryotic, O2-inhibited process that reduces N2 gas to biomass, is of paramount importance in biogeochemical cycling of nitrogen. We analyzed the levels of nif transcripts of Synechococcus ecotypes, NifH subunit and nitrogenase activity over the diel cycle in the microbial mat of an alkaline hot spring in Yellowstone National Park. The results showed a rise in nif transcripts in the evening, with a subsequent decline over the course of the night. In contrast, immunological data demonstrated that the level of the NifH polypeptide remained stable during the night, and only declined when the mat became oxic in the morning. Nitrogenase activity was low throughout the night; however, it exhibited two peaks, a small one in the evening and a large one in the early morning, when light began to stimulate cyanobacterial photosynthetic activity, but O2 consumption by respiration still exceeded the rate of O2 evolution. Once the irradiance increased to the point at which the mat became oxic, the nitrogenase activity was strongly inhibited. Transcripts for proteins associated with energy-producing metabolisms in the cell also followed diel patterns, with fermentation-related transcripts accumulating at night, photosynthesis- and respiration-related transcripts accumulating during the day and late afternoon, respectively. These results are discussed with respect to the energetics and regulation of N2 fixation in hot spring mats and factors that can markedly influence the extent of N2 fixation over the diel cycle.

Probing the MgATP-bound conformation of the nitrogenase Fe protein by solution small-angle X-ray scattering.

The MgATP-bound conformation of the Fe protein of nitrogenase from Azotobacter vinelandii has been examined in solution by small-angle X-ray scattering (SAXS) and compared to existing crystallographically characterized Fe protein conformations. The results of the analysis of the crystal structure of an Fe protein variant with a Switch II single-amino acid deletion recently suggested that the MgATP-bound state of the Fe protein may exist in a conformation that involves a large-scale reorientation of the dimer subunits, resulting in an overall elongated structure relative to the more compact structure of the MgADP-bound state. It was hypothesized that the Fe protein variant may be a conformational mimic of the MgATP-bound state of the native Fe protein largely on the basis of the observation that the spectroscopic properties of the [4Fe-4S] cluster of the variant mimicked in part the spectroscopic signatures of the native nitrogenase Fe protein in the MgATP-bound state. In this work, SAXS studies reveal that the large-scale conformational differences between the native Fe protein and the variant observed by X-ray crystallography are also observed in solution. In addition, comparison of the SAXS curves of the Fe protein nucleotide-bound states to the nucleotide-free states indicates that the conformation of the MgATP-bound state in solution does not resemble the structure of the variant as initially proposed, but rather, at the resolution of this experiment, it resembles the structure of the nucleotide-free state. These results provide insights into the Fe protein conformations that define the role of MgATP in nitrogenase catalysis.

On the electronic structure of the hydrogenase H-cluster.

X-ray absorption spectroscopic measurements and density functional calculations suggest that the hydrogenase H-cluster is best described as an electronically inseparable 6Fe-cluster due to extensive delocalization of frontier molecular orbitals of the iron centres, sulfide and the non-innocent dithiolate ligands.

In situ analysis of nitrogen fixation and metabolic switching in unicellular thermophilic cyanobacteria inhabiting hot spring microbial mats.

Genome sequences of two Synechococcus ecotypes inhabiting the Octopus Spring microbial mat in Yellowstone National Park revealed the presence of all genes required for nitrogenase biosynthesis. We demonstrate that nif genes of the Synechococcus ecotypes are expressed in situ in a region of the mat that varies in temperature from 53.5 degrees C to 63.4 degrees C (average 60 degrees C); transcripts are only detected at the end of the day when the mat becomes anoxic. Nitrogenase activity in mat samples was also detected in the evening. Hitherto, N2 fixation in hot spring mats was attributed either to filamentous cyanobacteria (not present at >50 degrees C in these mats) or to heterotrophic bacteria. To explore how energy-generating processes of the Synechococcus ecotypes track natural light and O2 conditions, we evaluated accumulation of transcripts encoding proteins involved in photosynthesis, respiration, and fermentation. Transcripts from photosynthesis (cpcF, cpcE, psaB, and psbB) and respiration (coxA and cydA) genes declined in the evening. In contrast, transcripts encoding enzymes that may participate in fermentation fell into two categories; some (ldh, pdhB, ald, and ackA) decreased in the evening, whereas others (pflB, pflA, adhE, and acs) increased at the end of the day and remained high into the night. Energy required for N2 fixation during the night may be derived from fermentation pathways that become prominent as the mat becomes anoxic. In a broader context, our data suggest that there are critical regulatory switches in situ that are linked to the diel cycle and that these switches alter many metabolic processes within the microbial mat.

Immobilization of active hydrogenases by encapsulation in polymeric porous gels.

Hydrogenases encapsulated in porous polymeric silica gels retain significant levels of hydrogen production activity when compared to hydrogenases in solution using reduced methyl viologen as an electron donor. Encapsulated hydrogenases remain active after storage at room temperature for longer than four weeks and are less sensitive to proteolytic digestion. Nanoscopic confinement of active hydrogenases in solids paves the way for their potential use in hydrogen producing catalytic materials applications.