The 3C-like protease (3CLpro), which controls the severe acute respiratory syndrome (SARS) coronavirus replication, has been identified as a potential target for drug design in the treatment of SARS. A series of tetrapeptide phthalhydrazide ketones, pyridinyl esters, and their analogs have been designed, synthesized, and evaluated as potential SARS 3CLpro inhibitors. Some pyridinyl esters are identified as very potent inhibitors, with IC50 values in the nanomolar range (50-65 nM). Electrospray mass spectrometry indicates a mechanism involving acylation of the active site cysteine thiol for this class of inhibitors.

An 8w growth trial was conducted to determine the effect of partial replacement of fish meal (FM) by a blend of rendered animal protein (BAP, comprised of 40% meat and bone meal, 40% poultry by‐product meal, 20% hydrolyzed feather meal in diets for juvenile Siberia sturgeon (Acipenser baerii Brandt). Five experimental diets were formulated. The control diet (C) contained 48% FM, whereas in the remaining four diets, FM were replaced by BAP at 25% or 50% level balanced by crystallized amino acid (AA) or spray‐dried blood meal (BM), which named as BAP25‐AA, BAP25‐BM, BAP50‐AA and BAP50‐BM, respectively. Weight gain rate of BAP50‐AA group was significantly higher than that of C group, while other BAP diets did not show negative effect on growth performance. The BAP25‐BM group exhibited the lowest feed conversion rate (FCR) (P < 0.05). FM replacement by BAP did not affect chemical composition of the whole body and crude fat level of the liver, but significantly affect the fillet lipid content. Hepatic aspartate aminotransferase and serum total protein, total cholesterol and triglyceride were reduced in fish fed diet BAP50B. Siberian sturgeon showed high efficiency on utilization of crystalline amino acid as good as those from BM.

The 3C-like main peptidase 3CL pro is a viral polyprotein processing enzyme essential for the viability of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV). While it is generalized that 3CL pro and the structurally related 3C pro viral peptidases cleave their substrates via a mechanism similar to that underlying the peptide hydrolysis by chymotrypsin-like serine proteinases (CLSPs), some of the hypothesized key intermediates have not been structurally characterized. Here, we present three crystal structures of SARS 3CL pro in complex with each of two members of a new class of peptide-based phthalhydrazide inhibitors. Both inhibitors form an unusual thiiranium ring with the nucleophilic sulfur atom of Cys145, trapping the enzyme's catalytic residues in configurations similar to the intermediate states proposed to exist during the hydrolysis of native substrates. Most significantly, our crystallographic data are consistent with a scenario in which a water molecule, possibly via indirect coordination from the carbonyl oxygen of Thr26, has initiated nucleophilic attack on the enzyme-bound inhibitor. Our data suggest that this structure resembles that of the proposed tetrahedral intermediate during the deacylation step of normal peptidyl cleavage.

No abstract

Patient perceptions of PA benefits are strongly associated with improving PA levels after a cancer diagnosis. Clinician counseling should focus on patient education and changing patient perceptions.

The unbinding process of E2020 ((R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine) leaving from the long active site gorge of Torpedo californica acetylcholinesterase (TcAChE) was studied by using steered molecular dynamics (SMD) simulations on a nanosecond scale with different velocities, and unbinding force profiles were obtained. Different from the unbinding of other AChE inhibitors, such as Huperzine A that undergoes the greatest barrier located at the bottleneck of the gorge, the major resistance preventing E2020 from leaving the gorge is from the peripheral anionic site where E2020 interacts intensively with several aromatic residues (e.g., Tyr70, Tyr121, and Trp279) through its benzene ring and forms a strong direct hydrogen bond and a water bridge with Ser286 via its O24. These interactions cause the largest rupture force, approximately 550 pN. It was found that the rotatable bonds of the piperidine ring to the benzene ring and dimethoxyindanone facilitate E2020 to pass the bottleneck through continuous conformation change by rotating those bonds to avoid serious conflict with Tyr121 and Phe330. The aromatic residues lining the gorge wall are the major components contributing to hydrophobic interactions between E2020 and TcAChE. Remarkably, these aromatic residues, acting in three groups as "sender" and "receiver", compose a "conveyer belt" for E2020 entering and leaving the TcAChE gorge.

Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC 50 ≈19 nM) urea-based inhibitor at 2.1 and 2.4 Å resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical α/β hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the bindingsite residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120° about its C α -C β bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the α/β type. KeywordsMycobacterium tuberculosis; epoxide hydrolase; hydrolase fold; enzyme mechanism; inhibitor design *Corresponding author. Michael.James@ualberta.ca. Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jmb.2008.06.030 Protein Data Bank accession codesThe atomic coordinates and the structure factors for the free Mtb EHB and Mtb EHB bound to the inhibitor structures have been deposited in the Protein Data Bank|| with the accession codes 2E3J and 2ZJF, respectively.

The excessive activation of the N-methyl-D-aspartate receptor (NMDAR)/nitric oxide (NO) pathway has been proposed to be involved in the neuropathology of various neurodegenerative disorders. In this study, NO was found to mediate glutamateinduced excitotoxicity in primary cultured neurons. Compared with the NO synthase (NOS) inhibitor, N G -monomethyl-L-arginine (L-NMMA), and the NMDAR antagonist memantine, bis(7)-tacrine was found to be more potent in reducing NO-mediated excitotoxicity and the release of NO caused by glutamate. Moreover, like L-NMMA but not like 5H-dibenzo [a,d]cyclohepten-5,10-imine (MK-801) and memantine, bis(7)-tacrine showed greater neuroprotection and inhibition on NO release when neurons were pretreated for a prolonged time between 0 and 24 h and remained quite potent even when neurons were post-treated 1 h after the glutamate challenge. Bis(7)-tacrine was additionally found to be as moderately potent as memantine in competing with [ 3 H]MK-801, inhibiting NMDA-evoked currents and reducing glutamate-triggered calcium influx, which eventually reduced neuronal NOS activity. More importantly, at neuroprotective concentrations, bis(7)-tacrine substantially reversed the overactivation of neuronal NOS caused by glutamate without interfering with the basal activity of NOS. Furthermore, in vitro pattern analysis demonstrated that bis(7)-tacrine competitively inhibited both purified neuronal and inducible NOS with IC 50 values at 2.9 and 9.3 M but not endothelial NOS. This result was further supported by molecular docking simulations that showed hydrophobic interactions between bis(7)-tacrine and three NOS isozymes. Taken together, these results strongly suggest that the substantial neuroprotection against glutamate by bis(7)-tacrine might be mediated synergistically through the moderate blockade of NMDAR and selective inhibition of neuronal NOS.The precise mechanisms leading to the pathogenesis of chronic and acute neurodegenerative disorders have not yet been fully elucidated. However, increasing evidence has shown that these diseases may share a final common pathway to neuronal injury as a result of the excitotoxicity caused by the overstimulation of glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype (Yuan and Yankner, 2000;