Repression of early lateral root initiation events by transient water deficit in barley and maize.

The formation of lateral roots (LRs) is a key driver of root system architecture and developmental plasticity. The first stage of LR formation, which leads to the acquisition of founder cell identity in the pericycle, is the primary determinant of root branching patterns. The fact that initiation events occur asynchronously in a very small number of cells inside the parent root has been a major difficulty in the study of the molecular regulation of branching patterns. Inducible systems that trigger synchronous lateral formation at predictable sites have proven extremely valuable in Arabidopsis to decipher the first steps of LR formation. Here, we present a LR repression system for cereals that relies on a transient water-deficit treatment, which blocks LR initiation before the first formative divisions. Using a time-lapse approach, we analysed the dynamics of this repression along growing roots and were able to show that it targets a very narrow developmental window of the initiation process. Interestingly, the repression can be exploited to obtain negative control root samples where LR initiation is absent. This system could be instrumental in the analysis of the molecular basis of drought-responsive as well as intrinsic pathways of LR formation in cereals.

Magnetic resonance imaging detection and time course of cerebral microhemorrhages during passive immunotherapy in living amyloid precursor protein transgenic mice.

In recent years immunotherapy-based approaches for treating Alzheimer's disease have become the subject of intensive research. However, an important mechanistic-related safety concern is exacerbation of the risk of microhemorrhage that may be associated with fast removal of amyloid-ß (Aß) deposits found in blood vessels or brain parenchyma. Rapid in vivo detection of microhemorrhages in living amyloid precursor protein transgenic mice has not been described, and histological analysis can take several months before this risk is assessed. Aged transgenic mice were divided into two groups that would undergo longitudinal passive immunotherapy for 12 or 18 weeks. 6G1, a nonselective anti-Aß monoclonal antibody, and 8F5, a more selective antioligomeric Aß monoclonal antibody, were examined in both longitudinal studies. High-resolution T2*-weighted magnetic resonance microscopy (100 × 100 × 400 µm) was used for microhemorrhage detection in vivo. Cerebral microhemorrhages by magnetic resonance imaging were compared with histological hemosiderin staining in each animal; results showed that T2*-weighted magnetic resonance microscopy can reliably detect microhemorrhages of ≥60 µm in diameter at baseline and after 12 to 18 weeks of treatment in the same animals in vivo. This correlated significantly with histological readings. This new imaging safety biomarker can be readily applied to preclinical antibody screening in a longitudinal manner. 6G1 and 8F5, however, both increased microhemorrhage incidence in aged amyloid precursor protein transgenic mice compared with their baseline and vehicle treatment. A highly selective antibody for soluble Aß is needed to address the question of whether antibodies that do not bind to deposited Aß have microhemorrhage liability.

Non-peptide entry inhibitors of HIV-1 that target the gp41 coiled coil pocket.

The ectodomain of HIV-1 gp41 mediates the fusion of viral and host cellular membranes. The peptide-based drug Enfuvirtide(1) is precedent that antagonists of this fusion activity may act as anti HIV-agents. Here, NMR screening was used to discover non-peptide leads against this target and resulted in the discovery of a new benzamide 1 series. This series is non-peptide, low molecular weight, and analogs have activity in a cell fusion assay with EC50 values ranging 3-41microM. Structural work on the gp41/benzamide 1 complex was determined by NMR spectroscopy using a designed model peptide system that mimics an open pocket of the fusogenic form of the protein.

Toxicological evaluation of thiol-reactive compounds identified using a la assay to detect reactive molecules by nuclear magnetic resonance.

We have recently reported on the development of a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) to detect reactive false positive hits from high-throughput screening, in which we observed a surprisingly large number of compounds that can oxidize or form covalent adducts with protein thiols groups. In the vast majority of these cases, the covalent interactions are largely nonspecific (e.g., affect many protein targets) and therefore unsuitable for drug development. However, certain thiol-reactive species do appear to inhibit the target of interest in a specific manner. The question then arises as to the potential toxicology risks of developing a drug that can react with protein thiol groups. Here, we report on the evaluation of a large set of ALARM-reactive and -nonreactive compounds against a panel of additional proteins (aldehyde dehydrogenase, superoxide dismutase, and three cytochrome P450 enzymes). It was observed that ALARM-reactive compounds have significantly increased risks of interacting with one or more of these enzymes in vitro. Thus, ALARM NMR seems to be a sensitive tool to rapidly identify compounds with an enhanced risk of producing side effects in humans, including alcohol intolerance, the formation of reactive oxygen species, and drug-drug interactions. In conjunction with other toxicology assays, ALARM NMR should be a valuable tool for prioritizing compounds for lead optimization and animal testing.

Discovery and design of novel HSP90 inhibitors using multiple fragment-based design strategies.

The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.

Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery.

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

A high throughput fluorescent assay for measuring the activity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is the enzyme responsible for the rapid degradation of fatty acid amides such as the endocannabinoid anandamide. Inhibition of FAAH activity has been suggested as a therapeutic approach for the treatment of chronic pain, depression and anxiety, through local activation of the cannabinoid receptor CB1. We have developed a high throughput screening assay for identification of FAAH inhibitors using a novel substrate, decanoyl 7-amino-4-methyl coumarin (D-AMC) that is cleaved by FAAH to release decanoic acid and the highly fluorescent molecule 7-amino-4-methyl coumarin (AMC). This assay gives an excellent signal window for measuring FAAH activity and, as a continuous assay, inherently offers improved sensitivity and accuracy over previously reported endpoint assays. The assay was validated using a panel of known FAAH inhibitors and purified recombinant human FAAH, then converted to a 384 well format and used to screen a large library of compounds (>600,000 compounds) to identify FAAH inhibitors. This screen identified numerous novel FAAH inhibitors of diverse chemotypes. These hits confirmed using a native FAAH substrate, anandamide, and had very similar rank order potency to that obtained using the D-AMC substrate. Collectively these data demonstrate that D-AMC can be successfully used to rapidly and effectively identify novel FAAH inhibitors for potential therapeutic use.

Crystal structures of human adenosine kinase inhibitor complexes reveal two distinct binding modes.

Adenosine kinase (AK) is an enzyme responsible for converting endogenous adenosine (ADO) to adenosine monophosphate (AMP) in an adenosine triphosphate- (ATP-) dependent manner. The structure of AK consists of two domains, the first a large alpha/beta Rossmann-like nucleotide binding domain that forms the ATP binding site, and a smaller mixed alpha/beta domain, which, in combination with the larger domain, forms the ADO binding site and the site of phosphoryl transfer. AK inhibitors have been under investigation as antinociceptive, antiinflammatory, and anticonvulsant as well as antiinfective agents. In this work, we report the structures of AK in complex with two classes of inhibitors: the first, ADO-like, and the second, a novel alkynylpyrimidine series. The two classes of structures, which contain structurally similar substituents, reveal distinct binding modes in which the AK structure accommodates the inhibitor classes by a 30 degrees rotation of the small domain relative to the large domain. This change in binding mode stabilizes an open and a closed intermediate structural state and provide structural insight into the transition required for catalysis. This results in a significant rearrangement of both the protein active site and the orientation of the alkynylpyrimidine ligand when compared to the observed orientation of nucleosidic inhibitors or substrates.

Preparation and characterization of nitrilotriacetic-acid-terminated self-assembled monolayers on gold surfaces for matrix-assisted laser desorption ionization-time of flight-mass spectrometry analysis of proteins and peptides.

On-target affinity capture, enrichment and purification of biomolecules improve detection of specific analytes from complex biological samples in matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. In this paper, we report a simple method for preparation of a self-assembled nitrilotriacetic acid (NTA) monolayer on gold surface which can be used as a MALDI-TOF-MS sample target specifically for recombinant oligohistidine-tagged proteins/peptides and phosphorylated peptides. The NTA functional groups are immobilized to the gold surface via the linkage of 1,8-octanedithiol which forms a self-assembled monolayer on gold. Characterization by X-ray photoelectron spectroscopy and MALDI analysis of the modified surface are described. The chemically modified surface shows strong affinity toward the analytes of interest, which allows effective removal of the common interferences, e.g. salts and detergents, and therefore leads to improved signal/noise ratio and detection limit. The use of the modified surface simplifies the sample preparation for MALDI analysis of these targeted analytes.

Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization.

Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.

Chkl binds and phosphorylates BAD protein.

Chk1 (checkpoint kinase 1) is a serine-threonine kinase that is critical for G2/M arrest in response to DNA damage. Chk1 phosphorylates Cdc25C at serine-216, a major regulatory site, in response to DNA damage. Furthermore, Chk1 also phosphorylates Cdc25A on serine 123 which accelerates its degradation through the ubiquitin-proteasome pathway and arrests cells in late G2-phase after DNA damage. In the present study, we demonstrated that Chk1 phosphorylates pro-apoptotic protein BAD (Bcl-2/Bcl-XL-Antagonist, causing cell Death) in vitro. In vitro phosphorylation analysis with various mouse BAD peptides has revealed two phosphorylation sites for Chk1 at serine-155 and serine-170. When wild-type and mutant BAD (S155A) constructs were transfected into 293T cells, an association between BAD and Chk1 was observed by co-immunoprecipitation. In addition, there was an increase in the phosphorylation of serine-155 following DNA damage by adriamycin treatment. Our results suggest that Chk1 associates with BAD and phosphorylates the BAD protein at serine-155. Taken together, our results suggest that Chk1 may inactivate BAD by associating with and phosphorylating residues critical for BAD function in response to DNA damage.