Scytalone dehydratase (SD) is a molecular target of inhibitor design efforts aimed at protecting rice plants from the fungal disease caused by Magnaporthe grisea. As determined from X-ray diffraction data of an SD-inhibitor complex [Lundqvist et al. (1994) Structure (London) 2, 937-944], there is an extended hydrogen-bonding network between protein side chains, the inhibitor, and two bound water molecules. From models of SD complexed to quinazoline and benztriazine inhibitors, a new class of potent SD inhibitors involving the displacement of an active-site water molecule were designed. We were able to increase inhibitory potency by synthesizing compounds with a nitrile functionality displayed into the space occupied by one of the crystallographic water molecules. Sixteen inhibitors are compared. The net conversion of potent quinazoline and benztriazine inhibitors to cyanoquinolines and cyanocinnolines increased binding potency 2-20-fold. Replacement of the nitrile with a hydrogen atom lowered binding affinity 100-30,000-fold. X-ray crystallographic data at 1.65 A resolution on a SD-inhibitor complex confirmed that the nitrile functionality displaced the water molecule as intended and that a favorable orientation was created with tyrosines 30 and 50 which had been part of the hydrogen-bonding network with the water molecule. Additional data on inhibitors presented herein reveals the importance of two hydrogen-bonding networks toward inhibitory potency: one between Asn131 and an appropriately positioned inhibitor heteroatom and one between a bound water molecule and a second inhibitor heteroatom.

Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochemical potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were determined by X-ray crystallography, and the inhibitors were found to bind in an allosteric binding site separate from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compounds in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.

The high-resolution NMR solution structure of the catalytic fragment of human collagenase-3 (MMP-13) was used as a starting point for structure-based design of selective inhibitors for MMP-13. The major structural difference observed between the MMP structures is the relative size and shape of the S1′ pocket where this pocket is significantly longer for MMP-13, nearly reaching the surface of the protein. On the basis of the extended nature of the MMP-13 S1′ pocket an inhibitor potent and selective for MMP-13 was designed from an initial high throughput screening (HTS) lead. CL-82198 was identified as a weak (10 µM) inhibitor against MMP-13 while demonstrating no activity against MMP-1, MMP-9, or the related enzyme TACE. The drug-like properties of CL-82198 made it an ideal candidate for optimization of enzyme potency and selectivity. On the basis of NMR binding studies, it was shown that inhibitor CL-82198 bound within the entire S1′ pocket of MMP-13 which is the basis of its selectivity against MMP-1, MMP-9, and TACE. A strategy utilizing this information was devised for designing new inhibitors that showed enhanced selectivity toward MMP-13. Our design strategy combined the critical selectivity features of CL-82198 with the known potency features of a nonspecific MMP inhibitor (WAY-152177) to generate a potent and selective MMP-13 inhibitor (WAY-170523). WAY-170523 has an IC50 of 17 nM for MMP-13 and showed >5800-, 56-, and >500-fold selectivity against MMP-1, MMP-9, and TACE, respectively.A structure-based approach to designing potent and selective inhibitors has established itself as an important component of the drug development process (for reviews see refs 1 and 2). This is evident by the extensive structural data available for the matrix metalloproteinase (MMP) family of enzymes and the emergence of unique inhibitors based on this structural information (for reviews see refs 3-7). The MMPs are involved in the degradation of the extracellular matrix that is associated with normal tissue remodeling processes such as pregnancy, wound healing, and angiogenesis. MMP expression and activity is highly controlled because of the degradative nature of these enzymes where the apparent loss in this regulation results in the pathological destruction of connective tissue and the ensuing disease state. Thus, the MMPs are a highly active set of targets for the design of therapeutic agents for the disease areas of arthritis and oncology. The MMP family is composed of a number of enzymes where MMP-13 was recently identified on the basis of differential expression in normal breast tissues and in breast carcinoma. Recently, both an NMR and X-ray structure of inhibited MMP-13 have been reported. 8,9 The MMPs are generally categorized based on their substrate specificity, where the collagenase subfamily of MMP-1, MMP-8, and MMP-13 selectively cleaves native interstitial collagens (types I, II, and III). It is likely that only a subset of MMP enzymes will be involved in a particular disease as is evident by the overexpression o...

We examined whether the performance of the National Institute of Aging's Consortium to Establish a Registry for Alzheimer's Disease's 10-word list (CWL), part of the consortium's neuropsychological battery, can be improved for detecting Alzheimer's disease and related disorders early. We focused on mild cognitive impairment (MCI) and mild dementia because these stages often go undetected, and their detection is important for treatment. Using standardized diagnostic criteria combined with history, physical examination, and cognitive, laboratory, and neuroimaging studies, we staged 471 community-dwelling subjects for dementia severity by using the Clinical Dementia Rating Scale. We then used correspondence analysis (CA) to derive a weighted score for each subject from their item responses over the three immediate-and one delayed-recall trials of the CWL. These CA-weighted scores were used with logistic regression to predict each subject's probability of impairment, and receiver operating characteristic analysis was used to measure accuracy. For MCI vs. normal, accuracy was 97% [confidence interval (C.I.) 97-98%], sensitivity was 94% (C.I. 93-95%), and specificity was 89% (C.I. 88 -91%). For MCI͞mild dementia vs. normal, accuracy was 98% (C.I. 98 -99%), sensitivity was 96% (C.I. 95-97%), and specificity was 91% (C.I. 89 -93%). MCI sensitivity was 12% higher (without lowering specificity) than that obtained with the delayed-recall total score (the standard method for CWL interpretation). Optimal positive and negative predictive values were 100% and at least 96.6%. These results show that CA-weighted scores can significantly improve early detection of Alzheimer's disease and related disorders.

NRTI resistance in cells can consist of both altered binding or incorporation and altered excision of the NRTI. For K65R, the combination of these opposing mechanisms results in decreased susceptibility to most NRTI but increased susceptibility to ZDV.

The solution structure of the catalytic fragment of human fibroblast collagenase (MMP-1) complexed with a sulfonamide derivative of a hydroxamic acid compound (CGS-27023A) has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. The solution structure of the complex was calculated by means of hybrid distance geometry-simulated annealing using a combination of experimental NMR restraints obtained from the previous refinement of the inhibitor-free MMP-1 (1) and recent restraints for the MMP-1:CGS-27023A complex. The hydroxamic acid moiety of CGS-27023A was found to chelate to the "right" of the catalytic zinc where the p-methoxyphenyl sits in the S1' active-site pocket, the isopropyl group is in contact with H83 and N80, and the pyridine ring is solvent exposed. The sulfonyl oxygens are in hydrogen-bonding distance to the backbone NHs of L81 and A82. This is similar to the conformation determined by NMR of the inhibitor bound to stromelysin (2, 3). A total of 48 distance restraints were observed between MMP-1 and CGS-27023A from 3D 13C-edited/12C-filtered NOESY and 3D 15N-edited NOESY experiments. An additional 18 intramolecular restraints were observed for CGS-27023A from a 2D 12C-filtered NOESY experiment. A minimal set of NMR experiments in combination with the free MMP-1 assignments were used to assign the MMP-1 (1)H, 13C, and 15N resonances in the MMP-1:CGS-27023A complex. The assignments of CGS-27023A in the complex were obtained from 2D 12C-filtered NOESY and 2D 12C-filtered TOCSY experiments.

No abstract

Many brain diseases have been linked to abnormal oxygen metabolism and blood perfusion; nevertheless, there is still a lack of robust diagnostic tools for directly imaging cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow (CBF), as well as the oxygen extraction fraction (OEF) that reflects the balance between CMRO2 and CBF. This study employed the recently developed in vivo 17O MR spectroscopic imaging to simultaneously assess CMRO2, CBF and OEF in the brain using a preclinical middle cerebral arterial occlusion mouse model with a brief inhalation of 17O-labeled oxygen gas. The results demonstrated high sensitivity and reliability of the noninvasive 17O-MR approach for rapidly imaging CMRO2, CBF and OEF abnormalities in the ischemic cortex of the MCAO mouse brain. It was found that in the ischemic brain regions both CMRO2 and CBF were substantially lower than that of intact brain regions, even for the mildly damaged brain regions that were unable to be clearly identified by the conventional MRI. In contrast, OEF was higher in the MCAO affected brain regions. This study demonstrates a promising 17O MRI technique for imaging abnormal oxygen metabolism and perfusion in the diseased brain regions. This 17O MRI technique is advantageous because of its robustness, simplicity, noninvasiveness and reliability: features that are essential to potentially translate it to human patients for early diagnosis and monitoring of treatment efficacy.