Kinase inhibitors and the case for CH...O hydrogen bonds in protein-ligand binding

Although the hydrogen bond is known to be an important mediator of intermolecular interactions, there has yet to be an analysis of the role of CH...O hydrogen bonds in protein-ligand complexes. In this work, we present evidence for such nonstandard hydrogen bonds from a survey of aromatic ligands in 184 kinase crystal structures and 358 high-resolution structures from the Protein Data Bank. CH groups adjacent to the positively charged nitrogen of nicotinamide exhibit geometric preferences strongly suggestive of hydrogen bonding interactions, as do heterocyclic CH groups in kinase ligands, while other aromatic CH groups do not exhibit these characteristics. Ab initio calculations reveal a considerable range of CH...O hydrogen bonding potentials among different aromatic ring systems, with nicotinamide and heterocycles preferred in kinase inhibitors showing particularly favorable interactions. These results provide compelling evidence for the existence of CH...O hydrogen bonds in protein-ligand interactions, as well as information on the relative strength of various aromatic CH donors. Such knowledge will be of considerable value in protein modeling, ligand design, and structure-activity analysis.     

A minimalist approach to fragment-based ligand design using common rings and linkers: application to kinase inhibitors

We present a novel method for stepwise scaffold assembly that integrates fragment-by-fragment ligand design approaches with high-throughput virtual library screening (COREGEN). As an extension of our earlier studies of common features present in drug molecules, we investigate the hypothesis that most pharmaceutically interesting ligands can be expressed in terms of the ring-linker frameworks that comprise them. Analysis of 119 published kinase inhibitors from at least 18 different targets illustrates that a basis set of 4 rings and 8 linkers is sufficient to describe approximately 90% of ring and linker occurrences, respectively. A similar result was derived from a larger set of approximately 40,000 kinase inhibitors from curated patents. A method for ring-linker-based assembly of scaffold libraries that uses experimental information to guide the placement of anchor fragments is validated using a set of reported kinase inhibitors of Bcr-Abl, Cdk2, and Src. In every case, the predominant structural motif of reported ligand cores is reproduced and variations are suggested. To underscore generality of this approach, a novel scaffold for a cyclooxygenase-2 (COX-2) selective ligand is proposed.     

Kinetic analysis of LY320236: competitive inhibitor of type I and non-competitive inhibitor of type II human steroid 5alpha-reductase

Type I and type II steroid 5alpha-reductases (5alpha-R) catalyze the conversion of testosterone (T) to dihydrotestosterone (DHT). LY320236 is a benzoquinolinone (BQ) that inhibits 5alpha-R activity in human scalp skin (Ki(typeI)=28.7+/-1.87 nM) and prostatic homogenates (Ki(typeII)=10.6+/-4.5 nM). Lineweaver-Burk, Dixon, and non-linear analysis methods were used to evaluate the kinetics of 5alpha-R inhibition by LY320236. Non-linear modeling of experimental data evaluated V(max) in the presence or absence of LY320236. Experimental data modeled to the following equation 1v=+ fixing the In0c value equal to 1.0 or 0 are consistent with non-competitive or competitive inhibition, respectively. LY320236 is a competitive inhibitor of type I 5alpha-R (In0c=0, Ki=3.39+/-0.38, RMSE = 1.300) and a non-competitive inhibitor of type II 5alpha-R (In0c=1, Ki=29. 7+/-3.4, RMSE = 0.0592). These data are in agreement with linear transformation of the data using Lineweaver-Burk and Dixon analyses. These enzyme kinetic data support the contention that the BQ LY320236 is a potent dual inhibitor with differing modes of activity against the two known human 5alpha-reductase isozymes. LY320236 represents a class of non-steroidal 5alpha-R inhibitors with potential therapeutic utility in treating a variety of androgen dependent disorders.     

In vitro micronucleus assay scored by flow cytometry provides a comprehensive evaluation of cytogenetic damage and cytotoxicity

This laboratory has previously reported on the development of a flow cytometry-based method for scoring in vitro micronuclei in mouse lymphoma (L5178Y) cells [S.L. Avlasevich, S.M. Bryce, S.E. Cairns, S.D. Dertinger, In vitro micronucleus scoring by flow cytometry: differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability, Environ. Molec. Mutagen. 47 (2006) 56-66]. With this method, necrotic and mid/late stage apoptotic cells are labeled with the fluorescent dye ethidium monoazide. Cells are then washed, stripped of their cytoplasmic membranes, and incubated with RNase plus a pan-nucleic acid dye (SYTOX Green). This process provides a suspension of free nuclei and micronuclei that are differentially stained relative to chromatin associated with dead/dying cells. The current report extends this line of investigation to include the human cell line TK6. Additionally, methods are described that facilitate simultaneous quantitative analysis of cytotoxicity, perturbations to the cell cycle, and what we hypothesize is aneuploidization. This comprehensive cytogenetic damage assay was evaluated with the following diverse agents: etoposide, ionizing radiation, methyl methanesulfonate, vinblastine, ethanol, and staurosporine. Cells were harvested after 30h of continuous treatment (in the case of chemicals), or following graded doses of radiation up to 1Gy. Key findings include the following: (1) Significant discrepancies in top dose selection were found for five of the six agents studied when relative survival measurements were based on Coulter counting versus flow cytometry. (2) Both microscopy- and flow cytometry-based scoring methods detected dose-dependent micronucleus formation for the four genotoxic agents studied, whereas no significant increases were observed for the presumed non-genotoxicants ethanol and staurosporine when top dose selection was based on flow cytometric indices of cytotoxicity. (3) SYTOX and ethidium monoazide fluorescence signals conveyed cell cycle and cell death information, respectively, and appear to represent valuable aids for interpreting micronucleus data. (4) The frequency of hypodiploid nuclei increased in response to each of the genotoxic agents studied, but not following exposure to ethanol or staurosporine. Collectively, these results indicate that a comprehensive assessment of genotoxicity and other test article-induced toxicities can be acquired simultaneously using a simple two-color flow cytometry-based technique.     

Tissue heterogeneity of the mammalian mitochondrial proteome

The functionality of the mitochondrion is primarily determined by nuclear encoded proteins. The mitochondrial functional requirements of different tissues vary from a significant biosynthetic role (liver) to a primarily energy metabolism-oriented organelle (heart). The purpose of this study was to compare the mitochondrial proteome from four different tissues of the rat, brain, liver, heart, and kidney, to provide insight into the extent of mitochondrial heterogeneity and to further characterize the overall mitochondrial proteome. Mitochondria were isolated, solubilized, digested, and subjected to quantitative liquid chromatography-mass spectroscopy. Of the 16,950 distinct peptides detected, 8,045 proteins were identified. High-confidence identification threshold was reached by 1,162 peptides, which were further analyzed. Of these 1,162 proteins, 1,149 were significantly different in content (P and q values < 0.05) between at least 2 tissues, whereas 13 were not significantly different between any tissues. Confirmation of the mitochondrial origin of proteins was determined from the literature or via NH₂-terminal mitochondrial localization signals. With these criteria, 382 proteins in the significantly different groups were confirmed to be mitochondrial, and 493 could not be confirmed to be mitochondrial but were not definitively localized elsewhere in the cell. A total of 145 proteins were assigned to the rat mitochondrial proteome for the first time via their NH₂-terminal mitochondrial localization signals. Among the proteins that were not significantly different between tissues, three were confirmed to be mitochondrial. Most notable of the significantly different proteins were histone family proteins and several structural proteins, including tubulin and intermediate filaments. The mitochondrial proteome from each tissue had very specific characteristics indicative of different functional emphasis. These data confirm the notion that mitochondria are tuned by the nucleus for specific functions in different tissues. structural proteins; oxidative phosphorylation; liquid chromatography; mass spectrometry; electrophoresis; histone; liver; heart; kidney; brain     

The 20,000 dalton structural variant of recombinant DNA-derived methionyl human growth hormone has early insulin-like effects in hypophysectomized rats

The 20,000 dalton variant of recombinant DNA-derived methionyl human growth hormone (20K-Met-hGH) induced decreases in blood glucose and free fatty acid concentrations one hour after intraperitoneal injection into fasted, hypophysectomized rats. Similar results were obtained using the 22,000 dalton form of recombinant DNA-derived methionyl human growth hormone (22K-Met-hGH). The data reported show that 20K-Met-hGH induces early insulin-like effects similar to the responses produced by 22K-Met-hGH in fasted hypophysectomized rats.     

Morphology and function of the feeding apparatus of the lungfish, Lepidosiren paradoxa (Dipnoi)

The feeding mechanism of the South American lungfish, Lepidosiren paradoxa retains many primitive teleostome characteristics. In particular, the process of initial prey capture shares four salient functional features with other primitive vertebrates: 1) prey capture by suction feeding, 2) cranial elevation at the cranio-vertebral joint during the mouth opening phase of the strike, 3) the hyoid apparatus plays a major role in mediating expansion of the oral cavity and is one biomechanical pathway involved in depressing the mandible, and 4) peak hyoid excursion occurs after maximum gape is achieved. Lepidosiren also possesses four key morphological and functional specializations of the feeding mechanism: 1) tooth plates, 2) an enlarged cranial rib serving as a site for the origin of muscles depressing the hyoid apparatus, 3) a depressor mandibulae muscle, apparently not homologous to that of amphibians, and 4) a complex sequence of manipulation and chewing of prey in the oral cavity prior to swallowing. The depressor mandibulae is always active during mouth opening, in contrast to some previous suggestions. Chewing cycles include alternating adduction and transport phases. Between each adduction, food may be transported in or out of the buccal cavity to position it between the tooth plates. The depressor mandibulae muscle is active in a double-burst pattern during chewing, with the larger second burst serving to open the mouth during prey transport. Swallowing is characterized by prolonged activity in the hyoid constrictor musculature and the geniothoracicus. Lepidosiren uses hydraulic transport achieved by movements of the hyoid apparatus to position prey within the oral cavity. This function is analogous to that of the tongue in many tetrapods.     

Evolution of the branchiostegal membrane and restricted gill openings in Actinopterygian fishes

A phylogenetic survey is a powerful approach for investigating the evolutionary history of a morphological characteristic that has evolved numerous times without obvious functional implications. Restricted gill openings, an extreme modification of the branchiostegal membrane, are an example of such a characteristic. We examine the evolution of branchiostegal membrane morphology and highlight convergent evolution of restricted gill openings. We surveyed specimens from 433 families of actinopterygians for branchiostegal membrane morphology and measured head and body dimensions. We inferred a relaxed molecular clock phylogeny with branch length estimates based on nine nuclear genes sampled from 285 species that include all major lineages of Actinopterygii. We calculated marginal state reconstructions of four branchiostegal membrane conditions and found that restricted gill openings have evolved independently in at least 11 major actinopterygian clades, and the total number of independent origins of the trait is likely much higher. A principal component analysis revealed that fishes with restricted gill openings occupy a larger morphospace, as defined by our linear measurements, than do fishes with nonrestricted openings. We used a decision tree analysis of ecological data to determine if restricted gill openings are linked to certain environments. We found that fishes with restricted gill openings repeatedly occur under a variety of ecological conditions, although they are rare in open‐ocean pelagic environments. We also tested seven ratios for their utility in distinguishing between fishes with and without restricted gill openings, and we propose a simple metric for quantifying restricted gill openings (RGO), defined as a ratio of the distance from the ventral midline to the gill opening relative to half the circumference of the head. Functional explanations for this specialized morphology likely differ within each clade, but its repeated evolution indicates a need for a better understanding of diversity of ventilatory morphology among fishes. J. Morphol. 276:681–694, 2015. © 2015 Wiley Periodicals, Inc.     

Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with 3H-naloxone or 3H-D-Ala2-D-Leu5-enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties.