Structure-based optimization of 1H-imidazole-2-carboxamides as Axl kinase inhibitors utilizing a Mer mutant surrogate

Axl has been a target of interest in the oncology field for several years based on its role in various oncogenic processes. To date, no wild-type Axl crystal structure has been reported. Herein, we describe the structure-based optimization of a novel chemotype of Axl inhibitors, 1H-imidazole-2-carboxamide, using a mutated kinase homolog, Mer(I650M), as a crystallographic surrogate. Iterative optimization of the initial lead compound (1) led to compound (21), a selective and potent inhibitor of wild-type Axl. Compound (21) will serve as a useful compound for further in vivo studies.     

Hyperbolic Modeling of Starburst Dendrimers

Starburst dendrimers are highly branched oligomers. A rigid dendritic hydrocarbon, C₁₁₃₄H₁₁₄₆, has recently been synthesized. It consists of 94 phenylacetylene units displayed in a self-similar two-dimensional skeleton isomorphous to the three-coordinated Bethe lattice. The three-dimensional representation of phenylacetylene dendrimer shows a globular architecture with large voids and niches in its interior, characteristic of hyperbolic surfaces. This work investigates the geometrical scaling behavior of this starburst dendrimer using the symmetry properties of a Bethe lattice embedded in the hyperbolic plane. The results for C₁₁₃₄H₁₁₄₆ provide its density profile and an upper bound for its macromolecular size. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pulmonary type II cell hypertrophy and pulmonary lipoproteinosis are features of chronic IL-13 exposure

Interleukin (IL)-13, a key mediator of Th2-mediated immunity, contributes to the pathogenesis of asthma and other pulmonary diseases via its ability to generate fibrosis, mucus metaplasia, eosinophilic inflammation, and airway hyperresponsiveness. In these studies, we compared surfactant accumulation in wild-type mice and mice in which IL-13 was overexpressed in the lung. When compared with littermate controls, transgenic animals showed alveolar type II cell hypertrophy under light and electron microscopy. Over time, their alveoli also filled with surfactant in a pulmonary alveolar proteinosis pattern. At the same time, prominent interstitial fibrosis occurs. Bronchoalveolar lavage fluid from these mice had a three- to sixfold increase in surfactant phospholipids. Surfactant proteins (SP)-A, -B, and -C showed two- to threefold increases, whereas SP-D increased 70-fold. These results indicate that IL-13 is a potent stimulator of surfactant phospholipid and surfactant accumulation in the lung. IL-13 may therefore play a central role in the broad range of chronic pulmonary conditions in which fibrosis, type II cell hypertrophy, and surfactant accumulation occur.     

Uptake and fate of IAA in apple callus tissues: metabolism of IAA-2-14C

Young and old apple callus tissues were incubated in light ordarkness with IAA-2-¹⁴C. A large portion of the IAA disappearedfrom the medium with both young and old calluses. Whereas withold calluses the loss was mainly due to IAA destruction, youngcalluses accumulated IAA to a level which exceeded the externalconcentration and, in addition, seemed to protect it from breakdown.After 24 hr the level of IAA-2-¹⁴C in the medium dropped to50% with old calluses both in the dark and light, and with youngcalluses to 20% in the light and 50% in the dark. Chromatographyand scanning of the media and calluses showed that IAA was convertedinto two compounds (comp. A and comp. B). The amounts and proportionsof these metabolites in the medium and tissue were dependenton the different treatments and callus age. The breakdown ofIAA by old tissue gave rise to a higher level of comp. B bothin the tissue and medium, particularly after 6 hr of incubation.In the medium of young tissues the level of comp. A was higherthan comp. B while equal amounts of the two compounds were detectedin the tissue, itself. The origin of the IAA products in thetissue was probably endogenous and not via absorption from themedium. The IAA metabolism of apple callus tissues seems toproceed via the oxindole pathway and it is proposed that compoundsA and B are 3-hydroxymethyloxindole and 3-methylene oxindole,respectively. ¹ Contribution from the Agricultural Research Origanization,The Volcani Center, Bet Dagan, Israel. 1973 Series No. 275-E. (Received May 30, 1974; )     

E,E,E-1-(4-Arylamino-4-oxo-2-butenoyl)-3,5-bis(arylidene)-4-piperidones: a topographical study of some novel potent cytotoxins

A series of E,E,E-3,5-bis(arylidene)-1-(4-arylamino-4-oxo-2-butenoyl)-4-piperidones 4 (phenylidene) and 5 (4-nitrophenylidene) were prepared in order to explore the structural features of the N-acyl group which affects the cytotoxic potency. Evaluation toward human Molt 4/C8 and CEM T-lymphocytes revealed that many of the IC(50) figures were submicromolar and lower than melphalan. Marked inhibitory potencies toward murine leukemia L1210 cells were also noted. When evaluated against a panel of human tumor cell lines, three representative compounds in series 4 displayed selective toxicity to leukemia and colon cancer cell lines and were significantly more potent than the reference drug melphalan. Molecular modeling of representative compounds in both series 4 and the analogs, in which the configuration of the olefinic double bond was changed from E to Z (series 3), revealed that the torsion angles of the arylidene aryl rings and locations of the terminal arylaminocarbonyl groups may have contributed to the greater cytotoxic properties displayed in 3. Compounds 4c (3,4-dichlorophenylamino), d (4-methylphenylamino) and 5c (3,4-dichlorophenylamino), d (4-methylphenylamino) inhibited the activity of human N-myristoyltransferase by approximately 50% at concentrations of 50-100 microM. The compounds in series 4 and 5 were well tolerated in a short-term toxicity study in mice.     

Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

Morinda lucida reduces contractility of isolated uterine smooth muscle of pregnant and non-pregnant mice.

The present work investigated the effect of Morinda lucida (M. lucida) extract on isolated uterine smooth muscle of pregnant and non-pregnant mice. Pregnant and non-pregnant mice were pretreated with oral stilboesterol (0.1 mg/kg body weight) and killed by cervical dislocation. Thin strips of the uterus were cut and mounted in a 20ml organ bath containing De Jalon solution bubbled with 95%O2-5% CO2 gas mixture. The strips were connected to a force transducer coupled to a Grass 7D Polygraph for the recording of isometric tension. Effects of graded concentrations of oxytocin (OXY; 10-5-10-2 mol/L), acetylcholine (ACh; 10-9-10-5 mol/L) and M. lucida extract (0.015-1.5 mg/ml) were recorded. Fresh uterine strips were then incubated with M. lucida extract for 5mins and cumulative response to OXY was repeated. Another set of fresh strips was incubated in L-NAME for 15mins and the cumulative responses to M.lucida extract were repeated. OXY resulted in increased contractile responses in both pregnant and non-pregnant uterine muscles. M. lucida resulted in relaxation of the uterine smooth muscle in both pregnant and non-pregnant mice at all doses. However, at 1.5mg/ml, M. lucida completely blocked spontaneous uterine contractions. Following incubation with L-NAME, M. lucida extract led to a slightly greater relaxation of the uterine strips. In conclusion, M. lucida reduced contractility of uterine smooth muscle in both pregnant and non-pregnant mice as well as blocking contractile responses to OXY and Ach in uterine smooth muscle of pregnant and non-pregnant mice. There was no significant alteration of M. lucida activity by L-NAME suggesting that the action of the compound on uterine muscle is not associated with impaired nitric oxide synthase.     

Synthesis and in vitro evaluation of novel small molecule inhibitors of bacterial arylamine N-acetyltransferases (NATs)

The synthesis and inhibitory activity of a series of 5-substituted-(1,1-dioxo-2,3-dihydro-1H-1 lambda(6)-benzo[e][1,2]thiazin-4-ylidene)-thiazolidine-2,4-dione derivatives as competitive inhibitors of recombinant bacterial arylamine-N-acetyltransferases (NATs) are described. The most potent NAT inhibitors are those that contain planar hydrophobic substituents on the sultam nitrogen.     

Maurocalcine and peptide A stabilize distinct subconductance states of ryanodine receptor type 1, revealing a proportional gating mechanism

Maurocalcine (MCa) isolated from Scorpio maurus palmatus venom shares 82% sequence identity with imperatoxin A. Both scorpion toxins are putative mimics of the II-III loop peptide (termed peptide A (pA)) of alpha(1s)-dihydropyridine receptor and are thought to act at a common site on ryanodine receptor type 1 (RyR1) important for skeletal muscle EC coupling. The relationship between the actions of synthetic MCa (sMCa) and pA on RyR1 were examined. sMCa released Ca(2+) from SR vesicles (EC(50) = 17.5 nm) in a manner inhibited by micromolar ryanodine or ruthenium red. pA (0.5-40 microm) failed to induce SR Ca(2+) release. Rather, pA enhanced Ca(2+) loading into SR and fully inhibited Ca(2+)-, caffeine-, and sMCa-induced Ca(2+) release. The two peptides modified single channel gating behavior in distinct ways. With Cs(+)-carrying current, 10 nm to 1 microm sMCa induced long lived subconductances having 48% of the characteristic full open state and occasional transitions to 29% at either positive or negative holding potentials. In contrast, pA stabilized long lived channel closures with occasional burst transitions to 65% (s1) and 86% (s2) of the full conductance. The actions of pA and sMCa were observed in tandem. sMCa stabilized additional subconductance states proportional to pA-induced subconductances (i.e. 43% of pA-modified s1 and s2 substates), revealing a proportional gating mechanism. [(3)H]Ryanodine binding and surface plasmon resonance analyses indicated that the peptides did not interact by simple competition for a single class of mutually exclusive sites on RyR1 to produce proportional gating. The actions of sMCa were also observed with ryanodine-modified channels and channels deficient in immunophilin 12-kDa FK506-binding protein. These results provide evidence that sMCa and pA stabilize distinct RyR1 channel states through distinct mechanisms that allosterically stabilize gating states having proportional conductance.