Synthesis and characterization of tritylthioethanamine derivatives with potent KSP inhibitory activity

Assembly of a bipolar mitotic spindle requires the action of class 5 kinesins, and inhibition or depletion of this motor results in mitotic arrest and apoptosis. S-Trityl-l-cysteine is an allosteric inhibitor of vertebrate Kinesin Spindle Protein (KSP) that has generated considerable interest due to its anti-cancer properties, however, poor pharmacological properties have limited the use of this compound. We have modified the triphenylmethyl and cysteine groups, guided by biochemical and cell-based assays, to yield new cysteinol and cysteamine derivatives with increased inhibitory activity, greater efficacy in model systems, and significantly enhanced potency against the NCI60 tumor panel. These results reveal a promising new class of conformationally-flexible small molecules as allosteric KSP inhibitors for use as research tools, with activities that provide impetus for further development as anti-tumor agents.     

Endogenously expressed estrogen receptors mediate neuroprotection in hippocampal cells (HT22)

Discovery of estrogen receptors (ER) in the central nervous system and the ability of estrogens to modulate neural circuitry and act as neurotrophic factors, suggest a therapeutic role of this steroid. To gain better understanding of the specificity and cellular mechanisms involved in estrogen-mediated neuroprotection, a mouse hippocampal neuronal cell line (HT22) was evaluated. Earlier reports indicated this cell line was devoid of ERs. Contrary to these findings, characterization of HT22 cells using RT-PCR, immunoblot, immunocytochemical, and radioligand binding techniques revealed endogenous expression of ER. The predominant subtype appeared to be ERalpha with functional activity confirmed using an ERE-tk-luciferase assay. The ability of an ER antagonist, ICI-182780, to block the neuroprotective effects of estrogens confirmed ER was involved mechanistically in neuroprotection. In conclusion, HT22 cells express functional ERalpha or a closely related ER enabling this cell line to be used to profile estrogens for neuroprotective properties acting via an ER-dependent mechanism.     

Origin of human chromosome 2 revisited

Similarities in chromosome banding patterns and hornologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chrornosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low-stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all three great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.     

Asoprisnil (J867): a selective progesterone receptor modulator for gynecological therapy

Asoprisnil is a novel selective steroid receptor modulator that shows unique pharmacodynamic effects in animal models and humans. Asoprisnil, its major metabolite J912, and structurally related compounds represent a new class of progesterone receptor (PR) ligands that exhibit partial agonist and antagonist activities in vivo. Asoprisnil demonstrates a high degree of receptor and tissue selectivity, with high-binding affinity for PR, moderate affinity for glucocorticoid receptor (GR), low affinity for androgen receptor (AR), and no binding affinity for estrogen or mineralocorticoid receptors. In the rabbit endometrium, both asoprisnil and J912 induce partial agonist and antagonist effects. Asoprisnil induces mucification of the guinea pig vagina and has pronounced anti-uterotrophic effects in normal and ovariectomized guinea pigs. Unlike antiprogestins, asoprisnil shows only marginal labor-inducing activity during mid-pregnancy and is completely ineffective in inducing preterm parturition in the guinea pig. Asoprisnil exhibits only marginal antiglucocorticoid activity in transactivation in vitro assays and animal models. In male rats, asoprisnil showed weak androgenic and anti-androgenic properties. In toxicological studies in female cynomolgus monkeys, asoprisnil treatment abolished menstrual cyclicity and endometrial atrophy. Early clinical studies of asoprisnil in normal volunteers demonstrated a dose-dependent suppression of menstruation irrespective of the effects on ovulation, with no change in basal estrogen concentrations and no antiglucocorticoid effects. Unlike progestins, asoprisnil does not induce breakthrough bleeding. With favorable safety and tolerability profiles thus far, asoprisnil appears promising as a novel treatment of gynecological disorders, such as uterine fibroids and endometriosis.     

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.     

The Arabidopsis gain-of-function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease

We describe the characterization of a novel gain-of-function Arabidopsis mutant, dll1 (disease-like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection. Despite the constitutive expression of defense-related responses, dll1 is unable to suppress the growth of virulent pathogens. However, dll1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dll1 is not defective in the induction of normal resistance responses. The lesion+ leaves of dll1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non-pathogenic to wild-type Col-0 plants. This suggests that dll1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease. Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dll1, suggesting that significant overlap exist between the signalling pathways leading to resistance- and disease-associated cell death. Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process.     

Identification of regional mechanical anisotropy in soft tissue analogs

In a previous work (Raghupathy and Barocas, 2010, "Generalized Anisotropic Inverse Mechanics for Soft Tissues,"J. Biomech. Eng., 132(8), pp. 081006), a generalized anisotropic inverse mechanics method applicable to soft tissues was presented and tested against simulated data. Here we demonstrate the ability of the method to identify regional differences in anisotropy from full-field displacements and boundary forces obtained from biaxial extension tests on soft tissue analogs. Tissue heterogeneity was evaluated by partitioning the domain into homogeneous subdomains. Tests on elastomer samples demonstrated the performance of the method on isotropic materials with uniform and nonuniform properties. Tests on fibroblast-remodeled collagen cruciforms indicated a strong correlation between local structural anisotropy (measured by polarized light microscopy) and the evaluated local mechanical anisotropy. The results demonstrate the potential to quantify regional anisotropic material behavior on an intact tissue sample.     

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.