3-Oxo-2-piperazinyl acetamides as potent bradykinin B1 receptor antagonists for the treatment of pain and inflammation

The discovery of novel and highly potent oxopiperazine based B1 receptor antagonists is described. Compared to the previously described arylsulfonylated (R)-3-amino-3-phenylpropionic acid series, the current compounds showed improved in vitro potency and metabolic stability. Compound 17, 2-((2R)-1-((4-methylphenyl)sulfonyl)-3-oxo-2-piperazinyl)-N-((1R)-6-(1-piperidinylmethyl)-1,2,3,4-tetrahydro-1-naphthalenyl)acetamide, showed EC₅₀ of 10.3 nM in a rabbit biochemical challenge model. The practical syntheses of chiral arylsulfonylated oxopiperazine acetic acids are also described.     

Divergent Protein Kinase A isoforms co-ordinately regulate conidial germination, carbohydrate metabolism and virulence in Aspergillus fumigatus

The genome of Aspergillus fumigatus encodes two isoforms of the catalytic subunit of the cAMP-dependent Protein Kinase (PKA). Although deletion of the class I isoform, pkaC1, leads to an attenuation of virulence, the function of the class II subunit, PkaC2, was previously uninvestigated. In this report, we demonstrate that both isoforms act in concert to support various physiologic processes that promote the virulence of this pathogen. Whereas pkaC1 and pkaC2 single-deletion mutants display wild-type conidial germination, a double-deletion mutant is delayed in germination in response to environmental nutrients. Furthermore, PkaC1 and PkaC2 interact to positively regulate flux through the carbohydrate catabolic pathway and, consequently, the ΔpkaC1ΔpkaC2 mutant is unable to grow on low glucose concentrations. Importantly, the reduced germinative capacity and inability to utilize glucose observed for the ΔpkaC1ΔpkaC2 strain correlated with an inability of the mutant to establish infection in a murine model. Conversely, overexpression of pkaC2 both promotes the in vitro growth on glucose, and restores the fungal burden and mortality associated with the ΔpkaC1 to that of the wild-type organism. Taken together, these data demonstrate the functional capacity of pkaC2 and emphasize the importance of PKA-mediated metabolic control in the pathogenic potential of A. fumigatus.     

Reconstructing community assembly in time and space reveals enemy escape in a Western Palearctic insect community

How geographically widespread biological communities assemble remains a major question in ecology. Do parallel population histories allow sustained interactions (such as host-parasite or plant-pollinator) among species, or do discordant histories necessarily interrupt them? Though few empirical data exist, these issues are central to our understanding of multispecies evolutionary dynamics. Here we use hierarchical approximate Bayesian analysis of DNA sequence data for 12 herbivores and 19 parasitoids to reconstruct the assembly of an insect community spanning the Western Palearctic and assess the support for alternative host tracking and ecological sorting hypotheses. We show that assembly occurred primarily by delayed host tracking from a shared eastern origin. Herbivores escaped their enemies for millennia before parasitoid pursuit restored initial associations, with generalist parasitoids no better able to track their hosts than specialists. In contrast, ecological sorting played only a minor role. Substantial turnover in host-parasitoid associations means that coevolution must have been diffuse, probably contributing to the parasitoid generalism seen in this and similar systems. Reintegration of parasitoids after host escape shows these communities to have been unsaturated throughout their history, arguing against major roles for parasitoid niche evolution or competition during community assembly.     

An in vitro simulation study of impulsive force transmission along the lower skeletal extremity

Several investigators have revealed that a relationship exists between articular cartilage deterioration and the mechanical stress that results from transient impulsive forces created in the lower extremity during gait. This study is an investigation of the transmission of impulse waves through the lower extremity and the effect of knee pathology and prosthetic knee replacement on their transmission. An in vitro experiment is performed using human cadaver specimens that are instrumented with accelerometers. The distal end of the tibia is impacted with a vibration shaker to simulate heel strike. The results indicate that the normal knee joint is able to attenuate 59% of the transient peak force applied to it by the tibia. This attenuation capacity is reduced by knee pathology and decreases further with implantation of a knee prosthesis. The results indicate that abnormalities at the knee may increase the risk of degenerative changes at the ankle, hip and in the spine due to increased transient impulsive forces.     

Autophagy: a role in metal ion homeostasis?

Nutrient limitation is one of the most common forms of stress encountered by microorganisms in the environment. Surviving this stress depends upon a number of integrated responses, one of the most important of which is autophagy. When the filamentous fungus Aspergillus fumigatus becomes nutrient deprived it undergoes two important processes: the developmental pathway for asexual sporulation (conidiation), and a foraging response that promotes the migration of the hyphal tips into new substrate. To determine the contribution of autophagy to these two functions, we disrupted the A. fumigatus atg1 gene. The data reveal that Atg1 is required for wild-type conidiation of A. fumigatus, but only when nitrogen is limiting. Secondly, we demonstrate that metal ion availability limits the extent to which A. fumigatus can grow without a carbon/nitrogen source and that autophagy is necessary for growth under conditions of metal ion deficiency. These findings indicate that autophagy is responsible for maintaining an adequate supply of nitrogen to support conidiophore development, and provide intriguing new evidence that autophagy is linked to metal ion homeostasis.     

Discovery of dihydroquinoxalinone acetamides containing bicyclic amines as potent Bradykinin B1 receptor antagonists

Replacement of the core beta-amino acid in our previously reported piperidine acetic acid and beta-phenylalanine-based Bradykinin B1 antagonists by dihydroquinoxalinone acetic acid increases the in vitro potency and metabolic stability. The most potent compounds from this series have IC(50)s<0.2 nM in a human B1 receptor functional assay. A molecular modeling study of the binding modes of key compounds, based on a B1 homology model, explains the structure-activity relationship (SAR) for these analogs.     

SERPINB12 is a novel member of the human ov-serpin family that is widely expressed and inhibits trypsin-like serine proteinases

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 +/- 1.6 x 10(5) and 1.6 +/- 0.2 x 10(4) M(-1) S(-1), respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.     

Expression of the Aspergillus fumigatus rheb homologue,rhbA, is induced by nitrogen starvation

A gene encoding a ras protein with homology to the rheb family was cloned from Aspergillus fumigatus. Although conserved ras domains are present, the predicted RhbA protein sequence deviates from the ras consensus in a manner that is characteristic of rheb proteins. The invariant Gly-Gly in the first GTP-binding domain of ras proteins is replaced by Arg-Ser in RhbA, and a conserved Asp in the effector region of ras proteins is replaced by Asn in RhbA. The rhbA mRNA was detected throughout the A. fumigatus asexual developmental cycle, and accumulated over 5-fold in response to nitrogen starvation. The rhbA gene was able to complement the canavanine hypersensitivity of Saccharomyces cerevisiae Deltarhb1 mutants, suggesting that the two proteins share overlapping function.     

Pharmacologic inhibition of tpl2 blocks inflammatory responses in primary human monocytes, synoviocytes, and blood

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.