Efforts toward oral bioavailability in factor VIIa inhibitors

Efforts toward developing orally bioavailable factor VIIa inhibitors starting from parenteral lead compound 1 are described. SAR resulted in improved physicochemical properties, leading to enhanced oral absorption in rat.     

Small molecule inhibitors of plasma kallikrein

Plasma kallikrein is a serine protease that is involved in pathways of inflammation, complement fixation, coagulation, and fibrinolysis. Herein, we describe the SAR and structural binding modes of a series of inhibitors of plasma kallikrein as well as the pharmacokinetics of a lead analog 11 in rat.     

Factor VIIa inhibitors: gaining selectivity within the trypsin family

Within the trypsin family of coagulation proteases, obtaining highly selective inhibitors of factor VIIa has been challenging. We report a series of factor VIIa (fVIIa) inhibitors based on the 5-amidino-2-(2-hydroxy-biphenyl-3-yl)-benzimidazole (1) scaffold with potency for fVIIa and high selectivity against factors IIa, Xa, and trypsin. With this scaffold class, we propose that a unique hydrogen bond interaction between a hydroxyl on the distal ring of the biaryl system and the backbone carbonyl of fVIIa lysine-192 provides a basis for enhanced selectivity and potency for fVIIa.     

Physical characterization of MxiH and PrgI, the needle component of the type III secretion apparatus from Shigella and Salmonella

Shigella and Salmonella use similar type III secretion systems for delivering effector proteins into host cells. This secretion system consists of a base anchored in both bacterial membranes and an extracellular "needle" that forms a rod-like structure exposed on the pathogen surface. The needle is composed of multiple subunits of a single protein and makes direct contact with host cells to facilitate protein delivery. The proteins that make up the needle of Shigella and Salmonella are MxiH and PrgI, respectively. These proteins are attractive vaccine candidates because of their essential role in virulence and surface exposure. We therefore isolated, purified, and characterized the monomeric forms of MxiH and PrgI. Their far-UV circular dichroism spectra show structural similarities with hints of subtle differences in their secondary structure. Both proteins are highly helical and thermally unstable, with PrgI having a midpoint of thermal unfolding (Tm) near 37 degrees C and MxiH having a value near 42 degrees C. The two proteins also have comparable intrinsic stabilities as measured by chemically induced (urea) unfolding. MxiH, however, with a free energy of unfolding (DeltaG degrees 0,un) of 1.6 kcal/mol, is slightly more stable than PrgI (1.2 kcal/mol). The relatively low m-values obtained for the urea-induced unfolding of the proteins suggest that they undergo only a small change in solvent-accessible surface area. This argues that when MxiH and PrgI are incorporated into the needle complex, they obtain a more stable structural state through the introduction of protein-protein interactions.     

Modeling the Hydraulics of Root Growth in Three Dimensions with Phloem Water Sources

Primary growth is characterized by cell expansion facilitated by water uptake generating hydrostatic (turgor) pressure to inflate the cell, stretching the rigid cell walls. The multiple source theory of root growth hypothesizes that root growth involves transport of water both from the soil surrounding the growth zone and from the mature tissue higher in the root via phloem and protophloem. Here, protophloem water sources are used as boundary conditions in a classical, three-dimensional model of growth-sustaining water potentials in primary roots. The model predicts small radial gradients in water potential, with a significant longitudinal gradient. The results improve the agreement of theory with empirical studies for water potential in the primary growth zone of roots of maize (Zea mays). A sensitivity analysis quantifies the functional importance of apical phloem differentiation in permitting growth and reveals that the presence of phloem water sources makes the growth-sustaining water relations of the root relatively insensitive to changes in root radius and hydraulic conductivity. Adaptation to drought and other environmental stresses is predicted to involve more apical differentiation of phloem and/or higher phloem delivery rates to the growth zone.Plant growth involves water uptake by the cells and expansion of the cell walls under the resultant turgor (internal hydrostatic pressure). The water uptake and increase in cell volume are accompanied by nutrient and metabolite deposition. Thus, hydraulics of growth (i.e. the energies, conductivities, and fluxes of water in growing tissue) are fundamental to understanding primary plant growth. Quantitatively, the driving force for water movement in the plant, as in other porous media, is considered to be the gradient in water potential (Ψ), an energy per unit volume given in MPa. Thus, primary growth can be modeled by considering plant tissue to be a distributed sink for water, with low Ψ and/or high hydraulic conductivity driving water deposition into rapidly expanding regions. Molz and Boyer (1978) developed the theoretical basis for predicting the radial water flux in one dimension within the intercalary meristem of growing soybean (Glycine max) hypocotyls. In this aerial tissue, water moves from the xylem both outward to the epidermis and inward to the pith. Thus, in the growing hypocotyls, Ψ is predicted to be least negative in the xylem and to decrease toward the epidermis and the pith. These predictions for growth-induced or growth-sustaining Ψ were confirmed when the experimental technology became sensitive enough to detect the gradients in Ψ (Nonami and Boyer, 1993). Passioura and Boyer (2003) expanded the theory to incorporate anatomical detail and corresponding spatial patterns of hydraulic conductivity. Their model explains experimental results on water relations during growth transients for many areas of the plant.The hydraulics of root growth differ from shoot growth because of differences in xylem anatomy. Root xylem becomes functional perhaps 1 cm behind the tip and well behind the growth zone. To enter the growing cells near the maize (Zea mays) root tip, externally supplied metabolites must move several millimeters without phloem (Fig. 1), and any water supplied by functional xylem would need to move more than 1 cm. Silk and Wagner (1980) provided a theoretical framework for a two-dimensional treatment of the growth-sustaining Ψ gradients in maize roots. They assumed that the water source was external (the soil or root-bathing medium) and that the root surface was in equilibrium with the soil or bathing medium, so that the flow path to growing cells in the root was predicted to be primarily inward. As in the shoot model, growing tissue was seen as a distributed sink for water. However, since the publication of that theory, experimental studies have revealed that the root tip is not in equilibrium with the bathing medium (Pritchard et al., 1996, 2000; Gould et al., 2004; Shimazaki et al., 2005). Pressure probes combined with osmotic potential determinations have shown that the Ψ of exterior root cells ranges from −0.17 to −0.6 MPa, depending on environmental conditions. This range is more negative than in the nutrient medium. Furthermore, evidence has accumulated that at least some water for root growth comes from the phloem. The most obvious evidence is perhaps the growth of nodal (adventitious) roots of maize, rice (Oryza sativa), and other gramineous plants (Westgate and Boyer, 1985). This growth is a normal part of crop development. The nodal roots grow through air and then dry layers of surface soil, making it unlikely that the expanding root cells obtain water from the dry media surrounding the root. Empirical and theoretical studies have concluded that the phloem probably provides water for growth of the primary maize root (Bret-Harte and Silk, 1994; Frensch and Hsiao, 1995; Pritchard, 1996; Pritchard et al., 1996, 2000; Hukin et al., 2002; Gould et al., 2004).Open in a separate windowFigure 1.Primary root growth zone. The tip of the seedling root of maize showing the meristem as part of the apical third of the elongation zone. The boundary of this root section was digitized to provide the computational body-fit grid used for the model. [See online article for color version of this figure.]The model described here follows the concepts of Pritchard and colleagues (1996, 2000) in assuming a pressure-driven bulk flow of solution through the phloem to the region where phloem is beginning to be functional (1–4 mm from the apex; Fig. 1). Water movement can occur from both the surrounding soil and the developing phloem. Henceforth, we refer to the “external water source equilibrium” or EE model, for which the boundary condition is solely an exterior medium of fairly high Ψ (−0.005 to −0.05 MPa) and no conditions are placed on the phloem Ψ (Silk and Wagner (1980), that the exterior of the root is in equilibrium with its bathing solution. Empirical studies have shown that this model is not realistic, because the root maintains peripheral cells at more negative Ψ than the bathing medium. Since this is hypothesized to occur by deposition of apoplastic solutes, we will refer to a model with external water source and apoplastic solutes near the exterior as the EASE model.

Table I.

Acronyms for models and definitions of symbols used in mathematical modeling

Modifier gene study of meconium ileus in cystic fibrosis: statistical considerations and gene mapping results

Cystic fibrosis (CF) is a monogenic disease due to mutations in the CFTR gene. Yet, variability in CF disease presentation is presumed to be affected by modifier genes, such as those recently demonstrated for the pulmonary aspect. Here, we conduct a modifier gene study for meconium ileus (MI), an intestinal obstruction that occurs in 16–20% of CF newborns, providing linkage and association results from large family and case–control samples. Linkage analysis of modifier traits is different than linkage analysis of primary traits on which a sample was ascertained. Here, we articulate a source of confounding unique to modifier gene studies and provide an example of how one might overcome the confounding in the context of linkage studies. Our linkage analysis provided evidence of a MI locus on chromosome 12p13.3, which was segregating in up to 80% of MI families with at least one affected offspring (HLOD = 2.9). Fine mapping of the 12p13.3 region in a large case–control sample of pancreatic insufficient Canadian CF patients with and without MI pointed to the involvement of ADIPOR2 in MI (p = 0.002). This marker was substantially out of Hardy–Weinberg equilibrium in the cases only, and provided evidence of a cohort effect. The association with rs9300298 in the ADIPOR2 gene at the 12p13.3 locus was replicated in an independent sample of CF families. A protective locus, using the phenotype of no-MI, mapped to 4q13.3 (HLOD = 3.19), with substantial heterogeneity. A candidate gene in the region, SLC4A4, provided preliminary evidence of association (p = 0.002), warranting further follow-up studies. Our linkage approach was used to direct our fine-mapping studies, which uncovered two potential modifier genes worthy of follow-up.     

Biochemical characterization,localization, and tissue distribution of the longer form of mouse SIRT3

SIRT3 is a key mitochondrial protein deacetylase proposed to play key roles in regulating mitochondrial metabolism but there has been considerable debate about its actual size, the sequences required for activity, and its subcellular localization. A previously cloned mouse SIRT3 has high sequence similarity with the C‐terminus of human SIRT3 but lacks an N‐terminal mitochondrial targeting sequence and has no detectable deacetylation activity in vitro. Using 5′ rapid amplification of cDNA ends, we cloned the entire sequence of mouse SIRT3, as well as rat and rabbit SIRT3. Importantly, we find that full‐length SIRT3 protein localizes exclusively to the mitochondria, in contrast to reports of SIRT3 localization to the nucleus. We demonstrate that SIRT3 has no deacetylation activity in vitro unless the protein is truncated, consistent with human SIRT3. In addition, we determined the inhibition constants and mechanism of action for nicotinamide and a small molecule SIRT3 inhibitor against active mouse SIRT3 and show that the mechanisms are different for the two compounds with respect to peptide substrate and NAD⁺. Thus, identification and characterization of the actual SIRT3 sequence should help resolve the debate about the nature of mouse SIRT3 and identify new mechanisms to modulate enzymatic activity.     

Reproductive skew in female common marmosets: what can proximate mechanisms tell us about ultimate causes?

Common marmosets are cooperatively breeding monkeys that exhibit high reproductive skew: most subordinate females fail to reproduce, while others attempt to breed but produce very few surviving infants. An extensive dataset on the mechanisms limiting reproduction in laboratory-housed and free living subordinate females provides unique insights into the causes of reproductive skew. Non-breeding adult females undergo suppression of ovulation and inhibition of sexual behaviour; however, they receive little or no aggression or mating interference by dominants and do not exhibit behavioural or physiological signs of stress. Breeding subordinate females receive comparable amounts of aggression to non-breeding females but are able to conceive, gestate and lactate normally. In groups containing two breeding females,however, both dominant and subordinate breeders kill one another's infants. These findings suggest that preconception reproductive suppression is not imposed on subordinate females by dominants, at a proximate level, but is instead self-imposed by most subordinates, consistent with restraint models of reproductive skew. In contrast to restraint models, however, this self-suppression probably evolved not in response to the threat of eviction by dominant females but in response to the threat of infanticide. Thus,reproductive skew in this species appears to be generated predominantly by subordinate self-restraint, in a proximate sense, but ultimately by dominant control over subordinates' reproductive attempts.