Development of alpha-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease

Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T. cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of alpha-ketoamide-, alpha-ketoacid-, alpha-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1' residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different alpha-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3A crystallographic structure of cruzain bound with one of the alpha-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization.     

Expression of the mRNA encoding truncated PPARα does not correlate with hepatic insensitivity to peroxisome proliferators

Two alternatively spliced forms of human PPAR mRNA, PPAR1 and PPAR2, have been identified. PPAR1 mRNA gives rise to an active PPAR protein while PPAR2 mRNA gives rise to a form of PPAR which lacks the ligand-binding domain. PPAR2 is unable to activate a peroxisome proliferator response element (PPRE) reporter gene construct in transient transfection assays. Both PPAR1 and PPAR2 mRNA are present in human liver, kidney, testes, heart, small intestine, and smooth muscle. In human liver, PPAR2 mRNA abundance is approximately half that of PPAR1 mRNA; a correlation analysis of PPAR1 and PPAR2 mRNA mass revealed an r-value of 0.75 (n = 18). Additional studies with intact liver from various species, showed that the PPAR2/PPAR1 mRNA ratios in rat, rabbit, and mouse liver were less than 0.10; significantly lower than the 0.3 and 0.5 ratios observed in monkey and human livers, respectively. To determine if a high PPAR2/PPAR1 mRNA ratio was associated with insensitivity to peroxisome proliferators, we treated human, rat, and rabbit hepatocytes with WY14643, a potent PPARa activator, and measured acyl CoA oxidase (ACO) mRNA levels. Rat ACO mRNA levels increased markedly in response to WY14643 while human and rabbit hepatocytes were unresponsive. Thus, although the PPAR2/PPAR1 mRNA ratio is low in rabbits, this species is not responsive to peroxisome proliferators. Further studies with male and female rats, which vary significantly in their response to peroxisome proliferators, showed little difference in the ratio of PPAR2/PPAR1 mRNA. These data suggest that selective PPAR2 mRNA expression is not the basis for differential species or gender responses to peroxisome proliferators.     

Downregulation of RKIP is associated with poor outcome and malignant progression in gliomas

Malignant gliomas are highly infiltrative and invasive tumors, which precludes the few treatment options available. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying gliomas aggressive phenotype and poor prognosis. The Raf Kinase Inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. Its role in the prognosis and tumourigenesis of gliomas remains unclear. In the present study, we found that RKIP protein is absent in a low frequency (10%, 20/193) of glioma tumors. Nevertheless, the absence of RKIP expression was an independent prognostic marker in glioma. Additionally, by in vitro downregulation of RKIP, we found that RKIP inhibition induces a higher viability and migration of the cells, having no effect on cellular proliferation and angiogenesis, as assessed by in vivo CAM assay. In conclusion, this is the largest series studied so far evaluating the expression levels of this important cancer suppressor protein in glioma tumors. Our results suggest that in a subset of tumors, the absence of RKIP associates with highly malignant behavior and poor survival of patients, which may be a useful biomarker for tailored treatment of glioma patients.     

T.H. Huxley's Criticism of German Cell Theory: An Epigenetic and Physiological Interpretation of Cell Structure

In 1853, the young Thomas Henry Huxley published a long review of German cell theory in which he roundly criticized the basic tenets of the Schleiden-Schwann model of the cell. Although historians of cytology have dismissed Huxley’s criticism as based on an erroneous interpretation of cell physiology, the review is better understood as a contribution to embryology. “The Cell-theory” presents Huxley’s “epigenetic” interpretation of histological organization emerging from changes in the protoplasm to replace the “preformationist” cell theory of Schleiden and Schwann (as modified by Albert vonKölliker), which posited the nucleus as the seat of organic vitality. Huxley’s views influenced a number of British biologists, who continued to oppose German cell theory well into the twentieth century. Yet Huxley was pivotal in introducing the new German program of “scientific zoology” to Britain in the early 1850s,championing its empiricist methodology as a means to enact broad disciplinary and institutional reforms in British natural history.     

Preliminary investigation of morphological differences between ten breeds of horses suggests selection for paedomorphosis

Paedomorphosis is the retention of juvenile morphology at maturity and is important in generating evolutionary change in domestic species and species in the wild. When comparing dogs with the wolf, this preliminary study saw paedomorphosis in their physical and behavioral traits (Goodwin, Bradshaw, & Wickens, 1997). This preliminary study compared morphological characteristics of 10 breeds from northern regions (Shetland ponies) and southern regions (Arabians) with the Exmoor pony. Twenty-three respondents from the United Kingdom and Australia rated the breeds for 7 physical traits. As evidenced by low standard deviations, the respondents demonstrated a high degree of agreement. The study ranked breeds from the most similar (Highland pony) to the least similar (Arabian) to the Exmoor pony. The least similar breeds had physical traits suggestive of paedomorphosis: small heads, long legs, and a low head-to-body ratio. This preliminary study suggests that morphological, behavioral, and physiological differences between the breeds-plus morphometric comparisons of extant breeds and faunal remains of predomestication horses-warrant further study.