Biomedical subjects

C S Ring

Publications and source records attributed to C S Ring.

8 recordsLinked to original sources

PubMed1994

Characterization of a Plasmodium vivax cysteine proteinase gene identifies uniquely conserved amino acids that may mediate the substrate specificity of malarial hemoglobinases.

The gene encoding a cysteine proteinase of the human malaria parasite Plasmodium vivax has been identified and characterized. The sequence predicted by the proteinase gene shares several unique features with the sequences of two recently characterized cysteine proteinases of other malarial species. These features include the conservation of a number of amino acids that are predicted, based on a recently devised model for the related Plasmodium falciparum cystine proteinase, to be located near the enzyme's active site. We hypothesize that these residues have been conserved to maintain optimal proteolytic specificity in the hydrolysis of globin by malaria parasites.

Amino Acid Sequence↗

PubMed1994

Anti-malarial drug development using models of enzyme structure.

BACKGROUND: The trophozoite stage of the malaria parasite infects red blood cells. During this phase of their life-cycle, the parasites use hemoglobin as their principal source of amino acids, using a cysteine protease to degrade it. We have previously reported a three-dimensional model of this cysteine protease, based on the structures of homologous proteases, and the use of the program DOCK to identify a ligand for the malaria protease. RESULTS: Here we describe the design of improved ligands starting from this lead. Ligand design was based on the predicted configuration of the lead compound docked to the model three-dimensional structure of the protease. The lead compound has an IC50 of 6 microM, and our design/synthesis strategy has resulted in increasingly potent derivatives that block the ability of the parasites to infect and/or mature in red blood cells. The two best derivatives to date have IC50(s) of 450 nM and 150 nM. CONCLUSIONS: A new class of anti-malarial chemotherapeutics has resulted from a computational search that was based on a model of the target protease. Despite the lack of a detailed experimental structure of the target enzyme or the enzyme-inhibitor complex, we have been able to identify compounds with increased potency. These compounds approach the activity of chloroquine (IC50 = 20 nM), but have a distinct mechanism of action. This series of compounds could thus lead to new therapies for chloroquine-resistant malaria.

Animals↗

PubMed1993

Structure-based inhibitor design by using protein models for the development of antiparasitic agents.

The lack of an experimentally determined structure of a target protein frequently limits the application of structure-based drug design methods. In an effort to overcome this limitation, we have investigated the use of computer model-built structures for the identification of previously unknown inhibitors of enzymes from two major protease families, serine and cysteine proteases. We have successfully used our model-built structures to identify computationally and to confirm experimentally the activity of nonpeptidic inhibitors directed against important enzymes in the schistosome [2-(4-methoxybenzoyl)-1-naphthoic acid, Ki = 3 microM] and malaria (oxalic bis[(2-hydroxy-1-naphthylmethylene)hydrazide], IC50 = 6 microM) parasite life cycles.

Animals↗

PubMed1993

Modeling protein structures: construction and their applications.

Although no general solution to the protein folding problem exists, the three-dimensional structures of proteins are being successfully predicted when experimentally derived constraints are used in conjunction with heuristic methods. In the case of interleukin-4, mutagenesis data and CD spectroscopy were instrumental in the accurate assignment of secondary structure. In addition, the tertiary structure was highly constrained by six cysteines separated by many residues that formed three disulfide bridges. Although the correct structure was a member of a short list of plausible structures, the "best" structure was the topological enantiomer of the experimentally determined conformation. For many proteases, other experimentally derived structures can be used as templates to identify the secondary structure elements. In a procedure called modeling by homology, the structure of a known protein is used as a scaffold to predict the structure of another related protein. This method has been used to model a serine and a cysteine protease that are important in the schistosome and malarial life cycles, respectively. The model structures were then used to identify putative small molecule enzyme inhibitors computationally. Experiments confirm that some of these nonpeptidic compounds are active at concentrations of less than 10 microM.

Amino Acid Sequence↗

PubMed1992

Taxonomy and conformational analysis of loops in proteins.

We propose a general classification scheme for loops, aperiodic segments of protein structure. In an effort to avoid the geometric complexity created by non-repeating phi psi angles, a morphologic definition that focuses upon the linearity and planarity of loops is utilized. Out of 432 loops (4 to 20 residues in length) extracted from 67 proteins, 205 are classified as linear (straps), 133 as non-linear and planar (omegas), and 86 as non-linear and non-planar (zetas). The remaining 8 are classified as compound loops because they contain a combination of strap, omega, and zeta morphologies. We introduce a structural alphabet as a shorthand notation for describing local conformation. The symbols of this alphabet are based on the virtual dihedral angle joining four consecutive alpha carbons. The notation is used to provide a compact description of loop motifs in phosphate binding and calcium binding proteins. Since similar loop conformations form similar "words", the structural sequence facilitates the search for common structural motifs in a family of loops. Contrary to the view of loops as "random coils", we find loops to have positional preferences for amino acid residues analogous to those previously described for beta-turns.

Amino Acid Sequence↗

PubMed1989

Detection of an intrinsic marker in hypoxic cells.

An autoradiographic method is presented for detecting, within a cell population, those cells which have been subjected to chronic hypoxia. No radioisotope is administered; rather the photographic emulsion is chemically reduced by intrinsic constituents of the cells. Hypoxic regions in the sandwich system, a multicellular in vitro tumor model, were detected in this manner. These regions were then compared with hypoxic sandwich regions as demonstrated by [3H]misonidazole labeling. Auxiliary studies, including studies on hypoxic monolayers, were consistent with the sandwich results. In all cases, the intracellular distribution of the chemographic grains was found to be cytosolic. Often the grains were clustered near the nucleus, perhaps in the region of the endoplasmic reticulum and the Golgi. We conclude that cells in a state of hypoxia and nutrient deprivation similar to that found in solid tumors retain a detectably altered biology for a significant period after reoxygenation. Therefore systematic methods of detecting previous hypoxia in histological tumor sections are feasible.

Animals↗

PubMed1989

3H-misonidazole labeling and viability of hypoxic cells in the sandwich system, an in vitro tumor analogue.

3H-misonidazole was used as a marker of hypoxic cells in an in vitro tumor analogue, the sandwich system. MISO binding was assessed in situ, using autoradiography. Binding profiles indicate that there are regions of radiobiological hypoxia surrounding the necrotic center in sandwiches of the V79 cell line and in sandwiches of the 9L cell line. Grains per cell were counted and detailed statistics on the variation of intrinsic binding among cells in the same microenvironment are presented. There is a systematic decrease in the standard deviation of grains per cell as one examines populations of cells further and further from the nutrient and oxygen source. Kinetic studies show that the growth fraction of the cell population also decreases with distance from the nutrient source. These findings taken together suggest that MISO binding is proportional to cell size and cells in the inner noncycling portion of the sandwich are more nearly uniform in size. Sandwich cells which exhibit heavy MISO binding, and are presumably radiobiologically hypoxic, were shown to be still viable if restored to good nutrient and oxygen conditions.

Animals↗

PubMed1989

Reducing the hypoxic fraction of a tumour model by growth in low glucose.

The question of whether growth under low glucose conditions leads to a reduced amount of cell hypoxia was investigated using an in vitro tumour analogue, the sandwich system. In this multicellular system, the interplay between diffusion and consumption of oxygen and nutrients results in spatial gradients of these environmental factors. Gradients in the environment lead to biological heterogeneity within the cell population. A necrotic centre, surrounded by a viable cell border, subsequently develops. Cells adjacent to the necrotic centre in sandwiches are hypoxic and are in an environment somewhat analogous to that of cells adjacent to necrotic regions in solid tumours. Using sandwiches of the 9L and V79 cell lines, the effects of growth under low glucose conditions on the degree of hypoxia in regions adjacent to the necrotic centre were investigated. Per-cell binding of 3H-misonidazole, assessed by autoradiography, was used as an indicator of oxygen deprivation. It was found that the extent of the hypoxic region and the severity of hypoxia were considerably reduced by growing sandwiches in a glucose concentration of 0.6 mM rather than 6.5 mM. This reduction was found in conjunction with a smaller viable border; it occurred despite the fact that the average per-cell oxygen consumption is higher in the low glucose sandwiches. The data are qualitatively consistent with a joint oxygen-glucose deprivation model for cell necrosis.

Animals↗