David G. Covell

Englerin A, a selective inhibitor of renal cancer cell growth, from Phyllanthus engleri.

An extract from Phyllanthus engleri was identified in a bioinformatic analysis of NCI 60-cell natural product extract screening data that selectively inhibited the growth of renal cancer cell lines. Bioassay-guided fractionation yielded two new guaiane sesquiterpenes, englerins A (1) and B (2). Englerin A showed 1000-fold selectivity against six of eight renal cancer cell lines with GI(50) values ranging from 1-87 nM. The structures of 1 and 2 and their relative stereochemistry were established by spectroscopic methods.

Relating molecular flexibility to function: a case study of tubulin.

Microtubules (MT), along with a variety of associated motor proteins, are involved in a range of cellular functions including vesicle movement, chromosome segregation, and cell motility. MTs are assemblies of heterodimeric proteins, alpha beta-tubulins, the structure of which has been determined by electron crystallography of zinc-induced, pacilitaxel-stabilized tubulin sheets. These data provide a basis for examining relationships between structural features and protein function. Here, we study the fluctuation dynamics of the tubulin dimer with the aim of elucidating its functional motions relevant to substrate binding, polymerization/depolymerization and MT assembly. A coarse-grained model, harmonically constrained according to the crystal structure, is used to explore the global dynamics of the dimer. Our results identify six regions of collective motion, comprised of structurally close but discontinuous sequence fragments, observed only in the dimeric form, dimerization being a prerequisite for domain identification. Boundaries between regions of collective motions appear to act as linkages, found primarily within secondary-structure elements that lack sequence conservation, but are located at minima in the fluctuation curve, at positions of hydrophobic residues. Residue fluctuations within these domains identify the most mobile regions as loops involved in recognition of the adjacent regions. The least mobile regions are associated with nucleotide binding sites where lethal mutations occur. The functional coupling of motions between and within regions identifies three global motions: torsional and wobbling movements, en bloc, between the alpha- and beta-tubulin monomers, and stretching longitudinally. Further analysis finds the antitumor drug pacilitaxel (TaxotereR) to reduce flexibility in the M loop of the beta-tubulin monomer; an effect that may contribute to tightening lateral interactions between protofilaments assembled into MTs. Our analysis provides insights into relationships between intramolecular tubulin movements of MT organization and function.

Antimitotic peptides and depsipeptides.

Tubulin is the target for an ever increasing number of unusual peptides and depsipeptides that were originally isolated from a wide variety of organisms. Since tubulin is the major component of cellular microtubules, which maintain cell shape in interphase and form the mitotic spindle, most of these compounds are highly toxic to mammalian cells. These peptides and depsipeptides disrupt cellular microtubules and prevent formation of a functional spindle, resulting in the accumulation of cultured cells in the G2/M phase of the cell cycle through specific inhibition of mitosis. At the biochemical level, the compounds all inhibit the assembly of tubulin into polymer and, in the cases where it has been studied, strongly suppress microtubule dynamics at low concentrations. In most cases the peptides and depsipeptides inhibit the binding of vinblastine and vincristine to tubulin in a noncompetitive manner, inhibit tubulin-dependent GTP hydrolysis, and interfere with nucleotide turnover at the exchangeable GTP site on beta-tubulin. Most of the peptides and depsipeptides induce tubulin to form oligomers of aberrant morphology, including tubulin rings that vary in diameter depending on the (depsi) peptide under study. The purpose of this review is to give an overview of the cellular, biochemical, in vivo, and SAR aspects of this group of compounds. We also summarize initial efforts by computer modeling to decipher a pharmacophore among the diverse structures of these peptides and depsipeptides.

Measurement of True Calcium Absorption in Premature Infants Using Intravenous 46Ca and Oral 44Ca

ABSTRACT: We have developed a method for measuring true fractional calcium absorption (α) in premature infants using two stable isotopes of calcium and tested it in seven studies in seven infants (birth weight 1543 ± 65 g, gestation 32.8 ± 7 wk). A total of 7.5 μg/kg 46Ca was given as a single intravenous bolus. Immediately thereafter 1.25 mg/kg of 44CA was given in a single gavage feeding of standard infant formula (Enfamil). A metabolic isolette was used to obtain 4-h collections of urine for 24 h total. 46Ca and 44Ca were measured in urine by thermal ionization mass spectroscopy and expressed as the ratio to naturally occurring 48Ca. The differences in the 46Ca/48Ca and 44Ca/48Ca ratios from natural levels (Δ% excess 46Ca and Δ% excess 44Ca) were calculated. Percent absorption (α) equals a constant times cumulative Δ% excess 44Ca/A% excess 46Ca. The calculation of α is independent of urine volume or concentration. The Δ% excess 46Ca, showed the expected multiexponential decline as a function of time, and Δ% excess 44Ca usually peaked during a 4− to 8-h urine collection. Calculations of α using increasingly long sampling times showed that a plateau had been reached by 12 h. α values calculated after 16–24 h in the seven infants at 2 wk of age were 41, 48, 45, 46, 25, 55, and 51%. Repeat studies at 3 wk of age were 46, 60, and 54%. These values are somewhat higher than net percent calcium absorption values reported for standard formula and thus appear very appropriate. This methodology will be very valuable in studying factors that may affect true calcium absorption in premature infants.

Enantioselective Synthesis of (+)-Chamaecypanone C, a Novel Microtubule Inhibitor

A bicycle built for tubulin: The total synthesis of (+)-chamaecypanone C has been achieved by using a tandem retro-Diels-Alder/Diels-Alder cascade reaction (see scheme). Initial biological studies demonstrate that (+)-chamaecypanone C is an inhibitor of tubulin assembly and binds at the colchicine site.

Structural investigation of C4b-binding protein by molecular modeling: Localization of putative binding sites

C4b‐binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one β‐chain and seven α‐chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy‐terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its α‐chains and with protein S through its β‐chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP α‐chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the α‐chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary‐structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein–protein interactions. Proteins 31:391–405, 1998.

Conformational Analysis of the Tachykinins in Solution: Substance P and Physalaemin

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.

Docking enzyme-inhibitor complexes using a preference-based free-energy surface

We present a docking scheme that utilizes both a surface complementarity screen as well as an energetic criterion based on surface area burial. Twenty rigid enzyme/inhibitor complexes with known coordinate sets are arbitrarily separated and reassembled to an average all‐atom rms (root mean square) deviation of 1.0 Å from the native complexes. Docking is accomplished by a hierarchical search of geometrically compatible triplets of surface normals on each molecule. A pruned tree of possible bound configurations is built up using successive consideration of larger and larger triplets. The best scoring configurations are then passed through a free‐energy screen where the lowest energy member is selected as the predicted native state. The free energy approximation is derived from observations of surface burial by atom pairs across the interface of known enzyme/inhibitor complexes. The occurrence of specific atom‐atom surface burial, for a set of complexes with well‐defined secondary structure both in the bound and unbound states, is parameterized to mimic the free energy of binding. The docking procedure guides the inhibitor into its native state using orientation and distance‐dependent functions that reproduce the ideal model of free energies with an average rms deviation of 0.9 kcal/mol. For all systems studied, this docking procedure identifies a single, unique minimum energy configuration that is highly compatible with the native state.

Linking tumor cell cytotoxicity to mechanism of drug action: an integrated analysis of gene expression, small-molecule screening and structural databases.

An integrated, bioinformatic analysis of three databases comprising tumor‐cell‐based small molecule screening data, gene expression measurements, and PDB (Protein Data Bank) ligand–target structures has been developed for probing mechanism of drug action (MOA). Clustering analysis of GI50 profiles for the NCIs database of compounds screened across a panel of tumor cells (NCI60) was used to select a subset of unique cytotoxic responses for about 4000 small molecules. Drug–gene–PDB relationships for this test set were examined by correlative analysis of cytotoxic response and differential gene expression profiles within the NCI60 and structural comparisons with known ligand–target crystallographic complexes. A survey of molecular features within these compounds finds thirteen conserved Compound Classes, each class exhibiting chemical features important for interactions with a variety of biological targets. Protein targets for an additional twelve Compound Classes could be directly assigned using drug‐protein interactions observed in the crystallographic database. Results from the analysis of constitutive gene expressions established a clear connection between chemo‐resistance and overexpression of gene families associated with the extracellular matrix, cytoskeletal organization, and xenobiotic metabolism. Conversely, chemo‐sensitivity implicated overexpression of gene families involved in homeostatic functions of nucleic acid repair, aryl hydrocarbon metabolism, heat shock response, proteasome degradation and apoptosis. Correlations between chemo‐responsiveness and differential gene expressions identified chemotypes with nonselective (i.e., many) molecular targets from those likely to have selective (i.e., few) molecular targets. Applications of data mining strategies that jointly utilize tumor cell screening, genomic, and structural data are presented for hypotheses generation and identifying novel anticancer candidates. Proteins 2005. Published 2005 Wiley‐Liss, Inc.

Mean residence time—theoretical development, experimental determination, and practical use in tracer analysis☆

Abstract This paper examines the relationship between calculations of kinetic quantities from moments of tracer data with calculations of the same quantities based on the rate constants of a compartmental model describing the data. Most kinetic measures obtained from moments of the data are shown to be simple algebraic functions of compartmental mean residence times. New, relatively simple expressions for calculating the zeroth, first and higher order moments of the residence time of material in the system from the compartmental rate constants are developed. Under certain circumstances the moments of the data yield kinetic quantities different from those obtained using compartmental mean residence times. This analysis illustrates the additional kinetic insight that can be achieved through compartmental analysis.