Current Chemical Biology - Volume 2, Issue 1, 2008

DNA triple helices are formed when a third nucleic acid strand binds within the major groove of a DNA duplex. The formation of these structures can be used to achieve selective recognition of extended DNA sequences, which may be useful in several medical and biotechnological applications. Although triplex formation is relatively straightforward in vitro, there are several problems that limit its use in cellular contexts, including a low stability at physiological pH and a requirement for oligopurine.oligopyrimidine target sites. There are also concerns about the uptake, localisation and degradation of triplex-forming oligonucleotides (TFOs) in cells, as well as the accessibility of chromosomal DNA. Major advances in the chemistry of DNA triplex formation have been made in the last few years and this review highlights the current status of this approach, with an emphasis on the use of chemically modified TFOs for gene targeting.

Prohibitins comprise a family of highly conserved ubiquitous eukaryotic proteins that localise to different compartments of the cell. They have been implicated in important cellular processes such as cellular signalling and transcriptional control, apoptosis, cellular senescence, early development of Caenorhabditis elegans and mitochondrial biogenesis. In yeast, mammals and C. elegans there exist at least two homologous prohibitin proteins (yeast: PHB1, PHB2; human: BAP32, BAP37), which assemble into high molecular weight complexes of about 1.2 MDa in the inner mitochondrial membrane. Experimentally determined structural information about these proteins has been elusive for a long time. Recently, however, the biogenesis and architecture of the yeast prohibitin complex has been analysed and yielded ringshaped structures as visualised by single particle electron microscopy. Structural details at atomic level remain to be determined, but a first step into this direction is provided by modelling approaches. Prohibitins consist of three domains, an N-terminal transmembrane helix, a middle (PHB) domain and a C-terminal coiled coil domain. The PHB domain is the landmark feature within the super-family of SPFH (stomatin/prohibitin/flotillin/HflK/C) domain proteins. The recently determined NMR structure of mouse flotillin-2 provides a first access to structural details of prohibitins. While the first functional role attributed to prohibitins was the regulation of cellular senescence, DNA transcription and tumour cell growth, there is recent evidence that they also can act as markers for adipose tissue. In a mouse model, an apoptotic peptide targeted at prohibitin was successful in reversing obesity. An extracellular complex containing both BAP32 and BAP37 was found to bind to the Vi capsular polysaccharide, first identified as a virulence antigen of Salmonella typhi, suggesting a key role for both proteins in infection with S. typhi. Furthermore, the interaction of prohibitin with compounds activating melanin production has placed these proteins at a central position in melanogenesis, and further implicates mitochondria in signalling pathways of the pigmentation process. Accumulating evidence suggests that prohibitins are implicated in mitochondrial, age and oxidative stress-related diseases, as well as in immunity and inflammation, cancer and cancer-like diseases, obesity, and drug resistance. The complementary interplay between structural and chemical biology will provide important insights into the molecular mechanisms of prohibitins and, more generally, the functions of mitochondria in living cells. This review discusses the current state of knowledge about prohibitins, and provides a vision for further developments in the field of these eminently important proteins.

Small molecule inhibitors of multisubunit RNA polymerases (RNAP) have emerged as powerful tools for the study of transcriptional mechanisms in both prokaryotes and eukaryotes. Here, we review studies in which RNAP inhibitors such as α-amanitin, rifampicin and streptolydigin were used to reveal key molecular aspects of the transcription reaction and catalytic mechanisms of RNAP. Many of the most significant findings, that represent the main focus of this review, are related to the identification and characterization of RNAP mutants that confer resistance to inhibitors and crystallographic data of RNAP-inhibitor complexes. Understanding the mechanism of action of RNAP inhibitors may lead to the design of better drugs targeting bacterial and fungal infections as well as other human diseases such as cancer.

Chemical rescue is an experimental strategy whereby the activity of a mutant enzyme is restored upon the addition of small exogenous compounds, which somehow surrogate the function of the mutated residue. These molecules become in effect “probes” of the chemical and structural requirements for efficient catalysis by the mutant enzyme. Entire batteries of small compounds can be tested for rescue, making it easier to implement the methods of physical organic chemistry (such as Brønsted analysis) to the study of enzymatic catalysis. To date, chemical rescue has been employed to address enzyme mechanisms in over a hundred studies, helping to identify catalytic residues, to better outline their roles and to probe the structural and functional context in which catalysis occurs. Recently, some researchers have explored the use of this strategy to modulate the activity of specific enzymes in vivo, as a tool for dissecting complex cellular processes. These studies have also raised the possibility that chemical rescue might one day be applied in therapy, for the reactivation of genetically defective enzymes. The present review illustrates the power, the pitfalls and the perspectives of this approach.

One of the convergence points of chemistry and biology is the synthesis of fine chemicals using enzymes as catalysts. Since many of these catalysts are not very efficient in typical conditions for organic synthesis, directed evolution has emerged in the last fifteen years as a powerful tool to improve the activity, selectivity and stability of enzymes. Directed evolution methods have been widely and successfully applied in the development of catalysts with improved properties. Nevertheless, this methodology is neither powerful enough to meet every catalyst properties, nor is suitable for the creation of new enzymatic activities. The recent advances and challenges in directed evolution, highlighting the problems of introducing genetic diversity coupled to Darwinian positive selection, are reviewed.

Glucose-dependent insulinotropic polypeptide (GIP or gastric inhibitory polypeptide) is a gut-derived incretin hormone which regulates glucose-induced insulin secretion. In addition to its actions on pancreatic beta-cells, GIP exerts a range of secondary extrapancreatic activities, which further augments its antihyperglycaemic properties. As such, GIP has attracted attention as a potential therapeutic agent for the treatment of diabetes, obesity and related metabolic disorders. However, a major drawback in utilising GIP as a therapeutic is its relatively short biological half-life due to degradation by the ubiquitous enzyme dipeptidylpeptidase-IV (DPP-IV) and rapid renal clearance. Consequently, efforts are presently focused on developing more stable and longer-acting forms of GIP which are resistant to DPP-IV-mediated degradation and have improved pharmacokinetic properties. In essence, structural modifications of GIP through N-terminal modification, amino acid substitution and/or fatty acid derivatisation have been shown to generate analogues which exhibit a range of activities from potent agonist action to specific antagonism of native GIP. The purpose of this review is to highlight recent advances in the development of GIP-based therapeutics and their potential in the treatment of type 2 diabetes and obesity.

The central metabolism of a cell can determine its short- and long-term structure and function. When a disease state arises, the metabolism (i.e., the transportation of nutrients into the cells, the overall substrate utilization and production, synthesis and accumulation of intracellular metabolites, etc.) is altered in a way that may permit organisms to survive under the changing physiologic constraints. Although the response of cells to injury was studied thoroughly using molecular biology and structural morphology techniques, the knowledge regarding the metabolic signatures of the disease is limited. However, recent advances in analytical methods and mathematical tools have led to new approaches to those questions with the concept of computational biology which relies on the integration of experimentation, data processing and modeling. The attempt to formulate current knowledge in mathematical terms has led to the development of several mathematical modeling tools (i.e., metabolic flux analysis, metabolic control analysis, etc.) that helps us to understand an entire biological system from basic structure to dynamic interactions. This review provides an overview and summarizes the current status of applications of mathematical models for the quantification of fluxes. A specific example of kidney podocyte cells illustrates how metabolic alterations, which occur during injury, can be used to aid in future therapeutic development.

Fundamental to the behavioral biology, organisms have the ability to detect and respond to chemical stimuli. Olfactory signal transduction and information processing in insects (e.g., moths) is a prime example of chemical communication found in nature for its exquisite sensitivity and selectivity. Although not completely understood yet, extensive research on the biology and chemistry of this complex event has revealed many facets of olfaction where donors, recipients, enormous pool of chemicals/stimulators, binding/carrier proteins and cellular receptors play their respective role with high precision, selectivity and sensitivity. Pheromone-binding proteins (PBPs), present in the antenna of male moth and other insect species, bind the volatile hydrophobic pheromone molecules and transport them across the aqueous sensillar lymph to the membrane-bound G protein-coupled receptor proteins. Recent structural studies on the PBP and PBP-pheromone complex have advanced our knowledge about the likely mode of ligand release and activation of pheromone receptors/odorant receptors. The pH-dependent conformational changes of the PBP play the key role in binding to the selective ligand, then shuttling and ultimately releasing the ligand to the receptor at the target cell membrane. Both the relatively higher pH of the sensillar lymph and the lower pH at the dendritic membrane are physiologically very important to foster the binding to and release of the ligand from PBP respectively. The NMR structures of the PBP at high and low pH provide evidence in support of this mechanism. However, the pH-induced structural change of pheromone-binding protein is quite different between two moths (B. mori and A. polyphemus) thus far studied.