Structural principles of insulin formulation and analog design: A century of innovation.

BACKGROUND: The discovery of insulin in 1921 and its near-immediate clinical use initiated a century of innovation. Advances extended across a broad front, from the stabilization of animal insulin formulations to the frontiers of synthetic peptide chemistry, and in turn, from the advent of recombinant DNA manufacturing to structure-based protein analog design. In each case, a creative interplay was observed between pharmaceutical applications and then-emerging principles of protein science; indeed, translational objectives contributed to a growing molecular understanding of protein structure, aggregation and misfolding. SCOPE OF REVIEW: Pioneering crystallographic analyses-beginning with Hodgkin's solving of the 2-Zn insulin hexamer-elucidated general features of protein self-assembly, including zinc coordination and the allosteric transmission of conformational change. Crystallization of insulin was exploited both as a step in manufacturing and as a means of obtaining protracted action. Forty years ago, the confluence of recombinant human insulin with techniques for site-directed mutagenesis initiated the present era of insulin analogs. Variant or modified insulins were developed that exhibit improved prandial or basal pharmacokinetic (PK) properties. Encouraged by clinical trials demonstrating the long-term importance of glycemic control, regimens based on such analogs sought to resemble daily patterns of endogenous ß-cell secretion more closely, ideally with reduced risk of hypoglycemia. MAJOR CONCLUSIONS: Next-generation insulin analog design seeks to explore new frontiers, including glucose-responsive insulins, organ-selective analogs and biased agonists tailored to address yet-unmet clinical needs. In the coming decade, we envision ever more powerful scientific synergies at the interface of structural biology, molecular physiology and therapeutics.

Of Molecules and Mechanisms.

Without question, molecular biology drives modern neuroscience. The past 50 years has been nothing short of revolutionary as key findings have moved the field from correlation toward causation. Most obvious are the discoveries and strategies that have been used to build tools for visualizing circuits, measuring activity, and regulating behavior. Less flashy, but arguably as important are the myriad investigations uncovering the actions of single molecules, macromolecular structures, and integrated machines that serve as the basis for constructing cellular and signaling pathways identified in wide-scale gene or RNA studies and for feeding data into informational networks used in systems biology. This review follows the pathways that were opened in neuroscience by major discoveries and set the stage for the next 50 years.

Enzyme Development for Human Islet Isolation: Five Decades of Progress or Stagnation?

In comparison to procedures used for the separation of individual cell types from other organs, the process of human pancreatic islet isolation aims to digest the pancreatic exocrine matrix completely without dispersing the individual cells within the endocrine cell cluster. This objective is unique within the field of tissue separation, and outlines the challenge of islet isolation to balance two opposing priorities. Although significant progress has been made in the characterization and production of enzyme blends for islet isolation, there are still numerous areas which require improvement. The ultimate goal of enzyme production, namely the routine production of a consistent and standardized enzyme blend, has still not been realized. This seems to be mainly the result of a lack of detailed knowledge regarding the structure of the pancreatic extracellular matrix and the synergistic interplay between collagenase and different supplementary proteases during the degradation of the extracellular matrix. Furthermore, the activation of intrinsic proteolytic enzymes produced by the pancreatic acinar cells, also impacts on the chance of a successful outcome of human islet isolation. This overview discusses the challenges of pancreatic enzymatic digestion during human islet isolation, and outlines the developments in this field over the past 5 decades.

Achievements and Challenges in Computational Protein Design.

Computational protein design (CPD), a yet evolving field, includes computer-aided engineering for partial or full de novo designs of proteins of interest. Designs are defined by a requested structure, function, or working environment. This chapter describes the birth and maturation of the field by presenting 101 CPD examples in a chronological order emphasizing achievements and pending challenges. Integrating these aspects presents the plethora of CPD approaches with the hope of providing a "CPD 101". These reflect on the broader structural bioinformatics and computational biophysics field and include: (1) integration of knowledge-based and energy-based methods, (2) hierarchical designated approach towards local, regional, and global motifs and the integration of high- and low-resolution design schemes that fit each such region, (3) systematic differential approaches towards different protein regions, (4) identification of key hot-spot residues and the relative effect of remote regions, (5) assessment of shape-complementarity, electrostatics and solvation effects, (6) integration of thermal plasticity and functional dynamics, (7) negative design, (8) systematic integration of experimental approaches, (9) objective cross-assessment of methods, and (10) successful ranking of potential designs. Future challenges also include dissemination of CPD software to the general use of life-sciences researchers and the emphasis of success within an in vivo milieu. CPD increases our understanding of protein structure and function and the relationships between the two along with the application of such know-how for the benefit of mankind. Applied aspects range from biological drugs, via healthier and tastier food products to nanotechnology and environmentally friendly enzymes replacing toxic chemicals utilized in the industry.

Fulfilling Koch's postulates in glycoscience: HCELL, GPS and translational glycobiology.

Glycoscience-based research that is performed expressly to address medical necessity and improve patient outcomes is called "translational glycobiology". In the 19th century, Robert Koch proposed a set of postulates to rigorously establish causality in microbial pathogenesis, and these postulates can be reshaped to guide knowledge into how naturally-expressed glycoconjugates direct molecular processes critical to human well-being. Studies in the 1990s indicated that E-selectin, an endothelial lectin that binds sialofucosylated carbohydrate determinants, is constitutively expressed on marrow microvessels, and investigations in my laboratory indicated that human hematopoietic stem cells (HSCs) uniquely express high levels of a specialized glycoform of CD44 called "hematopoietic cell E-/L-selectin ligand" (HCELL) that functions as a highly potent E-selectin ligand. To assess the role of HCELL in directing HSC migration to marrow, a method called "glycosyltransferase-programmed stereosubstitution" (GPS) was developed to custom-modify CD44 glycans to enforce HCELL expression on viable cell surfaces. Human mesenchymal stem cells (MSCs) are devoid of E-selectin ligands, but GPS-based glycoengineering of CD44 on MSCs licenses homing of these cells to marrow in vivo, providing direct evidence that HCELL serves as a "bone marrow homing receptor". This review will discuss the molecular basis of cell migration in historical context, will describe the discovery of HCELL and its function as the bone marrow homing receptor, and will inform on how glycoengineering of CD44 serves as a model for adapting Koch's postulates to elucidate the key roles that glycoconjugates play in human biology and for realizing the immense impact of translational glycobiology in clinical medicine.

Pharmaceutical and industrial protein engineering: where we are?

The huge amount of information, the big number of scientists and their efforts, labs, man/hrs, fund, companies all and others factors build the success of the amazing new branch of genetic engineering the 'protein engineering' (PE). It concerns with the modification of protein structure/function(s) or building protein from scratch. The engineered proteins usually have new criteria(s). Engineering proteins can be mediated on the level of genes or proteins. PE fined its way in different important sectors including industrial, pharmaceutical and medicinal ones. Aspects about PE and its applications will be discussed with this review. The concept, tools, and the industrial applications of the protein, engineered proteins and PE will be under focus. In order to get up to date knowledge about the applications of PE in basic protein and molecular biology, several examples are discussed. PE can play a significant role in different industrial and pharmaceutical sectors if used wisely and selectively.

Three decades of human monoclonal antibodies: past, present and future developments.

The hybridoma technique has been shown to be a most reproducible method for producing rodent monoclonal antibodies but poor results were obtained when it was used for generating human monoclonal antibodies. For immunotherapy, murine monoclonal antibodies are inadequate, whereas human monoclonal antibodies are virtually indispensable. Cellular, chemical, genetic and molecular methods to generate human monoclonal antibodies have been developed. Most often, the monoclonal antibodies for therapy are selected after deliberate vaccination, according to their high affinity towards an arbitrarily-chosen epitope of a pathogen or cellular antigen and therefore the selection is obviously skewed. A major hindrance of the production of therapeutic human monoclonal antibodies is the lack of an appropriate strategy to define and select the antibodies that would be effective in vivo. In contrast to antibodies induced by vaccination, there has been only a marginal interest in monoclonal antibodies which reflect antibodies of the innate immunity. In the future, human monoclonal antibodies that resemble antibodies that are ubiquitously present in sera of healthy individuals might serve as novel therapies in diseases such as Alzheimer's disease, where no other therapy exists.

From the first protein structures to our current knowledge of protein folding: delights and scepticisms.

Every breakthrough that opens new vistas also removes the ground from under the feet of other scientists. The scientific joy of those who have seen the new light is accompanied by the dismay of those whose way of life has been changed for ever. The publication of the first structures of proteins at atomic resolution 50 years ago astounded and inspired scientists in every field, but caused others to flee or scoff. That advance and every subsequent paradigm-shifting breakthrough in protein science have met with some resistance before universal acceptance. I relate these events and their impact on the field of protein folding.

Historical development of monoclonal antibody therapeutics.

Since the first publication by Kohler and Milstein on the production of mouse monoclonal antibodies (mAbs) by hybridoma technology, mAbs have had a profound impact on medicine by providing an almost limitless source of therapeutic and diagnostic reagents. Therapeutic use of mAbs has become a major part of treatments in various diseases including transplantation, oncology, autoimmune, cardiovascular, and infectious diseases. The limitation of murine mAbs due to immunogenicity was overcome by replacement of the murine sequences with their human counterpart leading to the development of chimeric, humanized, and human therapeutic antibodies. Remarkable progress has also been made following the development of the display technologies, enabling of engineering antibodies with modified properties such as molecular size, affinity, specificity, and valency. Moreover, antibody engineering technologies are constantly advancing to enable further tuning of the effector function and serum half life. Optimal delivery to the target tissue still remains to be addressed to avoid unwanted side effects as a result of systemic treatment while achieving meaningful therapeutic effect.

The trials and tribulations of producing the first genetically engineered drug.

Fifty years ago, the determination of the structure of DNA sparked a genetic revolution. Here, I give a personal perspective of the challenges involved in the development of the first biological therapeutic resulting from this revolution: recombinant human insulin.