Current Pharmaceutical Biotechnology - Volume 7, Issue 4, 2006

Small Molecule Versus Peptides The following is an excerpt from C&E News Volume 83, Number 11 pp. 17-24. Imagine a conversation between a small molecule and a peptide on a make-believe pharmacological playground. The small molecule would tout its virtues of small size, low price, oral availability, ability to cross membranes, and straightforward synthesis. The peptide would respond: "True, I may be bigger, more expensive to synthesize, and less stable than you. I may clear faster from the body and usually need to be injected rather than swallowed as a pill. But I can be much more potent, show higher specificity, and have few toxicology problems. I also don't accumulate in organs or face drug-drug interaction challenges like you do. So there. Small molecules have been the choice of drug for two important reasons. 1) ease and low cost of synthesis and 2) stability and therefore longer half life. However, toxicity is a major cause of failure for new drug candidate and Pharma Industry productivity. Many promising new drug candidates that show excellent efficacy in preclinical animal models fail when it is found that they cause toxic effects in humans. This is one of the reasons that the pipelines of many pharmaceutical companies are quite thin; and in contrast the pipeline of biologics is steadily growing. Although it is hard to pin point the reasons for such adverse effects, it is conceivable that small molecules by virtue of their size could potentially interact with multiple targets and accumulate in tissues. Despite applying stringent screens and rational drug design it is often difficult to predict how a small molecule interacts with different proteins in real life scenario. This is especially true if target protein belongs to a family of closely related proteins, e.g. kinases, matrix metalloproteinases. Such promiscuity often a cause for concern from safety view point however, may turn out to be useful when results in pleiotropic effects. Aspirin for example has many pharmacological effects most of which are beneficial. Statins, although targeted for HMG-CoA reductase inhibition and cholesterol lowering, interact with other targets which may explain some of their anti-inflammatory effects [1]. Methotrexate a widely used drug in cancer and inflammatory diseases has multiple mechanisms of action [2, 3]. Selectivity/high specificity may be the greatest advantage of proteins (peptides) over small molecule drugs. Biologics traditionally have not shown non-specific effects typical of small molecule drugs. Successful examples include TNFα blockers used in rheumatoid arthritis and related inflammatory diseases (e.g. Enbrel, Remicade and Humira), VEGF inhibitor Avastin for colorectal cancer, therapeutic proteins such as insulin, granulocyte-colony stimulating factor and erythropoietin. Not surprisingly there has been a shift towards biologics in the pharmaceutical industry. Although Protein therapeutics do not have the disadvantages of small molecule drugs, they have their own issues. They are all injectable drugs and therefore injection site reactions are a common occurrence. Immunogenicity is often a problem with many protein therapeutics. Protein Activity can be Mimicked by Peptides Although proteins are large molecules, the active site/moiety of a protein involve only a few amino acids. Thus a peptide derived from this region could potentially act as an agonist or antagonist. It is also possible to mimic the function of a protein by a stretch of amino acids that have a different sequence yet retain conformational similarity to the active site of the native protein. Phage display is a technology platform now widely used to identify peptide agonists and antagonists. Phage display makes large-peptide diversity libraries readily attainable for identifying novel peptide ligands for receptors and other protein or non-protein targets [4]. This technology is based on the idea that large protein-protein interaction surfaces (epitopes) can be distilled down to small pharmacophores. These may be accessible to scaffolding, yielding new orally active drugs that might otherwise have taken greater time and effort to be discovered through chemical-library screening (see Fig. 1). Vast libraries of peptides can be created through cloning of complex mixtures of combinatorially synthesized oligonucleotides into specialized expression display vectors. An example is the filamentous phage display system whereby the expressed peptides are displayed as fusion to phage coat proteins. Affinity purification of the phage on the target protein is then used to recover peptides with binding activity. Sequencing the appropriate segment of the DNA of each captured phage provides the primary sequence of peptides that bind the target. This technology has been used to generate peptide mimetics of structural proteins, hormones and growth factors and peptide inhibitors of enzymes [5,6].........

Cationic antimicrobial peptides serve as critical defense molecules protecting the host from invading bacteria, viruses and fungi. These antimicrobial peptides are widely distributed in nature and in vertebrates they have been localized to numerous tissues and cells. Cationic antimicrobial peptides can be expressed constitutively or under certain circumstances they can be induced in response to infection, inflammatory mediators, and cytokines. Although, their original and primary function was believed to be antimicrobial, it is now becoming clear that these antimicrobial peptides have a wide repertoire of functions with interesting ramifications on the immune system that are not solely antimicrobial. An area of active research is the determination of the mechanism(s) of action of antimicrobial peptides which have yet to be clarified. However, current consensus is that the mechanism is sufficiently different from conventional antibiotics that the development of resistance could be remote. Their broad spectrum activity, low potential to induce resistance and diverse functions make antimicrobial peptides an attractive family of compounds that have potential to be developed as therapeutics for treating certain infections.

Levels of high density lipoprotein (HDL) and its major protein component, apolipoprotein (apo) A-I, are strongly inversely correlated to risk of atherosclerosis and other vascular diseases. A number of properties of apo A-I may contribute to this protection, including removal of cholesterol from peripheral tissues to the liver (reverse cholesterol transport), anti-inflammatory and anti-oxidative activities, and modulation of vascular function. Apo A-I has lipidassociating domains that form class A amphipathic helices. Peptide analogs that have no sequence homology to the domains in apo A-I but possess the class A motif have been shown to not only associate with phospholipid but also mimic several of the functional properties of apo A-I. Peptide 4F, with four phenylalanines on the non-polar face, was found to be maximally effective in mimicking the positive qualities of apo A-I; this peptide inhibited atherosclerosis, reduced inflammation and oxidation, and improved vascular function in a number of animal models, and when synthesized with Damino acids is orally bioavailable. Several other classes of peptide mimetics are now being studied, and may contribute to our understanding of the functions of apo E and apo J. The use of peptide mimetics to study apolipoprotein function has proved to be a powerful tool, and may lead to novel therapeutic agents in the prevention of atherosclerosis and other vascular diseases.

Steroids are the best known anti-inflammatory drugs and have been in use for more than 50 years. Their chronic use however was limited by safety concerns. Non-steroidal anti-inflammatory drugs (NSAIDs) including COX-2 inhibitors although devoid of steroid side effects often possess gastrointestinal side effects. In addition recent data suggest that chronic use of some Cox inhibitors is associated with cardiovascular risk. Currently biologics represent the best option for many inflammatory diseases where TNFα is the main culprit. These include rheumatoid arthritis, ulcerative colitis, inflammatory bowel disease and psoriasis. A wealth of information is now available on the role of different cytokines and adhesion molecules in the origin and progression of inflammatory diseases. With the success of protein therapeutics such as Etanercept (Enbrel), which binds TNFα and inhibits its activity, research has been focused on developing small peptides that can interfere with cytokines or specific cell surface molecules and inhibit the inflammatory reactions. Here we review these peptides that are in discovery and development phases and their potential in the treatment of inflammatory diseases.

This review focuses on the synthetic progress of some naturally occurring cyclic peptides and depsipeptides apart from the development of peptidomimetics incorporating unnatural amino acids that have not been covered in the earlier reviews.

The protein and peptide therapeutics have become an important class of drugs due to advancement in molecular biology and recombinant technology. There are more than 100 biopharmaceutical products approved and generating revenue of more than US $56 billion. A safe, effective and patient friendly delivery of these agents is the key to commercial success. Currently, most of therapeutic proteins are administered by the parenteral route which has many drawbacks. Various delivery strategies and specialized companies have evolved over the past few years to improve delivery of proteins and peptides. Polymeric depot and PEGylation technologies have overcome some of the issues associated with parenteral delivery. A considerable research has been focused on non-invasive routes such as pulmonary, per oral and transdermal for delivery of proteins and peptides, in order to increase patient compliance yet their delivery via non-invasive routes remains challenge due to their poor absorption and enzymatic instability. Pulmonary route has shown some success evidenced by recent FDA approval of inhalable insulin. Development of an oral dosage form for protein therapeutics is still the most desirable one but with greater challenge. This review presents the issues of delivery of proteins and peptides, current and potential formulation technologies to improve delivery and current market trends.

Recent efforts combining nanotechnology and magnetic properties resulted in the development and commercialization of magnetic nanoparticles that can be used as carriers for nucleic acids for in vitro transfection and for gene therapy approaches including DNA-based vaccination strategies. The efficiency of intracellular delivery is still a limiting factor for basic cell biological research and also for emerging technologies such as temporary gene silencing based on inhibitory RNA/siRNA. Nanotechnology has resulted in a variety of different nanostructures and especially nanoparticles as carriers in a wide range of new drug delivery systems for conventional drugs, recombinant proteins, vaccines and more recently nucleic acids. It is possible to combine superparamagnetic nanoparticles with magnetic forces to increase, direct and optimize intracellular delivery of biomolecules. This article discusses the main approaches in the field of magnet assisted transfection (MATra) focusing on the transfection or intracellular delivery of nucleic acids, although also suitable to improve the intracellular delivery of other biomolecules.

Fluorescence correlation spectroscopy (FCS) is an increasingly important tool for determining low concentrations and dynamics of molecules in solution. Oftentimes triplet transitions give rise to fast blinking effects, which are accounted for by including an exponential term in the fitting of the autocorrelation function (ACF). In such cases, concomitant saturation effects also modify the amplitude and shape of the remaining parts of the ACF. We review studies of triplet and saturation effects in FCS and present a simple procedure to obtain more accurate results of particle concentrations and diffusional dynamics in experiments where triplet kinetics are evident, or where moderate laser powers approaching saturation levels are used, for example, to acquire sufficient photon numbers when observation times are limited. The procedure involves use of a modified function for curve-fitting the ACF, but there are no additional fitting parameters. Instead, a simple calibration of the total fluorescence count rate as a function of relative laser power is fit to a polynomial, and the non-linear components of this fit, together with the relative laser power used for the FCS measurement, are used to specify the magnitude of additional terms in the fitting function. Monte Carlo simulations and experiments using Alexa dyes and quantum dots, with continuous and pulsed laser excitation, demonstrate the application of the modified fitting procedure with first order correction terms, in the regime where distortions in the ACF due to photobleaching and detector dead time are small compared to those of fluorescence saturation and triplet photophysics.

The congenital CMV infection is the most common intrauterine transmitted viral infection. Since the first description in 1904 a lot of knowledge on epidemiology has been gained. Socio-economic maternal factors play a major role, and seem to be one of the major reasons for the differences of prevalence of congenital CMV infection between Europe and North America. Concerning the interaction of CMV and placental cells, reactions of the host immune system have a dual effect - protection against the virus on the one hand, and supporting of virus production and release of CMV on the other hand. In the last years new strategies for prevention and therapy of congenital CMV infection have been investigated. Passive immunization for prevention of transmission of the virus seems to be promising, but also therapy with ganciclovir of congenital infected newborns showed good results. Taking the side effects of antiviral therapy into consideration, active and passive immunization may ultimately be the best control strategy for this important public health problem.