The Pharmaceutical Journal Vol 265 No 7114 p411-415
September 16, 2000 The Conference

Science chairman's address

From genomics to the clinic: the challenge for molecular science?

By Martyn Davies, BPharm, PhD, MRPharmS, MRSC

In his address at the British Pharmaceutical Conference on September 11, the Science Chairman, Professor Martyn Davies, spoke of the importance of molecular-scale science in the advancement of pharmaceutical sciences. He presented information about the genomic revolution, the importance of molecular biology in the development of new antibiotics and chemotherapeutic agents, drug delivery and nanotechnology. He gave a detailed view of the use of scanning probe microscopes, illustrating his address with slides of DNA seen using this technology. Professor Davies concluded that there were major scientific challenges ahead and that it was an exciting time to be in the pharmaceutical sciences

Impact of the genomic revolution on medicine

The new millennium began with one of the most extraordinary scientific feats in the history of medicine - the publication of the first draft of the human genome.¹ While the complete sequencing of all 3.15 billion bases is yet to be finished (the proposed date for the final draft is 2001),¹ scientists everywhere recognise that this achievement opens up a new era for biology and medicine. Leaving aside the complex politics and the sometimes outright antagonism of the public and private funded teams who have contributed to the sequencing of the genome, this vast databank is likely to have a profound effect on health care as we know it. The challenge for modern molecular genetics will be in translating this sequence of data into an understanding of biological systems.² To date, we know the function of relatively few genes and it is the challenge for so-called "functional genomics" to help us attribute biological functions to individual genes. "Structural genomics" will further assist us in finding the molecular structure of molecules important in disease and, by providing vital information on proteins linked by genomics to disease states, "proteomics" will reveal important targets for new drug therapies. All these tools are a major challenge for informatics to allow the processing of extraordinarily large and complex sets of data.
The field of "pharmacogenomics" is predicted to revolutionise drug discovery. As pharmaceutical scientists, we are aware of the extraordinary differences in patients' responses to medicines both in efficacy and toxicity. While there are many factors which influence the human processing of drugs, such as diet, age and disease state, the major impact of inherited genes on therapeutic profiles is now apparent. We already know that genetic differences in drug-metabolising enzymes can influence the level and severity of toxic side effects experienced by patients undergoing chemotherapy for cancer.³ An understanding of the genetic-mediated differences² in the patient population in terms of such enzymes, and transporters, receptors and other drug targets, is now accessible through the Human Genome Project. The optimisation of drug therapy based on each patient's genetic code may be within our grasp in a decade or so, but we must not lose sight of the fact that a considerable investment will be required for pharmacogenomics to have a major impact in understanding the genetic basis of cancer and other diseases. Worldwide, government funding bodies and industry are strategically funding major initiatives in this area to realise the potential for future health care.
The genomic revolution will have a significant impact on health care in many other ways. Major advances have been made in gene technologies that underpin the programming of stem cells, allowing them to differentiate in a number of possible pathways from blood cells to nervous tissue to whole organ function.⁴ This technology has recently attracted significant political and media attention because it has the potential to provide replacement for damaged or defective tissue or, looking further ahead, to generate complete organs for transplantation. The control of gene expression has been exploited in the breeding of transgenic animals which provide an opportunity for large scale production of human protein pharmaceuticals. The genetic manipulation of plants is a fruitful area for natural product research because increased yields of medicines such as alkaloids can be achieved. One of the most controversial areas is the development of genetic modification technology for improved food production. This field has been the subject of intense public and media debate and has been portrayed as both ill-informed, new-age scare mongering with little scientific credibility and as big-business, profit-driven technology with little concern for the environment. What is clear is that while such novel technologies raise concerns over ethical, moral, environmental and medical issues, they provide significant opportunities for medicine. Given the often knee-jerk hostile reaction of the media to scientific advances in this area, they also pose, together with modern science in general, a considerable challenge to ensure the public understanding of science.

World-wide threat of multi-resistant bacteria

Understanding biological systems at a molecular level is essential in developing new antibiotics to combat the emergence of multi-resistant bacteria. Such bacteria have been at the forefront of public and media health care concerns over the past decade.⁵ The widespread inappropriate use of antibiotics and the ability of bacteria to exchange genes coding for antibiotic resistance has made many antibiotics clinically redundant. To overcome this alarming problem, the discovery of new effective drugs is a priority. To achieve this objective, a greater understanding of the molecular biology of bacterial cellular and molecular processes is required.⁵ Some of the areas which are receiving significant attention include the replication of DNA, proteins that control cell division, and unique cell wall/membrane biomolecules and structures. A wide range of strategies, including combinatorial methods linked to high-throughput mass screening, are being exploited in the search for new therapeutic agents. Genomics is also an active area for identifying virulence genes in bacteria and there is great interest in the specific targeting of resistance mechanisms to generate new antibacterial opportunities. One intriguing concept⁶ is the discovery that bacterial cells communicate with each other via small diffusible signalling molecules to control virulence gene expression, a phenomenon known as "quorum sensing". Interference with this communication process would limit the virulence of pathogenic bacteria, offering a novel target for anti-infective therapy.

Fighting cancer: novel chemotherapeutic agents

An understanding of the molecular biology of disease is at the forefront of the design of new chemical entities for treating cancer which are more selective, potent and have fewer side effects. Enzymes such as polymerase and telomerase and transmembrane receptors are some of the key targets to be reviewed at a major symposium at this year's Conference. In addition, the development of small molecules,⁷ including oligonucleotides,⁸ capable of binding to DNA or RNA in a highly sequence-specific manner has considerable potential for down- or up-regulation of individual genes which are associated with disease. For example, the promotion of activity of tumour suppressor gene proteins is a potential target for gene therapy.

Delivery of difficult macromolecules

The molecular approach is also a key feature for the future development of a new generation of drugs being discovered through combinatorial chemistry, rapid screening methods and the Human Genome Project. However, the selective delivery of a therapeutic molecule to the correct site of action in sufficient quantity and duration is an awesome obstacle for many drugs, particularly those of biological origin.⁹ A better molecular understanding of the biological barriers that influence the transport and processing of drugs in cells and tissues, together with the molecular basis of disease, are key targets for future fundamental research. This information will be of considerable benefit for strategies which deliver complex labile biologicals, such as nucleic acids, to target cells in emerging therapies, eg, gene therapy.
Considerable innovation is also being displayed in the development of novel delivery systems designed to overcome biological barriers.¹⁰ Mimicking nature is the inspiration for the development of a whole host of "smart materials"¹¹ that respond to subtle changes in the local cellular environment such as pH, light, electric or magnetic fields. While such concepts may seem fanciful and futuristic, a new range of novel systems is fast approaching the market, including needle-free injection systems for proteins and new inhaler devices which use novel particle technology for the pulmonary delivery of insulin. This year sees a call for a national initiative in drug delivery led by Professor Tony Moffat (chief scientist, Royal Pharmaceutical Society), with support from UK research councils, to drive fundamental research to underpin the technological developments required in this important field.

A view of the molecular world of pharmaceuticals

In my own research field of surface and biophysical chemistry, a new generation of microscopes has opened up a whole new vision of the molecular world of biomolecules and pharmaceutical materials.¹² Known generically as the scanning probe microscopes (SPMs), and the size of a large coffee cup, they detect the contours of a surface by scanning a sharp probe across the interface. This micromachined probe or tip is attached to a very fine and sensitive spring cantilever which deflects up and down and from side to side as it passes over the surface. Such is the sensitivity of these spring cantilevers that nanometer resolution, similar to that attained using state-of-the-art electron microscopes, is routinely achievable when the SPM is operated in air or under water. Such high resolution allows direct access into the molecular imaging of biological systems and processes which are of importance in disease and drug therapy.
The DNA of a single bacterial plasmid is shown in Figure 1. It has a relaxed and open structure that contrasts significantly with the image of the same plasmid in Figure 2. Here, the plasmid has been exposed to a model compound, similar to an anticancer drug, designed to insert itself between the bases of the DNA.^13-14 The resulting change in overall conformation, which leads to a supercoiling of the DNA, will inhibit its ability to be translated during cell replication, leading ultimately to cell death. Figure 3 shows the molecular structure of a DNA polymer complex, designed as a novel delivery system for gene therapy.¹⁵ Similar studies on many biomolecular structures can be found in the literature (ranging from fundamental studies on single enzyme reactions through to technologically important issues such as the molecular imaging of the packing and activity of antibodies in immunosensor assays).¹⁶

Figure 1: The molecule of life: an SPM image of DNA

Figure 2: DNA on drugs! The relaxed and open form of DNA seen in Figure 1 has been transformed by the DNA binding molecule causing it to change its conformation or shape. Two different forms of supercoiled DNA are seen, toroidal and plectonemic (see references 13 and 14 for further examples)

It quickly became apparent that the SPM could provide more than just images of molecules. The SPM cantilever can measure minute forces in the pico-Newton range, allowing us to detect the forces involved in molecular interactions between a single receptor-ligand pair.¹⁷ This is achieved by attaching the ligand to the SPM tip and the complementary receptor to a suitable surface. As the tip is brought into contact with the surface, a receptor-ligand complex is formed. On removal of the tip from the surface, the force required to break this association is measured by the cantilever. This approach is providing a fundamental understanding of important molecular recognition events such as antibody-antigen interactions^18,19 and nucleic acid chains binding (Figure 4). In recent work in our laboratory, we have extended these molecular force studies into areas such as understanding protein folding and conformation changes in stimuli-sensitive biomimetic materials.

Figure 3: DNA all wrapped and ready for delivery. Charged polymer DNA complexes are being investigated as potential DNA delivery systems for gene therapy. In this case, the complex forms a ring-like structure and the wrapping up of the DNA with the polymer can be watched in real time using the SPM (see reference 15 for another example)

The applications of SPM systems are not limited to biomolecules but also have considerable potential for the study of pharmaceutical materials such as drug crystals. High-resolution images of drug crystal formation or dissolution can be achieved where single molecular layers are seen to move along the crystal planes. The ability of the SPM system to measure surface forces opens up a whole new field of material science where one can measure the surface properties, such as hardness, hydrophobicity, adhesion and friction, of pharmaceutical materials. Such an approach has been used to distinguish different polymorphic forms of the drug cimetidine from single crystals²⁰ and the different chemical and dissolution properties of different crystal faces of aspirin²¹ (Figure 5).

Figure 4: Measuring biomolecular forces with an SPM. A schematic of an experiment measuring the force required to separate complementary strands of oligonucleotides grafted to the SPM tip and the surface

Figure 5: A schematic of dynamic SPM measurements on the (001) and (100) planes of aspirin. The measurements reveal a strong and extended attractive interaction between the CH3 probe and the (001) plane and similarly between a -COOH probe and the (100) plane. We ascribe such differences to the surface chemical structure of the crystal planes. The CH3 probes show a strong hydrophobic interaction with the phenyl, methyl dominated (001) plane. The opposite is true for -COOH probes, where hydrogen bonding between the acid group and the oxygen of the ester group exposed at crystal plane (100) is believed to be responsible for the long attractive region (see reference 21 for further details)

Use of the SPM system has further been extended by replacing the inert tip with probes that can detect specific properties at a nanometer level. For example, sharpened fibre-optic tips have been used to measure fluorescence of single molecules, and thermocouples have been fashioned as tips to measure the local thermal properties such as glass transition temperatures.²² SPMs have also been exploited in a diverse range of novel scientific developments, for example, as nanoplotters by using the SPM tip to write chemical patterns in surfaces²³ and also by detecting the presence of gene-specific protein markers on DNA molecules as a potential method for the rapid screening of gene function. Exploited as advanced biophysical tools operating at the molecular level, these remarkable microscopes have much to offer in the field of medicine.

Lab-on-a-chip

Nanotechnology is also causing a quiet revolution in analytical science. Rapid advances in microtechnology are allowing the development of a whole new generation of surface engineered analytical devices based on semiconductor chips. These "lab-on-a-chip" systems,^24,25 no bigger than a microscope slide, are fabricated with nano-scale chemical patterns on surfaces where a single layer of biomolecules can be immobilised in spatially defined regions. Such substrates have many biomedical applications, including the separation of proteins and DNA, the synthesis of libraries of drug molecules and as immunoassays to detect markers for disease. The key advantages for these chips are not only their small size but also that reducing everything down to the nano-scale speeds up the analysis time up to 100-fold, producing results within just a few seconds!¹⁹ Such is the pace of these developments that it is a far from fanciful idea that patients could visit their local hospital or pharmacy for rapid analysis of drug levels or genetic markers to establish their ability to metabolise a particular drug or to identify an appropriate course of therapy.

Nanotechology in the pharmaceutical sciences

Nanoscale science is also making an important contribution to the understanding of the physicochemical and material properties of pharmaceuticals. UK scientists, including future and past BPC science chairmen, Peter York and Ray Rowe, respectively, are pioneering the exploitation of advanced characterisation tools and computational methods to determine the molecular structure and packing of drug molecules within crystals. These approaches are leading to a better understanding and potential prediction of industrially-relevant issues such as drug polymorphism.
New industrial methods of crystal engineering are being developed, including technologies to prepare stable crystals in the nanometer range which could significantly improve the solubility kinetics of poorly soluble drug compounds. Nanotechnology has been used in the design of a host of drug delivery systems, including implantable controlled release microchips which are loaded with drugs for delivery on demand, a so-called "pharmacy on a chip".²⁶ As noted above, the emergence of the scanning probe microscope has allowed the measurement of material surface characteristics, such as adhesion and friction, at the molecular level.
In the exciting new field of tissue engineering, a multidisciplinary approach is being exploited in the modification of polymer surfaces by the precise micropatterning of biological molecules intended to promote cell adhesion and growth in defined structures.²⁷ Such nano-scale bioscience is an interesting mix of cell biology, material science and nanotechnology, and is a key feature of novel cell engineering strategies in the development of polymer scaffolds for tissue engineering applications such as nerve regeneration and whole organ creation in the laboratory.²⁸

Overview

It is clear that there are major scientific challenges and opportunities ahead. It is a remarkable time to be in the pharmaceutical sciences. The multidisciplinary environment is well suited to our training. Future activities will be based at the biological, chemical and physical interface where our broad-based knowledge will ensure that significant contributions can be made. It falls upon us collectively to influence and inform Foresight schemes to ensure that funding bodies are aware of the key issues for future strategic programmes in UK science. The continued encouragement of the technology transfer of fundamental scientific gains to industry is a clear demonstration of the value of the academic base in the UK. The greatest asset is the international quality of the people engaged by curiosity-driven research within UK academia and industry who lead the scientific breakthroughs and technical innovations. The pharmaceutical sciences are, as ever, rapidly changing and evolving. As attendees at the Millennium BPC will, I hope, see, there are exciting times ahead.

ACKNOWLEDGMENTS I wish to acknowledge the great debt to all past and current research students, postdoctorate fellows, academic and industrial colleagues with whom I have had the pleasure to work and who have helped make science fun and interesting. In particular, I would like to add my greatest thanks to my collaborators within the laboratory of biophysics and surface analysis at the University of Nottingham, namely Professor Saul Tendler and Drs Phil Williams, Clive Roberts and Stephanie Allen.

References