BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY - PART 2 potx

penicillin acylase). Downward adjustment of the reaction pH to 4.3 results in precipitation of the resultant 6-amino penicillanic acid ring, which can then be easily harvested. Novel side- chains can subsequently be attached, yielding semi-synthetic penicillins. Examples of the latter include phenethicillin, propicillin and oxacillin. Some semi-synthetic penicillins are effective against bacterial pathogens that have become resistant to natural penicillins. Others are acid- stable, allowing their oral administration. Cephalosporins display an antibiotic mechanism of action identical to that of the penicillins. Cephalosporin C (Figure 1.14) is the prototypic natural cephalosporin and is produced by the fungus Cephalosporium acrem onium. Most other members of this family are semi-synthetic derivatives of cephalosporin C. Chemical modification normally targets side-chains at posit ion 3 (the acetoxymethyl group) or 7 (derived from D-a-aminoadipic acid). Tetracyclines are a family of an tibiotics which display a characteristic 4-fused-core ring structure (Figure 1.16). They exhibit broad antimicrobial activity and induce their effect by inhibiting protein synthesis in sensitive microorganisms. Chlortetracycline was the first member of this family to be discovered (in 1948). Penicillin G and streptomycin were the only antibiotics in use at that time, and chlortetracycline was the first antibiotic employed therapeutically that retained its antimicrobial properties upon oral administration. Since then, a number of additional tetracyclines have been discovered (all produced by various strains of Streptomyces), and a variety of semi-synthetic derivatives have also been prepared (Table 1.18). Tetracyclines gained widespread medical use due to their broad spectrum of activity, which includes not only Gram-negative and Gram-positive bacteria, but also mycoplasmas, rickettsias, chlamydias and spirochaetes. However, adverse effects (e.g. staining of teeth and gastro- intestinal disturbances), along with the emergence of resistant strains, now somewhat limits their therapeutic applications. PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS 37 Figure 1.16. Chemical structure of the antibiotic tetracycline. Other members of the tetracycline family (see also Table 1.18) also display this characteristic 4-ring structure Table 1.18. Natural and semi-synthetic tetracyclines which have gained medical application Natural Semi-synthetic Chlortetracycline Methacycline Oxytetracycline Doxycycline Tetracycline Minocycline Demeclocycline The aminoglycosides are a closely related family of antibiotics produced almost exclus ively by members of the genus Streptomyces and Micromonospora (Table 1.19). Most are polycationic compounds, composed of a cyclic amino alcohol to which amino sugars are attached. They all induce their bacteriocidal effect by inhibiting protein synthesis (apparently by binding to the 30 S and, to some extent, the 50 S, ribosomal subunits). Most are orally inactive, generally necessitating their parenteral administration. The aminoglycosides are most active against Gram-negative rods. Streptomycin was the first aminoglycoside to be used clinically. Another notable member of this family, gentamicin, was first purified from a culture of Micro monospora purpurea in 1963. Its activity against Pseudomonas aeruginosa and Serratia marcescens renders it useful in the treatment of these (often life-threatening) infections. The macrolides and ansamycins The macrolides are a large group of antibiotics. They are characterized by a core ring structure containing 12 or more carbon atoms (closed by a lactone group), to which one or more sugars are attached. The core ring of most anti-bacterial macrolides consists of 14 or 16 carbon atoms, while that of the larger anti-fungal and anti-protozoal macrolides contain up to 30 carbons. This family of antibiotics are produced predominantly by various species of Streptomyces. Antibacterial macrolides induce their effects by inhibiting bacterial synthesis (anti-fungal/ protozoal macrolides appear to function by interfering with sterols, thus compromising membrane structure). The only member of this family that enjoys widespread therapeutic use is erythromycin, which was discovered in 1952. Ansamycins, like the macrolides, are synthesized by condensation of a number of acetate and propionate units. These antibiotics, which are produced by several genera of the Actinomy- cetales, display a characteristic core aromatic ring structure. Amongst the best-known family members are the rifamycins, which are particularly active against Gram-positive bacteria and mycobacteria. They have been used, for example, in the treatment of Mycobacterium tuberculosis. 38 BIOPHARMACEUTICALS Table 1.19. Some aminoglycoside antibiotics which have gained significant therapeutic application. Producer microorganisms are listed in brackets. In addition to naturally produced aminoglyco- sides, a number of semi-synthetic derivatives have also found medical application. Examples include amikacin, a semi-synthetic derivative of kanamycin and netilmicin, an N-ethyl derivative of sissomicin Streptomycin (Streptomyces griseus) Tobramycin (Streptomyces tenebrarius) Framycetin (Streptomyces spp.) Neomycin (Streptomyces spp.) Kanamycin (Streptomyces spp.) Paromycin (Streptomyces spp.) Gentamicin (Micromonospora purpurea) Sissomicin (Micromonospora spp.) Peptide and other antibiotics Peptide antibiotics consist of a chain of amino acids which often have cyclized, forming a ring- like structure. The first such antibiotics isolated were bacitracin and gramicidin, although neither are used clinically due to their toxicity. While a number of microbes produce peptide antibiotics, relatively few such antibiotics are applied therape utically. Polymyxins are the most common exception. Vancomycin, a glycopeptide, has also gained therapeutic application. It functions by interfering with bacterial cell wall synthesis, and is particularly active against Gram-positive cocci. A variety of additional antibiotics are known that, based on their chemical structure, do not fit into any specific antibiotic family. Perhaps the most prominent such antibiotic is chloramphenicol (Figure 1.17). Chloramphenicol was first isolated from a culture of Streptomyces venezuelae in 1947, but it is now obtained by direct chemical synthesis. It was the first truly broad-spectrum antibiotic to be discovered, and was found effective against Gram- negative and Gram-positive bacteria, rickettsias and chlamydias. It retains activity when administered orally and functions by inhibiting protein synthesis. However, due to its adverse effects upon bone marrow function, clinical application of chloramphenicol is undertaken with caution. Its main use has been to combat Salmonella typhi, Haemophilus influenzae (especially in cases of meningitis) and Bactero ides fragilis (an anaerobe which can cause cerebral abscess formation). Some semi-synthetic derivatives of chloramphenicol (e.g. thiamphenicol) have also been developed for clinical use. CONCLUSION Most major life form families (microorganisms, plants and animals) have each yielded a host of valuable therapeutic substances. Many pharmaceutical companies and other institutions continue to screen plants and microbes in the hope of discovering yet more such therapeutic agents. However, in recent years, more and more emphasis is being placed upon developing the ‘body’s own drugs’ as commercially produced pharmaceutical substances. Most such drugs are protein-based, and these biopharmaceuticals represent an exciting new family of pharmaceutical products. The number of such drugs gaining approval for general medical use continues to grow, as does their range of therapeutic applications. A fuller discussion of these biopharmaceuticals forms the basis of the remaining chapters of this text. In addition, the reader’s attention is drawn to Appendix 2 of this book, which contains a list of Internet sites of relevance to the biopharmaceutical sector. Much additional valuable information may be downloaded from these sites. PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS 39 Figure 1.17. Chemical structure of chloramphenicol, the first broad-spectrum antibiotic to gain clinical use FURTHER READING Books Buckingham, J. (1996). Dictionary of Natural Products. Chapman & Hall, London. Crommelin, D. & Sindelar, R. (2002) Pharmaceutical Biotechnology, 2nd Edn. Taylor & Francis, London. Goldberg, R. (2001). Pharmaceutical Medicine, Biotechnology and European Law. Cambridge University Press, Cambridge. Grindley, J. & Ogden, J. (2000). Understanding Biopharmaceuticals. Manufacturing and Regulatory Issues. Interpharm Press, Denver, CO. Kucers, A. (1997). Use of Antibiotics: A Clinical Review of Antibacterial, Antifungal and Antiviral Drugs, 5th edn. Butterworth–Heinemann, Oxford. Lincini, G. (1994). Biotechnology of Antibiotics and Other Bioactive Microbial Metabolites. Plenum, New York. Lubiniecki, A. (1994). Regulatory Practice for Biopharmaceutical Production. Wiley, Chichester. Mann, J. (1998). Bacteria and Antibacterial Agents. Oxford University Press, Oxford. Manske, R. (1999). Alkaloids. Academic Press, London. Oxender, D. & Post, L. (1999). Novel Therapeutics from Modern Biotechnology. Springer-Verlag, Berlin. Pezzuto, J. (1993). Biotechnology and Pharmacy. Chapman & Hall, London. Reese, R. (2000). Handbook of Antibiotics. Lippincott, Williams & Wilkins, Philadelphia, PA. Roberts, M. (1998). Alkaloids. Plenum, New York. Smith, E. et al. (1983). Mammalian biochemistry. In Principles of Biochemistry. McGraw Hill, New York. Strohl, W. (1997). Biotechnology of Antibiotics. Marcel Dekker, New York. Walsh, G. & Murphy, B. (Eds) (1998). Biopharmaceuticals: An Industrial Perspective. Kluwer Academic, Dordrecht. Articles General biopharmaceutical Drews, J. (1993). Into the twenty-first century. Biotechnology and the pharmaceutical industry in the next 10 years. Bio/ Technology 11, 516–520. Olson, E. & Ratzkin, B. (1999). Pharmaceutical biotechnology. Curr. Opin. Biotechnol. 10, 525–527. Walsh, G. (2000). Biopharmaceutical benchmarks. Nature Biotechnol. 18, 831–833. Walsh, G. (2002). Biopharmaceuticals and biotechnology medicines: an issue of nomenclature. Eur. J. Pharmaceut. Sci. 15, 135–138. Weng, Z. & DeLisi, C. (2000). Protein therapeutics: promises and challenges of the twenty-first century. Trends Biotechnol. 20(1), 29–36. Natural products Attaurrahman-Choudhary, M. (1997). Diterpenoid and steroidal alkaloids. Nat. Prod. Rep. 14(2), 191–203. Billstein, S. (1994). How the pharmaceutical industry brings an antibiotic drug to the market in the United States. Antimicrob. Agents Chemother. 38(12), 2679–2682. Bruce, N. et al. (1995). Engineering pathways for transformations of morphine alkaloids. Trends Biotechnol. 13, 200–205. Chopra, I. et al. (1997). The search for anti-microbial agents effective against bacteria resistant to multiple antibiotics. Antimicrob. Agents Chemother. 41(3), 497–503. Cordell, G. et al. (2001). The potential of alkaloids in drug discovery. Phytother. Res. 15(3), 183–205. Dougherty, T. et al. (2002). Microbial genomics and novel antibiotic discovery. New Technol. New Drugs Curr. Pharmaceut. Design 8(13), 1119–1135. Facchini, P. (2001). Alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation and metabolic engineering applications. Ann. Rev. Plant Physiol. Plant Mol. Biol. 52, 29–66. Flieger, M. et al. (1997). Ergot alkaloids — sources, structures and analytical methods. Folia Microbiol. 42(1), 3–29. Glass, J. et al. (2002). Streptococcus pneumoniae as a genomics platform for broad spectrum antibiotic discovery. Curr. Opin. Microbiol. 5(3), 338–342. Gournelis, D. et al. (1997). Cyclopeptide alkaloids. Nat. Prod. Rep. 14(1), 75–82. Hancock, R. (1997). Peptide antibiotics. Lancet 349(9049), 418–422. Kalant, H. (1997). Opium revisited— a brief review of its nature, composition, non-medical use and relative risks. Addiction 92(3), 267–277. 40 BIOPHARMACEUTICALS Kaufmann, C. & Carver, P. (1997). Antifungal agents in the 1990s —current status and future developments. Drugs 53(4), 539–549. Krohn, K. & Rohr, J. (1997). Angucyclines — total synthesis, new structures and biosynthetic studies of an emerging new class of antibiotics. Topics Curr. Chem. 188, 127–195. McManus, M. (1997). Mechanisms of bacterial resistance to anti-microbial agents. Am. J. Health Syst. Pharm. 54(12), 1420–1433. Marston, A. et al. (1993). Search for antifungal, molluscicidal and larvicidal compounds from African medicinal plants. J. Ethnopharmacol. 38, 215–233. Michael, J.P. (1997). Quinoline, quinazoline and acridone alkaloids. Nat. Prod. Rep. 14(1), 11–20. Nicolas, P. & Mor, A. (1995). Peptides as weapons against microorganisms in the chemical defence system of vertebrates. Ann. Rev. Microbiol. 49, 277–304. Normark, B. & Normark, S. (2002). Evolution and spread of antibiotic resistance. J. Intern. Med. 252(2), 91–106. Tudzynski, P. et al. (2001). Biotechnology and genetics of ergot alkaloids. Appl. Microbiol. Biotechnol. 57(5–6), 593–605. Walsh, C. (2002). Combinatorial biosynthesis of antibiotics, challenges and opportunities. Chembiochem. 3(2–3), 125–134. PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS 41 Chapter 2 The drug development process In this chapter, the life history of a successful drug will be outlined (summarized in Figure 2.1). A number of different strategies are adopted by the pharmaceutical industry in their efforts to identify new drug products. These approaches range from random screening of a wide range of biological materials to knowledge-based drug identification. Once a potential new drug has been identified, it is then subjected to a range of tests (both in vitro and in animals) in order to characterize it in terms of its likely safety and effectiveness in treating its target disease. After completing such pre-clinical trials, the developing company apply to the appropriate government-appointed agency (e.g. the FDA in the USA) for approval to commence clinical trials (i.e. to test the drug in humans). Clinical trials are required to prove that the drug is safe and effective when administered to human patients, and these trials may take 5 years or more to complete. Once the drug has been characterized, and perhaps early clinical work is under way, the drug is normally patented by the developing company, in order to ensure that it receives maximal commercial benefit from the discovery. Upon completion of clinical trials, the developing company collates all the pre-clinical and clinical data they have generated, as well as additional pertinent informat ion, e.g. details of the exact production process used to make the drug. They submit this information as a dossier (a multi-volume work) to the regulatory authorities. Regulatory scientific officers then assess the information provided and decide (largely on criteria of drug safety and efficacy) whether the drug should be approved for general medical use. If marketing approval is granted, the company can sell the product from then on. As the drug has been patented, they will have no competition for a number of years at least. However, in order to sell the product, a manufacturing facility is required, and the company will also have to gain manufacturing approval from the regulatory authorities. In order to gain a manufacturing licence, a regulatory inspector will review the proposed manufacturing facility. The regulatory authority will only grant the company a manufacturing licence if they are satisfied that every aspect of the manufacturing process is conducive to consistently producing a safe and effective product. Regulatory involvement does not end even at this point. Post-marketing surveillance is generally undertaken, with the company being obliged to report any subsequent drug-induced side effects/adverse reactions. The regulatory authority will also inspect the manufacturing facility from time to time in order to ensure that satisfactory manufacturing standards are maintained. Biopharmaceuticals: Biochemistry and Biotechnology, Second Edition by Gary Walsh John Wiley & Sons Ltd: ISBN 0 470 84326 8 (ppc), ISBN 0 470 84327 6 (pbk) DRUG DISCOVERY The discovery of virtually all the biopharmaceuticals discussed i n this t ext was a knowledge-based one. Continuing advances in the molecular sciences have deepened our understanding of the molecular mechanisms which underline health and disease. An understanding at the molecular level of how the body functions in health, and the deviations that characterize the development of a disease, often renders obvious potential strategies likely to cure/control that disease. Simpl e examples illustrating this include the use of insulin to treat diabetes, or the use of growth hormone to treat certain forms of dwarfism (Ch apter 8). The underlining causes of these types of disease are relatively straightforward, in that they are essentially p romo ted by the d efi ciency/absence of a single regulatory molecule. Other diseases, however, may be multifactorial and, hence, more complex. Examples here include cancer and inflammation. Neverthel ess, cytokines such as 44 BIOPHARMACEUTICALS Figure 2.1. An overview of the life history of a successful drug. Patenting of the product is usually also undertaken, often during the initial stages of clinical trial work interferons and interleukins, known to stimulate the immune response/regulate inflammation, have proved to be therapeutically useful in treating several such complex diseases (Chapters 4 and 5). An understanding, at the molecular level, of the actions of various regulatory proteins, or the progression of a specific disease does not, however, automatically translate into pinpointing an effective treatment strategy. The physiological responses induced by the potential biopharma- ceutical in vitro (or in animal models) may not accurately predict the physiological responses seen when the product is administered to a diseased human. For example, many of the most promising biopharmaceutical therapeutic agents (e.g. virtually all the cytokines; Chapter 4), display multiple activities on different cell populations. This makes it difficult, if not impossible, to predict what the overall effect administration of any biopharmaceutical will have on the whole body, hence the requirement for clinical trials. In other cases, the widespread application of a biopharmaceutical may be hindered by the occurrence of relatively toxic side effe cts (as is the case with tumour necrosis factor a (TNF-a), Chapter 5). Finally, some biomolecules have been discovered and purified because of a characteristic biological activity which, subsequently, was found not to be the molecule’s primary biological activity. TNF-a again serves as an example. It was first noted because of its cytotoxic effects on some cancer cell types in vitro. Subsequently, trials assessing its therapeutic application in cancer proved disappointing, due not only to its toxic side effects but also to its moderate, at best, cytotoxic effect on many cancer cell types in vivo. TNF’s major biological activity in vivo is now known to be as a regulator of the inflammatory response. In summary, the ‘discovery’ of biopharmaceuticals, in most cases, merely relat es to the logic al application of our rapidly increasing knowledge of the biochemical basis of how the body functions. These substances could be accurately described as being the body’s own pharmaceuticals. Moreover, rapidly expanding areas of research, such as genomics and proteomics, will likely hasten the discovery of many more such products, as discussed below. While biopharmaceuticals are typically proteins derived from the human body, most conventional drugs have been obtained from sources outside the body (e.g. plant and microbial metabolites, synthetic chemicals, etc.). Although they do not form the focus of this text, a brief overview of strategies adopted in the discovery of such ‘non-biopharmaceutical’ drugs is appropriate, and is summarized later in this chapter. The impact of genom ics and related technologies upon drug discovery The term ‘genomics’ refers to the systematic study of the entire genome of an organism. Its core aim is to sequence the entire DNA complement of the cell and to physically map the genome arrangement (assign exact positions in the genome to the various genes/non-coding regions). Prior to the 1990s, the sequencing and study of a single gene represented a significant task. However, improvements in sequencing technologies and the development of more highly automated hardware systems now render DNA sequencing considerably faster, cheaper and more accurate. Modern sequencing systems can sequence in excess of 1000 bases/h. Such innovations underpin the ‘high-throughput’ sequencing necessary to evaluate an entire genome sequence within a reasonable time frame. As a result, the genomes of almost 70 microorganisms have thus far been completely or almost completely sequenced (Table 2.1). In addition, various public and private bodies are currently sequencing the genomes of various plants and animals, including those of wheat, barley, chicken, dog, cow, pig, sheep, mouse and rat. The human genome project commenced in 1990, with an initial target completion date set at 2005. A ‘rough draft’ was published in February 2001, with the final completed draft expected in 2003. The total h uman THE DRUG DEVELOPMENT PROCESS 45 genome size is in the regio n of 3.2 gigabases (Gb), approximately 1000 times larger than a typical bacterial geno me (Table 2.1). Less than one-third of the genome is transcrib ed into RNA. Only 5% of that RNA is believed to encode polypeptides and the number of polypeptide-encoding genes is estimated to be of the order of 30 000 — well below the initial 100 000–120 000 estimates. From a drug discovery/development perspective, the significance of genome data is that it provides full sequence information of every protein the organism can produce. This should result in the identification of previously undiscovered proteins which will have potential therapeutic application, i.e. the process should help identify new potential biopharmaceuticals. The greatest pharmaceutical impact of sequence data, however, will almost certainly be the identification of numerous additional drug targets. It has been estimated that all drugs currently on the market target one (or more) of a maximum of 500 targets. The majority of such targets are proteins (mainly enzymes, hormones, ion channels and nuclear receptors). Hidden in the human genome sequence data is believed to be anywhere between 3000 and 10 000 new protein- based drug targets. Additionally, present in the sequence data of many human pathogens (e.g. Helicobacter pylori, Mycobact erium tuberculosis and Vibrio cholerae; Table 2.1) is sequence data of hundreds, perhaps thousands, of pathogen proteins that could serve as drug targets against those pathogens (e.g. gene products essential for pathogen viability or infectivity). While genome sequence data undoubtedly harbours new drug leads/drug targets, the problem now has beco me one of specifically identifying such genes. Impeding this process is the fact that (at the time of writing) the biological function of between one-third and half of sequenced gene products remains unknown. The focus of genome research is therefore now shifting towards elucidating the biological function of these gene products, i.e. shifting towards ‘functional genomics’. Assessment of function is critical to understanding the relationship between genotype and phenotype and, of course, for the direct identification of drug leads/targets. The term ‘function’ traditionally has been interpreted in the narrow sense of what isolated biological role/activity the gene product displays (e.g. is it an enzyme and, if so, what specific reaction does it catalyse?). In the context of genomics, gene function is assigned a bro ader meaning, incorporating not only the isolated biological function/activity of the gene product, but also relating to: . where in the cell that product acts and, in particular, what other cellular elements it influences/interacts with; . how such influences/interactions contribute to the overall physiology of the organism. 46 BIOPHARMACEUTICALS Table 2.1. Genome size (expressed as the number of nucleotide base pairs present in the entire genome of various microorganisms whose genome sequencing is complete/near complete). MB (megabase)= million bases Microbe Genome size (MB) Microbe Genome size (MB) Agrobacterium tumefaciens 5.3 Neisseria meningitidis 2.27 Aquifex aeolicus 1.5 Salmonella typhi 4.8 Bacillus subtilis 4.2 Streptococcus pneumoniae 2.2 Campylobacter jejuni 1.64 Sulfolobus solfataricus 2.99 Clostridium acetobutylicum 4.1 Thermoplasma acidophilum 1.56 Escherichia coli 4.6 Vibrio cholerae 4.0 Haemophilus influenzae 1.83 Yersinia pestis 4.65 Helicobacter pylori 1.66 Saccharomyces cerevisiae 13.0 Mycobacterium leprae 3.26 Candida albicans 15.0 Mycobacterium tuberculosis 4.4 [...]... point can be particularly significant in the context of drug/target identification, as most such targets are likely to be kinases and other regulatory proteins which are generally expressed within cells at very low levels More recently, high-resolution chromatographic techniques (particularly reverse-phase and ion exchanged-based HPLC) have been applied in the separation of proteome proteins and highresolution... both native and mutated/engineered forms from various species, as well as insulins in various polymeric forms and in the presence of various stabilizers and other chemicals) In reality, by the year 20 00, the 3-D structure of approximately 20 00 truly different proteins had been resolved Until quite recently, X-ray crystallography was the technique used almost exclusively to resolve the 3-D structure... species (usually rats and rabbits) The animals are sacrificed close to term and a full autopsy on the mother and fetus ensues Post-natal toxicity evaluation often forms an extension of such studies This entails administration of the drug to females both during and after pregnancy, with assessment of mother and progeny not only during pregnancy, but also during the lactation period  72 BIOPHARMACEUTICALS. .. drugs and how they interact with/affect the body Within this broad discipline exist (somewhat artificial) sub-disciplines, including pharmacokinetics and pharmacodynamics 70 BIOPHARMACEUTICALS Table 2. 4 The range of major tests undertaken on a potential new drug during pre-clinical trials The emphasis at this stage of the drug development process is upon assessing safety Satisfactory pharmacological, and. .. technical and legal argument — ethical and political issues, including public opinion, also impinge on the decision-making process The increasing technical complexity and sophistication of the biological principles and processes upon which biotechnological innovations are based also render resolution of legal patenting issues more difficult A major step in clarifying EU-wide law with regard to patenting in biotechnology. .. systematic and comprehensive analysis of the proteins expressed in the cell and their function Classical proteomic studies generally entailed initial extraction of the total protein content from the target cell/tissue, followed by separation of the proteins therein using two-dimensional ( 2- D) electrophoresis (Chapter 3) Isolated protein ‘spots’ could then be eluted from the electrophoretic gel and subjected... used non-human guides in targeting certain plants, e.g some have studied the plant types fed to sick monkeys by other members of the monkey troop) The collection procedure itself is straightforward After cataloguing and identification, 1 2 kg of the plant material is dried, or stored in alcohol and brought back to the lab The plant material is crushed and extracted with various solvents (most plant-derived... in the biosynthesis of new nucleic acids within the cell Synthetic analogues of purine nucleosides are used medically as anti-cancer or anti-viral agents (they interfere with normal synthesis of nucleic acids and, hence, retard cell growth/viral infection; an example is 2 -3 ’-dideoxynosine, used to treat AIDS) PNP, however, can cleave these drugs, thus negating their therapeutic effect PNP also appears... diabetes, rheumatoid arthritis, psoriasis and multiple sclerosis — all of which are characterized by excessive T cell activity X-ray crystallographic analysis illustrated that three amino acids in the purine-binding pocket of PNP form H-bonds with the purine rings, while the sugar residue interacts with additional active-site amino acids via hydrophobic bonds (Figure 2. 5) Design of an effective inhibitor... and engaged in stronger H-bonding or additional hydrophobic interactions with the active site residues Without using computer modelling, identification of a potent inhibitor would, on average, require screening of hundreds of thousands of candidates, take up to 10 years and cost several million dollars With computer modelling, the time and cost are cut to a fraction of this and, in this case, less than . Microbiol. 49, 27 7–304. Normark, B. & Normark, S. (20 02) . Evolution and spread of antibiotic resistance. J. Intern. Med. 25 2 (2) , 91–106. Tudzynski, P. et al. (20 01). Biotechnology and genetics. biotechnology. Curr. Opin. Biotechnol. 10, 525 – 527 . Walsh, G. (20 00). Biopharmaceutical benchmarks. Nature Biotechnol. 18, 831–833. Walsh, G. (20 02) . Biopharmaceuticals and biotechnology medicines: an issue. 57(5–6), 593–605. Walsh, C. (20 02) . Combinatorial biosynthesis of antibiotics, challenges and opportunities. Chembiochem. 3 (2 3), 125 –134. PHARMACEUTICALS, BIOLOGICS AND BIOPHARMACEUTICALS 41 Chapter 2 The drug