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International
Tables for Crystallography Volume F Crystallography of biological macromolecules Edited by M. G. Rossmann and E. Arnold © International Union of Crystallography 2006 |
International Tables for Crystallography (2006). Vol. F. ch. 1.3, pp. 12-24
Section 1.3.4. Crystallography and development of novel pharmaceuticals
aBiomolecular Structure Center, Department of Biological Structure, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195-7742, USA |
The impact of detailed knowledge of protein and nucleic acid structures on the design of new drugs has already been significant, and promises to be of tremendous importance in the next decades. The first structure of a known major drug bound to a target protein was probably that of methotrexate bound to dihydrofolate reductase (DHFR) (Matthews et al., 1977![[link]](/graphics/greenarr.gif)
). Even though the source of the enzyme was bacterial while methotrexate is used as a human anticancer agent, this protein–drug complex structure was nevertheless a hallmark achievement. It is generally accepted that the first protein-structure-inspired drug actually reaching the market was captopril, which is an antihypertensive compound blocking the action of angiotensin-converting enzyme, a metalloprotease. In this case, the structure of zinc-containing carboxypeptidase A was a guide to certain aspects of the chemical modification of lead compounds (Cushman & Ondetti, 1991). This success has been followed up by numerous projects specifically aimed at the design of new inhibitors, or activators, of carefully selected drug targets.
Structure-based drug design (SBDD) (Fig. 1.3.4.1) is the subject of several books and reviews that summarize projects and several success stories up until the mid-1990s (Kuntz, 1992; Perutz, 1992; Verlinde & Hol, 1994; Whittle & Blundell, 1994; Charifson, 1997; Veerapandian, 1997). Possibly the most dramatic impact made by SBDD has been on the treatment of AIDS, where the development of essentially all of the protease inhibitors on the market in 1999 has been guided by, or at least assisted by, the availability of numerous crystal structures of protease–inhibitor complexes.
![[Figure 1.3.4.1]](/figures/Fafig1o3o4o1thm.gif) |
The need for a large number of structures is common in all drug design projects and is due to several factors. One is the tremendous challenge for theoretical predictions of the correct binding mode and affinity of inhibitors to proteins. The current force fields are approximate, the properties of water are treacherous, the flexibility of protein and ligands lead quickly to a combinatorial explosion, and the free-energy differences between various binding modes are small. All this leads to the need for several experimental structures in a structure-based drug design cycle (Fig. 1.3.4.1). In this cycle, numerous disciplines are interacting in multiple ways. Many institutions, small and large, are following in one way or another this paradigm to speed up the lead discovery, lead optimization and even the bioavailability improvement steps in the drug development process. Moreover, a very powerful synergism exists between combinatorial chemistry and structure-based drug design. Structure-guided combinatorial libraries can utilize knowledge of ligand target sites in the design of the library [see e.g. Ferrer et al. (1999), Eckert et al. (1999) and Minke et al. (1999)]. Once tight-binding ligands are found by combinatorial methods, crystal structures of library compound–target complexes provide detailed information for new highly specific libraries.
The fate of a drug candidate during clinical tests can hinge on a single methyl group – just as a point mutation can alter the benefit of a wild-type protein molecule into the nightmare of a life-long genetic disease. Hence, many promising inhibitors eventually fail to be of benefit to patients. Nevertheless, knowledge of a series of protein structures in complex with inhibitors is of immense value in the design and development of future pharmaceuticals. In the following sections some examples will be looked at.
Some icosahedral pathogenic viruses have all their capsid proteins elucidated, while for the more complex viruses like influenza virus, hepatitis C virus (HCV) and HIV, numerous individual protein structures have been solved (Table 1.3.4.1). However, not all 14 native proteins of the HIV genome have yet surrendered to the crystallographic community, nor to the NMR spectroscopists or the high-resolution electron microscopists, our partners in experimental structural biology (Turner & Summers, 1999). Nevertheless, the structures of HIV protease, reverse transcriptase and fragments of HIV integrase and of HIV viral core and surface proteins are of tremendous value for developing novel anti-AIDS therapeutics [Arnold et al., 1996; Lin et al., 1998; Wlodawer & Vondrasek, 1998; see also references in Table 1.3.4.1(a)]. A similar situation occurs for hepatitis C virus. The protease structure of this virus has been solved recently (simultaneously by four groups!), as well as its helicase structure, providing platforms on the basis of which the design of novel drugs is actively pursued (Le et al., 1998).
References: [1] Zuccola et al. (1998) ; [2] Weissenhorn et al. (1998); [3] Murthy et al. (1999); [4] Love et al. (1996); [5] Yan et al. (1998); [6] Yao et al. (1997); [7] Rey et al. (1995); [8] Varghese et al. (1983); [9] Wilson et al. (1981); [10] Sha & Luo (1997); [11] Allaire et al. (1994); [12] Hogle et al. (1985); [13] Hansen et al. (1997); [14] Rossmann et al. (1985); [15] Matthews et al. (1994); [16] Filman et al. (1998); [17] Worthylake et al. (1999); [18] Hill et al. (1996); [19] Navia, Fitzgerald et al. (1989); [20] Wlodawer et al. (1989); [21] Erickson et al. (1990); [22] Rodgers et al. (1995); [23] Ding et al. (1995); [24] Dyda et al. (1994); [25] Kwong et al. (1998); [26] Lee et al. (1996); [27] Chan et al. (1997); [28] Ding et al. (1996); [29] Roberts et al. (1986); [30] Xia et al. (1994); [31] Wynne et al. (1999); [32] Tong et al. (1996); [33] Qiu et al. (1996); [34] Shieh et al. (1996); [35] Bochkarev et al. (1995); [36] Zdanov et al. (1997); [37] Hoog et al. (1997); [38] Wild et al. (1995); [39] Savva et al. (1995); [40] Liu et al. (1999); [42] Qiu et al. (1997); [43] Hegde & Androphy (1998); [44] Harris & Botchan (1999); [45] Hodel et al. (1996); [46] Sharma et al. (1994); [47] Kohlstaedt et al. (1992); [48] Jacobo-Molina et al. (1993); [49] Ren et al. (1995).
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A quite spectacular example of how structural knowledge can lead to the synthesis of powerful inhibitors is provided by influenza virus neuraminidase. The structure of a neuraminidase–transition-state analogue complex suggested the addition of positively charged amino and guanidinium groups to the C4 position of the analogue, which resulted, in one step, in a gain of four orders of magnitude in binding affinity for the target enzyme (von Itzstein et al., 1993).
A very large number of structures of important drug target proteins of bacterial origin have been solved crystallographically (Table 1.3.4.2). Currently, the most important single infectious bacterial agent is Mycobacterium tuberculosis, with three million deaths and eight million new cases annually (Murray & Salomon, 1998). The crystal structures of several M. tuberculosis potential and proven drug target proteins have been elucidated (Table 1.3.4.2). The complete M. tuberculosis genome has been sequenced recently (Cole et al., 1998), and this will undoubtedly have a tremendous impact on future drug development.
References: [1] Song et al. (1996) ; [2] Banbula et al. (1998); [3] Herzberg & Moult (1987); [4] Symersky et al. (1997); [5] Hennig et al. (1998); [6] Hampele et al. (1997); [7] Sundstrom et al. (1996); [8] Papageorgiou et al. (1998); [9] Papageorgiou et al. (1995); [10] Fields et al. (1996); [11] Vath et al. (1997); [12] Silvian et al. (1999); [13] Pedersen et al. (1995); [14] Pedelacq et al. (1999); [15] Loll & Lattman (1989); [16] Hofmann et al. (1993); [17] Rabijns et al. (1997); [18] Prasad et al. (1993); [19] Yeh et al. (1996); [20] Jia et al. (1993); [21] Cobessi et al. (1999); [22] Pares et al. (1996); [23] Tran et al. (1998); [24] Zhang, Evans et al. (1999); [25] Roussel et al. (1997); [26] Petosa et al. (1997); [27] Mikami et al. (1999); [28] Carfi et al. (1995); [29] Pauptit et al. (1988); [30] Watanabe et al. (1997); [31] Hough et al. (1989); [32] Lacy et al. (1998); [33] Naylor et al. (1998); [34] Rossjohn, Feil, McKinstry et al. (1997); [35] Umland et al. (1997); [36] Choe et al. (1992); [37] Qiu et al. (1995); [38] Moser et al. (1997); [39] Parge et al. (1995); [40] Huang, Xue et al. (1998); [41] Li de la Sierra et al. (1997); [42] Stein et al. (1994); [43] Emsley et al. (1996); [44] Lobkovsky et al. (1993); [45] Schonbrunn et al. (1996); [46] Sixma et al. (1991); [47] Ozaki et al. (1991); [48] Stein et al. (1992); [49] Choudhury et al. (1999); [50] Sauer et al. (1999); [51] Cirilli et al. (1993); [52] Hennig et al. (1999); [53] Bruns et al. (1997); [54] Kuzin et al. (1999); [55] Gouet et al. (1995); [56] Rossjohn, Polekhina et al. (1998); [57] Gong et al. (1997); [58] Athanasiadis et al. (1994); [59] Isupov et al. (1998); [60] Baumann (1994); [61] Wolf et al. (1998); [62] Perrakis et al. (1994); [63] Tews et al. (1996); [64] Miller et al. (1994); [65] Arnoux et al. (1999); [66] Yoshimoto et al. (1999); [67] Murray et al. (1995); [68] Ling et al. (1998); [69] Merritt et al. (1994); [70] Zhang et al. (1995); [71] Hu et al. (1997); [72] Stuckey et al. (1994); [73] Miyatake et al. (1995); [74] Pearl et al. (1994); [75] Adman et al. (1978); [76] Almassy & Dickerson (1978); [77] Fulop et al. (1995); [78] Allured et al. (1986); [79] Gatti et al. (1994); [80] Beaman et al. (1998); [81] Kallarakal et al. (1995); [82] Nurizzo et al. (1997); [83] Villeret et al. (1995); [84] Frankenberg et al. (1999); [85] Thayer et al. (1991); [86] Han et al. (1995); [87] Hulsmeyer et al. (1998); [88] Toney et al. (1993); [89] Kim et al. (1997); [90] Correll et al. (1992); [91] Concha et al. (1996); [92] Mande et al. (1996); [93] Roe et al. (1998); [94] Gourley et al. (1999); [95] Li et al. (2000); [96] Baca et al. (2000); [97] Dessen et al. (1995); [98] Chang, Spencer et al. (1998); [99] Sharma et al. (1998); [100] Cooper et al. (1995); [102] Beaman et al. (1997); [103] Li et al. (1997); [104] Crennell et al. (1994).
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The crystal structures of many bacterial dihydrofolate reductases, the target of several antimicrobials including trimethoprim, have also been reported. Recently, the atomic structure of dihydropteroate synthase (DHPS), the target of sulfa drugs, has been elucidated, almost 60 years after the first sulfa drugs were used to treat patients (Achari et al., 1997; Hampele et al., 1997).
A very special set of bacterial proteins are the toxins. Some of these have dramatic effects, with even a single molecule able to kill a host cell. These toxins outsmart and (mis)use many of the defence systems of the host, and their structures are often most unusual and fascinating, as recently reviewed by Lacy & Stevens (1998). The structures of the toxins are actively used for the design of prophylactics and therapeutic agents to treat bacterial diseases [see e.g. Merritt et al. (1997), Kitov et al. (2000) and Fan et al. (2000)]. It is remarkable that the properties of these devastating toxins are also used, or at least considered, for the treatment of disease, such as in the engineering of diphtheria toxin to create immunotoxins for the treatment of cancer and the deployment of cholera toxin mutants as adjuvants in mucosal vaccines. Knowledge of the three-dimensional structures of these toxins assists in the design of new therapeutically useful proteins.
A major cause of death and worldwide suffering is due to infections by several protozoa, including:
Drug resistance, combined with other factors, has been the cause of a major disappointment for the early hopes of a `malaria eradication campaign'. Fortunately, new initiatives have been launched recently under the umbrella of the `Malaria roll back' program and the `Multilateral Initiative for Malaria' (MIM). We are facing a formidable challenge, however, since the parasite is very clever at evading the immune response of the human host. Drugs are the mainstay of current treatments and may well be so for a long time to come. Protein crystallographic studies of Plasmodium proteins are hampered by the unusual codon usage of the Plasmodium species, coupled with a tendency to contain insertions of numerous hydrophilic residues in the polypeptide chain (Gardner et al., 1998) which provide sometimes serious obstacles to obtaining large amounts of Plasmodium proteins for structural investigations.
However, the structures of an increasing number of potential drug targets from these protozoan parasites are being unravelled. These include the variable surface glycoproteins (VSGs) and glycolytic enzymes of Trypanosoma brucei, crucial malaria proteases, and the remarkable trypanothione reductase (Table 1.3.4.3). The structures of nucleotide phosphoribosyl transferases of a variety of protozoan parasites have also been elucidated. Moreover, the structure of DHFR from Pneumocystis carinii, the major opportunistic pathogen in AIDS patients in the United States, has been determined. Several of these structures are serving as starting points for the development of new drugs.
References: [1] Leonard et al. (1997) ; [2] Liu et al. (1998); [3] Phillips et al. (1999); [4] Knighton et al. (1994); [5] Kim et al. (1995); [6] Schlagenhauf et al. (1998); [7] Shi, Schramm & Almo (1999); [8] Rigden et al. (1999); [9] Williams et al. (1999); [10] Kim et al. (1998); [11] Read et al. (1999); [12] Chitarra et al. (1999); [13] Silva et al. (1996); [14] Shi, Li et al. (1999); [15] Velanker et al. (1997); [16] Champness et al. (1994); [17] Schumacher et al. (1996); [18] Schumacher et al. (1998); [19] Chudzik et al. (2000); [20] Vellieux et al. (1993); [21] Phillips et al. (1998); [22] Bernstein et al. (1998); [23] Wierenga et al. (1987); [24] Freymann et al. (1990); [25] McGrath et al. (1995); [26] Souza et al. (1998); [27] Focia et al. (1998); [28] Maldonado et al. (1998); [29] Lantwin et al. (1994); [30] Blankenfeldt et al. (1999); [31] Yang & Moffat (1995); [32] Whitlow et al. (1997); [33] Weston et al. (1998); [34] Cleasby et al. (1996); [35] Cutfield et al. (1995); [36] Kohno et al. (1996); [37] Suguna et al. (1987); [38] Kurihara et al. (1992); [39] Rossjohn, Feil, Wilce et al. (1997); [40] McTigue et al. (1995); [41] Mulichak et al. (1998); [42] Yang et al. (1995); [43] Bullock et al. (1996); [44] Huang et al. (1994); [45] Mikol et al. (1998); [46] Taylor et al. (1998).
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In general, the human immune system is able to keep the growth of fungi under control, but in immuno-compromised patients (e.g. as a result of cancer chemotherapy, HIV infection, transplantation patients receiving immunosuppressive drugs, genetic disorders) such organisms are given opportunities they usually do not have. Candida albicans is an opportunistic fungal organism which causes serious complications in immuno-compromised patients. Several of its proteins have been structurally characterized (Table 1.3.4.3) and provide opportunities for the development of selectively active inhibitors.
Worms affect the lives of billions of human beings, causing serious morbidity in many ways. Onchocerca volvolus is the cause of river blindness, which resulted in the virtual disappearance of entire villages in West Africa, until ivermectin appeared. This remarkable compound dramatically reduced the incidence of the disease, even though it does not kill the adult worms. Therefore, a biological clock is ticking, waiting until resistance occurs against this single compound available for treatment. Schistosoma species are responsible for a wide variety of liver diseases and are spreading continuously since irrigation schemes provide a perfect environment for the intermediate snail vector. Other medically important helminths are Wuchereria bancrofti and Brugia malayi. Only a few protein structures from these very important disease-causing organisms have been unravelled so far (Table 1.3.4.3).
Resistance to drugs in infectious organisms, as well as in cancers, is a fascinating subject, since it demonstrates the action and reaction of biological systems in response to environmental challenges. Life, of course, has been evolving to do just that – and the arrival of new chemicals, termed `drugs', on the scene is nothing new to organisms that are the result of evolutionary processes involving billions of years of chemical warfare. Populations of organisms span a wide range of variation at the genetic and protein levels, and the chance that one of the variants is able to cope with drug pressure is nonzero. The variety of mechanisms observed to be responsible for drug resistance is impressive (Table 1.3.4.4).
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Crystallography has made major contributions to the detailed molecular understanding of resistance in the case of detoxification, mutation and enzyme replacement mechanisms. Splendid examples are:
Clearly, the structural insight gained from these studies provides us with avenues towards methods for coping with the rapid and alarming spread of resistance against available antibiotics that threatens the effective treatment of bacterial infections of essentially every person on this planet. This implies that we will constantly have to be aware of the potential occurrence of mono- and also multi-drug resistance, which has profound consequences for drug-design strategies for essentially all infectious diseases. It requires the development of many different compounds attacking many different target proteins and nucleic acids in the infectious agent. It appears to be crucial to use, from the very beginning, several new drugs in combination so that the chances of the occurrence of resistance are minimal. Multi-drug regimens have been spectacularly successful in the case of leprosy and HIV. Obviously, the development of vaccines is by far the better solution, but it is not always possible. Antigenic variation, see e.g. the influenza virus, requires global vigilance and constant re-engineering of certain vaccines every year. Moreover, for higher organisms, and even for many bacterial species like Shigella (Levine & Noriega, 1995), with over 50 serotypes per species, the development of successful vaccines has, unfortunately, proved to be very difficult indeed. For sleeping sickness, the development of a vaccine is generally considered to be impossible. It is most likely, therefore, that world health will depend for centuries on a wealth of therapeutic drugs, together with many other measures, in order to keep the immense number of pathogenic organisms under control.
Of this large and diverse category of human afflictions we have already touched upon genetic disorders in Section 1.3.3 above. Other major types of non-communicable diseases include cancer, aging disorders, diabetes, arthritis, and cardiovascular and neurological illnesses. The field of non-communicable diseases is immense. Describing in any detail the current projects in, and potential impact of, protein and nucleic acid crystallography on these diseases would need more space than this entire volume on macromolecular crystallography. Hence, only a few selected examples out of the hundreds which could be described can be discussed here. Table 1.3.4.5 lists many examples of human protein structures elucidated without any claim as to completeness – it is simply impossible to keep up with the fountain of structures being determined at present. Yet, such tables do provide, it is hoped, an overview of what has been achieved and what needs to be done.
References: [1] Borkakoti et al. (1994) ; [2] Lovejoy, Cleasby et al. (1994); [3] Lovejoy, Hassell et al. (1994); [4] Spurlino et al. (1994); [5] Libson et al. (1995); [6] Gohlke et al. (1996); [7] Becker et al. (1995); [8] Dhanaraj et al. (1996); [9] Esser et al. (1997); [10] Gomis-Ruth et al. (1997); [11] Finzel et al. (1998); [12] Browner et al. (1995); [13] Bode et al. (1994); [14] Reinemer et al. (1994); [15] Stams et al. (1994); [16] Betz et al. (1997); [17] Gomis-Ruth et al. (1996); [18] Bode et al. (1986); [19] Wei et al. (1988); [20] Navia, McKeever et al. (1989); [21] Walker et al. (1994); [22] Rano et al. (1997); [23] Wilson et al. (1996); [24] Tong et al. (1997); [25] Scott et al. (1991); [26] Wery et al. (1991); [27] Kitadokoro et al. (1998); [28] Sielecki et al. (1989); [29] Ghosh et al. (1995); [30] Breton et al. (1996); [31] Zhang et al. (1998); [32] Nam et al. (1996); [33] Musil et al. (1991); [34] Baldwin et al. (1993); [35] Metcalf & Fusek (1993); [36] De Bondt et al. (1993); [37] Russo et al. (1998); [38] Oefner et al. (1988); [39] Davies et al. (1990); [40] Blaber et al. (1996); [41] McTigue et al. (1999); [42] Varney et al. (1997); [43] Karpusas et al. (1997); [44] Cho et al. (1994); [45] Gorina & Pavletich (1996); [46] Xu et al. (1997); [47] Ealick et al. (1990); [48] DeVos et al. (1988); [49] Pai et al. (1989); [50] Krengel et al. (1990); [51] Scheffzek et al. (1997); [52] Renwick et al. (1998); [53] Ekstrom et al. (1999); [54] Schiffer et al. (1995); [55] Redinbo et al. (1998); [56] Stewart et al. (1998); [57] Banner et al. (1993); [58] Graves et al. (1990); [59] Priestle et al. (1988); [60] Schreuder et al. (1997); [61] Vigers et al. (1997); [62] Baldwin et al. (1991); [63] Kissinger et al. (1995); [64] McGrath et al. (1998); [65] Kallen et al. (1991); [66] Ke et al. (1991); [67] Pfuegl et al. (1993); [68] Van Duyne, Standaert, Karplus et al. (1991); [69] Van Duyne, Standaert, Schreiber & Clardy (1991); [70] Van Duyne et al. (1993); [71] Colby et al. (1999); [72] Ealick et al. (1991); [73] Walter et al. (1995); [74] Zhu et al. (1999); [75] Futterer et al. (1998); [76] Meng et al. (1999); [77] Brandhuber et al. (1987); [78] Milburn et al. (1993); [79] Livnah et al. (1996); [80] Livnah et al. (1998); [81] Carrell et al. (1994); [82] Schreuder et al. (1994); [83] Skinner et al. (1997); [84] Skinner et al. (1998); [85] Muller et al. (1994); [86] Muller et al. (1996); [87] Banner et al. (1996); [88] Rao et al. (1995); [89] Padmanabhan et al. (1993); [90] Yee et al. (1994); [91] Mather et al. (1996); [92] Pratt et al. (1997); [93] Spraggon et al. (1997); [94] Mottonen et al. (1992); [95] Aertgeerts et al. (1995); [96] Xue et al. (1998); [97] Bode et al. (1989); [98] Rydel et al. (1990); [99] Rydel et al. (1994); [100] Laba et al. (1996); [101] Spraggon et al. (1995); [102] Bienkowska et al. (1997); [103] Huizinga et al. (1997); [104] Emsley et al. (1998); [105] Ciszak & Smith (1994); [106] Hubbard et al. (1994); [107] Hubbard (1997); [108] DeVos et al. (1992); [109] Schwabe et al. (1993); [110] Brzozowski et al. (1997); [111] Williams & Sigler (1998); [112] Somers et al. (1994); [113] Kannan et al. (1975); [114] He & Carter (1992); [115] Curry et al. (1998); [116] Sinning et al. (1993); [117] Cameron et al. (1995); [118] Bruns et al. (1999); [119] Tskovsky et al. (1999); [120] Raghunathan et al. (1994); [121] Wilson et al. (1992); [122] Xie et al. (1998); [123] Thompson et al. (1997); [124] Radhakrishnan et al. (1996); [125] Muchmore et al. (1996); [126] Waksman et al. (1993).
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Over a hundred different cancers have been described and clearly the underlying defect, loss of control of cell division, can be the result of many different shortcomings in different cells. The research in this area proceeds at a feverish pace, yet the development, discovery and design of effective but safe anti-cancer agents are unbelievably difficult challenges. The modifications needed to turn a normal cell into a malignant one are very small, hence the chance of arriving at `true' anti-cancer drugs that exploit such small differences between normal and abnormal cells is exceedingly small. Nevertheless, such selective anti-cancer agents would leave normal cells essentially unaffected and are therefore the holy grail of cancer therapy. Few if any such compounds have been found so far, but cancer therapy is benefiting from a gradual increase in the number of useful compounds. Many have serious side effects, weaken the immune system and are barely tolerated by patients. However, they rescue large numbers of patients and hence it is of interest that many targets of these compounds, proteins and DNA molecules, have been structurally elucidated by crystallographic methods – often in complex with the cancer drug. The mode of action of many anti-cancer compounds is well understood, e.g. methotrexate targeting dihydrofolate reductase, and fluorouracil targeting thymidilate synthase. These are specific enzyme inhibitors acting along principles well known in other areas of medicine. Several anti-cancer drugs display unusual modes of action, such as:
Meanwhile, it is sad that crystallography has not yet made any contribution to the molecular understanding of multi-drug resistance in cancer. The resistance is caused by cellular pumps that efficiently pump out the drugs, often leading to failed chemotherapy (Borst, 1999). On the other hand, the structures of major oncogenic proteins such as p21 (DeVos et al., 1988; Pai et al., 1989; Krengel et al., 1990; Scheffzek et al., 1997) and p53 (Cho et al., 1994; Gorina & Pavletich, 1996) are of tremendous importance for future structure-based design of anti-neoplastic agents.
The hallmark characteristic of type I diabetes is a lack of insulin. A major therapeutic approach to this problem is insulin replacement therapy. Unfortunately, the insulin requirements of the body vary dramatically during the course of a day, with high concentrations needed at meal times and a basal level during the rest of the day. Only monomeric insulin is active at the insulin receptor level, but insulin has a natural tendency to form dimers and hexamers that dissociate upon dilution. Thanks to the three-dimensional insight obtained from dozens of insulin crystal structures, as wild-type (Hodgkin, 1971), mutants (Whittingham et al., 1998) and in complex with zinc ions and small molecules such as phenol (Derewenda et al., 1989), it has been possible to fine-tune the kinetics of insulin dissociation. The resulting availability of a variety of insulin preparations with rapid or prolonged action profiles has improved the quality of life of millions of people (Brange, 1997).
The main causes of blindness worldwide are cataract, trachoma, glaucoma and onchocerciasis (Thylefors et al., 1995). Trachoma and onchocerciasis are parasitic diseases that destroy the architecture of the eye; they were discussed in Section 1.3.4.1. The other two are discussed here. During cataract development, the lens of the eye becomes non-transparent as a result of aggregation of crystallins, preventing image formation. Crystal structures of several mammalian beta- and gamma-crystallins are known, but no human ones yet. In glaucoma, the optic nerve is destroyed by high intra-ocular pressure. One way to lower the pressure is to inhibit carbonic anhydrase II, a pivotal enzyme in maintaining the intra-ocular pressure. On the basis of the carbonic anhydrase crystal structure, researchers at Merck Research Laboratories were able to guide the optimization of an S-thienothiopyran-2-sulfonamide lead into a marketed drug for glaucoma: dorzolamide (Baldwin et al., 1989).
Thrombosis is a major cause of morbidity and mortality, especially in the industrial world. Hence, major effort is expended by pharmaceutical industries in the development of new classes of anti-coagulants with fewer side effects than available drugs, such as heparins and coumarins. Because blood coagulation is the result of an amplification cascade of enzymatic reactions, many potential targets are available. At present most of the effort is directed towards thrombin (Weber & Czarniecki, 1997) and factor Xa (Ripka, 1997), responsible for the penultimate step and the step immediately preceding it in the cascade, respectively. Thrombin is especially fascinating owing to the presence of at least three subsites: a primary specificity pocket with the catalytic serine-protease machinery, an exosite for recognizing extended fibrinogen and an additional pocket for binding heparin. This knowledge has led to the design of bivalent inhibitors which occupy two sites with ultra-high affinity and exquisite specificity. Several of these agents are in clinical trials (Pineo & Hull, 1999).
Even a quick glance at Table 1.3.4.5 shows that crystallography contributes to new therapeutics for numerous human afflictions and diseases. Yet there are major gaps in our understanding of protein functions, in particular of those involved in development and in neurological functions. These proteins are the target of many drugs obtained by classical pre-crystal-structure methods. These proven drug targets are very often membrane proteins involved in neuronal functions, and the diseases concerned are some of the most prevalent in mankind. A non-exhaustive list includes cerebrovascular disease (strokes), Parkinson's, epilepsy, schizophrenia, bipolar disease and depression.
Some of these diseases are heart-breaking afflictions, where parents have to accept the suicidal tendencies of their children, often with fatal outcomes; where partners have to endure the tremendous mood swings of their bipolar spouses and have to accept extreme excesses in behaviour; where a happy evening of life is turned into the gradual and sad demise of human intellect due to the progression of Alzheimer's, or to the loss of motor functions due to Parkinson's, or into the tragic stare of a victim of deep depression. Human nature, in all its shortcomings, has the tendency to try to help such tragic victims, but drugs for neurological disorders are rare, drug regimens are difficult to optimize and the commitment to follow a drug regimen – often for years, and often with major side effects – is a next to impossible task in many cases. New, better drugs are urgently needed and hence the structure determinations of the `molecules of the brain' are major scientific as well as medical challenges of the next decades. Such molecules will shed light on some of the deepest mysteries of humanity, including memory, cognition, desire, sleep etc. At the same time, such structures will provide opportunities for treating those suffering from neurodegenerative diseases due to age, genetic disposition, allergies, infections, traumas and combinations thereof. Such `CNS protein structures' are one of the major challenges of biomacromolecular crystallography in the 21st century.
As soon as a drug enters the body, an elaborate machinery comes into action to eliminate this foreign and potentially harmful molecule as quickly as possible. Two steps are usually distinguished in this process: phase I metabolism, in which the drug is functionalized, and phase II metabolism, in which further conjugation with endogenous hydrophilic molecules takes place, so that excretion via the kidneys can occur. Whereas this `detoxification' process is essential for survival, it often renders promising inhibitors useless as drug candidates. Hence, structural knowledge of the proteins involved in metabolism could have a significant impact on the drug development process.
Thus far, only the structures of a few proteins crucial for drug distribution and metabolism have been elucidated. Human serum albumin binds hundreds of different drugs with micromolar dissociation constants, thereby altering drug levels in the blood dramatically. The structure of this important carrier molecule has been solved in complex with several drug molecules and should one day allow the prediction of the affinity of new chemical entities for this carrier protein, and thereby deepen our understanding of the serum concentrations of new candidate drugs (Carter & Ho, 1994; Curry et al., 1998; Sugio et al., 1999). Human oxidoreductases and hydrolases of importance in drug metabolism with known structure are: alcohol dehydrogenase (EC 1.1.1.1) (Hurley et al., 1991), aldose reductase (EC 1.1.1.21) (Wilson et al., 1992), glutathione reductase (NADPH) (EC 1.6.4.2) (Thieme et al., 1981), catalase (EC 1.11.1.6) (Ko et al., 2000), myeloperoxidase (EC 1.11.1.7) (Choi et al., 1998) and beta-glucuronidase (EC 3.2.1.31) (Jain et al., 1996). Recently, the first crystal structure of a mammalian cytochrome P-450, the most important class of xenobiotic metabolizing enzymes, has been reported (Williams et al., 2000).
Of the conjugation enzymes, only glutathione S-transferases (EC 2.5.1.18) have been characterized structurally: A1 (Sinning et al., 1993), A4-4 (Bruns et al., 1999), MU-1 (Patskovsky et al., 1999), MU-2 (Raghunathan et al., 1994), P (Reinemer et al., 1992) and THETA-2 (Rossjohn, McKinstry et al., 1998). Tens of structures await elucidation in this area (Testa, 1994).
The development of drugs is a major undertaking and one of the hallmarks of modern societies. However, once a safe and effective therapeutic agent has been fully tested and approved, manufacturing the compound on a large scale is often the next major challenge. Truly massive quantities of penicillin and cephalosporin are produced worldwide, ranging from 2000 to 7000 tons annually (Conlon et al., 1995). In the production of semi-synthetic penicillins, the enzyme penicillin acylase plays a very significant role. This enzyme catalyses the hydrolysis of penicillin into 6-aminopenicillanic acid. Its crystal structure has been elucidated (Duggleby et al., 1995) and may now be used for protein-engineering studies to improve its properties for the biotechnology industry. The production of cephalosporins could benefit in a similar way from knowing the structure of cephalosporin acylase (CA), since the properties of this enzyme are not optimal for use in production plants. Therefore, the crystal structure determination of CA could provide a basis for improving the substrate specificity of CA by subsequent protein-engineering techniques. Fortunately, a first CA structure has been solved recently (Kim et al., 2000), with many other structures expected to be solved essentially simultaneously. Clearly, crystallography can be not only a major player in the design and optimization of therapeutic drugs, but also in their manufacture.
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