International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 1.3, pp. 12-24   | 1 | 2 |

Section 1.3.4. Crystallography and development of novel pharmaceuticals

W. G. J. Hola* and C. L. M. J. Verlindea

aBiomolecular Structure Center, Department of Biological Structure, Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195-7742, USA
Correspondence e-mail:  hol@gouda.bmsc.washington.edu

1.3.4. Crystallography and development of novel pharmaceuticals

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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]). 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[link]). 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[link]) is the subject of several books and reviews that summarize projects and several success stories up until the mid-1990s (Kuntz, 1992[link]; Perutz, 1992[link]; Verlinde & Hol, 1994[link]; Whittle & Blundell, 1994[link]; Charifson, 1997[link]; Veerapandian, 1997[link]). 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]

Figure 1.3.4.1 | top | pdf |

The structure-based drug design cycle.

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[link]). 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[link]), Eckert et al. (1999[link]) and Minke et al. (1999[link])]. 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.

1.3.4.1. Infectious diseases

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1.3.4.1.1. Viral diseases

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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[link]). 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[link]). 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[link]; Lin et al., 1998[link]; Wlodawer & Vondrasek, 1998[link]; see also references in Table 1.3.4.1(a)[link]]. 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[link]).

Table 1.3.4.1| top | pdf |
Important human pathogenic viruses and their proteins

(a) RNA viruses

(i) Single-stranded

FamilyExampleProtein structures solvedReference
Arenaviridae Lassa fever virus None  
Bunyaviridae Hantavirus None  
Caliciviridae Hepatitis E virus, Norwalk virus None  
Coronaviridae Corona virus None  
Deltaviridae Hepatitis D virus Oligomerization domain of antigen [1]
Filoviridae Ebola virus GP2 of membrane fusion glycoprotein [2]
Flaviviridae Dengue NS3 protease [3]
Hepatitis C NS3 protease [4], [5]
RNA helicase [6]
Yellow fever None  
Tick-borne encephalitis virus Envelope glycoprotein [7]
Orthomyxoviridae Influenza virus Neuraminidase [8]
Haemagglutinin [9]
Matrix protein M1 [10]
Paramyxoviridae Measles, mumps, parainfluenza, respiratory syncytial virus None  
Picornaviridae Hepatitis A virus 3C protease [11]
Poliovirus Capsid [12]
RNA-dependent polymerase [13]
Rhinovirus Capsid [14]
3C protease [15]
Echovirus Capsid [16]
Retroviridae HIV Capsid protein [17]
Matrix protein [18]
Protease [19], [20], [21]
Reverse transcriptase [22], [23], [47], [48], [49]
Integrase [24]
gp120 [25]
NEF [26]
gp41 [27]
Rhabdovirus Rabies virus None  
Togaviridae Rubella None  

(ii) Double-stranded

FamilyExampleProtein structures solvedReference
Reoviridae Rotavirus None  

(b) DNA viruses

(i) Single-stranded

FamilyExampleProtein structures solvedReference
Parvoviridae B 19 virus None  

(ii) Double-stranded

FamilyExampleProtein structures solvedReference
Adenoviridae Adenovirus Protease [28]
Capsid [29]
Knob domain of fibre protein [30]
Hepadnaviridae Hepatitis B Capsid [31]
Herpesviridae Cytomegalovirus Protease [32], [33], [34]
Epstein–Barr virus Domains of nuclear antigen 1 [35]
BCRF1 [36]
Herpes simplex Protease [37]
Thymidine kinase [38]
Uracyl-DNA glycosylase [39]
Core of VP16 [40]
Varicella zoster Protease [42]
Papovaviridae Papillomavirus DNA-binding domain of E2 [43]
Activation domain of E2 [44]
Poxviridae Smallpox virus None  
Vaccinia virus (related to smallpox but non-pathogenic) Methyltransferase VP39 [45]
Domain of topoisomerase [46]

References: [1] Zuccola et al. (1998)[link]; [2] Weissenhorn et al. (1998)[link]; [3] Murthy et al. (1999)[link]; [4] Love et al. (1996)[link]; [5] Yan et al. (1998)[link]; [6] Yao et al. (1997)[link]; [7] Rey et al. (1995)[link]; [8] Varghese et al. (1983)[link]; [9] Wilson et al. (1981)[link]; [10] Sha & Luo (1997)[link]; [11] Allaire et al. (1994)[link]; [12] Hogle et al. (1985)[link]; [13] Hansen et al. (1997)[link]; [14] Rossmann et al. (1985)[link]; [15] Matthews et al. (1994)[link]; [16] Filman et al. (1998)[link]; [17] Worthylake et al. (1999)[link]; [18] Hill et al. (1996)[link]; [19] Navia, Fitzgerald et al. (1989)[link]; [20] Wlodawer et al. (1989)[link]; [21] Erickson et al. (1990)[link]; [22] Rodgers et al. (1995)[link]; [23] Ding et al. (1995)[link]; [24] Dyda et al. (1994)[link]; [25] Kwong et al. (1998)[link]; [26] Lee et al. (1996)[link]; [27] Chan et al. (1997)[link]; [28] Ding et al. (1996)[link]; [29] Roberts et al. (1986)[link]; [30] Xia et al. (1994)[link]; [31] Wynne et al. (1999)[link]; [32] Tong et al. (1996)[link]; [33] Qiu et al. (1996)[link]; [34] Shieh et al. (1996)[link]; [35] Bochkarev et al. (1995)[link]; [36] Zdanov et al. (1997)[link]; [37] Hoog et al. (1997)[link]; [38] Wild et al. (1995)[link]; [39] Savva et al. (1995)[link]; [40] Liu et al. (1999)[link]; [42] Qiu et al. (1997)[link]; [43] Hegde & Androphy (1998)[link]; [44] Harris & Botchan (1999)[link]; [45] Hodel et al. (1996)[link]; [46] Sharma et al. (1994)[link]; [47] Kohlstaedt et al. (1992[link]); [48] Jacobo-Molina et al. (1993[link]); [49] Ren et al. (1995[link]).

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[link]).

1.3.4.1.2. Bacterial diseases

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A very large number of structures of important drug target proteins of bacterial origin have been solved crystallographically (Table 1.3.4.2[link]). Currently, the most important single infectious bacterial agent is Mycobacterium tuberculosis, with three million deaths and eight million new cases annually (Murray & Salomon, 1998[link]). The crystal structures of several M. tuberculosis potential and proven drug target proteins have been elucidated (Table 1.3.4.2[link]). The complete M. tuberculosis genome has been sequenced recently (Cole et al., 1998[link]), and this will undoubtedly have a tremendous impact on future drug development.

Table 1.3.4.2| top | pdf |
Protein structures of important human pathogenic bacteria

OrganismDisease(s)Protein structures solvedReference
Staphylococcus aureus Abscesses Alpha-haemolysin [1]
Endocarditis Aureolysin [2]
Gastroenteritis Beta-lactamase [3]
Toxic shock syndrome Collagen adhesin [4]
7,8-Dihydroneopterin aldolase [5]
Dihydropteroate synthetase [6]
Enterotoxin A [7]
Enterotoxin B [8]
Enterotoxin C2 [9]
Enterotoxin C3 [10]
Exfoliative toxin A [11]
Ile-tRNA-synthetase [12]
Kanamycin nucleotidyltransferase [13]
Leukocidin F [14]
Nuclease [15]
Staphopain [16]
Staphylokinase [17]
Toxic shock syndrome toxin-1 [18]
Staphylococcus epidermidis Implant infections None  
Enterococcus faecalis Urinary tract and biliary tract infections NADH peroxidase [19]
(Streptococcus faecalis)   Histidine-containing phosphocarrier protein [20]
Streptococcus mutans Endocarditis Glyceraldehyde-3-phosphate dehydrogenase [21]
Streptococcus pneumoniae Pneumonia Penicillin-binding protein PBP2x [22]
Meningitis, upper respiratory tract infections Dpnm DNA adenine methyltransferase [23]
Streptococcus pyogenes Pharyngitis Inosine monophosphate dehydrogenase [24]
Scarlet fever, toxic shock syndrome, immunologic disorders (acute glomerulonephritis and rheumatic fever) Pyrogenic exotoxin C [25]
Bacillus anthracis Anthrax Anthrax protective antigen [26]
Bacillus cereus Food poisoning Beta-amylase [27]
Beta-lactamase II [28]
Neutral protease [29]
Oligo-1,6-glucosidase [30]
Phospholipase C [31]
Clostridium botulinum Botulism Neurotoxin type A [32]
Clostridium difficile Pseudomembranous colitis None  
Clostridium perfringens Gas gangrene Alpha toxin [33]
Food poisoning Perfringolysin O [34]
Clostridium tetani Tetanus Toxin C fragment [35]
Corynebacterium diphtheriae Diphtheria Toxin [36]
Toxin repressor [37]
Listeria monocytogenes Meningitis, sepsis Phosphatidylinositol-specific phospholipase C [38]
Actinomyces israelii Actinomycosis None  
Nocardia asteroides Nocardiosis None  
Neisseria gonorrhoeae Gonorrhea Type 4 pilin [39]
Carbonic anhydrase [40]
Neisseria meningitidis Meningitis Dihydrolipoamide dehydrogenase [41]
Bordetella pertussis Whooping cough Toxin [42]
Virulence factor P.69 [43]
Brucella sp. Brucellosis None  
Campylobacter jejuni Enterocolitis None  
Enterobacter cloacae Urinary tract infection, pneumonia Beta-lactamase: class C [44]
UDP-N-acetylglucosamine enolpyruvyltransferase [45]
Escherichia coli      
 ETEC (enterotoxigenic) Traveller's diarrhoea Heat-labile enterotoxin [46]
Heat-stable enterotoxin (is a peptide) [47]
 EHEC (enterohaemorrhagic) HUS Verotoxin-1 [48]
 EPEC (enteropathogenic) Diarrhoea None  
 EAEC (enteroaggregative) Diarrhoea None  
 EIEC (enteroinvasive) Diarrhoea None  
 UPEC (uropathogenic)   FimH adhesin [49]
FimC chaperone [49]
PapD [50]
 NMEC (neonatal meningitis) Meningitis None  
Franciscella tularensis Tularemia None  
Haemophilus influenzae Meningitis, otitis media, pneumonia Diaminopimelate epimerase [51]
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase [52]
Ferric iron binding protein Mirp [53]
Klebsiella pneumoniae Urinary tract infection, pneumonia, sepsis β-Lactamase SHV-1 [54]
Legionella pneumophila Pneumonia None  
Pasteurella multocida Wound infection None  
Proteus mirabilis Pneumonia, urinary tract infection Catalase [55]
Glutathione S-transferase [56]
Proteus vulgaris Urinary tract infections Pvu II DNA-(cytosine N4) methyltransferase [57]
Pvu II endonuclease [58]
Tryptophanase [59]
Salmonella typhi Typhoid fever None, but many for S. typhimurium linked with zoonotic disease  
Salmonella enteridis Enterocolitis None  
Serratia marcescens Pneumonia, urinary tract infection Serralysin [60]
Aminoglycoside 3-N-acetyltransferase [61]
Chitinase A [62]
Chitobiase [63]
Endonuclease [64]
Hasa (haemophore) [65]
Prolyl aminopeptidase [66]
Shigella sp. Dysentery Chloramphenicol acetyltransferase [67]
Shiga-like toxin I [68]
Vibrio cholerae Cholera Cholera toxin [69], [70]
DSBA oxidoreductase [71]
Neuraminidase [104]
Yersinia enterocolitica Enterocolitis Protein-Tyr phosphatase YOPH [72]
Yersinia pestis Plague None  
Pseudomonas aeruginosa Wound infection, urinary tract infection, pneumonia, sepsis Alkaline metalloprotease [73]
Amidase operon [74]
Azurin [75]
Cytochrome 551 [76]
Cytochrome c peroxidase [77]
Exotoxin A [78]
p-Hydroxybenzoate hydroxylase [79]
Hexapeptide xenobiotic acetyltransferase [80]
Mandelate racemase [81]
Nitrite reductase [82]
Ornithine transcarbamoylase [83]
Porphobilinogen synthase [84]
Pseudolysin [85]
Burkholderia cepacia Wound infection, urinary tract infection, pneumonia, sepsis Biphenyl-cleaving extradiol dioxygenase [86]
cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase [87]
Dialkylglycine decarboxylase [88]
Lipase [89]
Phthalate dioxygenase reductase [90]
Stenotrophomonas maltophilia (= Pseudomonas maltophilia) Sepsis None  
Bacteroides fragilis Intra-abdominal infections Beta-lactamase type 2 [91]
Mycobacterium leprae Leprosy Chaperonin-10 (GroES homologue) [92]
RUVA protein [93]
Mycobacterium tuberculosis Tuberculosis 3-Dehydroquinate dehydratase [94]
Dihydrofolate reductase [95]
Dihydropteroate synthase [96]
Enoyl acyl-carrier-protein reductase (InhA) [97]
Mechanosensitive ion channel [98]
Quinolinate phosphoribosyltransferase [99]
Superoxide dismutase (iron dependent) [100]
Iron-dependent repressor  
Mycobacterium bovis Tuberculosis Tetrahydrodipicolinate-N-succinyltransferase [102]
Chlamydia psitacci Psittacosis None  
Chlamydia pneumoniae Atypical pneumonia None  
Chlamydia trahomatis Ocular, respiratory and genital infections None  
Coxiella burnetii Q fever None  
Rickettsia sp. Rocky Mountain spotted fever None  
Borrelia burgdorferi Lyme disease Outer surface protein A [103]
Leptospira interrogans Leptospirosis None  
Treponema pallidum Syphilis None  
Mycoplasma pneumoniae Atypical pneumonia None  

References: [1] Song et al. (1996)[link]; [2] Banbula et al. (1998)[link]; [3] Herzberg & Moult (1987)[link]; [4] Symersky et al. (1997)[link]; [5] Hennig et al. (1998)[link]; [6] Hampele et al. (1997)[link]; [7] Sundstrom et al. (1996)[link]; [8] Papageorgiou et al. (1998)[link]; [9] Papageorgiou et al. (1995)[link]; [10] Fields et al. (1996)[link]; [11] Vath et al. (1997)[link]; [12] Silvian et al. (1999)[link]; [13] Pedersen et al. (1995)[link]; [14] Pedelacq et al. (1999)[link]; [15] Loll & Lattman (1989)[link]; [16] Hofmann et al. (1993)[link]; [17] Rabijns et al. (1997)[link]; [18] Prasad et al. (1993)[link]; [19] Yeh et al. (1996)[link]; [20] Jia et al. (1993)[link]; [21] Cobessi et al. (1999)[link]; [22] Pares et al. (1996)[link]; [23] Tran et al. (1998)[link]; [24] Zhang, Evans et al. (1999)[link]; [25] Roussel et al. (1997)[link]; [26] Petosa et al. (1997)[link]; [27] Mikami et al. (1999)[link]; [28] Carfi et al. (1995)[link]; [29] Pauptit et al. (1988)[link]; [30] Watanabe et al. (1997)[link]; [31] Hough et al. (1989)[link]; [32] Lacy et al. (1998)[link]; [33] Naylor et al. (1998)[link]; [34] Rossjohn, Feil, McKinstry et al. (1997)[link]; [35] Umland et al. (1997)[link]; [36] Choe et al. (1992)[link]; [37] Qiu et al. (1995)[link]; [38] Moser et al. (1997)[link]; [39] Parge et al. (1995)[link]; [40] Huang, Xue et al. (1998)[link]; [41] Li de la Sierra et al. (1997)[link]; [42] Stein et al. (1994)[link]; [43] Emsley et al. (1996)[link]; [44] Lobkovsky et al. (1993)[link]; [45] Schonbrunn et al. (1996)[link]; [46] Sixma et al. (1991)[link]; [47] Ozaki et al. (1991)[link]; [48] Stein et al. (1992)[link]; [49] Choudhury et al. (1999)[link]; [50] Sauer et al. (1999)[link]; [51] Cirilli et al. (1993)[link]; [52] Hennig et al. (1999)[link]; [53] Bruns et al. (1997)[link]; [54] Kuzin et al. (1999)[link]; [55] Gouet et al. (1995)[link]; [56] Rossjohn, Polekhina et al. (1998)[link]; [57] Gong et al. (1997)[link]; [58] Athanasiadis et al. (1994)[link]; [59] Isupov et al. (1998)[link]; [60] Baumann (1994)[link]; [61] Wolf et al. (1998)[link]; [62] Perrakis et al. (1994)[link]; [63] Tews et al. (1996)[link]; [64] Miller et al. (1994)[link]; [65] Arnoux et al. (1999)[link]; [66] Yoshimoto et al. (1999)[link]; [67] Murray et al. (1995)[link]; [68] Ling et al. (1998)[link]; [69] Merritt et al. (1994)[link]; [70] Zhang et al. (1995)[link]; [71] Hu et al. (1997)[link]; [72] Stuckey et al. (1994)[link]; [73] Miyatake et al. (1995)[link]; [74] Pearl et al. (1994)[link]; [75] Adman et al. (1978)[link]; [76] Almassy & Dickerson (1978)[link]; [77] Fulop et al. (1995)[link]; [78] Allured et al. (1986)[link]; [79] Gatti et al. (1994)[link]; [80] Beaman et al. (1998)[link]; [81] Kallarakal et al. (1995)[link]; [82] Nurizzo et al. (1997)[link]; [83] Villeret et al. (1995)[link]; [84] Frankenberg et al. (1999)[link]; [85] Thayer et al. (1991)[link]; [86] Han et al. (1995)[link]; [87] Hulsmeyer et al. (1998)[link]; [88] Toney et al. (1993)[link]; [89] Kim et al. (1997)[link]; [90] Correll et al. (1992)[link]; [91] Concha et al. (1996)[link]; [92] Mande et al. (1996)[link]; [93] Roe et al. (1998)[link]; [94] Gourley et al. (1999)[link]; [95] Li et al. (2000[link]); [96] Baca et al. (2000[link]); [97] Dessen et al. (1995)[link]; [98] Chang, Spencer et al. (1998)[link]; [99] Sharma et al. (1998)[link]; [100] Cooper et al. (1995)[link]; [102] Beaman et al. (1997)[link]; [103] Li et al. (1997)[link]; [104] Crennell et al. (1994[link]).

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[link]; Hampele et al., 1997[link]).

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[link]). 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[link]), Kitov et al. (2000[link]) and Fan et al. (2000[link])]. 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.

1.3.4.1.3. Protozoan infections

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A major cause of death and worldwide suffering is due to infections by several protozoa, including:

  • (a) Plasmodium falciparum and related species, causing various forms of malaria;

  • (b) Trypanosoma cruzi, the causative agent of Chagas' disease in Latin America;

  • (c) Trypanosoma brucei, causing sleeping sickness in Africa;

  • (d) Some eleven different Leishmania species, responsible for several of the most horribly disfiguring diseases known to mankind.

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[link]) 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[link]). 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.

Table 1.3.4.3| top | pdf |
Protein structures of important human pathogenic protozoa, fungi and helminths

(a) Protozoa

OrganismDiseaseProtein structures solvedReference
Acanthamoeba sp. Opportunistic meningoencephalitis, corneal ulcers Actophorin [1]
Profilin [2]
Cryptosporidium parvum Cryptosporidiosis None  
Entamoeba histolytica Amoebic dysentery, liver abscesses None  
Giardia lamblia Giardiasis None  
Leishmania sp. Leishmaniasis Adenine phosphoribosyltransferase [3]
Dihydrofolate reductase-thymidylate synthase [4]
Glyceraldehyde-3-phosphate dehydrogenase [5]
Leishmanolysin [6]
Nucleoside hydrolase [7]
Pyruvate kinase [8]
Triosephosphate isomerase [9]
Plasmodium sp. Malaria Fructose-1,6-bisphosphate aldolase [10]
Lactate dehydrogenase [11]
MSP1 [12]
Plasmepsin II [13]
Purine phosphoribosyltransferase [14]
Triosephosphate isomerase [15]
Pneumocystis carinii Pneumonia Dihydrofolate reductase [16]
Toxoplasma gondii Toxoplasmosis HGXPRTase [17]
UPRTase [18]
Trichomonas vaginalis Trichomoniasis None  
Trypanosoma brucei Sleeping sickness Fructose-1,6-bisphosphate aldolase [19]
Glyceraldehyde-3-phosphate dehydrogenase [20]
6-Phosphogluconate dehydrogenase [21]
Phosphoglycerate kinase [22]
Triosephosphate isomerase [23]
VSG [24]
Trypanosoma cruzi Chagas' disease Cruzain (cruzipain) [25]
Glyceraldehyde-3-phosphate dehydrogenase [26]
Hypoxanthine phosphoribosyltransferase [27]
Triosephosphate isomerase [28]
Trypanothione reductase [29]
Tyrosine aminotransferase [30]

(b) Fungi

OrganismDiseaseProtein structures solvedReference
Aspergillus fumigatus Aspergillosis Restrictocin [31]
Blastomyces dermatidis Blastomycosis None  
Candida albicans Candidiasis Dihydrofolate reductase [32]
N-Myristoyl transferase [33]
Phosphomannose isomerase [34]
Secreted Asp protease [35]
Coccidiodes immitis Coccidioidomycosis None  
Cryptococcus neoformans Cryptococcosis None  
Histoplasma capsulatum Histoplasmosis None  
Mucor sp. Mucormycosis None  
Paracoccidioides brasiliensis Paracoccidioidomycosis None  
Rhizopus sp. Phycomycosis Lipase II [36]
Rhizopuspepsin [37]
RNase Rh [38]

(c) Helminths

OrganismDiseaseProtein structures solvedReference
Clonorchis sinensis Clonorchiasis None  
Fasciola hepatica Fasciolasis Glutathione S-transferase [39]
Fasciolopsis buski Fasciolopsiasis None  
Paragominus westermani Paragonimiasis None  
Schistosoma sp. Schistosomiasis Glutathione S-transferase [39], [40]
Hexokinase [41]
Diphyllobotrium latum Diphyllobothriasis None  
Echinococcus granulosus Unilocular hydatid cyst disease None  
Taenia saginata Taeniasis None  
Taenia solium Taeniasis None  
Ancylostoma duodenale Old World hookworm disease None  
Anisakis Anisakiasis None  
Ascaris lumbricoides Ascariasis Haemoglobin [42]
Major sperm protein [43]
Trypsin inhibitor [44]
Enterobius vermicularis Pinworm infection None  
Necator New World hookworm disease None  
Strongyloides stercoralis Strongyloidiasis None  
Trichinella spiralis Trichinosis None  
Trichuris trichiura Whipworm infection None  
Brugia malayi Filariasis Peptidylprolyl isomerase [45], [46]
Dracunculus medinensis Guinea worm disease None  
Loa loa Loiasis None  
Onchocerca volvulus River blindness None  
Toxocara canis Visceral larva migrans None  
Wuchereria bancrofti Lymphatic filariasis (elephantiasis) None  

References: [1] Leonard et al. (1997)[link]; [2] Liu et al. (1998)[link]; [3] Phillips et al. (1999)[link]; [4] Knighton et al. (1994)[link]; [5] Kim et al. (1995)[link]; [6] Schlagenhauf et al. (1998)[link]; [7] Shi, Schramm & Almo (1999)[link]; [8] Rigden et al. (1999)[link]; [9] Williams et al. (1999)[link]; [10] Kim et al. (1998)[link]; [11] Read et al. (1999)[link]; [12] Chitarra et al. (1999)[link]; [13] Silva et al. (1996)[link]; [14] Shi, Li et al. (1999)[link]; [15] Velanker et al. (1997)[link]; [16] Champness et al. (1994)[link]; [17] Schumacher et al. (1996)[link]; [18] Schumacher et al. (1998)[link]; [19] Chudzik et al. (2000[link]); [20] Vellieux et al. (1993)[link]; [21] Phillips et al. (1998)[link]; [22] Bernstein et al. (1998)[link]; [23] Wierenga et al. (1987)[link]; [24] Freymann et al. (1990)[link]; [25] McGrath et al. (1995)[link]; [26] Souza et al. (1998)[link]; [27] Focia et al. (1998)[link]; [28] Maldonado et al. (1998)[link]; [29] Lantwin et al. (1994)[link]; [30] Blankenfeldt et al. (1999)[link]; [31] Yang & Moffat (1995)[link]; [32] Whitlow et al. (1997)[link]; [33] Weston et al. (1998)[link]; [34] Cleasby et al. (1996)[link]; [35] Cutfield et al. (1995)[link]; [36] Kohno et al. (1996)[link]; [37] Suguna et al. (1987)[link]; [38] Kurihara et al. (1992)[link]; [39] Rossjohn, Feil, Wilce et al. (1997)[link]; [40] McTigue et al. (1995)[link]; [41] Mulichak et al. (1998)[link]; [42] Yang et al. (1995)[link]; [43] Bullock et al. (1996)[link]; [44] Huang et al. (1994)[link]; [45] Mikol et al. (1998)[link]; [46] Taylor et al. (1998)[link].

1.3.4.1.4. Fungi

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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[link]) and provide opportunities for the development of selectively active inhibitors.

1.3.4.1.5. Helminths

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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[link]).

1.3.4.2. Resistance

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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[link]).

Table 1.3.4.4| top | pdf |
Mechanisms of resistance

Overexpress target protein
Mutate target protein
Use other protein with same function
Remove target altogether
Overexpress detoxification enzyme
Mutate detoxification enzyme
Create new detoxification enzyme
Mutate membrane porin protein
Remove or underexpress membrane porin protein
Overexpress efflux pumps
Mutate efflux pumps
Create/steal new efflux pumps
Improve DNA repair
Mutate prodrug conversion enzyme

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:

  • (a) The beta-lactamases: These beta-lactam degrading enzymes, of which there are four classes, are produced by many bacteria to counteract penicillins and cephalosporins, the most widely used antibiotics on the planet. No less than 53 structures of these enzymes reside in the PDB.

  • (b) HIV protease mutations: Tens of mutations have been characterized structurally. Many alter the active site at the site of mutation, thereby preventing drug binding. Other mutations rearrange the protein backbone, reshaping entire pockets in the binding site (Erickson & Burt, 1996[link]).

  • (c) HIV reverse transcriptase mutations: Via structural studies, at least three mechanisms of drug resistance have been elucidated: direct alteration of the binding sites for the nucleoside analogue or non-nucleoside inhibitors, mutations that change the position of the DNA template, and mutations that induce conformational changes that propagate into the active site (Das et al., 1996[link]; Hsiou et al., 1998[link]; Huang, Chopra et al., 1998[link]; Ren et al., 1998[link]; Sarafianos et al., 1999[link]).

  • (d) Resistance to vancomycin: In non-resistant bacteria, vancomycin stalls the cell-wall synthesis by binding to the D-Ala-D-Ala terminus of the lipid–PP-disaccharide–pentapeptide substrate of the bacterial transglycosylase/transpeptidase, thereby sterically preventing the approach of the substrate. Resistant bacteria, however, have acquired a plasmid-borne transposon encoding for five genes, vanS, vanR, vanH, vanA and vanX, that allows them to synthesise a substrate ending in D-Ala-D-lactate. This minute difference, an oxygen atom replacing an NH, leads to a 1000-fold reduced affinity for vancomycin, explaining the resistance (Walsh et al., 1996[link]). Thus far, the structures of vanX (Bussiere et al., 1998[link]) and D-Ala-D-Ala ligase as a model for vanA (Fan et al., 1994[link]) have been solved. They provide an exciting basis for arriving at new antibiotics against vancomycin-resistant bacteria.

  • (e) DHFR: Some bacteria resort to the `ultimate mutation' in order to escape the detrimental effects of antibiotics. They simply replace the entire targeted enzyme by a functionally identical but structurally different enzyme. A prime example is the presence of a dimeric plasmid-encoded DHFR in certain trimethoprim-resistant bacteria. The structure proved to be unrelated to that of the chromosomally encoded monomeric DHFR (Narayana et al., 1995[link]).

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[link]), 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.

1.3.4.3. Non-communicable diseases

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Of this large and diverse category of human afflictions we have already touched upon genetic disorders in Section 1.3.3[link] 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[link] 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.

Table 1.3.4.5| top | pdf |
Important human protein structures in drug design

Proteins from other species that might have been studied as substitutes for human ones were left out because of space limitations. We apologize to the researchers affected.

Pharmacological categoryProteinReference
Synaptic and neuroeffector junctional function None  
Central nervous system function None  
Inflammation Fibroblast collagenase (MMP-1) (also important in cancer) [1], [2], [3], [4]
Gelatinase [5], [6]
Stromelysin-1 (MMP-3) (also important in cancer) [7], [8], [9], [10], [11]
Matrilysin (MMP-7) (also important in cancer) [12]
Neutrophil collagenase (MMP-8) (also important in cancer) [13], [14], [15], [16]
Collagenase-3 (MMP-13) [17]
Human neutrophil elastase (also important for cystic fibrosis) [18], [19], [20]
Interleukin-1 beta converting enzyme (ICE) [21], [22]
p38 MAP kinase [23], [24]
Phospholipase A2 [25], [26], [27]
Renal and cardiovascular function Renin [28]
Gastrointestinal function None  
Cancer 17-Beta-hydroxysteroid dehydrogenase [29], [30]
BRCT domain (BRCA1 C-terminus) [31]
Bcr-Abl kinase [32]
Cathepsin B [33]
Cathepsin D [34], [35]
CDK2 [36]
CDK6 [37]
DHFR [38], [39]
Acidic fibroblast growth factor (FGF) [40]
FGF receptor tyrosine kinase domain [41]
Glycinamide ribonucleotide formyl transferase [42]
Interferon-beta [43]
MMPs: see Inflammation  
p53 [44], [45]
p60 Src [46]
Purine nucleoside phosphorylase [47]
ras p21 [48], [49], [50], [51]
Serine hydroxymethyltransferase [52]
S-Adenosylmethionine decarboxylase [53]
Thymidylate synthase [54]
Topoisomerase I [55], [56]
Tumour necrosis factor [57]
Interleukin 1-alpha [58]
Interleukin 1-beta [59]
Interleukin 1-beta receptor [60], [61]
Interleukin 8 [62]
Immunomodulation Calcineurin [63]
Cathepsin S [64]
Cyclophilin [65], [66], [67]
Immunophilin FKBP12 [68], [69], [70]
Inosine monophosphate dehydrogenase [71]
Interferon-gamma [72], [73]
Lymphocyte-specific kinase Lck [74]
PNP [47]
Syk kinase [75]
Tumour necrosis factor [57]
ZAP Tyr kinase [76]
Interleukin 2 [77]
Interleukin 5 [78]
Haematopoiesis Erythropoietin receptor [79], [80]
Coagulation AT III [81], [82], [83], [84]
Factor III [85], [86]
Factor VII [87]
Factor IX [88]
Factor X [89]
Factor XIII [90]
Factor XIV [91]
Fibrinogen: fragment [92], [93]
Plasminogen activator inhibitor (PAI) [94], [95], [96]
Thrombin [97], [98], [99]
tPA [100]
Urokinase-type plasminogen activator [101]
von Willebrand factor [102], [103], [104]
Hormones and hormone receptors Insulin [105]
Insulin receptor [106], [107]
Human growth hormone + receptor [108]
Oestrogen receptor [109], [110]
Progesterone receptor [111]
Prolactin receptor [112]
Ocular function Carbonic anhydrase [113]
Genetic diseases See Table 1.3.3.1[link]  
Drug binding Human serum albumin [114], [115]
Drug metabolism Glutathione S-transferase A-1 [116], [117]
Glutathione S-transferase A4–4 [118]
Glutathione S-transferase Mu-1 [119]
Glutathione S-transferase Mu-2 [120]
Neurodegeneration Aldose reductase [121]
JNK3 [122]
Osteoporosis Cathepsin K [123], [64]
Src SH2 [126]
Various Interferon-alpha 2b [124]
Bcl-xL [125]

References: [1] Borkakoti et al. (1994)[link]; [2] Lovejoy, Cleasby et al. (1994)[link]; [3] Lovejoy, Hassell et al. (1994)[link]; [4] Spurlino et al. (1994)[link]; [5] Libson et al. (1995)[link]; [6] Gohlke et al. (1996)[link]; [7] Becker et al. (1995)[link]; [8] Dhanaraj et al. (1996)[link]; [9] Esser et al. (1997)[link]; [10] Gomis-Ruth et al. (1997)[link]; [11] Finzel et al. (1998)[link]; [12] Browner et al. (1995)[link]; [13] Bode et al. (1994)[link]; [14] Reinemer et al. (1994)[link]; [15] Stams et al. (1994)[link]; [16] Betz et al. (1997)[link]; [17] Gomis-Ruth et al. (1996)[link]; [18] Bode et al. (1986)[link]; [19] Wei et al. (1988)[link]; [20] Navia, McKeever et al. (1989)[link]; [21] Walker et al. (1994)[link]; [22] Rano et al. (1997)[link]; [23] Wilson et al. (1996)[link]; [24] Tong et al. (1997)[link]; [25] Scott et al. (1991)[link]; [26] Wery et al. (1991)[link]; [27] Kitadokoro et al. (1998)[link]; [28] Sielecki et al. (1989)[link]; [29] Ghosh et al. (1995)[link]; [30] Breton et al. (1996)[link]; [31] Zhang et al. (1998)[link]; [32] Nam et al. (1996)[link]; [33] Musil et al. (1991)[link]; [34] Baldwin et al. (1993)[link]; [35] Metcalf & Fusek (1993)[link]; [36] De Bondt et al. (1993)[link]; [37] Russo et al. (1998)[link]; [38] Oefner et al. (1988)[link]; [39] Davies et al. (1990)[link]; [40] Blaber et al. (1996)[link]; [41] McTigue et al. (1999)[link]; [42] Varney et al. (1997)[link]; [43] Karpusas et al. (1997)[link]; [44] Cho et al. (1994)[link]; [45] Gorina & Pavletich (1996)[link]; [46] Xu et al. (1997)[link]; [47] Ealick et al. (1990)[link]; [48] DeVos et al. (1988)[link]; [49] Pai et al. (1989)[link]; [50] Krengel et al. (1990)[link]; [51] Scheffzek et al. (1997)[link]; [52] Renwick et al. (1998)[link]; [53] Ekstrom et al. (1999)[link]; [54] Schiffer et al. (1995)[link]; [55] Redinbo et al. (1998)[link]; [56] Stewart et al. (1998)[link]; [57] Banner et al. (1993)[link]; [58] Graves et al. (1990)[link]; [59] Priestle et al. (1988)[link]; [60] Schreuder et al. (1997)[link]; [61] Vigers et al. (1997)[link]; [62] Baldwin et al. (1991)[link]; [63] Kissinger et al. (1995)[link]; [64] McGrath et al. (1998)[link]; [65] Kallen et al. (1991)[link]; [66] Ke et al. (1991)[link]; [67] Pfuegl et al. (1993)[link]; [68] Van Duyne, Standaert, Karplus et al. (1991)[link]; [69] Van Duyne, Standaert, Schreiber & Clardy (1991)[link]; [70] Van Duyne et al. (1993)[link]; [71] Colby et al. (1999)[link]; [72] Ealick et al. (1991)[link]; [73] Walter et al. (1995)[link]; [74] Zhu et al. (1999)[link]; [75] Futterer et al. (1998)[link]; [76] Meng et al. (1999)[link]; [77] Brandhuber et al. (1987)[link]; [78] Milburn et al. (1993)[link]; [79] Livnah et al. (1996)[link]; [80] Livnah et al. (1998)[link]; [81] Carrell et al. (1994)[link]; [82] Schreuder et al. (1994)[link]; [83] Skinner et al. (1997)[link]; [84] Skinner et al. (1998)[link]; [85] Muller et al. (1994)[link]; [86] Muller et al. (1996)[link]; [87] Banner et al. (1996)[link]; [88] Rao et al. (1995)[link]; [89] Padmanabhan et al. (1993)[link]; [90] Yee et al. (1994)[link]; [91] Mather et al. (1996)[link]; [92] Pratt et al. (1997)[link]; [93] Spraggon et al. (1997)[link]; [94] Mottonen et al. (1992)[link]; [95] Aertgeerts et al. (1995)[link]; [96] Xue et al. (1998)[link]; [97] Bode et al. (1989)[link]; [98] Rydel et al. (1990)[link]; [99] Rydel et al. (1994)[link]; [100] Laba et al. (1996)[link]; [101] Spraggon et al. (1995)[link]; [102] Bienkowska et al. (1997)[link]; [103] Huizinga et al. (1997)[link]; [104] Emsley et al. (1998)[link]; [105] Ciszak & Smith (1994)[link]; [106] Hubbard et al. (1994)[link]; [107] Hubbard (1997)[link]; [108] DeVos et al. (1992)[link]; [109] Schwabe et al. (1993)[link]; [110] Brzozowski et al. (1997)[link]; [111] Williams & Sigler (1998)[link]; [112] Somers et al. (1994)[link]; [113] Kannan et al. (1975)[link]; [114] He & Carter (1992)[link]; [115] Curry et al. (1998)[link]; [116] Sinning et al. (1993)[link]; [117] Cameron et al. (1995)[link]; [118] Bruns et al. (1999)[link]; [119] Tskovsky et al. (1999)[link]; [120] Raghunathan et al. (1994)[link]; [121] Wilson et al. (1992)[link]; [122] Xie et al. (1998)[link]; [123] Thompson et al. (1997)[link]; [124] Radhakrishnan et al. (1996)[link]; [125] Muchmore et al. (1996)[link]; [126] Waksman et al. (1993[link]).

1.3.4.3.1. Cancers

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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:

  • (a) the DNA intercalators daunomycin (Wang et al., 1987[link]) and adriamycin (Zhang et al., 1993[link]);

  • (b) cisplatin, which forms DNA adducts (Giulian et al., 1996[link]);

  • (c) taxol, which not only binds to tubulin but also to bcl-2, thereby blocking the machinery of cancer cells in two entirely different ways (Amos & Lowe, 1999[link]);

  • (d) camptothecin analogues, such as irinotecan and topotecan, which have the unusual property of prolonging the lifetime of a covalent topoisomerase–DNA complex, generating major road blocks on the DNA highway and causing DNA breakage and cell death;

  • (e) certain compounds function as minor-groove binders, e.g. netropsin and distamycin (Kopka et al., 1985[link]);

  • (f) completely new drugs which were developed based on the structures of matrix metalloproteinases, purine nucleotide phosphorylase and glycinamide ribonucleotide formyltransferase and which are in clinical trials (Jackson, 1997[link]).

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[link]). On the other hand, the structures of major oncogenic proteins such as p21 (DeVos et al.[link], 1988; Pai et al., 1989[link]; Krengel et al., 1990[link]; Scheffzek et al., 1997[link]) and p53 (Cho et al., 1994[link]; Gorina & Pavletich, 1996[link]) are of tremendous importance for future structure-based design of anti-neoplastic agents.

1.3.4.3.2. Diabetes

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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[link]), mutants (Whittingham et al., 1998[link]) and in complex with zinc ions and small molecules such as phenol (Derewenda et al., 1989[link]), 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[link]).

1.3.4.3.3. Blindness

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The main causes of blindness worldwide are cataract, trachoma, glaucoma and onchocerciasis (Thylefors et al., 1995[link]). Trachoma and onchocerciasis are parasitic diseases that destroy the architecture of the eye; they were discussed in Section 1.3.4.1[link]. 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[link]).

1.3.4.3.4. Cardiovascular disorders

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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[link]) and factor Xa (Ripka, 1997[link]), 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[link]).

1.3.4.3.5. Neurological disorders

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Even a quick glance at Table 1.3.4.5[link] 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.

1.3.4.4. Drug metabolism and crystallography

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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[link]; Curry et al., 1998[link]; Sugio et al., 1999[link]). Human oxidoreductases and hydrolases of importance in drug metabolism with known structure are: alcohol dehydrogenase (EC 1.1.1.1) (Hurley et al., 1991[link]), aldose reductase (EC 1.1.1.21) (Wilson et al., 1992[link]), glutathione reductase (NADPH) (EC 1.6.4.2) (Thieme et al., 1981[link]), catalase (EC 1.11.1.6) (Ko et al., 2000[link]), myeloperoxidase (EC 1.11.1.7) (Choi et al., 1998[link]) and beta-glucuronidase (EC 3.2.1.31) (Jain et al., 1996[link]). 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.[link], 2000).

Of the conjugation enzymes, only glutathione S-transferases (EC 2.5.1.18) have been characterized structurally: A1 (Sinning et al., 1993[link]), A4-4 (Bruns et al., 1999[link]), MU-1 (Patskovsky et al., 1999[link]), MU-2 (Raghunathan et al., 1994[link]), P (Reinemer et al., 1992[link]) and THETA-2 (Rossjohn, McKinstry et al., 1998[link]). Tens of structures await elucidation in this area (Testa, 1994[link]).

1.3.4.5. Drug manufacturing and crystallography

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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[link]). 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[link]) 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[link]), 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.

References

Achari, A., Somers, D. O., Champness, J. N., Bryant, P. K., Rosemond, J. & Stammers, D. K. (1997). Crystal structure of the anti-bacterial sulfonamide drug target dihydropteroate synthase. Nature Struct. Biol. 4, 490–497.
Amos, L. A. & Lowe, J. (1999). How Taxol stabilises microtubule structure. Chem. Biol. 6, R65–R69.
Arnold, E., Das, K., Ding, J., Yadav, P. N., Hsiou, Y., Boyer, P. L. & Hughes, S. H. (1996). Targeting HIV reverse transcriptase for anti-AIDS drug design: structural and biological considerations for chemotherapeutic strategies. Drug Des. Discov. 13, 29–47.
Baldwin, J. J., Ponticello, G. S., Anderson, P. S., Christy, M. E., Murcko, M. A., Randall, W. C., Schwam, H., Sugrue, M. F., Springer, J. P., Gautheron, P., Grove, J., Mallorga, P., Viader, M. P., McKeever, B. M. & Navia, M. A. (1989). Thienothiopyran-2-sulfonamides: novel topically active carbonic anhydrase inhibitors for the treatment of glaucoma. J. Med. Chem. 32, 2510–2513.
Borst, P. (1999). Multidrug resistance: a solvable problem? Ann. Oncol. 10, S162–S164.
Brange, J. (1997). The new era of biotech insulin analogues. Diabetologia, 40, S48–S53.
Bruns, C. M., Hubatsch, I., Ridderstrom, M., Mannervik, B. & Tianer, J. A. (1999). Human glutathione transferase A4-4 crystal structures and mutagenesis reveal the basis of high catalytic efficiency with toxic lipid peroxidation products. J. Mol. Biol. 288, 427–439.
Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T. & Park, C. H. (1998). The structure of VanX reveals a novel amino-dipeptidase involved in mediating transposon-based vancomycin resistance. Mol. Cell, 2, 75–84.
Carter, D. C. & Ho, J. X. (1994). Structure of serum albumin. Adv. Protein Chem. 45, 153–203.
Charifson, P. S. (1997). Practical application of computer-aided drug design. New York: Marcel Dekker Inc.
Cho, Y., Gorina, S., Jeffrey, P. D. & Pavletich, N. P. (1994). Crystal structure of a p53 tumor suppressor–DNA complex: understanding tumorigenic mutations. Science, 265, 346–355.
Choi, H. J., Kang, S. W., Yang, C. H., Rhee, S. G. & Ryu, S. E. (1998). Crystal structure of a novel human peroxidase enzyme at 2.0 Å resolution. Nature Struct. Biol. 5, 400–406.
Cole, S. T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E. III, Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M. A., Rajandream, M. A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J. E., Taylor, K., Whitehead, S. & Barrell, B. G. (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature (London), 393, 537–544.
Conlon, H. D., Baqai, J., Baker, K., Shen, Y. Q., Wong, B. L., Noiles, R. & Rausch, C. W. (1995). 2-step immobilized enzyme conversion of cephalosporin-c to 7-aminocephalosporanic acid. Biotechnol. Bioeng. 46, 510–513.
Curry, S., Mandelkow, H., Brick, P. & Franks, N. (1998). Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites. Nature Struct. Biol. 5, 827–835.
Cushman, D. W. & Ondetti, M. A. (1991). History of the design of captopril and related inhibitors of angiotensin converting enzyme. Hypertension, 17, 589–592.
Das, K., Ding, J., Hsiou, Y., Clark, A. D. Jr, Moereels, H., Koymans, L., Andries, K., Pauwels, R., Janssen, P. A. J., Boyer, P. L., Clark, P., Smith, R. H. Jr, Kroeger Smith, M. B., Michejda, C. J., Hughes, S. H. & Arnold, E. (1996). Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant. J. Mol. Biol. 264, 1085–1100.
DeVos, A. M., Tong, L., Milburn, M. V., Matias, P. M., Jancarik, J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E. & Kim, S. H. (1988). Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21. Science, 239, 888–893.
Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C. & Swenson, D. (1989). Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer. Nature (London), 338, 594–596.
Duggleby, H. J., Tolley, S. P., Hill, C. P., Dodson, E. J., Dodson, G. & Moody, P. C. (1995). Penicillin acylase has a single-amino-acid catalytic centre. Nature (London), 373, 264–268.
Eckert, D. M., Malashkevish, V. N., Hong, L. H., Carr, P. A. & Kim, P. S. (1999). Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket. Cell, 99, 103–115.
Erickson, J. W. & Burt, S. K. (1996). Structural mechanisms of HIV drug resistance. Annu. Rev. Pharmacol. Toxicol. 36, 545–571.
Fan, C., Moews, P. C., Walsh, C. T. & Knox, J. R. (1994). Vancomycin resistance: structure of D-alanine:D-alanine ligase at 2.3 Å resolution. Science, 266, 439–443.
Fan, E., Zhang, Z., Minke, W. E., Hou, Z., Verlinde, C. L. M. J. & Hol, W. G. J. (2000). A 105 gain in affinity for pentavalent ligands of E. coli heat-labile enterotoxin by modular structure-based design. J. Am. Chem. Soc. 122, 2663–2664.
Ferrer, M., Kapoor, T. M., Strassmaier, T., Weissenhorn, W., Skehel, J. J., Oprian, D., Schreiber, S. L., Wiley, D. C. & Harrison, S. C. (1999). Selection of gp41-mediated HIV-1 cell entry inhibitors from biased combinatorial libraries of non-natural binding elements. Nature Struct. Biol. 6, 953–960.
Gardner, M. J., Tettelin, H., Carucci, D. J., Cummings, L. M., Aravind, L., Koonin, E. V., Shallom, S., Mason, T., Yu, K., Fujii, C., Pederson, J., Shen, K., Jing, J., Aston, C., Lai, Z., Schwartz, D. C., Pertea, M., Salzburg, S., Zhou, L., Sutton, G. G., Clayton, R., White, O., Smith, H. O., Fraser, C. M., Adams, M. D., Venter, J. C. & Hoffman, S. L. (1998). Chromosome 2 sequence of the human malaria parasite Plasmodium falciparum. Science, 282, 1126–1132.
Giulian, D., Corpuz, M., Richmond, B., Wendt, E. & Hall, E. R. (1996). Activated microglia are the principal glial source of thromboxane in the central nervous system. Neurochem. Int. 29, 65–76.
Gorina, S. & Pavletich, N. P. (1996). Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science, 274, 1001–1005.
Hampele, I. C., D'Arcy, A., Dale, G. E., Kostrewa, D., Nielsen, J., Oefner, C., Page, M. G., Schonfeld, H. J., Stuber, D. & Then, R. L. (1997). Structure and function of the dihydropteroate synthase from Staphylococcus aureus. J. Mol. Biol. 268, 21–30.
Hodgkin, D. C. (1971). Insulin molecules: the extent of our knowledge. Pure Appl. Chem. 26, 375–384.
Hsiou, Y., Das, K., Ding, J., Clark, A. D. Jr, Kleim, J. P., Rosner, M., Winkler, I., Riess, G., Hughes, S. H. & Arnold, E. (1998). Structures of Tyr188Leu mutant and wild-type HIV-1 reverse transcriptase complexed with the non-nucleoside inhibitor HBY 097: inhibitor flexibility is a useful design feature for reducing drug resistance. J. Mol. Biol. 284, 313–323.
Huang, H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998). Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science, 282, 1669–1675.
Hurley, T. D., Bosron, W. F., Hamilton, J. A. & Amzel, L. M. (1991). Structure of human beta 1 beta 1 alcohol dehydrogenase: catalytic effects of non-active-site substitutions. Proc. Natl Acad. Sci. USA, 88, 8149–8153.
Itzstein, M. von, Wu, W. Y., Kok, G. B., Pegg, M. S., Dyason, J. C., Jin, B., Van Phan, T., Smythe, M. L., White, H. F., Oliver, S. W., Colman, P. M., Varghese, J. N., Ryan, D. M., Woods, J. M., Bethell, R. C., Hotham, V. J., Cameron, J. M. & Penn, C. R. (1993). Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature (London), 363, 418–423.
Jackson, R. C. (1997). Contributions of protein structure-based drug design to cancer chemotherapy. Semin. Oncol. 24, 164–172.
Jain, S., Drendel, W. B., Chen, Z. W., Mathews, F. S., Sly, W. S. & Grubb, J. H. (1996). Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs. Nature Struct. Biol. 3, 375–381.
Kim, Y., Yoon, K.-H., Khang, Y., Turley, S. & Hol, W. G. J. (2000). The 2.0 Å crystal structure of cephalosporin acylase. Struct. Fold. Des. 8, 1059–1068.
Kitov, P. I., Sadowska, J. M., Mulvey, G., Armstrong, G. D., Ling, H., Pannu, N. S., Read, R. J. & Bundle, D. R. (2000). Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands. Nature (London), 403, 669–672.
Ko, T.-P., Safo, M. K., Musayev, F. N., Di Salvo, M. L., Wang, C., Wu, S.-H. & Abraham, D. J. (2000). Structure of human erythrocyte catalase. Acta Cryst. D56, 241–245.
Kopka, M. L., Yoon, C., Goodsell, D., Pjura, P. & Dickerson, R. E. (1985). The molecular origin of DNA-drug specificity in netropsin and distamycin. Proc. Natl Acad. Sci. USA, 82, 1376–1380.
Krengel, U., Petsko, G. A., Goody, R. S., Kabsch, W. & Wittinghofer, A. (1990). Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35-Å resolution: implications for the mechanism of GTP hydrolysis. EMBO J. 9, 2351–2359.
Kuntz, I. D. (1992). Structure-based strategies for drug design and discovery. Science, 257, 1078–1082.
Lacy, D. B. & Stevens, R. C. (1998). Unraveling the structure and modes of action of bacterial toxins. Curr. Opin. Struct. Biol. 8, 778–784.
Le, H. V., Yao, N. & Weber, P. C. (1998). Emerging targets in the treatment of hepatitis C infection. Emerg. Ther. Targets, 2, 125–136.
Levine, M. M. & Noriega, F. (1995). A review of the current status of enteric vaccines. Papua New Guinea Med. J. 38, 325–331.
Lin, J. H., Ostovic, D. & Vacca, J. P. (1998). The integration of medicinal chemistry, drug metabolism, and pharmaceutical research and development in drug discovery and development. The story of Crixivan, an HIV protease inhibitor. Pharm. Biotechnol. 99, 233–255.
Matthews, D. A., Alden, R. A., Bolin, J. T., Freer, S. T., Hamlin, R., Xuong, N., Kraut, J., Poe, M., Williams, M. & Hoogsteen, K. (1977). Dihydrofolate reductase: X-ray structure of the binary complex with methotrexate. Science, 197, 452–455.
Merritt, E. A., Sarfaty, S., Feil, I. K. & Hol, W. G. J. (1997). Structural foundation for the design of receptor antagonists targeting E. coli heat-labile enterotoxin. Structure, 5, 1485–1499.
Minke, W. E., Hong, F., Verlinde, C. L. M. J., Hol, W. G. J. & Fan, E. (1999). Using a galactose library for exploration of a novel hydrophobic pocket in the receptor binding site of the E. coli heat-labile enterotoxin. J. Biol. Chem. 274, 33469–33473.
Murray, C. J. & Salomon, J. A. (1998). Modeling the impact of global tuberculosis control strategies. Proc. Natl Acad. Sci. USA, 95, 13881–13886.
Narayana, N., Matthews, D. A., Howell, E. E. & Nguyen-huu, X. (1995). A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site. Nature Struct. Biol. 2, 1018–1025.
Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J. & Wittinghofer, A. (1989). Structure of the guanine-nucleotide-binding domain of the Ha-ras oncogene product p21 in the triphosphate conformation. Nature (London), 341, 209–214.
Patskovsky, Y. V., Patskovska, L. N. & Listowsky, I. (1999). Functions of His107 in the catalytic mechanism of human glutathione s-transferase hGSTM1a-1a. Biochemistry, 38, 1193–1202.
Perutz, M. (1992). Protein structure. New approaches to disease and therapy. New York: W. H. Freeman & Co.
Pineo, G. F. & Hull, R. D. (1999). Thrombin inhibitors as anticoagulant agents. Curr. Opin. Hematol. 6, 298–303.
Raghunathan, S., Chandross, R. J., Kretsinger, R. H., Allison, T. J., Penington, C. J. & Rule, G. S. (1994). Crystal structure of human class mu glutathione transferase GSTM2–2. Effects of lattice packing on conformational heterogeneity. J. Mol. Biol. 238, 815–832.
Reinemer, P., Dirr, H. W., Ladenstein, R., Huber, R., Lo Bello, M., Federici, G. & Parker, M. W. (1992). Three-dimensional structure of class pi glutathione S-transferase from human placenta in complex with S-hexylglutathione at 2.8 Å resolution. J. Mol. Biol. 227, 214–226.
Ren, J., Esnouf, R. M., Hopkins, A. L., Jones, E. Y., Kirby, I., Keeling, J., Ross, C. K., Larder, B. A., Stuart, D. I. & Stammers, D. K. (1998). 3-Azido-3′-deoxythymidine drug resistance mutations in HIV-1 reverse transcriptase can induce long range conformational changes. Proc. Natl Acad. Sci. USA, 95, 9518–9523.
Ripka, W. C. (1997). Design of antithrombotic agents directed at factor Xa. In Structure-based drug design, edited by P. Veerapandian, pp. 265–294. New York: Marcel Dekker.
Rossjohn, J., McKinstry, W. J., Oakley, A. J., Verger, D., Flanagan, J., Chelvanayagam, G., Tan, K. L., Board, P. G. & Parker, M. W. (1998). Human theta class glutathione transferase: the crystal structure reveals a sulfate-binding pocket within a buried active site. Structure, 6, 309–322.
Sarafianos, S. G., Das, K., Ding, J., Boyer, P. L., Hughes, S. H. & Arnold, E. (1999). Touching the heart of HIV-1 drug resistance: the fingers close down on the dNTP at the polymerase active site. Chem. Biol. 6, R137–R146.
Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmuller, L., Lautwein, A., Schmitz, F. & Wittinghofer, A. (1997). The Ras–RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science, 277, 333–338.
Sinning, I., Kleywegt, G. J., Cowan, S. W., Reinemer, P., Dirr, H. W., Huber, R., Gilliland, G. L., Armstrong, R. N., Ji, X., Board, P. G., Olin, B., Mannervik, B. & Jones, T. A. (1993). Structure determination and refinement of human alpha class glutathione transferase A1-1, and a comparison with the mu and pi class enzymes. J. Mol. Biol. 232, 192–212.
Sugio, S., Kashima, A., Mochizuki, S., Noda, M. & Kobayashi, K. (1999). Crystal structure of human serum albumin at 2.5 Å resolution. Protein Eng. 12, 439–446.
Testa, B. (1994). Drug metabolism. In Burger's medicinal chemistry and drug discovery, 5th ed., edited by M. E. Wolf, Vol. 1. New York: John Wiley & Sons.
Thieme, R., Pai, E. F., Schirmer, R. H. & Schulz, G. E. (1981). Three-dimensional structure of glutathione reductase at 2 Å resolution. J. Mol. Biol. 152, 763–782.
Thylefors, B., Negrel, A. D., Pararajasegaram, R. & Dadzie, K. Y. (1995). Global data on blindness. Bull. WHO, 73, 115–121.
Turner, B. G. & Summers, M. F. (1999). Structural biology of HIV. J. Mol. Biol. 285, 1–32.
Veerapandian, P. (1997). Editor. Structure-based drug design. New York: Marcel-Dekker.
Verlinde, C. L. M. J. & Hol, W. G. J. (1994). Structure-based drug design: progress, results and challenges. Structure, 2, 577–587.
Walsh, C. T., Fisher, S. L., Park, I. S., Prahalad, M. & Wu, Z. (1996). Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story. Chem. Biol. 3, 21–28.
Wang, A. H., Ughetto, G., Quigley, G. J. & Rich, A. (1987). Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2 Å resolution. Biochemistry, 26, 1152–1163.
Weber, P. C. & Czarniecki, M. (1997). Structure-based design of thrombin inhibitors. In Structure-based drug design, edited by P. Veerapandian, pp. 247–264. New York: Marcel Dekker.
Whittingham, J. L., Edwards, D. J., Antson, A. A., Clarkson, J. M. & Dodson, G. G. (1998). Interactions of phenol and m-cresol in the insulin hexamer, and their effect on the association properties of B28 pro Asp insulin analogues. Biochemistry, 37, 11516–11523.
Whittle, P. J. & Blundell, T. L. (1994). Protein structure-based drug design. Annu. Rev. Biophys. Biomol. Struct. 23, 349–375.
Williams, P. A., Cosme, J., Sridhar, V., Johnson, E. F. & McRee, D. E. (2000). Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol. Cell, 5, 121–131.
Wilson, D. K., Bohren, K. M., Gabbay, K. H. & Quiocho, F. A. (1992). An unlikely sugar substrate site in the 1.65 Å structure of the human aldose reductase holoenzyme implicated in diabetic complications. Science, 257, 81–84.
Wlodawer, A. & Vondrasek, J. (1998). Inhibitors of HIV-1 protease: a major success of structure-assisted drug design. Annu. Rev. Biophys. Biomol. Struct. 27, 249–284.
Zhang, H., Gao, Y. G., van der Marel, G. A., van Boom, J. H. & Wang, A. H. (1993). Simultaneous incorporations of two anticancer drugs into DNA. The structures of formaldehyde-cross-linked adducts of daunorubicin-d(CG(araC)GCG) and doxorubicin-d(CA(araC)GTG) complexes at high resolution. J. Biol. Chem. 268, 10095–10101.








































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