Size does not matter: a molecular insight into the biological activity of chemical fragments utilizing computational approaches.

Masters Degree. University of KwaZulu-Natal, Durban.Insight into the functional and physiological state of a drug target is of essential importance in the drug discovery process, with the lack of emerging (3D) drug targets we propose the integration of homology modeling which may aid in the accurate yet efficient construction of 3D protein structures. In this study we present the applications of homology modeling in drug discovery, a conclusive route map and detailed technical guideline that can be utilised to obtain the most accurate model. Even with the presence of available drug targets and substantial advancements being made in the field of drug discovery, the prevalence of incurable diseases still remains at an all-time high. In this study we explore the biological activity of chemically derived fragments from natural products utilising a range of computational approaches and implement its use in a new route towards innovative drug discovery. A potential avenue referred to as the reduce to maximum concept recently proposed by organic chemists, entails reducing the size of a chemical compound to obtain a structural analogs with retained or enhanced biological activity, better synthetic approachability and reduced toxicity. Displaying that size may not in fact matter. Molecular dynamic simulations along with toxicity profiling were comparatively performed, on natural compound Anguinomycin D and its derived analog SB 640 each in complex with the CRM1 protein which plays an avid role in cancer pathogenesis. Each system was post-dynamically studied to comprehend structural dynamics adopted by the parent compound to that exhibited by the analog. Although being reduced by 60% the analog SB 640 displayed an overall exhibition of attractive pharmacophore properties which include minimal reduction in binding affinity, enhanced synthetic approachability and reduced toxicity in comparison to the parent compound. Potent inhibitor of CRM1, Leptomycin B (LMB) displayed substantial inhibition of the CRM1 export protein by binding to four of the PKIαNES residues (ϕ0, ϕ1, ϕ2, ϕ3, and ϕ4) present within the hydrophobic binding groove of CRM1. Although being drastically reduced in size and lacking the presence of the polyketide chain present in the parent compound Anguinomycin D and LMB the analog SB 640 displaced three of these essential NES residues. The potential therapeutic activity of the structural analog remains undeniable, however the application of this approach in drug design still remains ambiguous as to which chemical fragments must be retained or truncated to ensure retention or enhanced pharmacophore properties. In this study we aimed to the use of thermodynamic calculations, which was accomplished by incorporating a MM/GBSA per-residue energy contribution footprint from molecular dynamics simulation. The proposed approach was generated for each system. Anguinomycin D and analog SB 640 each in complex with CRM1 protein, each system formed interactions with the conserved active site residues Leu 536, Thr 575, Val 576 and Lys 579. These residues were highlighted as the most energetically favourable amino acid residues contributing substantially to the total binding free energy. Thus implying a conserved selectivity and binding mode adopted by both compounds despite the omission of the prominent polyketide chain in the analog SB 640, present in the parent compound. A strategic computational approach presented in this study could serve as a beneficial tool to enhance novel drug discovery. This entire work provides an invaluable contribution to the understanding of the phenomena underlying the reduction in the size of a chemical compound to obtain the most beneficial pharmacokinetic properties and could largely contribute to the design of potent analog inhibitors for a range of drug targets implicated in the orchestration of diseases

Computational simulations of enzyme dynamics and the modelling of their reaction mechanisms

Proteins and enzymes are large and complex biological molecules, characterized by unique three-dimensional structure are highly flexible and dynamic nature. Thorough understanding of protein and enzyme function requires studying of their conformational flexibility, because important physiological processes, such as ligand binding and catalysis rely on an enzyme’s dynamic nature and their ability to adopt a variety of conformational states. Computational methods are widely applied in studying enzymes and proteins structure and function providing a detailed atomistic-level of resolution data about the dynamics and catalytic processes, mechanisms in biomolecules, therefore even more nowadays a term ‘computational enzymology’ has emerged. Experimental methods often have difficulty in predicting dynamic motions of proteins. Computational simulations techniques, such as Molecular Dynamics simulations, have proven successful in simulating the conformational flexibility of proteins in studying structure-function relationships. Additionally, the binding events between two molecules, e.g. an enzyme and its substrate, can be computationally predicted with molecular docking methods. Enzymes are proteins that catalyse almost all biochemical reactions and metabolic processes in all organisms. In order to study the conformational flexibility of proteins we apply molecular dynamics simulations, and in order to simulate their reaction mechanisms we apply quantum mechanical simulations. Quantum mechanical simulations can also be used to predict the electronic structure of organic compounds, by calculating their electronic structures we perform orbital analyses and predict their optical properties. The results gained from our computational simulations can give new insights into explanation of experimental findings and data and can inspire and guide further experiments

Oxydifficidin-Producing \u3ci\u3eBacillus\u3c/i\u3e Presents Novel Antimicrobial Activity Against \u3ci\u3eNeisseria gonorrhoeae\u3c/i\u3e Involving the Deda Protein

Bacterial human pathogens cause severe infectious diseases which are the second most common cause of death next to cancer and cardiovascular diseases in the world, especially in developing countries. Gonorrhea particularly, is the second most common sexually transmitted infection (STI) which is caused by the microorganism Neisseria gonorrhoeae (GC). Centers for Disease Control and Prevention (CDC) estimates that more than 1.6 million new gonorrhea cases emerged in USA in 2018 (“Detailed STD Facts - Gonorrhea” n.d.). Also, the WHO (World Health Organization) shows that gonorrhea is the most antibiotic resistant STI (“PAHO/WHO | Gonorrhea” n.d.), highlighting the shortage of efficient antibiotics act against N. gonorrhoeae nowadays. Gonorrhea is a growing public health concern worldwide due to the rapid development of antibiotic resistance. At present, the only recommended way to efficiently cure or control the condition of gonorrhea is the combination usage (dual therapy) of ceftriaxone and azithromycin (Unemo and Nicholas 2012). Nonetheless, there are some recently reported “superbugs” which are resistant in a high level to antibiotics known to act against gonorrhoeae, such as GC strains F89 (Unemo et al. 2012) and H041 (Tomberg et al. 2013). This means gonorrhea may become untreatable if no novel antibiotics or substitute therapy is developed in the immediate future and a superbug gains resistance to all those antibiotic and spreads in the population. Thus, the most urgent effort that we should make is to find or design alternative drugs to solve the gonorrhea crisis that people are facing or get it under control even temporarily. However, the discovery of new antibiotics is usually challenging and slow, especially after the “golden era” stimulated by Selman Waksman in the 1940s, not only for large-scale screening of natural products, but also for synthetic compounds (K. Lewis 2020). As a serendipity, Dr. Nicolas Biais and I noticed a contaminant on a bacterial survival plate, which shows specifically strong inhibition activity to N. gonorrhoeae compared to other strains. This “penicillin-discovery-like” observation triggered my central hypothesis of the thesis: this contaminant produces a narrow spectrum antimicrobial compound which effectively acts against Neisseria gonorrhoeae due to a molecular mechanism to be elucidated. My thesis has been organized in the following way: In Chapter 1, I will introduce the context and familiarizing the reader with N. gonorrhoeae, antimicrobial products, and species’ interaction, as well as presenting the aims and broad experimental design of this research. I isolated the strain and named it as “Bacillus S” based on phylogenetic study, allowing me to characterize it, identify the active compound, and investigate its interaction with N. gonorrhoeae. Chapter 2 begins by characterizing Bacillus S. I used genomic sequencing and phylogenetic study to identify that it is a species of the Bacillus genus, close to the subtilis and amyloliquefaciens species. The disc diffusion assay of its bacterial supernatant indicates that the compound that inhibits gonorrhoeae (anti-GC) can be excreted to the extracellular environment. The following part is determining the active compound, by using a combination of molecular biology assays, bioinformatic analysis, mass spectrometry, and nuclear magnetic resonance (NMR). I finally identified that anti-GC compound is oxydifficidin produced by Bacillus S, this new antimicrobial activity has not been published before. Lastly, I will turn to the questions of target of oxydifficidin in N. gonorrhoeae and the potential mechanism of action. In Chapter 3, I used transposon mutagenesis and insertion site mapping to successfully find that the target of this compound is an ancient trans-membrane protein – DedA. This protein family is poorly investigated since it has been discovered. Surprisingly, previous research shows DedA promotes antibiotic resistance in bacterial cells, which shows discrepancy to what I describe in this study as DedA is promoting sensitivity to oxydifficidin in N. gonorrhoeae. Through the membrane integrity assay, membrane potential assay, and molecular modeling of oxydifficidin – DedA interaction, I propose that oxydifficidin may bind to the trans-membrane DedA protein on N. gonorrhoeae, subsequently modifying cell structure and metabolism which then motivates cell death. In addition, pili dynamic assays show that the type IV pili dynamics of N. gonorrhoeae is relative to the DedA protein, the annex of this thesis provides a deeper understanding of type IV pili as a side project. This work starts with isolating an antibiotic producing strain, aims to identify and characterize the active compound, and understand the interaction between this small molecule and N. gonorrhoeae cell. For the reason that new treatments of gonorrhea are urgently needed, I suggest that this molecule’s type and inhibition method can be considered as a novel or alternative direction for future drug development of gonorrhea, and even other pathogens

Dehydrodiconiferyl Alcohol의 항염증 및 항골다공증 활성에 대한 분자 기전 연구

학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 생명과학부, 2018. 2. 김선영.Dehydrodiconiferyl alcohol (DHCA) is a lignan compound isolated from Cucurbita moschata. Synthetic DHCA has previously been shown to contain anti-adipogenic, anti-oxidative stress and anti-inflammatory activities. In an effort to understand the underlying mechanisms of such multiple bioactivities of this lignan molecule, my thesis research was focused on the effects of DHCA on IL-17-mediated inflammation and also on its potential estrogenic activities, both in vitro and in vivo. The effects of DHCA on IL-17-mediated inflammation were investigated using HaCaT keratinocyte cell line. DHCA significantly inhibited the IL-17-mediated cell proliferation and suppressed the expression of various inflammatory mediators, such as TNF-α, IL-6, IL-1β and various chemokines by inhibiting p38 MAPK signaling pathway. Consistent with these in vitro data, in the imiquimod-induced psoriasis-like skin inflammation mouse model, DHCA ameliorated psoriatic symptoms, histological phenotypes and expression of various inflammatory mediators. Data from immunohistochemical analysis and ex vivo culture experiments suggested that DHCA reduced the infiltration of IL-17 producing inflammatory cells by suppressing various chemokines such as CXCL1, CXCL8 and CCL20. Being a lignan molecule, DHCA is a member of the phytoestrogen family. Therefore, it was investigated whether DHCA contains anti-osteoporotic activities similar to estrogen. The effects of DHCA were studied on RANKL-induced osteoclastogenesis using RAW264.7 pre-osteoclast cell line. DHCA effectively inhibited the RANKL-induced differentiation and function of osteoclast in a dose-dependent manner. DHCA also suppressed the expression of various osteoclastogenic genes, including NFATc1, TRAP, c-Fos, DC-STAMP, MMP-9, and Cathepsin K, through the inhibition of NF-ĸB and p38 MAPK signaling pathways. These anti-osteoclastogenic effects of DHCA were suppressed when cells were transfected with siRNAs for AMPKα1 or ERα, whereas ERβ siRNA did not have any effect. The effects of DHCA on BMP-2-induced osteoblastogenesis were also studied using MC3T3-E1 pre-osteoblast cell line. DHCA promoted BMP-2-induced differentiation of osteoblast in a dose-dependent manner. This lignan molecule further up-regulated the BMP-2 mediated activation of Smad1/5/9 and AMPK signaling pathways, the expression of RUNX2 and subsequently that of ALP, osteocalcin and OPG. Gene knockdown analysis, involving specific siRNAs for ERα or ERβ, indicated that DHCA might interact with either ERα or ERβ to promote the BMP-2-induced osteoblast differentiation. Above data indicated that DHCA might produce anti-osteoporotic activities through its agonistic effect on estrogen receptor. When an ovariectomized mouse model was used, DHCA indeed improved a variety of bone morphometric parameters as determined by 3D-structure analysis. DHCA also reduced the blood level of NTx and CTx, biochemical markers for bone degradation which also regulate the expression of osteoclastogenic and osteoblastogenic genes in the bone marrow. Together with our previous findings, data from my thesis work demonstrated that DHCA had a wide range of bioactivities including anti-adipogenic, anti-oxidative stress, anti-inflammatory, anti-osteoclastogenic and osteoblastogenic activities. Such multiple bioactivities of DHCA could be best explained if this molecule acts like estrogen. Data from molecular docking simulation suggested that DHCA could bind to both ERα and ERβ with a similar binding pose to estrogen. Furthermore, the 2D ligand-receptor interaction diagram showed that intermolecular forces and MM-GBSA binding energy were analogous to the case of estradiol. Indeed, results from estrogen receptor competition assay indicated that DHCA could efficiently bind to ERα and ERβ. In conclusion, high therapeutic effects of DHCA observed in psoriasis and osteoporosis mouse models could be explained with DHCA acting as an estrogen receptor agonist, and thus producing anti-inflammatory, anti-osteoclastogenic and osteoblastogenic activities. Taken together, with previous findings, DHCA may be developed as a safe and effective therapeutic agent for the treatment of various diseases where inflammation and/or estrogen play prominent role(s).I. Introduction 1 1. Background information 2 2. Phytoestrogens 2 3. Psoriasis 5 4. Osteoporosis 9 5. Rationale and purpose of this study 15 II. Material and Methods 17 1. Cell culture and reagents 18 2. Molecular cellular biological techniques 20 3. Mouse disease models 27 III. Suppressive Effects of DHCA on IL-17 mediated Inflammatory Responses using Psoriasis as a Model Disease 30 1. Background 31 2. Results 32 3. Discussion 42 IV. Anti-osteoclastogenic Effects of DHCA in RAW264.7 cells 46 1. Background 47 2. Results 48 3. Discussion 64 V. Effects of DHCA on BMP-2-induced Osteoblastogenesis and Osteoporosis Mouse Model 67 1. Background 68 2. Results 69 3. Discussion 81 VI. Concluding Remarks 86 References 92 Abstract in Korean 110Docto

Design, synthesis and biological evaluation of small molecules for controlling cellular development

Cellular differentiation is a process directed by a wide range of controlling signaling molecules and pathways. All-trans-retinoic acid (ATRA) is one such compound that shows a wide range of biological activity. The endogenous effects of ATRA have the potential to be translated into many in vitro and in vivo applications; however, its administration is associated with many drawbacks. Consequently, a large group of synthetic analogues known as synthetic retinoids - that are structurally similar to ATRA have been prepared and tested in vitro in the search for higher stability and more potency. A small library of stable synthetic retinoids known as EC and GZ derivatives were prepared and their biological activity investigated using TERA2.cl.SP12 human embryonal carcinoma (EC) stem cells and SHSY5Y neuroblastoma cells. Two compounds, EC23 and GZ25 were found to inhibit cellular proliferation and induce neural differentiation in both cell lines. EC50s showed higher binding affinity of these two analogues to all RAR types and was confirmed by how they fit into the binding pocket of the different RARs. They bind into the binding pocket through a hydrophilic network of carboxylate group with Arg (salt bridge) and Ser (two hydrogen bonds) residues similar to ATRA. These effects were thoroughly characterized and quantified by monitoring the phenotypic changes of both cell lines and the gene expression markers such as RAR-β, PAX6, NeuroD1 which showed higher order of efficacy for induction of neuronal differentiation.In this study, the combined use of calculated chemical structures, molecular docking tools with receptor binding assays and biological characterization was useful to probe, and hence, understand the biological activity of certain synthetic retinoids with the ultimate goal of designing more specific synthetic retinoic acid derivatives

Small molecule-protein interactions exemplified on short-chain dehydrogenases/reductases

The short-chain dehydrogenase/reductase (SDRs) family represents one of the largest enzyme superfamilies, with over 80 members in the human genome. Even though the human genome project has sequenced and mapped the entire human genome, the physiological functions of more than 70% of all SDRs are currently unexplored or insufficiently characterized. To start to fill this gap, the present thesis aimed to employ a combination of molecular modeling approaches and biological assessments for the identification and characterization of novel inhibitors and/or potential substrates of different SDRs. Due to their involvement in steroid biosynthesis and metabolism, SDRs are potential targets of endocrine disrupting chemicals (EDCs). To test the use of pharmacophore-based virtual screening (VS) applications and subsequent in vitro evaluation of virtual hits for the identification and characterization of potential inhibitors, 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) was selected as an example. 11β-HSD2 has an important role in the placenta by inactivating cortisol and protecting the fetus from high maternal glucocorticoid levels. An impaired placental 11β-HSD2 function has been associated with altered fetal growth and angiogenesis as well as a higher risk for cardio-metabolic diseases in later life. Despite this vital function, 11β-HSD2 is not covered in common off-target screening approaches. Several azole fungicides were identified as 11β-HSD inhibitors amongst approved drugs by testing selected virtually retrieved hits for inhibition of cortisol to cortisone conversion in cell lysates expressing recombinant human 11β-HSD2. Moreover, a significant structure-activity relationship between azole scaffold size, 11β-HSD enzyme selectivity and potency was observed. The most potent 11β-HSD2 inhibition was obtained for itraconazole (IC50 139 ± 14 nM), for its active metabolite hydroxyitraconazole (IC50 223 ± 31 nM), and for posaconazole (IC50 460 ± 98 nM). Interestingly, substantially lower inhibitory 11β-HSD2 activity of these compounds was detected using mouse and rat kidney homogenate preparations, indicating species-specific differences. Impaired placental 11β-HSD2 function exerted by these compounds might, in addition to the known inhibition of P-glycoprotein efflux transport and cytochrome P450 enzymes, lead to locally elevated cortisol levels and thereby could affect fetal programming. Successful employment of pharmacophore-based VS applications requires suitable and reliable in vitro validation strategies. Therefore, the following study addressed the re-evaluation of a potential EDC, the widely used flame retardant tetrabromobisphenol A (TBBPA), on glucocorticoid receptor (GR) and androgen receptor (AR) function. TBBPA was reported earlier in yeast-based reporter assays to potently interfere with GR and moderately with AR function. Human HEK-293 cell-based reporter assays and cell-free receptor binding assays did not show any activity of TBBPA on GR function, which was supported by molecular docking calculations. The antiandrogenic effect, however, could be confirmed, although less pronounced than in the HEK-293 cell system. Nevertheless, the evaluation of the relevant concentrations of an EDC found in the human body is crucial for an appropriate safety assessment. Considering the rapid metabolism of TBBPA and the low concentrations observed in the human body, it is questionable whether relevant concentrations can be reached to cause harmful effects. Thus, it is vital to take the limitations of each testing system including the distinct sensitivities and specificities into account to avoid false positive or false negative results. To extend the applications of in silico tools with demonstrated proof-of-concept, they were further employed to investigate novel substrate specificities for three different SDR members: the two multi-functional enzymes, 11β-HSD1 and carbonyl reductase (CBR) 1 as well as the orphan enzyme DHRS7. A role for 11β-HSD1 in oxysterol metabolism by metabolizing 7-ketocholesterol (7kC) has already been described. However, in contrast to the known receptors for 7α,25-dihydroxycholesterol (7α25OHC), i.e. Epstein-Barr virus-induced gene 2 (EBI2), or 7β,27-dihydroxycholesterol (7β27OHC), i.e. retinoic acid related orphan receptor (ROR)γ, no endogenous receptor has been identified so far for 7kC or its metabolite 7β-hydroxycholesterol. To explore the underlying biosynthetic pathways of such dihydroxylated oxysterols, the role of 11β-HSD1 in the generation of dihydroxylated oxysterols was investigated. For the first time, the stereospecific and seemingly irreversible oxoreduction of 7-keto,25-hydroxycholesterol (7k25OHC) and 7-keto,27-hydroxycholesterol (7k27OHC) to their corresponding 7β-hydroxylated metabolites 7β25OHC and 7β27OHC by recombinant human 11β-HSD1 could be demonstrated in vitro in intact HEK-293 cells. Furthermore, 7k25OHC and 7k27OHC were found to be potently inhibited the 11β-HSD1-dependent oxoreduction of cortisone to cortisol. Molecular modeling experiments confirmed these results and suggested competition of 7k25OHC and 7k27OHC with cortisone in the enzyme binding pocket. For a more detailed enzyme characterization, 11β-HSD1 pharmacophore models were generated and employed for VS of the human metabolome database and the lipidmaps structure database. The VS yielded several hundred virtual hits, including the successful filtering of known substrates such as endogenous 11-ketoglucocorticoids, synthetic glucocorticoids, 7kC, and several bile acids known to inhibit the enzyme. Further hits comprised several eicosanoids including prostaglandins, leukotrienes, cyclopentenone isoprostanes, levuglandins or hydroxyeicosatetraenoic acids (HETEs) and compounds of the kynurenine pathway. The important role of these compounds as well as 11β-HSD1 in inflammation emphasizes a potential association. However, further biological validation is of utmost necessity to explore a potential link. The closest relative of 11β-HSD1 is the orphan enzyme DHRS7, which has been suggested to act as tumor suppressor. Among others, cortisone and 5α-dihydrotestosterone have been identified as substrates of DHRS7, although effects in functional assays could only be observed at high concentrations that may not be of physiological relevance. Hence, the existence of other yet unexplored substrates of DHRS7 can be assumed, and the generation of homology models to study the structural features of the substrate binding site of DHRS7 was employed. The predictivity of the constructed models is currently limited, due to a highly variable region comprising a part of the ligand binding site but particularly the entry of the binding pocket, and requires further optimizations. Nevertheless, the models generally displayed a cone-shaped binding site with a rather hydrophobic core. This may suggest larger metabolites to be converted by DHRS7. Moreover, the flexible loops surrounding the binding pocket may lead to the induction of an induced fit upon ligand binding. However, further studies are crucial to confirm these findings. CBR1 is well-known for its role in phase I metabolism of a variety of carbonyl containing xenobiotic compounds. Several endogenous substrates of CBR1 have been reported such as prostaglandins, S-nitrosoglutathione or lipid aldehydes. The physiological relevance of these endogenous substrates, however, is not fully understood. Thus, the physiological roles of CBR1 was further explored by identifying a novel function for CBR1 in the metabolism glucocorticoids. CBR1 was found to catalyze the conversion of cortisol into 20β-dihydrocortisol (20β-DHF), which was in turn detected as the major route of cortisol metabolism in horses and elevated in adipose tissue derived from obese horses, humans and mice. Additionally, 20β-DHF was demonstrated as weak endogenous agonist of the GR, suggesting a novel pathway to modulate GR activation by CBR1-depenent protection against excessive GR activation in obesity. In conclusion, this thesis emphasized the employment of molecular modeling approaches as an initial filter to identify toxicological relevant compound classes for the identification of potential EDCs and, moreover, as valuable tools to identify novel substrates of multifunctional SDRs and to unravel novel functions for the large majority of yet unexplored orphan SDR members, while carefully considering the limitations of this strategy

Towards detailed structural understanding of α-ʟ-fucosidase: insights through X-ray crystallography and inhibitor-binding

Carbohydrates are one of the most abundant biomolecules and are fundamental to the correct function of many biological processes. The monosaccharide ʟ-fucose is incorporated into biological polymers including oligo and polysaccharides, and glycoproteins. ʟ-fucose is often appended to the end of a glycan chain, and as such is recognised by lectins in a number of molecular recognition events. Due to this, the monosaccharide plays a critical role in the immune response, the colonisation of bacteria in mammals, and cancer. Two enzymes regulate homeostasis of ʟ-fucosylated biomolecules, GDP-ʟ-fucosyltransferases append the sugar to nascent biomolecules while α-ʟ-fucosidases catalyse its cleavage. Two α-ʟ-fucosidases exist in the human genome. Deficiency of one of these enzymes causes the lysosomal storage disorder fucosidosis, and the enzyme is upregulated in a number of cancers. Meanwhile, the other enzyme has been shown to play a critical role in enabling the adhesion of the pathogen Helicobacter pylori to mammals. Thus, inhibition of α-ʟ-fucosidase activity is clinically relevant. In this work, the 1.6 - 2.1 Å X-ray crystal structures of α-ʟ-fucosidase inhibitors complexed with a bacterial α-ʟ-fucosidase are presented and discussed. Of the inhibitors discussed, the majority comprise 5-membered iminocyclitols, a potent yet infrequently used framework for inhibition of glycoside hydrolases, and their mode of binding to the enzyme in an E3 conformation is elaborated from crystal structures. Further, the crystallographic observation of the interaction between a 6-membered ring inhibitor comprising an aziridine moiety as an electrophilic trap and a glycoside hydrolase is reported for the first time. Finally, efforts towards the purification and crystallisation of α-ʟ-fucosidases from Homo sapiens are documented. The results reported herein may aid in the rational design of more potent inhibitors of α-ʟ-fucosidase in the future and may help direct future efforts towards the crystallisation and structure solution of the clinically important human enzymes