Triazaspiroalkenes as potential inhibitors of dihydrofolate reductase 3

CHAPTER 1. Introduction 1.1 The Overall Drug Discovery Process Drug discovery is a long term and costly process. An approved medicine requires 1224 years of research and development.1 A typical drug discovery project involves three main stages, i.e. discovery, development and commercialization (Figure 1).2, Stage 3: Commercialization Stage 1: Discovery Therapeutic Target Selection Concept Target Validation Lead Finding Stage 2: Development Lead Optimization Preclinical Development Clinical Development Regulatory Product Approval Figure 1. Three main stages in the development of a new drug.2, Drug discovery process starts with defining a therapeutics need that is to be met and a drug target is selected. A drug target can be a receptor, enzyme or any biomolecule. In the early days, the targets were usually identified from an understanding of the pathophysiology of a disease or by serendipitous findings; however, nowadays, targets are identified by scientists’ understandings of the human genomics. Once a drug target is identified, it is subjected to huge number of compounds in high throughput screening (HTS) mode.4, These compounds can be derived from natural products libraries, combinatorial synthetic libraries or virtual libraries. HTS plays an increasing and major role in the drug discovery process to identify biological probes and to provide hits for further development at an accelerated rate.6 HTS is becoming routine in any drug discovery programme; this topic will be reviewed further in the next section. Compounds that elicit a positive response will be selected as “hits”. This hit compound will be further optimised to enhance its potency as well as physiological properties such ADME properties. In contemporary drug discovery, it is recognised that multiparametric optimization should begin as early as possible in the process. According to the empirical definition of drug-likeness and lead-likeness, careful selection of the type of compounds can assist in hit generation.7 A successful stage one in the drug discovery process will result in a lead compound to be evaluated clinically.1 Stage two of drug development starts from the preclinical trial of drugs in animal models to assess drug efficacy and safety.1 Drug candidates that pass the preclinical trial will be tested on human in clinical trial. Phase I clinical trial studies the tolerance of the drug candidate in healthy volunteers; Phase II trial studies the efficacy in patients; Phase III involves large scale trials in many patients. Drug candidates that pass this series of evaluation will be filed and reviewed as New Drug Application (NDA) by the Food and Drug Administration (FDA) in the United States or regulatory bodies in other countries.1 Only the approved drugs can be marketed and used in the patients. 1.2 Anticancer Drug Discovery Cancer is an unavoidable disease especially in this modern society. The cause of death attributed to cancer has continued to increase. For example, in Singapore, death due to cancer has increased from 14.8% in year 1968-1972 to 27.1% in year 1998-2002.8 Anticancer drug discovery is full of opportunities and challenges. It has undergone significant changes over the last decade. The discovery of anticancer drug remains a highly challenging endeavour where in comparison to the development of cardiovascular drugs, investigational anticancer drugs are found to be three time less successful.9 There are three main elements, which are emphasized in current cancer drug discovery.2, 10 Firstly, the molecular targets of contemporary drug discovery reflect the understanding of scientist on the gene and pathways that are associated with cancer development.11 Drug candidates are designed based on etiological process of specific cancer. Secondly, emphasis on the innovation and improvement of technologies lead to more efficient and faster rates of anticancer drug discovery.11 Powerful technologies such as HTS and combinatorial chemical synthesis have evolved as essential techniques in need. Thirdly, the introduction of new clinical treatments reflects the success of the two above-mentioned factors. As a result, anticancer drugs are leading the way in the development of personalised molecular medicines.11 One such successful example of small molecular cancer therapeutic which was derived from the emphasis of the three key points above was Gleevec (Imatinib.)4, 12 Gleevec was discovered by the scientists of Norvatis for the treatment of chronic myeloid leukaemia through high throughput screening.4 The primary target of Gleevec is the Abelson tyrosine kinase, which is activated by a chromosomal translocation that occurs in the chronic myeloid leukaemia. N H N N O H N N N N Gleevec, This thesis focuses on the lead discovery and development of potential anticancer drugs. It deals with the synthesis of targeted small molecules and advances biological screening. Hence, in the following sections, the above mentioned points one and two will be discussed in details. In particular, HTS and structured-based dihydrofolate reductase (DHFR) inhibitors design will be highlighted. 1.3 High Throughput Screening in Drug Discovery High throughput screening (HTS) is the extensive parallel testing of a large, structurally diverse set of compounds for pharmacological activity.13, 14 Since its advent in the early 1980s, HTS has developed and improved continuously not only in the technology but also the various requirements in hit discovery. In previous years, much effort has been put on increasing the number of input for HTS. However, it is well recognized that even compounds from HTS are not always suitable for the initiation of further medicinal exploration. More often, hits generated from HTS are known to be withdrawn at the stage of clinical trial. The phrase “garbage in, garbage out” is probably appropriate to describe this phenomenon.13 Thus, this leads us to question whether the HTS paradigm has really been successful. In the following sections, the development and emerging trend of HTS will be described. This includes various components of HTS, advantages and drawbacks of HTS, as well as the future of HTS. The reviews will lead us to the answer of the question above. 1.3.1 The Magic Triangle of High Throughput Screening Traditionally, two key components of HTS have been (i) the management of large number of compounds and (ii) the miniaturization of the sample size. In the 1990s, the concept of scale has been introduced. It was believed that screening a greater number of compounds should provide more leads of improved quality.15 The compound collection consisted of the library from natural products or from organic synthesis. Later on, combinatorial chemistry was added on to create large number of compounds to feed into the HTS system.16, 17 The number of compounds is consistently increasing. As such, effectively managing the enormous number of compound is very important in HTS. Hence, a strong driving force is committed towards the development of advanced technology to aid HTS, such as robotic liquid handling system, highly sensitive detection method, and automated assay platform. Improvement on the instrumentation served as the first degree of development for HTS assay. There are three major categories of new HTS technologies, i.e. assay methods and detection (bioware), liquid handling and robotics (hardware) and process flow and information management (software).14, 18 The instrumentation for HTS should be accurate, reliable and easily amenable to automation.19 Detection method in HTS depends on the type of biochemical pathway being explored. For example, enzymatic assays routinely use colorimetric, fluorescence and radiometric methods. Colorimetric and fluorescence methods are significantly safer and cheaper than the radiometric method. Cell-based assay technologies occurred over the past decades. For example, the FLIPRTM (a fluorescence imaging plate reader integrated with liquid handling) is capable of measuring intracellular calcium mobilization in real-time. The FLIPRTM can be used for measuring ion channel function, as well as to screen for ligand for G-protein coupled receptor in HTS mode.20 Hardware implementation and software controls are equally important in HTS. The speed and accuracy in dispensing of liquid are essentially the main challenges in this area. Most automated liquid handling is achieved by using syringe pump-based or peristaltic pump-based pipettors.21 These pipettors are capable of dispensing liquid accurately in the order of microliters range. With the non-contact technologies such as syringe-solenoid and piezoelectric, low nanoliter or picoliter droplets can be dispensed.21 Miniaturization is also another essential achievement in HTS. Miniaturization is necessary in HTS as this will result in major cost reduction by increasing throughput and reducing time and money spend on material. An example of cost-benefit analysis conducted by comparing the cost of screening compounds in 96-well microplates to 9600-well plates is shown in Table 1.21 Table 1. Comparative costs of screening compounds in 96-well or 9600-well plates. (Based on a 100,000-member compound library)21 96-well microplate (Single 9600-Well plate (Triplicate screening) Screening) (in US$) (in US$) Plate costs 4,000 200 Peptide reagents 25,000 150 Enzyme productions costs 150,000 50,000 Labour costs 30,000 2,500 Total costs 209,000 5,300 Saving per assay per year - 156,000 Miniaturization in to microtiter plate format is a norm in HTS. In recent years, HTS assay has been commonly run in 96-, 384- or 1,536-well microplates.13, 22 Higher density plate, such as 3,456-well microplate is also used, but its usage is still not popular as the liquid dispensing technology is still not fully automated with 3,456well microplate yet. It is also important to note that, ultra-low volume reactions will affect the surface-to-volume ratio, which will lead to the decrease of sensitivity on reagent absorption and stability. Statistic showed that 384-well microplate is currently the most acquired, with more than 60% of the campaigns in 2008 predicted to have been run in this format.13 Most of the bioassay could be adapted to 384-well microplate without problem. The usual working volume for 384-well microplate ranges from 30 to 100 µL. In Phase one HTS, the equipment focused on speed and quantity where contributions were made greatly to increase the number of libraries as well as the vast development of HTS instrumentations. However, the efforts devoted to expand the size of libraries for screening was not a fruitful strategy. More often, hits generated from HTS were withdrawn at the stage of clinical trial. Late stage attrition was extremely costly and therefore, such failures must be kept to minimum. Clearly, an understanding of the reason for drug attrition from clinical trial will allow the scientists to tackle this problem better. The reasons for drug attrition changed with time. In the early 1990s, the attrition of these compounds was always associated with poor pharmacokinetic properties and low bioavailability, such as poor ADME (absorption, distribution, metabolism, and excretion) properties and poor solubility. As such, ADME assay was initiated at the time of screening.11, 23, 24 The major cause of drug attrition in 2000 was associated with drug safety and efficacy. 23 The safety and efficacy testing in in vivo models did not guarantee the success of clinical trial.25 Thus, the pressing need for more predictive animal model had to be addressed. Besides, carefully selecting the targets could also minimize the late stage drug attrition. Unavoidably, the lower success could be also related to more stringent and demanding regulatory requirements.23, 25 Understanding of the reason for attrition also has led to a new phase in HTS. The awareness of generating better quality hits from high quality libraries increased. Successful lead generation requires a good hit as a starting point. In order to be able to produce high quality hits from screening libraries, it is more important to generate high quality libraries.26 Thus, recently there has been a trend of moving away from screening of huge and diverse “random” libraries towards more focused drug-like libraries.27 More stringent criteria have been applied in assessing the druggability of a library. One of them is the “Lipinski’s Rule of Five”.7, 28 According to this set of rules, an orally active drug should preferably have a molecular weight of less than 500, not more than hydrogen bond donors, not more than 10 hydrogen bond acceptors, and a logP (partition coefficient) value of less than 5. Considering these criteria as early as possible in the drug development process will aid in designing higher quality libraries, and, hence, reducing the attrition rate at the later phase of drug discovery. The concept of “synthesize and test” is unfavourable due to its low hits rate. Instead, nowadays, small and focused compound collections are fed into HTS. A few approaches have been applied in the synthesis of small and focused libraries as discussed below: (i) Guided by target26 A focused library can be built based on the information gathered for a specific biological target.29 Owing to the advances and improvements in analytical techniques, many of the crystal structures of biological targets are solved by either X-ray crystallography or NMR. Computational chemistry is also vital for formulating library proposal that fits the target structure. An understanding of the mechanism of ligandtarget interaction will assist in designing focused libraries of compounds that embrace the appropriate pharmacophores. (ii) Privileged structures or motifs26 The privileged structures or ligand-motif based library design is especially important for target with very limited or no biostructural information. The elements of known biological active molecules are used for building the libraries which consist these privileged motifs. These motifs can be potent against the whole target families; hence selectivity issue has to be addressed as early as possible. (iii) “Cherry picking” from virtual space26, 28, 30-32 Virtual screening has emerged as a reliable, cost-effective and time-saving technique for the discovery of lead compounds. Virtual screening tools search for possible hits compound from the known active structures.33 There are three main categories in virtual screening, specifically virtual filtering, virtual profiling and virtual screening. These categories are meant to eliminate compounds which are less appealing as potential lead compounds. The issues of pharmacokinetic properties or drug-likeness of the compounds can also be addressed prior to synthesis, and the non-compatible compounds can be eliminated. This has allowed a rapid evaluation of the vast chemical space. Hence, the highest-ranking molecules are more feasible in terms of druggability and synthetic-friendliness. In a small and focused library, the chances of finding hits are higher than a large and random library as the structural diversity is already positively biased for a given problem.15, 34 Small but carefully designed libraries are even better representation of the chemical space than the large and random libraries.10 Knowledge-based small libraries allow direct comparison of all libraries members, which are very essential in formulating the structure-activity relationship of a ligand-target interaction. Thus, for a HTS, rapid feedback screening of small and focused library is more realistic than the gigantic random library. A comparison of small but focused library to large but random library is shown in Table 2. Table 2. Comparison of small but focused libraries vs. large but random libraries.34 Small but focused libraries Large but random libraries Only limited number of library members Large number of different library members  Only limited diversity possible  Much larger diversity Locally separated library synthesis possible  no extra tagging required Only traditional split-mix-synthesis  Additional tagging necessary (marker, coding strand) -6 Micromol (10 ) level of each library member Only picomol (10-12) level of each library member available available  Qualitative and quantitative screening possible  Direct comparison of all library members possible  Only qualitative “yes” or “no” information can be obtained.  Selected hits have to be resynthesised for further analysis. Thus, the emerging trend of HTS is the integration of three components reviewed above: quality, quantity and cost-efficiency. The three key factors constitute the principles of “the magic triangle of HTS” (Figure 2) and optimization of each factor is the goal in successful HTS.35 The key success factors for modern HTS are quality, time and costs. All three factors are closely linked. Changes in anyone of these will influence the other factors as well. Finding a right balance between the three are of utmost importance for the success of HTS in drug discovery of the future. Time - time/well - wells/day -screens/year -project time HTS - few false positives - few false negatives - Signal/ noise - validated binders Quality Costs -reagents -consumables -instrumentation -personnel Figure 2. The magic triangle of HTS 1.3.2 High Throughput Screening for Enzyme Assay Enzymes offer unique opportunities for drug design that are not available to cell surface receptors, hormone receptors, ion channels or DNA. According to American Association for the Advancement of Science, enzymes form the second largest group (22%) of therapeutic target classes, after receptors.26 The FDA Orange Book documents that out of 1278 approved therapeutic chemical entities in the United States, 317 drugs are enzymes inhibitors. 36 This accounts for almost 25% of the drugs in use. These drugs inhibit 71 enzymes, including 48 human, 13 bacterial, viral, fungal, and one protozoal enzyme.36 This demonstrates the importance of enzymes as drug targets. 10 incubated at 37 °C for 24 hours in % CO2 to allow the cells to adhere to the surface. The medium of the plates was then replaced with mL of medium containing test compounds. Cells without compound served as control. After an incubation period of 72 hours, both adherent and floating cells were collected into a 15 mL falcon tube and then pelleted by centrifugation. Cell pellets were washed with PBS. Cells (in a concentration of 106 cells/mL) were fixed at ice-cold 70% ethanol for at least 18 hours in -20 °C freezer. 9.5.2 Staining of Cells PI staining buffer (4 mL) was prepared fresh prior to use from 200 µL of 50 µg/mL PI in mili-Q water, mL of mg/mL RNase A, 80 µL of 0.1% Triton X-100 in PBS and the rest were PBS (1.72 mL) and protected from light. Cells were spun down at 10K rpm for 60 seconds. Supernatant was removed and cells were washed with cold PBS. Cells were resuspended in 1mL PI staining buffer per 106 cells. Cells were incubated at 37 °C for 15 minutes, followed by hour incubation on ice before flow cytometry analysis. 9.5.3 Flow Cytometry Flow cytometry was performed on Epics Altra (Beckmann Coulter) Flow Cytometer. PMT4 filter was selected to capture PI, with an excitation at 488 nm and an emission at 600 nm. 10,000 events of each samples were analysed. Software Winmdi was used for data analysis. One way ANOVA with post test Tukey test were performed using GraftPad Software Prism 5.0. Mean of p[...]... fluorogenic substrates as solid support .38 Miniaturization of the enzyme-based assays in the form of microarray has no doubt bolstered the screening for inhibitors, biocatalysts or enzyme fingerprinting.41 Applications for microarray assays include examples such as fingerprinting of proteases in peptide microarrays and profiling of kinases.42 Adaptation of enzyme-based assay in HTS mode has contributed to... actively effluxed out of the cells, making it less toxic to the cancer cells.86 An understanding of the MTX resistance mechanism has also led to revolution in the design of DHFR inhibitors These studies led to the development of two distinct classes of DHFR inhibitors, i.e classical and non-classical DHFR inhibitors Classical DHFR inhibitors utilized the same transportation mode as MTX, in which they... outside the scope of this report to describe each and every one here Thus, the following review will be based on a particular enzyme of our interest The enzyme of interest here is dihydrofolate reductase (DHFR) DHFR catalyses the conversion of dihydrofolate to tetrahydrofolate It is an important enzyme in the folate pathway The inhibition of DHFR will eventually result in the termination of DNA replication... technology, enzyme-based assay in HTS mode will result in more diverse and better screening methods 11 1 .3. 3 High Throughput Screening for Dihydrofolate Reductase (DHFR) Inhibitors HTS has become a mainstay in pharmaceutical research It is clear that, with proper library design, HTS is a very powerful tool in hit and lead identification Applications of HTS in a variety of enzyme-based assays have been... Alimta, 33 31 The distinction between classical and non-classical DHFR inhibitors has somewhat blurred with the introduction of non-polyglutamylatable classical DHFR inhibitors Low polyglutamylation is one of the common resistance mechanisms for MTX treatment As follows, it is logical for the design of inhibitor which uses the RFC transporter but not polyglutamylated These non-polyglutamylatable inhibitors. .. library 4 consisted of analogues of hits identified in library 1 12 Table 3 Inhibitors of DHFR identified in the libraries by Wyss et al. 43 Library Selection procedure Size Inhibitors n (%) 1 Top-score in docking , structure-based 252 54 (21) 2 Low score or no docking solution, structure-based 269 4 (1) 3 Diversity-based 501 17 (3) 4 Analogues of hits identified in library 1 37 0 90 (24) On preliminary screening,... series as a model, a series of classical 2,4diaminofuro[2 ,3- d]pyrimidines were synthesised In the series, AAG120-292 -3 (32 ) stood out as a more potent human DHFR inhibitor than MTX, with an IC50 value of 0.22 µM.64 This compound has also demonstrated promising anticancer properties against more than 30 cancer cell lines in the NCI solid tumor panel.64 COOH NH2 HN N H2N O COOH O N AAG120-292 -3, 32 Pemetrexed... COOH Methotrexate, MTX, 27 MTX is used clinically for the treatment of various forms of cancers, such as lymphoma, germ cell tumors, breast cancer and head and neck cancer.68, 73 Low dose of MTX is also used for the treatment of autoimmune diseases such as rheumatoid arthritis and psoriasis and the prevention of graft-vs-host desease. 73 Besides, MTX is also used in combination with misoprostol to terminate... the expression of RFC are thus not sensitive to MTX.80, 82 (ii) Decreased retention as a consequence of lack of polyglutamate function.82 (iii) An increased level of a lysosomal enzyme, γ-glutamyl hydrolase, that hydrolyses MTX polyglutamates.82 (iv) Increased in DHFR MTX binds to DHFR in direct proportion manner An increased in DHFR due to gene amplification gradually increased the doses of MTX.82 28... ring of its paminobenzoyl. 63 Contradicting to DHF, the N1 of MTX is protonated and hydrogen bonded to one of the carboxylate oxygen of Asp26 residue N8 of MTX is hydrogen bonded to the water molecule 2 53 4-NH2 is bonded with two amino acid residues, namely Leu4 and Ala97 The only similarity to that of DHF is, the 2-NH2 of MTX forms two hydrogen bonding with water molecule 201 and with one of the 23 carboxylate . for microarray assays include examples such as fingerprinting of proteases in peptide microarrays and profiling of kinases. 42 Adaptation of enzyme-based assay in HTS mode has contributed. review will be based on a particular enzyme of our interest. The enzyme of interest here is dihydrofolate reductase (DHFR). DHFR catalyses the conversion of dihydrofolate to tetrahydrofolate. It. enzyme-based assay in HTS mode will result in more diverse and better screening methods. 12 1 .3. 3 High Throughput Screening for Dihydrofolate Reductase (DHFR) Inhibitors HTS has become