Fragment Based Drug Design: From Experimental to Computational Approaches

Transcription

1 Current Medicinal Chemistry, 2012, 19,????-???? 1 Fragment Based Drug Design: From Experimental to Computational Approaches A. Kumar, A. Voet and K.Y.J. Zhang* Zhang Initiative Research Unit, Advanced Science Institute, RIKE, 2-1 Hirosawa, Wako, Saitama , Japan Abstract: Fragment based drug design has emerged as an effective alternative to high throughput screening for the identification of lead compounds in drug discovery in the past fifteen years. Fragment based screening and optimization methods have achieved credible success in many drug discovery projects with one approved drug and many more compounds in clinical trials. The fragment based drug design starts with the identification of fragments or low molecular weight compounds that generally bind with weak affinity to the target of interest. The fragments that form high quality interactions are then optimized to lead compounds with high affinity and selectivity. The weak affinity of fragments for their target requires the use of biophysical techniques such as nuclear magnetic resonance, X-ray crystallography or surface plasmon resonance to identify hits. These techniques are very sensitive and some of them provide detailed protein fragment interaction information that is important for fragment to lead optimization. Despite the huge advances in technology in the past years, experimental methods of fragment screening suffer several challenges such as low throughput, high cost of instruments and experiments, high protein and fragment concentration requirements. To address challenges posed by experimental screening approaches, computational methods were developed that play an important role in fragment library design, fragment screening and optimization of initial fragment hits. The computational approaches of fragment screening and optimization are most useful when they are used in combination with experimental approaches. The use of virtual fragment based screening in combination with experimental methods has fostered the application of fragment based drug design to important biological targets including protein-protein interactions and membrane proteins such as GPCRs. This review provides an overview of experimental and computational screening approaches used in fragment based drug discovery with an emphasis on recent successes achieved in discovering potent lead molecules using these approaches. Keywords: Computational fragment based drug design, de novo design, fragment based drug design, fragment growing, fragment linking, ligand efficiency, molecular docking, scaffold based drug design, protein-protein interactions, small molecule protein-protein interaction inhibitors. 1. ITRDUCTI Drug discovery is a highly interdisciplinary endeavor that involves a multitude of specialty areas and can be characterized in multiple steps. Generally, it starts with target identification and validation, followed by lead identification and optimization, then progressing to preclinical studies on animals and ends up with clinical trials in humans. This review only covers the lead identification and optimization aspect of drug discovery, specifically focusing on a relatively new technique of fragmentbased drug design covering both experimental and computational approaches. The identification and optimization of lead compounds is critical in the drug discovery process and plentitude of methods, such as high-throughput screening (HTS)[1], QSAR[2], structurebased drug design [3], combinatorial chemistry [4-6], high content screening [7-9] have been developed to identify novel and potent chemical compounds against biological targets and to optimize them into leads. The hits identified from HTS screens of large corporate compound collections especially those of combinatorial chemistry origin tend to be large albeit potent. The chemical optimization of those compounds has led to some high profile failures of lead series. These have been attributed to the reduced productivity of pharmaceutical industry [10]. Partially motivated by searching for an answer to this question, the Lipinski s Rule of five was proposed that have highlighted some important properties that good lead compounds should possess [11]. ne of the factors identified is the correlation of high molecular weight (MW) with poor solubility. If one starts with very potent but high molecular weight lead compounds, optimization may result in molecules with even higher molecular weight with reduced solubility and this is generally associated with poor pharmacokinetic (PK) properties. To address this problem, a fragment-based drug design (FBDD) approach was proposed [12]. In the past fifteen years, FBDD has become an established strategy to discover novel chemical entities in both industry and academia [13]. The FBDD approach represents *Address correspondence to this author at the Zhang Initiative Research Unit, Advanced Science Institute, RIKE, 2-1 Hirosawa, Wako, Saitama , Japan; Tel: ; Fax: ; kamzhang@riken.jp a rapid, resource efficient and productive route to the identification of novel hits in the early phase of drug discovery process. This method was proposed by Fesik and co-workers in 1996 at Abbott Laboratories [12]. n a historical note, the FBDD concept was also proposed earlier in 1990 by Hol and co-workers [14]. FBDD approach focuses on the identification of compounds low in molecular-weight and chemical complexity, which target subpockets within the target binding site. These fragment hits are expected to be more suitable starting points for hit to lead optimization due to their reduced complexity, which leaves more freedom for multidimensional property optimization of the fragment hits. The optimization of fragments is an iterative process where the potency of the initial fragment is improved in each step by adding functional groups, or linking two independent fragments together. FBDD is hallmarked by three advantages compared with a conventional HTS drug discovery approach [10, 15]. The first advantage is that the chemical diversity space is better covered with FBDD. In FBDD, smaller fragment libraries are required to probe chemical space more effectively while generating the same amount of information as generated by screening a huge number of compounds. A theoretical analysis by Reymond and coworkers [16, 17] suggests that each fragment represents enormous number of bigger compounds. Their analysis suggests that each additional heavy atom added to a molecule increases its chemical space by approximately eight folds. Also, Roughley and Hubbard [18] analyzed this theory in the real world and found that the chemical space is indeed more efficiently sampled with fragments than with larger molecules in the case of Hsp90. The second advantage is that screening a fragment library achieves higher hit rates as compared to conventional HTS. This may be attributed to the fact that a fragment molecule can bind to various subsites of a target in many ways. Large molecules, on the other hand, contain more functional groups that may present more steric hindrance or electrostatic clashes than the fragment molecule in a binding site. These molecular incompatibilities prevent most large molecules from being accommodated in the protein pockets [15, 19]. Finally, the third advantage is that compounds optimized from fragments exhibit high binding efficiency per atom as compared to compounds optimized from HTS. FBDD has now evolved into a very successful drug discovery strategy since its conception in /12 $ Bentham Science Publishers

2 2 Current Medicinal Chemistry, 2012 Vol. 19, o. 1 Kumar et al. [12]. There is already an FDA approved drug [20] and at least 10 more compounds in clinical trials which originate from fragment screening and optimization approaches [21]. In recent years, FBDD has gained an important place as a screening tool for novel inhibitors. This is reflected in the number of publications on FBDD each year. In the year 2011 alone over 125 papers (of which 17 are reviews) were published. Furthermore in the last decade at least 5 books were published dealing with methods and successes in FBDD [19, 22-25]. There are two very good blogs dedicated to discussion about FBDD highlighting its importance [ practicalfragments.blogspot.com/ and A recent review by Hajduk and coworkers [26] listed 19 pharmaceutical companies that are using FBDD in their drug discovery efforts. Another list compiled on practicalfragments.blogspot.com/ blog included 44 companies involved in FBDD related projects. Realizing the significance of FBDD in drug discovery, many approaches were developed that either complement FBDD or improve FBDD to realize its full potential. In order to overcome its challenge of not being able to screen fragments with biochemical assays, also motivated by the search for chemical scaffolds that are well-known reoccurring motifs in marketed drugs, a scaffold-based drug design (SBDD) approach was proposed [27]. Instead of using basic chemical building blocks (MW < 150 Da) as fragments in classical FBDD, the SBDD method uses chemical scaffolds that are significantly larger (MW Da). These larger scaffold-like compounds are richer in functional groups that could form key interactions with the target protein - thus providing a more robust anchoring point for subsequent chemical optimization through substitution. There are several significant differences between classical FBDD and SBDD. First, the compounds used for SBDD are significantly larger with an average molecular weight of about 250 Da. Consequently, the size of the scaffold library is bigger with about 20,000 compounds instead of for FBDD. Secondly, these scaffold-like compounds could bind to target protein at low affinity and are detectable by biochemical assays. To reduce the high false positive rate associated with high compound concentration and low binding affinity, only compounds that show activity against multiple members in the same protein family are selected as hits. Thirdly, biophysical methods such as X-ray crystallography are used as a secondary filter instead of primary screen as in FBDD. Finally, only scaffolds with binding modes that are tolerant to small substitutions are selected for further optimization. This leads to more predictable SAR and more efficient chemical optimization. Complimentary to the classical experimental screening approach, is the computational fragment based drug design. Similar to the classical rational drug design strategies, computational tools are successfully used in different steps of FBDD from fragment library design to identification and optimization of fragment hits. Although computational fragment screening is in preliminary stage and yet to realize its full potential but this approach has shown early promises to become a widely used tool to complement experimental methods of fragment screening. 2. FRAGMET BASED DRUG DESIG: DEFIITI AD CCEPT 2.1. Fragment Definition Fragments are defined as low molecular weight, moderately lipophilic, highly soluble organic molecules. Fragments typically bind to their target protein with low affinity, generally in the μm to mm range, and can be grown, merged or linked with another fragment to improve the potency. In analogy to Lipinski s Rule of five [11], a Rule of three was proposed to define fragments by Congreve et al.[28]. This rule was derived after carefully analyzing hits from various fragment screens. The Rule of three states that fragments should have a molecular weight < 300 Da, clogp 3, number of hydrogen bond donors 3 and number of hydrogen bond acceptors 3. Their analysis also indicates that using additional filters, such as the number of rotatable bonds 3 and the polar surface area (PSA) 60 Å 2, would give more desirable fragmentlike compounds. Although the Rule of three is widely accepted, the guidelines vary according to the different interpretations by different research teams. Most of these modifications to the rule are particularly related with molecular weight. For example, researchers at Plexxikon screened a library of scaffold-like compounds with molecular weight ranges from 125 Da to 350 Da to identify hits for variety of drug targets including PPARs, PDE-4 and BRAF V600E [27, 29-31] Ligand Efficiency and ther Efficiency Indices Fragments generally have a weak binding affinity for their protein targets; thus typically the affinity needs to be optimized by adding new functional groups or by linking two hit fragments bound in adjacent pockets. An important question that needs to be addressed here is how to select the best fragment for optimization? as there are many fragment hits to start with. Ligand efficiency (LE) [32, 33] is a widely used concept and is generally used for comparing different hit fragments to guide the lead generation and optimization process. LE was proposed by Hopkins et al. [32] based on the concept of the Andrew binding energy [34] and the practice of using experimental binding affinity [33]. Ligand efficiency is the free energy of binding divided by the number of heavy (non-hydrogen) atoms and is defined by the following equation: G RT ln( K d ) RT ln( IC50 ) LE = = HAC HAC HAC which G is the binding free energy, R is the gas constant, T is the absolute temperature, K d, is the dissociation binding constant, IC 50 is the concentration of inhibitor required to inhibit 50% activity of the enzyme, and HAC is the number of heavy atoms. The LE is considered a very important parameter in FBDD. A fragment with a high LE is preferred for optimization because it provides an opportunity to obtain highly active compounds, without increasing the molecular weight too much. The commonly used lower acceptable threshold value for LE is 0.3 kcal/mol per heavy atom, which is derived from a hypothetical drug molecule with a high K d value of 10nM and a molecular weight of 500 Da (the upper molecular weight limit according to Lipinski s rule) and contains approximately 36 heavy atoms. A retrospective analysis of highly optimized inhibitors by Hajduk et al. [35] also indicated a linear relationship between the molecular weight and the potency during optimization of fragment hits. With each heavy atom added to the initial fragment, the binding energy is increased by 0.3 kcal/mol. Therefore, fragments with high LE are preferred over the fragments with lower LE for optimization. Although LE is a widely used metric, it also has some drawbacks. ne of them is that it does not display similar behavior with ligands of different sizes. LE works very well with smaller compounds, but is relatively insensitive to compounds with a high molecular weight. To overcome the shortcomings associated with LE, a number of other metrics have been proposed by various groups in recent years for evaluating fragment quality. Some of these metrics are listed in (Table 1) along with their definitions. These include closely related metrics, such as percent efficiency index (PEI) and binding efficiency index (BEI), developed by researchers at the Abbott Laboratories [36]. The PEI is the percentage of inhibition at a given concentration of compound, divided by the molecular weight. The BEI is the negative logarithm of the inhibition constant divided by the molecular weight. The

3 Fragment Based Drug Design Current Medicinal Chemistry, 2012 Vol. 19, o. 1 3 Table 1. Ligand Efficiency Indices ame Abbreviation Definition Reference Ligand Efficiency Percent Efficiency Index Binding Efficiency Index Surface Binding Efficiency Index LE PEI BEI SEI G RT ln( K d ) RT ln( IC50 ) LE = = Hopkins et al. (2004) HAC HAC HAC % inhibition at a givenconcentration of compound Abad-Zapatero and Metz MW (2005) pk i ( or pk d ) Abad-Zapatero and Metz MW (2005) pk i ( or pk d ) Abad-Zapatero and Metz PSA (2005) Fit Quality Score FQ, LE ( ) 2 3 ( HA) ( HA) ( HA) Reynolds et al. (2007) Percent Ligand Efficiency %LE, LE ( ) log HA rita et al. (2009) where = ( golden ratio) Ligand Lipophilicity Efficiency LLE pk i ( or IC50 ) c log P Leeson and Springthorpe (2007) Ligand Lipophilicity Efficiency at Astex Therapeutics LLE AT * G 0.11 HA * where G = G G lipo RT ln( IC 50 ) RT ln( P) Mortenson and Murray (2011) Ligand-Efficiency-Dependent Lipophilicity LELP log P LE Keseru and Makara (2009) Group Efficiency GE G = HA where G = G ( B) G ( A) and HA = HA( B) HA( A b a ) when a functional group added to a molecule Ato form molecule B Verdonk and Rees (2008) Kinetic Efficiency KE, t 1 2 = HA HA where is relaxation constant and t 1 2 is half life for dissociation Holdgate and Gill (2011) surface-binding efficiency index (SEI), calculated by dividing the pki by the polar surface area (PSA) was also proposed as an FBDD metric [36]. Some metrics, such as the fit quality score (FQ)[37] and percent ligand efficiency (%LE)[38], are size independent and allow the comparison of various fragments irrespective to their molecular weight. To address the challenges of high lipophilicity, which is one of the main causes of increased attrition rate in the clinical trials, Leeson and Springthorpe proposed the ligand lipophilicity efficiency (LLE) index, which subtracts logp from log 10 IC 50 [39]. Mortenson and Murray [40] from Astex Therapeutics proposed a similar metric Ligand Lipophilicity Efficiency AT Astex Therapeutics (LLE AT ) to account for molecule size along with lipophilicity. A related metric proposed by Keseru and Makara [41] is Ligand-Efficiency-Dependent Lipophilicity (LELP), which is simply logp/le. Verdonk and Rees [42] proposed the concept of Group Efficiency (GE) that estimates the binding efficiency of groups added to an existing lead. GE is considered analogous to LE where the change in binding energy is divided by the change in the number of heavy atoms. Recently, Holdgate and Gill [43] from AstraZeneca proposed the Kinetic Efficiency (KE) that is a metric to address the kinetics of ligand binding to a protein. The KE is devised to complement the LE and other metrics and is suitable for later stages of fragment optimization. However, KE usage is limited to the earlier stages because of the rapid kinetics involved with small low molecular weight fragments. 3. METHDS FR FRAGMET SCREEIG AD PTIMIZATI The process of FBDD consists of two steps: (a) the identification of the initial fragment hit with a weak binding affinity and (b) the optimization of the fragment hit into a high affinity lead compound Identification of Fragment Hit The first step in FBDD is the identification of the initial fragment hit that has sufficiently high LE and that can be used as an anchor for the development of large and potent lead compounds. Fragments are smaller in size, making few interactions with the protein and displaying low binding affinity. This makes them particularly difficult to be detected by standard biochemical assays. Instead, biophysical methods such as nuclear magnetic resonance (MR) and X-ray crystallography were commonly used to identify these low molecular weight compounds. In the past decade, there has been a considerable effort in the development of screening technologies for fragment detection and many new technologies were developed including native mass spectrometry, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), capillary electrophoresis, weak affinity chromatography, biolayer interferometry and ultra-filteration. The development efforts were

4 4 Current Medicinal Chemistry, 2012 Vol. 19, o. 1 Kumar et al. focused on two types of fragment screening methods: Methods that detect binding of fragments and methods that reveal binding interactions. Most of the fragment screening technologies like ligand-detected MR, SPR, ITC etc. belong to the former group and offer various degrees of binding affinity information. X-ray crystallography and protein-detected MR are the only two fragment screening methodologies that give protein ligand interaction information. The following paragraphs give an overview of these screening technologies. However, for a more thorough understanding of these fragment screening methodologies, the reader is referred to other in-depth reviews describing the advantages and disadvantages, which have been published in recent years [15, 21, 44-46] uclear Magnetic Resonance (MR) Spectroscopy MR spectroscopy is a method that exploits the magnetic properties of certain atomic nuclei to provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules in which they are contained. MR spectroscopy is widely used in fragment screening and was first described by Fesik and co-workers at Abbott Laboratories in their work SAR by MR [12], where they first demonstrated the feasibility and first practical success of FBDD. In their approach, they observed changes in protein amide chemical shifts obtained from 2D-MR spectra (specifically 1 H/ 15 HSQC) in the presence and absence of fragments. This screening resulted in two noncompetitive hits, which were first individually optimized into moderate affinity binders with a K d value of 2 and 100 M. In the last step, a high affinity binder with a K d value of 19 nm was obtained by linking the two moderate affinity binders. This type of fragment screening is known as protein-detected MR and is one of the two MR-based fragment screening approaches. The other MR-based fragment screening approach is ligand-detected MR in which changes in MR properties (such as uclear verhouser Effect (E), relaxation rates, and magnetization transfer) of fragment are detected instead of the target protein. The proteindetected MR can detect nm to mm interactions and yields precise information about the binding site and protein ligand interaction. This approach however is restricted to smaller proteins (< 50kDa) and requires large quantities (about mg) of isotopically labeled protein with a high solubility [47, 48]. To overcome the challenges associated with protein-detected MR, ligand-detected MR methods were developed by several groups that rely on changes in fragment signals while binding to the target protein [48]. Ligand-detected MR methods do not provide information about the ligand binding site and additional experiments need to be performed to obtain this information. The most commonly used ligand-detected MR technologies are Saturation Transfer Difference-MR (STD-MR)[49, 50] and Water Ligand ptimized Gradient Spectroscopy (Water-LGSY)[51, 52]. Both of these methods measure the 1 H MR signals of fragments that differ in their relaxation properties, signs and intensity for bound and unbound fragments. In STD-MR, the target protein becomes saturated after the excitation of selective hydrogens. Due to spin diffusion the saturation can spread over the hydrogens across target protein and onto the bound fragments, whereas unbound fragments remain unaffected. The 1 H MR difference spectrum is then recorded between on- and off-resonance excitation of the protein which results in positive signals for bound fragments. Water- LGSY is one of most sensitive ligand-detected MR technique that is based on the theory that binding of fragment displaces water from the binding site. Water-LGSY exploits the intermolecular magnetization transfer from bulk water to the protein binding site and onto the bound ligands. ther reported ligand-detected MR methods include FAXS (Fluorine chemical shift Anisotropy and exchange for Screening) method [53] and TIS (Target Immobilized MR Screening) method [54, 55]. FAXS is 19 F screening technique based on 19 F detection and allows the determination of K d and IC 50 of the identified binding ligands. A recent paper by Jordan et al. [56] demonstrates the practical application of FAXS in FBDD. In the TIS fragment screen method, a mixture of fragments is passed through a resin with immobilized target protein and a reference in an automated process. A 1D 1 H MR spectrum is recorded for each mixture of fragments while the fragment binding is determined by measuring the reduction in their MR amplitudes. The reference serves to cancel out the non-specific binding of fragments to protein surfaces. MR based fragment screening is now routinely used in various FBDD campaigns within the pharmaceutical companies like Abbott Laboratories, Merck, Vernalis, Astex Therapeutics and Evotec and many academic laboratories X-ray Crystallography X-ray crystallography is a commonly used method in structural biology. It can be applied to very large proteins, as well as other biopolymers, and provides very high-resolution structural data. Compounds with affinity for a pocket can be soaked into the crystallized protein and after diffraction the compound will be visible in the pocket as an electron density cloud. It has become one of the preferred methods for common structure based drug discovery efforts [57, 58]. X-ray crystallography however can also play an important role in the identification of hit fragments. During a crystallographic screening experiment, the investigated fragments will be soaked into the crystal. To speed up the process and reduce the costs, the screened fragments are usually pooled into cocktails containing (usually 10) different fragments. After solving the crystal structures of the receptor protein, one can easily identify bound fragments in the protein pockets [59]. There are several companies who used the cocktail-soaking method in their drug discovery programs. Astex Therapeutics identified fragments for the cyclin dependent kinase (CDK) 2 that resulted in the design of AT7519 [60], as well as fragments that evolved into the AT9283 auroraa kinase inhibitor [61]. Both of them are currently in clinical trials for cancer therapy. Furthermore the same company used this method to identify a urokinase inhibitor which exhibits a promising PK profile [62]. SGX (Eli Lilly) reported allosteric inhibitors for the Hepatitis C virus S5b RA polymerase [63], as well as the design of a JAK2 over JAK3 specific kinase inhibitor, after X-ray crystallography based screening and structure based optimization of the fragments [64]. Researchers at decde Biostructures Inc. developed DG-051, a leukotriene A4 hydrolase inhibitor, currently in Phase 2 clinical trials, after screening 1300 fragments using this crystallographic method [65]. While previous results clearly indicate the applicability of X- ray based fragment screening methods, there are some drawbacks associated with it. It is essential to be able to make a crystal of the target protein, consequently protein targets that haven t been crystallized are excluded from this procedure. Furthermore X-ray based crystallography cannot be used to determine the affinity of the bound fragments, for this purpose a secondary method should be employed. The major advantage however is the presence of a high resolution image of the complex, which can be directly used for structure based drug discovery efforts [58]. X-ray crystallography also plays an important role during the optimization phases of fragments (see section 3.2). nce the crystal structures with the fragments have been solved, the structural insights can be employed to evolve the fragments into potent druglike molecules. By exploiting the structural insights of the fragment bound to the receptor, fragments can be grown into larger molecules. Also linking simultaneously bound fragments can be facilitated by including the bound fragment geometry during the design of a linked molecule.

5 Fragment Based Drug Design Current Medicinal Chemistry, 2012 Vol. 19, o Surface Plasmon Resonance SPR is a generally used technique to study biomolecular interactions revealing both kinetic as well as binding affinity information. SPR is a highly sensitive technique and is becoming increasingly popular as a fast and cost effective primary screening technology to identify fragment hits. In most cases of SPR-based experiments, the target protein is immobilized on the chip (generally a gold coated glass slide) and fragments are passed through it. The binding of molecules to the target protein, which is immobilized on the gold layer, causes a change in the absorbance spectrum of the reflected light related to a change in the medium absorbed on the gold layer. These changes are related to the mass of fragment and protein and are efficiently measured by an SPR instrument [66]. Initial SPR based biosensors were not sensitive enough to measure interactions of small molecules and were limited to only macromolecules. But with recent technical advances, SPR biosensors are capable of detecting weak binding of fragments to the target protein and are suitable for fragment screening [67]. SPR was successfully used in various fragment screening projects to identify potent fragment hits against a number of protein targets including BACE-1 [68-70], Pim-1 [71], MMP-12 [72], HIV-1 reverse transcriptase [73], HIV-1 protease [74], carbonic anhydrase II [74, 75], human serum albumin [74], thrombin [74], chymase [76] and CCR5 [77]. In one of these screening projects, Xiang et al. [71] used SPR followed by a biochemical assay to screen a library of 1800 fragments at 75 μm concentration in order to identify Pim- 1 inhibitors. ne fragment hit with a benzofuran core that initially displayed IC 50 of 8.5 μm was optimized to 1nM by adding an aminocyclohexanylamino group at the 7-position of the benzofuran core. SPR has been also used by de Kloe et al. [78] to identify hotspots regions for small molecule binding in the protein. The researchers deconstructed potent ligands for nicotinic acetylcholine binding protein (AChBP) containing quinuclidine core into 20 fragments. The binding of these fragments was evaluated by an SPR biosensor assay and revealed LE hotspots regions that can be used to identify promising hits in a fragment screening campaign Biolayer Interferometry Biolayer interferometry (BLI) is a recent technique which measures changes in the interference pattern of light between the sensor and the solution, caused by fragment binding to an immobilized target protein on the surface of the sensor. In a recent paper by Wartchow et al. [79] its fragment screening capabilities are demonstrated for three proteins (Bcl-2, JK1, and eif4e) and compared with those of other fragment screening methods Isothermal Titration Calorimetry ITC is a thermodynamic technique that measure the heat released or absorbed during a biomolecular binding event. ITC allows the accurate determination of thermodynamic properties like binding constants (K B ), reaction stoichiometry (n), enthalpy ( H) and entropy ( S) in a single experiment [80, 81]. ITC is used by some researchers as a fragment screening tool and it measures the heat released when a fragment binds to a protein. Although ITC is a powerful screening tool, it is low throughput in nature and requires higher protein concentration than other screening approaches. Though with the use of recent technology like the AutoITC 200 ( samples can be processed in a day but still ITC is better suited for secondary screenings rather than primary fragment screening Mass Spectrometry Mass spectrometry is an analytical technique in which the gaseous ionic state is studied by transferring the analytes from the condensed phase to the gas phase followed by their ionization. Mass spectrometry then measures the mass-to-charge ratio in gas phase ionized molecule to detect its molecular weight. These days, mass spectrometry can be also used to effectively detect fragments binding to protein as it is high-throughput in nature and consumes little amount of sample. Moreover, fragment mixtures can be used and the stoichiometric information and dissociation constants can be determined. There are two types of approaches: (a) Electrospray ionization mass spectrometry (ESI-MS)[82] detects the covalently bound fragments; (b) on-covalent electrospray ionization mass spectrometry (C-ESI-MS), or simply native mass spectrometry [83], can detect fragments that bind non-covalently to the target protein with K d upto mm range. A French company ovalix ( has demonstrated the practical application of C-ESI-MS. They screened a fragment library of about 350 compounds against Hsp90 which resulted in 40 fragments binding to Hsp90 [83] Weak Affinity Chromatography Duong-Thi et al. [84] recently proposed weak affinity chromatography (WAC) as an alternative to other fragment screening methods. They used WAC to screen fragments against trypsin and thrombin. In WAC, the screened fragments are passed through a chromatography column with immobilized target protein. Fragments having affinity for protein stay in the columns and are later detected with either UV spectrometry or mass spectrometry. The current implementation of this technique allows the detection of fragments in the 1mM to 10μM range, with very low consumption of fragments and protein. Although WAC is a new technique and needs to be developed further for more diverse targets and larger fragment libraries, its simplicity, reproducible and adaptable nature defines it as a suited complementary fragment screening technique Capillary Electrophoresis Capillary electrophoresis was initially developed for HTS [85, 86] and has led to the discovery of multiple lead compounds. Capillary electrophoresis was modified for the screening of fragments (CEfrag TM ) by Selcia, a drug discovery screening service company. In this approach, fragments are detected by monitoring the changes in the electrophoretic profile of fragments displaying affinities in the mm to pm range Ultrafiltration Ultrafiltration is similar to capillary electrophoresis and weak affinity chromatography as all these methods involve affinity based separation of bound and unbound fragments. The principle of ultrafiltration is also simple in which fragments to be screened are mixed with protein and passed through a membrane which retains the protein (complexed with bound fragments). The composition of the initial mixture is then compared with the filtrate composition to identify the fragments that have affinity for the protein. This fragment screening technique was tested on two proteins [87]: riboflavin kinase and methionine aminopeptidase 1. The screening resulted into 3 and 9 fragment hits for riboflavin kinase and methionine aminopeptidase 1 respectively Biochemical Assays/High Concentration Screenings Standard biochemical assays are not the preferred method in FBDD due to their inability to detect fragments with a very weak binding affinity for the protein target. The higher concentration of fragments used during screening causes higher number of false positives and negatives due to aggregation, chemical reactivity and interference with the assay. Moreover, high concentration screening requires high solubility (about 1mM) of the fragments. Despite the disadvantages with the use of biochemical assays, recent studies showed that good fragment hits can be identified by screening fragment library at a higher concentration effectively, rapidly and cheaply. The higher number of false positives and negatives can be avoided by removing fragments with poor solubility, reactive fragments, frequent hitters and aggregators from the fragment library. Plexxikon, for example, has routinely used high-

6 6 Current Medicinal Chemistry, 2012 Vol. 19, o. 1 Kumar et al. concentration biochemical screening to look for inhibitors or activators of enzyme activity. They have screened scaffold-like compound libraries at a concentration ranging from 100 μm to 200μM using different functional assays. X-ray crystallography was used as a secondary screen to further prioritize hits after high concentration screening. Plexxikon s effort ultimately led to the identification of PLX4032 (Vemurafenib) (see section 4). ther groups also used high concentration biochemical screening as their primary fragment screening approach to identify inhibitors of phosphatidylinositol-3 kinase [88], phosphoinositide-dependent kinase-1 (PDK1)[89] and beta-secretase (BACE-1)[90]. owadays, with significant successes of using high concentration screening by biological assays reported, many groups in pharmaceutical industry are using fragments in HTS screening at higher concentration to develop target focused sets of fragments for further biophysical screening [15] Tethering Tethering or covalent tethering is a fragment based screening approach developed at Sunesis Pharmaceuticals and it allows the detection of very weakly binding fragments that cannot be detected by traditional means. Tethering uses the reversible disulfide bonds between cysteine residues in a protein and thiol linker containing fragments to capture and identify weakly binding fragments by mass spectrometry [91-93]. In tethering approach, single cysteine mutations are introduced in the target protein surface at the site of interest. A library of thiol linker containing fragments is then screened against this cysteine mutant protein. The thiol linker containing fragments compete for disulfide formation with the cysteine residue introduced in the protein. The fragments that have inherent affinity for the protein apart from disulfide bond are conjugated at equilibrium and are detected by mass spectrometry. Tethering approach has been used to identify inhibitor of interleukin-2 [94], caspase-3 [95, 96], GPCR [97] etc. Different versions of tethering also exist such as extended tethering and tethering with dynamic extenders. Extended tethering or tethering with extenders is a modification to covalent tethering by combining with aspects from dynamic combinatorial chemistry [96]. In extended tethering, cysteine residue is covalently linked using irreversible electrophile of extender which is a protected thiol group containing small molecule that has some inherent affinity for the protein. The thiol group is deprotected to screen a library of disulfide containing fragments. Fragments making favorable interactions with protein will form stable disulfide bonds with extender and are detected by mass spectrometry. Recently, Erlanson et al. [98] used tethering with extender to identify inhibitors of 3- phosphoinositide dependent protein kinase-1 (PDK1). Another modification to tethering is tethering with dynamic extenders, in which an irreversible electrophile of extender is replaced with disulfide that enables reversible cysteine modification. Cancilla et al. [99] used tethering with dynamic extenders to discover an Aurora kinase inhibitor Computational Methods Computational methods, such as molecular docking, have also been used alone or in combination with experimental fragment screening approaches to successfully identify fragment hits for optimization into lead-like compounds. The detailed discussion about these methods and practical applications is presented in Section 6 of this review Fragment ptimization There are two commonly used approaches for the optimization of fragment hits into lead-like compounds: (a) Fragment growing and (b) Fragment linking. Fragment growing is the stepwise addition of functional groups or substituents to the fragment core to maximize the favorable interactions with the binding site residues. The fragment linking approach is based on covalently linking two or more fragments bound independently in proximity with suitable linkers. A schematic overview of fragment growing and fragment linking is presented in Fig. (1) using the data derived from the study by Hung et al. [100] Both approaches are described in detail with examples in following paragraphs Fragment Growing Fragment growing is the most common and popular approach for the optimization of fragment hits into lead-like compounds [13]. Fragment growing is an iterative process and at each step additional features are added to the fragments core with the goal of improving potency and pharmacological properties Fig. (1a). The structural information derived from X-ray crystallography or MR is generally used to guide the substitution or addition of functional groups with the ultimate goal of improving potency. The most important consideration while growing from an initial fragment is that it conserves the binding mode of initial fragment in the optimized compound Fig. (1a). ne of the great advantages with the use of fragment growing is that subtle changes in binding mode with each step of the fragment optimization can be monitored [13]. A large number of successful examples of using growing approach for fragment optimization have been reported in literature. We cannot cover all of these studies and a number of recently published reviews can be consulted [10, 13, 15, 21, 101, 102]. Recent studies that utilized fragment growing as an optimization strategy include the discovery of Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1)[70], Acetylcholine-binding protein (AChBP)[103], Matrix metalloproteinases (MMPs)[104] and phosphatidylinositol-3 kinases (PI3Ks)[88] inhibitors. In one of these studies, Cheng et al. [70] screened a library of 4000 fragments against BACE1, using SPR and identified 2-aminoquinoline as initial fragment hit. This hit initially displayed a potency of 900 μm and was improved 10 6 fold by fragment growing based optimization to an IC 50 value of 11 nm on BACE1 and cellular activity of 80 nm. Edink et al. [103] also used fragment growing to optimize a fragment by growing into ligand induced subpocket of AChBP binding site. They started with the co-crystallization and structure solution of a moderately potent fragment with AChBP. The structure revealed absence of one sub-pocket which was present in co-crystal structure of AChBP with a natural product lobeline. The fragment binds in the same way as the natural product lobeline, but it lacks the hydroxyphenetyl group, which extends to a subpocket in AChBP binding site. To optimize this fragment hit, the researchers introduced hydroxyphenetyl group on the fragment which led to a 50 fold more potent compound. The crystal structure was also obtained for this molecule which confirms successful fragment growing and the hydroxyphenetyl group indeed binds into the lobeline subpocket. The authors also described structural and thermodynamic consequences of fragment growing in their paper Fragment Linking Fragment linking is less common than fragment growing, but linking fragments that bind in adjacent sites of target protein is a powerful fragment optimization approach to turn low affinity fragments into high affinity leads Fig. (1b). Fragment linking was first successfully demonstrated by Fesik and co-workers in their SAR by MR paper [12]. Since then, the fragment linking approach has been used by a number of groups [100, ] to link two weak affinity fragments to obtain highly potent compound. ne of these studies describes linking two low affinity fragments identified from a fragment screen against Hsp90 [111] to obtain a compound with 1000 fold higher affinity than initial fragments against Hsp90 while maintaining LE. X-ray crystallography of linked compound also revealed that it displays similar binding mode as two initial fragments. In another study, Petros et al. [110] identified through fragment linking approach a highly potent Bcl-2 inhibitor that was 1000 fold selective for Bcl-2 over Bcl-xL. They started with a less potent and moderately selective compound

7 Fragment Based Drug Design Current Medicinal Chemistry, 2012 Vol. 19, o. 1 7 identified from protein-detected MR screen of fragments. The researchers screened a smaller library in the presence of this less potent and moderately selective compound to identify a second fragment that can be linked and occupy an adjacent hydrophobic subsite. This study provided a very good example for improving potency and selectivity using fragment linking. Recently, Ichihara et al. [113] reviewed successful fragment linking reports and suggested a strategy to maximize the success in fragment linking approach. They proposed that super-additivity (when binding free energy of the linked fragments is more than the sum of the binding energies of individual fragments) can be achieved by carefully selecting the fragment pair for optimization. To achieve successful fragment linking, binding mode of individual fragments need to remain conserved during optimization. But it is very difficult to obtain or synthesize such an ideal linker. In such cases, fragment pair where one fragment form strong H-bonds with binding site residues and other fragment interact via hydrophobic or van der Waals interactions (more tolerant to changes in binding mode) may be chosen. Also, Yamane et al. [114] suggested incrystal chemical ligation for effectively linking two fragments. Their strategy involves first soaking the target protein apo-crystals with anchor molecules (in their study trypsin as target protein and benzamidine as anchor molecule) and then transferring these protein crystals into another solution of tuning molecules. The crystals are then analyzed for any bound ligand i.e. tuning molecules that can form stable ligated product with anchor molecule at binding site. Although their study did not result in any molecule that can bind with better affinity than initial fragment, superior binders however might be generated using a bigger tuning molecules library. Fragment in situ self assembly is another fragment linking approach used to optimize fragments into lead-like compounds. In this approach, the protein acts as a scaffold for the formation of highly potent compound through the reaction of two low affinity fragments (from a compound mixture) in close proximity to each other. The first application of fragment in situ self assembly in FBDD was demonstrated by Lewis et al. [115] where enzyme acetylcholinesterase assembles azide and alkyne fragments into an inhibitor of very high potency. In another example, Hu et al. [116] used fragment in situ self assembly to identify a small molecule protein-protein interaction inhibitor (SMPPII). They found that Bcl-XL serves as a template/scaffold for the amidation reaction between thio acids and sulfonyl azides to form a SMPPII. The latest example of fragment in situ self assembly for fragment optimization is from Suzuki et al. [117] where they have used this approach to identify histone deacetylase (HDAC) inhibitors. They have incubated HDAC8 with two hydroxamic-containing alkynes and 15 azide fragments with a goal to get a linked product able to inhibit the enzyme. They obtained one compound with greater inhibitory power than either of the individual fragments Fragment Growing Versus Linking ut of the two choices for fragment optimization, the fragment growing approach is more popular and it s a clear choice when there is an obvious place to grow. It gives more freedom to a medicinal chemist for multidimensional property optimization. Fragment linking has not received the same success as fragment growing, as this strategy is dependent upon the ability to chemically link adjacent fragments without disturbing the binding mode displayed by fragments alone. Although fragment linking provides a clear starting point for optimization, achieving a similar binding mode of the fragments in the final compound is very difficult considering the limited repertoire of linkers to tether the two fragments. Hung et al. [100] compared the two fragment optimization approaches by applying them on the same target pantothenate synthetase from Mycobacterium tuberculosis as illustrated in Fig. (1). They started with an indole fragment for fragment growing and an indole and a benzofuran fragment for fragment linking". These starting fragments were identified using a number of biophysical techniques including thermal shifts assay, WaterLGSY MR, ITC and X-ray crystallography. The optimization of initial fragment hits using two fragment optimization strategies resulted in similar compounds with similar potency Fig. (1) SAR by Catalog SAR by catalog is one of most common fragment optimization approach used by various researchers to optimize initial fragment hits. SAR by catalog is simply searching various commercially available chemical vendor s library for similar compounds that can be purchased and tested. Jahnke et al. [118] carried out the screening of a library of 400 fragments for binding to farnesyl pyrophosphate synthase (FPPS) using MR spectroscopy. To optimize 4 weakly binding hits obtained from fragment screening, they conducted similarity search in ovartis compound inventory. The similarity search resulted in 40 hits similar to weakly binding fragments. These hits were again tested by MR spectroscopy and some of the hits were further characterized by ITC and X-ray crystallography. Their efforts led to the development of a compound with comparable potency to approved drugs that target FPPS. Researchers at Vernalis [119] also used SAR by catalog approach to search compound containing resorcinol substructure in their in-house database as a means to identify lead compounds against Hsp90. ne of the hit compounds that initially showed IC 50 of 300nM was optimized by medicinal chemistry modification into a 9nM potency compound. This compound AUY922 is now in Phase II clinical trials [120]. 4. VEMURAFEIB, A RECET SUCCESS WITH EXPERIMETAL FRAGMET SCREEIG In this chapter we would like to highlight a recent success in FBDD by drawing the attention to the identification of PLX4032 (Vemurafenib) as a BRAF inhibitor, which was recently approved by the FDA and is marketed as Zelboraf [29, 31]. PLX4032 is one of the first approved drugs of which the origin can be traced back to a FBDD hit discovery. A more detailed account on the discovery of Vemurafenib is given in a recent review by Bollag [121]. In 2002, Davies et al. reported that activating mutations (V600E) in the BRAF encoding gene were present in a significant population of malignant melanoma patients [122]. This report caused several groups to embark on a drug discovery program targeting this oncogenic mutant BRAF kinase, including a research team at the Plexxikon Inc. (a member of Daiichi Sankyo group). They opted for a modified fragment based drug discovery approach, referred to as scaffold based drug discovery. In order to identify protein kinase scaffolds, a library of 20,000 compounds (of which the molecular mass ranged between 125 to 350 Da) was created. This library was screened at 200 M on a divergent set of structurally characterized kinases. Analysis of this data resulted in the selection 238 compounds, with at least 30% inhibitory activity at 200 M for three different kinases (Pim-1, p38, and CSK). In total over 100 structures were solved containing a small molecule. In particular a 7-azaindole drew the researchers attention since it was able to form key hydrogen bonding interactions within the active site and subsequently a set of derivatives were synthesized resulting in increased affinity. verlapping the hit molecule with the structure of multiple kinases indicated that the compound was able to maintain the hydrogen bonding pattern with the kinase hinge while also bearing several putative substitution sites for optimization of potency as well as specificity. Derivatives were designed based on structural information trying to grow the molecule so that it exploited key interactions to gain potency as well as specificity. The designed molecules were tested and excellent potency as well selectivity was identified for the BRAF V600E mutant. Guided by co-

8 8 Current Medicinal Chemistry, 2012 Vol. 19, o. 1 Kumar et al. Fig. (1). A schematic illustration of fragment optimization strategies using the data derived from the study by Hung et al. (2009) (a) Fragment growing: Initial fragment with low potency is optimized by stepwise addition of functional groups to obtain a compound with high potency. (b) Fragment linking: Two or more fragments bound independently in proximity are covalently linked with suitable linkers to obtain a compound with high potency while maintaining the binding mode. crystal structures the compounds were further optimized resulting in PLX4720 with 13nM potency for the BRAF V600E mutant [29] Fig. (2). The pharmacokinetic analysis in animal models of PLX4720 analogues led to the selection of PLX4032 (Vemurafenib), over PLX4720, for further clinical evaluation because of a more favorable PK profile [31]. Clinical trials conducted in collaboration with Roche indicated the efficacy and safety of Vemurafenib in treatment naive as well as pre-treated melanoma patients with the BRAF V600E mutation [123, 124], finally leading to the FDA approval of Vemurafenib for patients with unresectable or metastatic melanoma with BRAF V600E mutation [20, 29, 31]. f note, researchers at Plexxikon have also used a similar screening strategy for the discovery a pan-ppar inhibitor, indeglitazar, which has progressed to Phase II clinical trial [30] and selective PDE4 inhibitors [125]. As can be concluded from this successful FBDD case, FBDD approaches can indeed lead to approved drug molecules endorsing the FBDD method for drug discovery. 5. FRAGMET BASED DRUG DESIG: CHALLEGES WITH EXPERIMETAL APPRACHES Identification of initial fragment hits with various experimental fragment screening methods is pivotal to any FBDD campaign. Therefore, highly sensitive fragment screening technologies including MR based approaches [47-55], X-ray crystallography [59], surface plasmon resonance [75, 126] are used for this purpose. Recently, several new fragment screening technologies including electrospray ionization and native mass spectroscopy [82, 83], weak affinity chromatography [84], ultrafiltration [87] etc. have been developed to improve the efficiency and throughput of fragment identification and optimization. The overview of some commonly used experimental fragment screening technologies is presented in (Table 2). As can be seen from (Table 2), each one of the experimental fragment screening methods has advantages and disadvantages. For example, protein-detected MR method [47, 48] is a highly sensitive method for identifying very weakly binding fragments and it also provides 3D structural information of fragment binding to the target protein. However, protein-detected MR requires high quantities of isotope labeled protein that raises the cost of experiments by many folds. Ligand-detected MR methods such as STD-MR [49, 50], WaterLGSY [51, 52], FAXS [53] and TIS [54, 55] were developed to overcome challenges associated with protein-detected MR. Ligand-detected MR methods have their own problems such as high false positive rate and their inability to detect tight binders [47]. X-ray crystallography, a commonly used primary or secondary fragment screening technique provides detailed 3D structural information but requires high quality of protein and well diffracting crystals [127]. Also, crystal soaking with fragments requires high concentration of fragments which is detrimental to crystal. An important disadvantage with protein-detected MR and X-ray crystallography is that they consume large amount of target protein and fragments and provide no information on binding affinity. SPR based

9 Fragment Based Drug Design Current Medicinal Chemistry, 2012 Vol. 19, o. 1 9 Fig. (2). Growth evolution of Vemurafenib. High concentration screening of scaffold molecules against a kinase revealed a series of hit molecules. These hit molecules were co-crystallized with protein kinase domains (represented in white cartoon). ne of the first hit fragments, a 7-azaindole scaffold, was able to bind to the active site of the kinase domain (frame 1). This scaffold molecule could be modified (frame 2) and gained in potency. In several rounds of optimization the scaffold was grown into a more potent molecule (frame 3). In the final round several molecules with high affinity were identified. Frame 4 depicts Vemurafenib (PLX4032) which is approved for clinical usage. PLX4032 was selected for clinical development over a more potent derivative, PLX4720 (frame 5) due to its superior PK properties in animal models. Table 2. Strength and Weaknesses of Some of Commonly Used Experimental Fragment Screening Methods Screening Method Throughput Protein Requirement Sensitivity Advantages Disadvantages Ligand detected MR 1000s Medium-high (μm range) 100 nm to 10 mm Highly sensitive, do not require labeled protein Instrument is expensive, false positive rate is high, cannot detect tight binders Protein detected MR 100s High (50 to 200 mg) 100 nm to 10 mm Provides 3D structure information Instrument is expensive, require isotope labeled protein, expert required X-ray crystallography 100s High (10 to 50 mg) 100 nm to 10 mm Provides detailed 3D structure information Instrument is expensive, well diffracting high quality crystal requirement, Crystal have to survive high concentration of fragment while soaking, expert required Surface Plasmon Resonance 1000s Low (about 5 μg) 1nM to 100mM Provides kinetic data like association rate, dissociation rate alongwith binding affinity (K d) Protein immobilization on gold surface required Isothermal Titration calorimetry 10s Low ( μg) 1nM to 1mM Provides highly quantitative affinity data and mechanistic information about non covalent forces in binding Requires high sample concentration ative Mass Spectrometry 1000s Low (about few μg) 10 nm to 1mM Label free, no need protein immobilization or assay development Requires careful choice of buffers, problem small molecule aggregation High Concentration biochemical screening >10000 Low (< 100μg) ot available Simple and straightforward method Require knowledge of biochemical function, problem of false positives and negatives technology was developed to overcome these challenges and recent instrumentations like Biacore 4000 (GE Healthcare Life Sciences) consumes less protein and fragment and provides reasonable throughput. SPR is a fast and efficient technology now used for primary fragment screening courtesy of the technical advances. Although SPR is highly efficient and provides high quality kinetic data along with binding affinity, controlling the rate of false positives and false negatives is quite challenging [75, 126]. Additionally, SPR requires immobilized target protein and therefore depending upon the immobilization methodology, target could be adversely affected. As seen from (Table 2) experimental methods of fragment screening have several advantages but despite their immense utility, most of the experimental screening approach can test only up to several hundreds or thousands compounds. The commercially available fragments is much higher ( fragments in ZIC database [128]) than what can be tested by any experimental fragment screening approach. Experimental methods of fragment screening also involve huge investment in equipments such as an X-ray machine or beam line, high frequency MR spectroscopes or surface plasmon resonance equipment. Apart from instrument cost, there is the material cost as sample preparation is expensive for example isotope labeled protein for protein-detected