2.1 Assessing biological risk in target validation

Candidate targets come from a variety of sources that are either based on biological high-throughput screening efforts or on hypothesis-driven research. Whilst these approaches provide us with a range of proteins that show a link to tumour biology, these potential targets have to be regarded as candidates that will essentially always have to undergo further in-depth validation experiments that are designed to build sufficient confidence that modulation of the particular target would lead to the desired therapeutic effect.

Over the last decade or so, molecular therapeutic targets for cancer treatment have been identified as individual gene products following rigorous and focused assessment of their contribution to tumourigenesis and their potential as for drug discovery. Increasingly, more systematic approaches are being employed that involve genome wide unbiased cancer genome sequencing. Examples include the UK- based Cancer Genome project (http://www.sanger.ac.uk/genetics/CGP/) and the NCI Cancer Genome Atlas project (http://cancergenome.nih.gov/) in the USA. An exhaustive worldwide global cancer genome sequencing campaign across a wide range of cancer types is now underway under the auspices of The International Cancer Genome Consortium or ICGC (http://www.icgc.org). Indeed the goal of the ICGC is: ‘To obtain a comprehensive description of the genomic, transcriptomic and epigenomic changes in 50 different tumour types and/or subtypes which are of clinical and societal importance across the globe’. These large-scale efforts aim to characterise many tumours of different types comparing global genome abnormalities, gene mutations, gene expression and epigenetic changes. The scale and speed of these efforts is becoming ever more feasible with the recent advent of higher throughput and more cost-effective Next-Generation Sequencing technologies for DNA and RNA (Boehm and Hahn, 2011).

Cancer genome sequencing has identified some important and frequently mutated oncogenes for which addiction has been demonstrated and against which drugs have been discovered, developed and approved – the most notable example being BRAF. However, it is clear that oncogenes that are now being identified will be much less frequently mutated, and therefore represent smaller, though still potentially important patient populations.

As well as defining the full repertoire of cancer genes and other molecular abnormalities, a further objective of the comprehensive profiling campaigns will be to understand how cancer genes interact together in dynamic networks. These effects are likely to identify further new targets and certainly help to prioritize certain target loci that may be especially important in particular genetic systems and disease contexts. Also critical is to identify genes that are not necessarily themselves genetically altered but that are essential for viability in cancer cells. Alongside sequencing and expression profiling efforts, the complimentary approach of functional screening based on genome-wide RNA interference (RNAi) is currently being used to identify such targets (Brough et al., 2011; Mohr et al., 2010; Quon and Kassner, 2009).

It can be argued that the strongest evidence that a target is important for the biology of a certain tumour is of genetic nature, for example detection of activating mutations as with BRAF (Davies et al., 2002); gene amplification such as for ERBB2 (Slamon et al., 1989); or fusion genes as in the case of ALK (Soda et al., 2007). Importantly, it is essential to distinguish between passenger mutation/genetic alterations that do not contribute to tumour biology and the significantly less frequent driver mutations/genetic alterations to which a tumour is addicted (Sellers, 2011). Although statistical evidence is important in this respect, ‘wet biology lab’ experimentation is important in identifying and validating driver oncogene targets. This can be done firstly by showing that introduction of the genetic defect or increased expression of a particular gene is able to transform normal cells into those having a more cancerous phenotype. And secondly by showing that the reversal of the molecular abnormalities, for example by RNAi or a dominant negative construct, is able to block malignant transformation or kills the cancer cell. Such experiments are generally performed in cancer cell lines in tissue culture, but genetically-engineered mouse models (GEMMS) are increasingly being used to validate that introduction of a genetic aberration leads to the formation of tumours and that correction of it yields an anticancer effect (Politi and Pao, 2011).

Showing that a target is genetically activated leading to addiction certainly builds confidence in the target. But not all targets fall into this category. Recently a concept sometimes referred to as non-oncogene addiction has been proposed. This model suggests that a variety of non-oncogenic gene products/pathways that lack oncogenic activity in their own right may still be essential for supporting the transformed cancer phenotype and as such may also represent potential therapeutic targets (Luo et al., 2009).

This model of non-oncogenic addiction also encompasses the concept of synthetic lethality (Luo et al., 2009; Ashworth et al., 2011). According to this concept, cancer-associated genes can be identified that represent a vulnerability that results in cell death only when another gene is inactivated. In the cancer therapy context, this can mean the identification of a drug target that when inhibited pharmacologically results in the death of cells harbouring a particular tumour suppressor gene. The initial worked example is the dramatic observation that tumours with mutations in BRCA1 or BRCA2 become much more dependent on the activity of poly (ADP-ribose polymerase) or PARP and are much more highly sensitive to inhibitors of PARP compared to normal cells, even though PARP expression or activity is generally not increased in these tumours (Lord and Ashworth, 2008). Selective killing is due to the fact that BRCA1/2 mutant cells are deficient in DNA double-strand repair by homologous recombination and thus become extremely sensitive to cell death when a second DNA repair mechanism, mediated by PARP, is inhibited.

The identification of such synthetic lethal targets is attractive because it provides a means to exploit pharmacologically loss-of-function tumour suppressor gene defects, which are otherwise essentially undruggable. Use of PARP inhibitors in BRCA mutant patients has been validated clinically, initially with the drug olaparib (Fong et al., 2009). Another, albeit somewhat different example of indirectly attacking a tumour suppressor is inhibiting the protein kinase AKT/PKB that is activated upon mutation/deletion or epigenetic suppression of tumour suppressor gene PTEN in many cancers (Hollander et al., 2011). In this case, inhibition of the target antagonises the effect of tumour suppressor loss rather than normalising the activity of a target that is overactive due to a genetic activation. These examples, particularly the synthetic lethality approach, are generating increased interest in identifying targets that can be inhibited in cancers with defined loss of function mutations.

Having established that the target of interest is important in the biology of cancer, the second important step when validating a target is to increase confidence in the hypothesis that inhibition of the target will lead to the desired therapeutic effect in patients. These validation experiments are now frequently performed, as discussed above, by suppressing the expression of a protein through RNAi, since a suitable chemical inhibitor is often not available at this stage of the project. However, where such chemical tools are available, these can be very valuable, subject to a number ‘fitness factors’ and controls that we have set out (Workman and Collins, 2010).

The limitations of using RNAi to mimic a chemical inhibitor have recently received widespread attention (Palchaudhuri and Hergenrother, 2010; Quon and Kassner, 2009; Sigoillot and King, 2010). RNAi will rarely completely suppress target expression and it is now widely accepted that most if not all RNAi reagents can have off-target effects additional to inhibition of target gene expression. Multiple sequences and appropriate non-targeting controls should be used to mitigate the risk of off-target effects.

An additional complication is that inhibition of target gene expression by RNAi leads to the depletion of the entire protein which may influence the composition of protein complexes or protein scaffolding functions. In contrast, a small molecule inhibitor will generally only inhibit one particular function, e.g. the enzymatic activity. If the biological effects observed through RNAi knockdown is due to inhibition of a scaffolding function they may not be recapitulated by a small molecule inhibitor. These concerns can be addressed to some extent by suitable control experiments. For example, exogenous expression of an RNAi knockdown-resistant version of the target gene should rescue the effect of the RNAi unless the RNAi activity is a result of off-target effects (Falschlehner et al., 2010; Palchaudhuri and Hergenrother, 2010; Sigoillot and King, 2010). Conversely, the effect should not be rescued by exogenous expression of mutant that reflects inhibition through a small molecule, e.g. expression of a catalytically dead mutant in the cases of enzymes. Achieving the same biological effect with RNAi and with chemical tool compounds (especially using inhibitors from more than one chemical class or chemotype and incorporating inactive analogues) is especially persuasive (Workman and Collins, 2010).

Once confidence has been built that the effect of RNAi is a genuine on-target mechanism, the observed phenotype has to be closely scrutinised. In many cases, it is expected that the effect of target depletion or inhibition should be selective to cancer cell lines that are addicted to the particular target or pathway. Determination of whether this leads to cytostasis or cell death, e.g. through apoptosis or autophagy, is important – with cell death being a more desirable therapeutic outcome. It is important to demonstrate that target modulation leads to the expected effect in multiple cell lines which show that particular genetic lesion (Brough et al., 2011). It is equally important that no or minimal effect is observed in cancer cells with a different genetic background, and ideally also in normal, non-transformed cell lines from the same tissue, although the latter are challenging to work with in culture and to transduce.

The robustness and reproducibility of effects is important. A recent publication has shown that a high proportion of potential targets identified in the literature could not be validated in-house within the laboratories of a pharmaceutical company (Prinz et al., 2011). This emphasizes the importance of obtaining robust and reproducible data – ideally, as mentioned, in multiple cell lines and with multiple reagents and orthogonal technical approaches. Also important is our own experience of obtaining quantitative data, not only on the degree of target inhibition or knockdown but also on the extent of the resulting phenotype observed. Thus the amount of cell cycle arrest or cell death induced must be sufficient to result in a meaningful therapeutic effect.

In addition to target validation studies in vitro, there is an increasing need to look at the effects of target modulation in in vivo animal models, enabled by the increasing availability of validated short hairpin RNAs for target knockdown, ideally using an inducible system. Furthermore, it should be remembered that although inhibition of cancer cell proliferation and cell death predominate as desirable therapeutic endpoints, other malign cancer hallmarks that may need to be evaluated are properties such as motility, invasion, angiogenesis and metastasis (Hanahan et al., 2000; see De Palma and Hanahan, 2012).

2.2 Assessing technical risk

Even if a target shows strong biological validation, the discovery of a safe and effective small molecule drug candidate is still a scientifically challenging and risky endeavour that can fail at several stages. For this reason it is essential to think through the whole project to identify key roadblocks, challenges and risks that will threaten the identification and clinical development of the drug that will emerge. Key risks or challenges include that compound screening will not deliver chemical hits that act on the target or that a biological assay that is critical for the progression of the project cannot be made available. Many technical risks or gaps can be tackled and managed if they are recognised sufficiently early. Some typical questions and issues are discussed below.

As mentioned, a key question is whether the proposed target is druggable with a small molecule agent. By druggability we refer here to the question of whether a target can be modulated in the desired fashion by a small molecule (Hopkins and Groom, 2002; Russ and Lampel, 2005). To bind with strong potency, i.e. Kd ≤ 20 nM, a small molecule has to engage in a number of hydrophobic and polar interactions with its target. For drug-like molecules, this is generally only possible if the drug binds to an enclosed and hydrophobic pocket or cavity on the protein. Or to use Paul Ehrlich's classic lock and key analogy (Kaufmann, 2008), a druggable target must have a keyhole into which a small molecule key is able to fit. Evaluating druggability in the context of small molecules thus comes down to analysing if the target contains such a cavity or pocket. Different methods exist to evaluate druggability, a few important and topical examples of which will be reviewed briefly here.

One approach is to ask if the target is druggable by association. Thus if the target is a member of a protein family of which others members have already been drugged, as for example in the case of protein kinases, then there is a good chance that it will be druggable as well, especially if the sequence homology is high (Hopkins and Groom, 2002; Russ and Lampel, 2005). The publicly available database canSAR (https://cansar.icr.ac.uk/), which was developed in our Unit, is a powerful tool with which to search for homologous targets based on protein sequence and structure as well as to identify and download published chemical hit matter (Halling-Brown et al., 2012). Such a search, which we use ourselves extensively and which we recommend should always be performed for a novel target, has the added benefit that any inhibitors of a related target can serve as the launch pad with which to kick-start a new drug discovery project.

Structure-based assessment can be very valuable if 3-dimensional structural information is available on the target. Thus if an X-ray crystal or NMR structure is in hand this can be inspected for enclosed pockets suitable for the binding of a small molecule. The analysis can be done qualitatively by visually inspecting the structure or more quantitatively using computational tools that rank targets according to predicted druggability (Fuller et al., 2009).

It is increasingly recognised that pockets that are able to bind drug-like molecules are not necessarily fully present in static picture of an apo crystal structure (Lee and Craik, 2009). Instead these pockets may represent a particular conformation of the protein that does not crystallise but can be trapped by binding to a drug. These transient pockets represent an exciting opportunity for targets that are difficult to drug but are still very challenging to identify and explore (Surade and Blundell, 2012).

The druggability of a protein can never be predicted with absolute accuracy, especially if little information on the target is available. It is often possible to assess fairly accurately the extreme cases, i.e. the very druggable (for example protein kinase-like) or the essentially undruggable (such as the flat surfaces involved in many protein–protein interactions). But there is a considerable grey zone in between these two extremes. In these cases attempting to identify chemical hits is frequently the only way to find out if the target is likely to be druggable.

Whilst a high-throughput screening campaign using a large library of drug-like molecules is expensive to run, the screening of around 1000 very low molecular weight compounds known as ‘fragments’ has been proposed as a cost-effective test of druggability (Hajduk et al., 2005). Fragment screening will be discussed further below. But it is important to mention in this present context that fragment binding can only be taken as an indication of druggabilty if the fragments have been shown to bind to a site that is relevant for drug discovery.

There are a number of technical enablers that must be considered ahead of committing to a drug discovery campaign on a new target. Particularly important is the so-called ‘screening cascade’, a term that is often used to describe the series of biological assays that are needed and the relative order in which compounds are progressed through them. Figure 3 shows a typical example that we used for our HSP90 drug discovery programme (in collaboration with Vernalis) that ultimately led to the drug candidate NVP-AUY922 that is now in Phase II clinical trials (Brough et al., 2007). The test cascade assays are run on compounds generated in successive iterative make-and-test cycles, so that the desired properties of the drug, which are usually decided by the team at the outset, are progressively achieved.

The biological test cascade will usually be specific to a particular target and thus tailored to the drug discovery project concerned. Most projects will require a biochemical assay with recombinant protein(s). This assay needs to have sufficient throughput and fidelity, particularly if the screening of large compound libraries is planned.

Next, both a cancer cell proliferation assay or similar together with a mechanism-based cellular endpoint are pivotal to show that anticancer effects are mediated through inhibition of the target in the cellular setting (Collins and Workman, 2006). These assays are particularly relevant if the desired cellular outcome is cell death since cell killing can be induced by many (off-target) mechanisms. The observation that a compound induces apoptosis does not necessarily mean that it does so through inhibition of the desired target. Examples of mechanism-based cellular read outs are phosphorylation of downstream targets in the case of a target kinase or depletion of client proteins as in the case of HSP90 (Brough et al., 2009; Sharp et al., 2007). Several technologies now exist to perform these cellular mechanistic assays with significant throughput and with quantitation, e.g. high-content cellular imaging or ELISA-based assays.

Quantitation is important for ranking compounds and understanding their structure-activity relationships and activity is commonly expressed as the concentration of compound required to achieved a 50% inhibitory effect.

In the case where the target concerned belongs to a protein family, selectivity assays are commonly necessary to assess preferential activity for the desired target against other family members and to minimise off-target effects.

In addition to improving potency and selectivity on the desired target, medicinal chemists must also improve the physicochemical properties of a prototype drug lead, particularly solubility and lipophilicity, in order to achieve cell permeability and appropriate in vivo pharmacokinetic properties. Thus it is important to run assays that predict for appropriate characteristics with respect to ADME (Absorption, Distribution, Metabolism and Excretion).

Prior to testing for in vivo therapeutic activity in an animal model, most modern cancer drug discovery project teams will optimize pharmacokinetic (drug exposure) and pharmacodynamic (target engagement and downstream pathway inhibition) properties, the latter measured by appropriate target-specific biomarkers (Collins and Workman, 2006). Defining appropriate in vivo models with sufficient throughput to support the make-and-test cycles involved in medicinal chemistry optimisation – often requiring data turn around on a weekly or bi-weekly basis – is frequently a challenge. We will cover this topic further below.

We believe that it is essential for the project team to consider the likely clinical trial strategy early on in the project. In particular, key questions of how patients will be stratified using predictive biomarkers and which pharmacodynamic biomarkers will be used to demonstrate target modulation should be addressed. In our own Unit we have adopted a ‘no biomarker no project’ policy which can present a substantial hurdle, particularly for newer targets that lack a substantial body of background data. Nevertheless, we believe that it is critical to plan for these downstream activities at an early stage.

It is also important consider, both early on and then continuously after, the competitive landscape around the target. A new drug will only be successful if there are not already drugs available that adequately address the medical need. When evaluating a potential target it is thus important to obtain an overview of the competition. Assessing the competitive landscape is often not straightforward for several reasons. It involves the analysis of patent applications which are notoriously difficult to penetrate. In addition, patents are only published 18 months after submission and there is a significant time gap between patents that have been filed and patents that can be analysed. Moreover, the situation at the start of a drug discovery project has to be forecasted into the future when the project reaches clinical trials or even the market. These predictions can only be done with limited accuracy, yet assessing the competitive situation at an early stage will give important clues. For example, if there many competitors of which several are ahead, the chances are that the project will face strong competition when entering the market and clear advantages will need to be demonstrated.

On the other hand, it should be noted that embarking on a drug discovery project on a target for which competitors are somewhat ahead can be advantageous because a project team can learn from their progress as evident from patent applications and publications to generate drugs with a competitive edge. In addition, it has been shown that for novel targets, the first drug to reach clinical trials – although potentially important for a proof-of-concept – is often not the first to reach the market. Moreover, even if a drug is already approved, a follow-on drug can still receive priority review from the regulatory authority such as the US FDA (DiMasi and Faden, 2011). These considerations are more likely to be relevant for industry teams, as academic drug discovery groups are more inclined to focus on innovative high risk targets, although the competition may still be strong.

However, at the latest when any competitor compounds are reaching the clinic, these compounds will need to be prepared or sourced and then profiled in depth to define their properties (DiMasi and Faden, 2011; Giordanetto et al., 2011), hence allowing an assessment of whether the in-house project already has or can produce superior compounds.

In summary, choosing a promising target is a matter of assessing opportunities and risks, many of which we have discussed here. It goes without saying that eliminating all risks early on is impossible. Moreover, cancer is an area with high unmet medical need and thus, rather than shying away from risks, a more productive approach is to assess and manage them effectively from the start of the project. Also worth noting is that a non-profit drug discovery group like our own may frequently be prepared to take on higher levels of risk than a commercial company, since our raison d'etre is to be innovative.

3.1 High-throughput screening

Since the advent of targeted therapy, high-throughput screening (HTS) has been a cornerstone for hit generation. The first cancer drugs originating from HTS have reached the market, e.g. soranfinib (Macarron et al., 2011). Others are undergoing clinical trials, as in the case of inhibitors of HSP90 (Cheung et al., 2005; Brough et al., 2007), PI3 kinase (Folkes et al., 2008; Raynaud et al., 2009) and the Hedgehog pathway (Robarge et al., 2009).

One advantage of HTS is that it is a relatively unbiased approach and can identify compounds with novel binding modes. Recent examples include allosteric inhibitors of the kinase AKT/PKB (Figure 5). Initially compound 1 was identified by HTS and shown not to be competitive with ATP, in strong contrast to most other kinase inhibitors (Lindsley et al., 2005). Optimisation of this series of compounds eventually led to the drug candidate MK-2206 (2) (Hirai et al., 2010) which is now in Phase II clinical trials.

The mechanism of action was revealed by solving the crystal structure of AKT1 bound to 3, another potent inhibitor from this class of compounds (Wu et al., 2010). 3 binds to a pocket at the interface of the regulatory PH domain and the kinase domain, approximately 10Å away from adenine pocket where ATP competitive inhibitors bind. Binding to this allosteric pocket stabilises the kinase in an inactive (PH-in) conformation. This PH-in conformation is significantly different from the PH-out conformation observed with ATP competitive inhibitors. Importantly Wu et al. hypothesise that the PH-in conformation explains why the allosteric inhibitors potently inhibit the phosphorylation of AKT itself and do not trigger the well known feedback activation observed with ATP competitive inhibitors (Okuzumi et al., 2009). A key step of the activation of AKT is recruitment of the protein to the membrane. However, the phospholipid binding which is responsible for attachment of AKT to the membrane is blocked in the inactive PH-in conformation, thus keeping the kinase in the cytosol and preventing its phosphorylation. AKT competitive inhibitors on the other hand are hypothesised to bind to the active PH-out conformation thus triggering recruitment to the membrane, phosphorylation and feedback activation (Wu et al., 2010).

Binding to the allosteric pocket also resulted in inhibitors with unprecedented selectivity over closely related proteins from the AGC family of protein kinases (Lindsley et al., 2005). It is very unlikely that this series of allosteric inhibitors would have been identified by design or focused screening, as opposed to HTS.

Whilst most HTS campaigns are conducted using biochemical assays, large-scale screening can also be performed in a cellular context. Two different scenarios for cellular HTS can be distinguished. In the first scenario, a defined target is screened in a cellular context. A recent example is an assay developed for the screening of ninety thousand compounds to identify inhibitors of the epigenetic regulator histone demethylase JMJD3. A high-content cellular mechanistic imaging assay was used to determine both cellular viability and, as an on-target biomarker, the level of enzyme-dependent demethylation of the H3K27(Me)3 mark produced by an exogenously expressed JMJD3 (Mulji et al., 2012). Such screening of a defined target readout in a cellular context can be particular advantageous when a biochemical assay is challenging to generate, e.g. because the protein or substrate is not stable or multiple reagents are needed.

In the second scenario (sometimes referred to as chemical genetic screening) a cellular HTS is performed to identify compounds that induce a particular phenotype or inhibit a pathway that is relevant to say, the proliferation or survival of cancer cells. The aim of the chemical screening is thus twofold in this case: Firstly to discover chemical hits and secondly to identify a novel target that is modulated by these hits. However, the second step has proven very challenging even though different target identification technologies can be explored, as reviewed elsewhere (Sato et al., 2010). A recent example is the identification of inhibitors that antagonise WNT signalling (Huang et al., 2009). The authors performed screening using a WNT-responsive luciferase reporter assay. Through a series of experiments, including a quantitative chemical proteomics approach, they then showed that the obtained hits target the WNT pathway through inhibition of the poly-ADP-ribosylating enzymes tankyrase 1 and tankyrase 2, previously not known to be key regulators of the pathway. This is thus an elegant example of a chemical genetic screen that identified chemical hits and at the same time revealed a novel target for the WNT pathway.

The examples described here illustrate the potential of HTS to utilise biochemical and cellular assays to discover chemical compounds with novel and innovative modes of action. However, it is also important to point out that maintaining and screening a large screening collection is resource-intensive (Macarron et al., 2011). In addition, HTS assays have to be carefully optimised to be fit for the screening of hundreds of thousands of compounds, otherwise meaningless data may be produced. The process of screening and following up on the results can be time-consuming, not least because frequent false positives and undesired hits have to be eliminated. False positives often arise from compounds that aggregate (Shoichet, 2006) or interfere with the assay read out (Thorne et al., 2010). Undesired hits often have chemically reactive groups that react in an unspecific fashion with the target. Unfortunately, compounds that are prone to give false positives or represent chemically reactive compounds are still fairly prominent in commercial libraries but ways to identify them have recently been described (Shoichet, 2006; Baell and Holloway, 2010; Thorne et al., 2010). Lastly, the ideal size and composition of a compound screening file is still a matter of discussion that goes beyond the scope of this review (Akella and DeCaprio, 2010; Macarron et al., 2011).

The costs associated with HTS are obviated to some extent if smaller, more focused screening is performed. Focused screening is particularly relevant if the target is a member of a well-studied family of proteins, e.g. kinases or G protein-coupled receptors (GPCRs). The idea behind focused screening is that inhibitors of different members of a protein family, such as protein kinases, often have common molecular features. Focused libraries thus consist of compounds having these features and accordingly frequently show much higher hit rates compared to divers screening collections.

Similarly, cross-profiling of inhibitors generated for one particular kinase, has traditionally been a rich source for hits of other kinases. In the extreme case, one clinical candidate can be explored as an inhibitor of more than one kinase, as in the case of imatinib acting on ABL, PDGR and KIT (Kindler et al., 2004; Matei et al., 2004) and crizotinib inhibiting MET and ALK (Cui et al., 2011).

3.2 Fragment screening

A special application of the screening concept is fragment screening or fragment-based drug discovery (FBDD) (Hajduk and Greer, 2007). In sharp contrast to HTS, in FBDD only a limited number of compounds (typically 1000) of relatively low molecular weight is (<300) are screened with the aim of identifying weakly potent fragment hits (∼100 μM).

This approach addresses one of the key challenges of HTS: That is that an HTS hit must engage in several interactions with the target in order to have sufficient potency to be identified as a hit. To be able to engage in these interactions, the hit must feature the required chemical groups, e.g. hydrogen bond donors or acceptors and/or hydrophobic groups, positioned in the correct geometrical arrangement. Particularly for molecular targets that are challenging to drug, the chances are that such a compound is not present even in large screening sets, thus rendering the HTS campaign unsuccessful.

Fragment screening, on the other hand seeks deliberately to identify small fragment hits that are of lower potency. As a result only a few key interactions with the receptor are required, greatly reducing the number of compounds that have to be screened (just as it is easier to find a parking spot for an Austin Mini than for a 1970's Cadillac). However, the reduced screening effort comes at a price: The initial, weakly potent fragment hit has to be optimized through chemical modification to improve the potency into the range of a hit compound. The optimisation of a fragment will in most cases require the detailed knowledge of the binding mode to the receptor, e.g. through crystal or NMR structures, thus limiting FBDD to targets where this information can be derived.

A recent example of successful FBDD is the BRAF inhibitor vemurafenib (5), recently approved for the treatment of late-stage melanoma by the FDA (Bollag et al., 2010; Tsai et al., 2008). Screening yielded compound 4 as a low molecular weight fragment hit with weak potency (Figure 6). The co-crystal structure of this compound was then explored through several iterative rounds of design, synthesis and testing ultimately to identify vemurafenib. As can be seen in Figure 6, vemurafenib shows higher molecular weight and potency than 4 but retains its chemical core (depicted in red). This approach, where one fragment serves as starting point and is optimised through the appending of additional chemical groups is often referred to as fragment growing.

An alternative approach to fragment growing is known as fragment linking and has been explored in pioneering work to identify inhibitors of the BCL-2 family of proteins (Oltersdorf et al., 2005; Petros et al., 2005). These proteins are overexpressed in many cancers types and contribute to tumour initiation, progression, survival and in particular to resistance to therapy. Obtaining potent small molecule inhibitors proved difficult using alternative approaches such as HTS owing to the necessity of targeting what is a very challenging protein–protein interaction that does not score highly for conventional druggability. The activity of the BLC-2 protein family is mediated via the binding of the BH3 α-helix of one protein with a large hydrophobic pocket present on binding partners. A high-throughput NMR-based method called ‘SAR by NMR’ was used to screen a fragment library to identify small molecules that bind to the hydrophobic BH3-binding groove of the BCL-2 family member Bcl-X_L. In this case, two different fragments (Figure 7, compounds 6 and 7) were identified that bound to distinct but proximal pockets of the protein. These weakly binding fragments were then linked through several rounds of synthesis and design, ultimately resulting in ABT-737 (8). Although of relatively high molecular weight for a conventional small molecule drug, this agent proved to be an extremely potent inhibitor of BCL-X_L, BCL-2 and BCL-w that had oral activity and was progressed into clinical trials.

The power of the fragment linking approach lies in the fact that due to the entropic contributions to binding, the linking of two weak binders can lead to a much more potent inhibitor. However, this example also illustrates that linking two fragments in a productive manner and eventually discovering a drug candidate is a challenging endeavour that may require the synthesis and testing of hundreds of compounds and the commitment to the project over a longer than usual period.

3.3 Virtual screening

The approach of selecting compounds from large databases by using computational tools rather than physically screening them is generally referred to as virtual screening. Conceptually two different approaches can be followed. Ligand-based approaches select compounds from databases that are in one way or another similar to an already existing inhibitor of the target in question (Schneider, 2010). Structure-based approaches seek to evaluate computationally the fit of compounds to a binding pocket, e.g. by exploring crystal structures. The compounds are then ranked by the predicted affinity and only the top 100–1000 compounds are screened.

Virtual screening has obvious advantages over physical screening. It is significantly less resource-intensive and faster. In addition, even compounds that are not available can be evaluated by virtual screening and if found promising, can be bought or synthesised. Millions of compounds can thus be analysed by virtual screening.

It is important to keep in mind, however, that virtual screening is still a relatively coarse filter. That applies particularly to structure-based screening because prediction of binding affinities still remains one of the holy grails of computational chemistry (Schneider, 2010). Nevertheless, several successful examples have been published and recently reviewed (Ripphausen et al., 2010).

3.4 Knowledge-based design

Despite the advent of HTS and random screening, intelligent design remains an important component of the medicinal chemist's tool box. This is illustrated by the recent publication of ABL inhibitors that are effective against the imatinib-resistant mutant in which threonine is mutated to isoleucine at the gatekeeper position known as T315I that is also resistant to second generation inhibitors dasatinib and nilotinib (Chan et al., 2011; Packer and Marais, 2011). Currently available ABL inhibitors have shown impressive efficacy against CML. However, the T315I mutation frequently emerges and confers resistance to all of them, partially through steric interference of the mutant isoleucine with the inhibitor. The researchers (Chan et al., 2011) recognised that potent inhibition of the kinase can be achieved without binding to the space that is altered through the T315I mutant, particularly if an inactive (DFG out) conformation of the kinase is explored (Packer and Marais, 2011). The design started with substituted aminopyrazoles that were already known to bind to a region distant to the mutant gatekeeper (Figure 8, red part of compound 10). The same region of the ATP pocket is addressed by other ABL inhibitors, e.g. imatinib (Figure 8, red part of compound 9). Chan et al. took this concept further by recognising that the aminopyrazole moiety can also be explored to engage in additional interactions distant from the gatekeeper in particular with Arg386 and Glu282 of ABL1 (Figure 8, blue part of compound 9). These interactions also further stabilise the inactive conformation of the kinase. As a first proof of concept, compound 10 showed reasonable potency against the T315I mutant and wildtype kinase. To further improve the activity, the same researchers then extended the compound into the adenine binding pocket but in a manner that is not obstructed by the gatekeeper mutation, thus leading to very potent inhibitors of both wildtype and T315I mutant ABL, in particular the clinical candidate DCC-2036 (Figure 8). Interestingly, this appended moiety resembles the corresponding group in the marketed multi-kinase inhibitor sorafenib (Macarron et al., 2011). The work thus represents an elegant example how different and previously known components of a kinase inhibitor can be explored and extended to engage in an additional interaction, leading to an inhibitor with innovative properties.

3.5 Hit discovery strategy considerations

As described above, several approaches are available for hit finding and can be used either individually or in combination. Particularly when exploring challenging targets with low predicted druggability, it is often advisable to follow more than just one approach so as to increase the chances of finding hits that can be progressed by medicinal chemistry.

Whilst following different hit discovery approaches increases the likelihood of finding hit matter, it is also time- and resource-intensive. For druggable targets, e.g. the ATP pocket of a kinase, the extra effort might not be warranted and the screening can be limited to a focused collection to save time and resources. Designing a hit strategy is thus a question of carefully reviewing what is known about the target to assess which approaches will be followed so as to be as confident as possible that hits can be obtained.

3.6 Assessing the quality of the hit matter

The quality of the chemical hit matter will to a large extent determine how fast and efficiently it can be optimised to a preclinical candidate. A hit of high quality already shows many of the properties desired for a lead or even the future drug candidate.

However, it is equally important to consider properties generally referred to as lead-likeness (Hann and Oprea, 2004), in particular molecular weight and lipophilicity (Hann, 2011; Nadin et al., 2012). When comparing two hits with equal potency, it is generally the case that the one with lower molecular weight has an advantage. This due to the observation that both molecular weight and lipophilicity often increase during the optimisation of a hit towards a preclinical candidate. In addition, it has been shown – as a rule of thumb based on past experience – that oral drug candidates with high molecular weight (> 550 Da) and excessive lipophilicity have a higher chance of failing during development (Lipinski et al., 1997). When relationships between physicochemical drug properties and toxicity were inferred from a data set comprising animal in vivo tolerability studies on 245 preclinical Pfizer compounds, an increased likelihood of toxic events was found for less polar, more lipophilic compounds (Hughes et al., 2008). A smaller hit thus gives the medicinal chemist more room to add substituents in order to dial in properties like potency and selectivity. Increasingly, ligand efficiency indices are used to evaluate and compare screening hits which represent the activity against the target normalised to the molecular weight (Abad-Zapatero and Metz, 2005; Hopkins et al., 2004; Leeson and Springthorpe, 2007).

4.1 Drugs with prolonged residence time at the target and covalently binding agents

Most drugs have to modulate their target in a sustained fashion to be efficacious. This can be achieved by optimising the pharmacokinetic properties to achieve prolonged exposure to the drug. Alternatively, sustained target modulation can arise from a slow off-rate such that a drug will take several hours to dissociate from its target even when the compound is cleared from the organism. Whilst optimisation of pharmacokinetic properties has traditionally been a key focus of medicinal chemists, the beneficial effect of extended residence time of the drug at the target has only recently received wider attention (Copeland et al., 2006). This might partially be due to the availability and wider use of surface plasmon resonance (SPR) that enables measurements of off-rates with reasonable throughput (Rich et al., 2008).

A particularly good example is the dual EGFR/ERBB2 inhibitor lapatinib (Figure 10). It has been shown to have a very slow dissociative half-life of 300 min compared to less than 10 min for gefitinib and erlotinib, two other approved EFGR inhibitors (Wood et al., 2004). Interestingly, the available co-crystal structure of lapatinib shows that the drug binds to an unusual inactive conformation of EGFR. Based on this structure it has been hypothesised that dissociation from the protein requires a slow conformational change of EGFR, thus significantly prolonging the off-rate (Wood et al., 2004). The long half-life may potentiate drug efficacy by prolonging drug-induced down-regulation of receptor-mediated tyrosine kinase activity (Gilmer et al., 2008).

An obvious way to prolong the off-rate is to design a drug that engages in a covalent bond with the target leading to irreversible or slowly reversible binding (Singh et al., 2011). Examples are the pan-ERBB inhibitors neratinib and afatinib currently undergoing clinical trials. These drugs carry a reactive group (a so-called Michael acceptor) and engage in a covalent bond with Cys797 of the EGFR protein (Figure 10). Crucially, neratinib and afatinib have been shown to be active against the T790M/L858R double mutant that renders most reversible inhibitors ineffective. Covalent inhibitors in general have been proposed to be more resilient towards target mutation (Singh et al., 2011). Several examples of covalent drugs as well as the challenges of tuning the reactivity of the chemically reactive group to avoid toxicity have been reviewed elsewhere (Potashman and Duggan, 2009; Singh et al., 2011; Smith et al., 2008). The recent co-crystal structure of abiraterone shows that a ‘covalent’ interaction is formed between the pyridine moiety and the haem iron present in the active site of the CYP171A1 target (DeVore and Scott, 2012).

4.2 Preclinical cancer models

In therapeutic development a major hurdle is translating efficacy determined during the drug discovery phase into efficacy in the clinic, as there are often discrepancies between drug efficacy demonstrated in the current preclinical experimental models and final efficacy, or lack thereof, in patients. Preclinical models need to take account both of the molecular nature of the target and also of how the chemical compound will behave. Different models will be required for compounds targeting genetic dependency, as compared to those exploiting host-tumour interaction through stromal or hormonal signalling or compounds targeting non-oncogenic addiction, perhaps through synthetic lethal interactions (Caponigro and Sellers, 2011).

During lead optimisation, tumour cell lines grown in vitro culture are frequently used as the first line of study as cell proliferation, apoptosis and other cellular endpoints can easily be measured using different technologies in high-throughput formats as required. Cancer cell line-based platforms have recently been reviewed (Sharma et al., 2010). As already mentioned above, it is valuable to use proliferation/apoptosis assays in combination with mechanism-based cell assays for target engagement and pathway inhibition. Using both types of assays together ensures that the target is modulated at concentrations similar to or lower than the levels required for cell growth inhibition and thus that the growth inhibitory effect is achieved by the anticipated mechanism. Confidence is increased by seeing a correlation between cancer cell growth inhibition and target/pathway modulation in the structure-activity relationships across a range of analogues.

There are, however, some caveats with regard to the use of tissue culture cell lines. In vitro culture of cancer cell lines can result in substantial phenotypic, genetic, and epigenetic alterations induced by artificial environment of cell culture, which may not reflect the biology of original tumours in situ. Moreover, prolonged culture of tumour cells will inevitably result in genetic drift with time. In addition, cancer cells from some tumour types or genetic backgrounds fail to grow under in vitro culture conditions and will be under-represented in cell line panels. As adherent cell growth on a plastic dish has limitations and may not really reflect tumour biology, 3-dimensional growth conditions are now being more frequently used as in vitro models that may be more representative of tumour biology in the host organism. Cells are either grown as spheroids in suspension, or in matrices that may represent the extra-cellular environment of the tumour. However, it is currently quite challenging to use these 3-dimensional models in sufficiently high throughput, although methodologies to increase throughput are in development.

Compounds that show promising activity in cell-based assays will progress to in vivo animal studies, provided that their ADME properties are suitable for such studies. Human tumour xenografts have been used extensively and when selected to exhibit the relevant molecular characteristics and pathogenic drivers can mimic many of the features and response to targeted drugs of the corresponding human cancers (Caponigro and Sellers, 2011; Ocana et al., 2011; Workman et al., 2010). However, the above reviews also highlight limitations of human tumour xenografts: They do not replicate human stromal–tumour cell interactions, they grow much faster than human tumours in patients and hence can be more sensitive to targeted drugs. The human tumour xenograft approach also generally relies on the use of established cancer cell lines with all their limitations that are described above. Finally, the tumours are usually not implanted at their native site. Recently, the latter two shortcomings have been mitigated by using orthotopic models in which the tumour is transplanted into the correct organ site and by relying on early passages of fresh biopsies from patients to maximize retention of the original patient characteristics (Caponigro and Sellers, 2011; Ocana et al., 2011; Workman et al., 2010). We believe that despite the limitations listed above, molecularly relevant human tumour xenografts will retain their role as workhorses in drug discovery since they allow testing of agents in a variety of genetically-defined human tumours with reasonable throughput. Sufficient throughput is critical once an advanced stage of lead optimisation has been reached and often 50–100 efficacy experiments per year may have to be performed. Our view is that the key factors in using human tumour xenografts successfully are to ensure molecular relevance and in addition to avoid overly optimistic interpretation of the outcome. Thus it seems likely that a higher hurdle for efficacy should be used than is often currently the case in the selection of clinical development candidates.

Referred to briefly earlier, GEMMs have now emerged as in vivo models for cancer drugs and are complimentary to the use of human tumour xenografts (Caponigro and Sellers, 2011; Ocana et al., 2011; Workman et al., 2010). In GEMMs, the tumours are initiated by inducing a specific genetic lesion, e.g. through activation or overexpression of oncogenes such as KRAS or MYC or via depletion of tumour suppressors, ideally in a tissue- and time-dependant manner. GEMMs thus have the advantage that the tumours arise in a more natural fashion and exist in their natural, albeit murine environment. In addition, they allow the investigation of drug effects on tumour development, as well as studies on established cancers. Crucially some GEMMs have shown patterns of sensitivity to chemotherapeutic agents and development of resistance that are similar to their human tumour counterparts.

On the other hand, GEMMs also have some limitations. From the drug discovery viewpoint, as distinct from more basic research, the logistics of maintaining sufficient stocks of tumour-bearing mice can be challenging, particularly for models with long tumour latencies which in most cases renders it impossible to use them routinely to support make-and-test cycles in the context of lead optimisation (Politi and Pao, 2011). They may also suffer from significant protein sequence differences between the human and the murine target and will likely also show limited genetic aberrations whereas human tumours generally exhibit very large numbers of mutations, although more sophisticated cancer gene-combination models are now emerging. Perhaps the most valuable role for GEMMS in molecularly targeted drug discovery is to show proof of concept for in vivo animal model activity in the context of clearly defined and clinically relevant genetic abnormalities. Of note in the present context, a promising and practical compromise for drug discovery is the use of transplantable lines derived from GEMMs.

The question whether human tumour xenografts or GEMMs are more predictive for the response of human cancers continues to be intensely debated. We believe that the predictive value of neither model is fully understood and that they are of complimentary value for drug discovery. Human tumour xenografts (and transplantable GEMM tumours) are the better suited for routine in vivo testing and GEMMs can be used to explore proof of concept once an advanced candidate has been identified. There are now many examples where both models give similar results to a targeted agent when the pathogenic driver is the same. Where this is seen it can build additional confidence in the target hypothesis.

4.3 Preclinical safety testing

Once a preclinical candidate has been identified, sufficient preclinical data have to be generated to support a clinical trial. A new guidance from ICH (International conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) topic (S9) provides recommendations for non-clinical evaluations to support clinical trials of cancer drugs. This new ICH topic has recently been reviewed (Joness and Jones, 2011). For safety testing of small molecule drug candidates, generally the use of one rodent and one non-rodent species is recommended but some exceptions exist where a single species may be possible, e.g. with cytotoxics. However, many organisations perform the more extensive studies required for a non-cancer agent (under the ICH topic M3).