Cancer Drug Design and Discovery

I

Basic Principles and Methodology

Outline

Chapter 1 Modern Cancer Drug Discovery

Chapter 2 Pharmacogenomics and Personalized Medicines in Cancer Treatment

Chapter 3 Natural Product Chemistry and Cancer Drug Discovery

Chapter 4 Structural Biology and Anticancer Drug Design

Chapter 1

Modern Cancer Drug Discovery

Integrating Targets, Technologies, and Treatments for Personalized Medicine

Paul Workman and Ian Collins,    Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London, UK

Abstract

We are living through an exciting era for cancer drug discovery and development—one that is full of enormous opportunities and challenges. In this chapter, we aim to capture the sense of excitement and to describe the opportunities and challenges in the discovery and design of molecularly targeted small-molecule cancer drugs. In particular, we stress the importance of integrating three key themes: targets, technologies, and treatments. First, the molecular targets of contemporary drug discovery projects reflect our increased understanding of the genetic or epigenetic dysregulation of cellular processes that leads to the initiation and malignant progression of cancer. We describe different ways in which potential new molecular targets can be identified, validated, and prioritized. Second, we highlight the integrated application of a range of powerful drug discovery technologies, including genomic methods, compound screening methodologies, and the application of structural biology. We describe the multidisciplinary endeavor of small-molecule cancer drug discovery and emphasize how the challenges of multiparameter lead optimization are being met. Third, we examine how technological innovations in drug design have led to the new treatments that have emerged over the past 15 years. We show how the development and use of such treatments depend on the identification of biomarkers to select appropriate patients and to monitor the effects of treatment. Finally, we reflect on the success to date of mechanism-based molecular therapeutics acting on cancer-causing targets and outline the future prospects for the enhanced development of personalized molecular medicines, highlighting the need for combinatorial drug treatments to overcome the enduring problem of drug resistance.

Keywords

Cancer; Cancer treatments; Drug discovery; Molecular medicine; Molecular targets; Molecularly targeted therapy

Acknowledgments

The authors' work in the Cancer Research UK Cancer Therapeutic Unit (http://www.icr.ac.uk/research/research_divisions/Cancer_Therapeutics/index.shtml) is supported primarily by Cancer Research UK program grant number C309/A11566 and by The Institute of Cancer Research. Paul Workman is a Cancer Research UK Life Fellow. We thank our colleagues in the Unit and The Institute of Cancer Research and our many external collaborators for valuable discussions.

Introduction: Changing Times

Cancer drug discovery has undergone a remarkable series of changes over the last 15 years. So what has changed so much? First, the molecular targets of contemporary cancer drug discovery projects are very different. Current targets reflect our increasing understanding of the genetic and epigenetic changes that are responsible for the initiation and malignant progression of cancer through the dysregulation of cell biochemistry and signaling networks [1,2]. Second, the integrated application of a range of powerful drug discovery technologies has had a major impact [3]. Third, many new treatments have emerged over the last 15 years based on this paradigm that are firmly established in the clinic [4]. The present volume brings together many of the important aspects of the discovery and design of new cancer drugs, emphasizing small molecules. In this chapter, we provide a scene-setting introduction to, and overview of, modern small-molecule cancer drug discovery. We will argue that success is dependent on the close integration of the three major themes: targets, treatments, and technologies. In this regard, cancer drugs are leading the way in the development of personalized medicine. On the other hand, it is clear that drug resistance is just as much a problem with molecularly targeted agents as it has been with cytotoxic drugs.

First, however, it is useful to assess at the overview level what progress has been made and what the current limitations are. This provides a firm foundation for understanding what needs to be done to move the field forward. Following this, we will review the drug discovery process in detail from the identification of the molecular target through to selection of a drug candidate. Specific examples and case histories will be provided. We end the chapter by drawing some conclusions and taking a look into the future, including the potential for combinatorial therapies to overcome the problem of drug resistance.

Successes and Limitations

Cytotoxic Agents

The first generation of cancer drugs, originating since the 1950s, were almost all cytotoxic agents. These frequently act by damaging DNA, inhibiting its synthesis or interfering with the mechanics of cell division, for example, by blocking topoisomerases or binding to microtubules [5,6]. Many of these agents were discovered by screening for chemical compounds that were able to kill cancer cells, as with a natural product like the microtubule inhibitor paclitaxel [7]. DNA-alkylating agents, originally based on sulfur and nitrogen mustards, were structurally modified so as to control their rates of chemical reactivity, leading to drugs like cyclophosphamide and ifosphamide [8]. Drugs developed in this first, cytotoxic era of cancer drug development were not designed to take advantage of our current knowledge of the genetic and molecular basis of cancer. Nevertheless, many of them were molecularly targeted, as in the case of the antifolate thymidylate synthase inhibitors, in the sense that they were designed according to the principles of contemporary medicinal chemistry and in some cases involved the application of structure–activity relationships and X-ray crystallography to a single, defined molecular target [9].

There have been many notable successes with cytotoxic drug treatments for cancer. The disease exists in a large number of forms, as defined anatomically under the light microscope and, more recently, at the molecular level. The effectiveness of drug treatment varies across these different anatomical, histological, and molecular types. Major improvements have been achieved in the treatment of leukemias, lymphomas, testicular cancer, and children's malignancies with cytotoxic drugs, leading to marked increases in survival (www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/document/acspc-031941.pdf). On the other hand, progress has been modest at best in the common adult epithelial tumors, although successful exceptions have included adjuvant cytotoxic chemotherapy in breast and prostate cancers. Despite some advances, cancer remains the second most frequent cause of death in the United States (http://www.cdc.gov/nchs/data/nvsr/nvsr61/nvsr61_06.pdf). Furthermore, despite a decrease in incidence for many cancers, incidence rates for other cancers are increasing, including human papillomavirus-related oropharyngeal cancer; melanoma; and cancers of the pancreas, liver, and kidney. The worldwide increase in cancer burden in part reflects the changing demography of both developed and developing countries, where older age groups with the highest cancer risk are increasing in size [10]. Together with tremendous opportunities afforded by the availability of new targets and technologies, the major unmet need for cancer treatment continues to drive the extensive contemporary efforts in cancer drug discovery.

New Molecular Cancer Therapeutics

From the late 1990s onward, it became clear that major gains in survival were unlikely to be made by fine-tuning the classical cytotoxic agents. Antiestrogen and antiandrogen therapies in breast and prostate cancers, respectively, continued to lead to improved outcomes in specific cancer subtypes, as exemplified by the recent approval of abiraterone (Zytiga) for the treatment of metastatic castration-resistant prostate cancer [11] (see Chapter 11), but new approaches were needed to tackle the wider forms of the disease. This view coincided with the arrival of new molecular target opportunities emerging from basic cancer research and large-scale genomics. Consistent with common usage, we will use the term molecular cancer therapeutics to refer to mechanism-based agents acting on drug targets involved in the molecular causation of cancer. Success with the new molecularly targeted approach is evidenced by the approval by the US Food and Drug Administration (FDA) of many innovative drugs, both antibodies and small molecules, since the introduction of trastuzumab (Herceptin) in 1998 (Table 1.1; and see http://www.centerwatch.com/patient/drugs/druglist.html).

TABLE 1.1

Examples of Targeted Molecular Cancer Therapeutics Receiving Marketing Approval by the US Food and Drug Administration (FDA), 1998–2012¹

¹See www.centerwatch.com.

²Patients who have been previously treated with multiple lines of therapy.

³In combination with trastuzumab and docetaxel.

⁴Patients who have received at least two prior therapies, including bortezomib and an immunomodulatory agent, and have demonstrated disease progression.

⁵After failure of one prior systemic therapy.

⁶In combination with prednisone.

⁷FDA-approved companion diagnostic test for the presence of BRAFV600E mutation.

⁸FDA-approved companion diagnostic test for the presence of ALK rearrangement.

⁹Unresectable locally advanced or metastatic disease.

¹⁰CD30-targeting antibody cleavably conjugated to monomethyl auristatin E.

¹¹In combination with cisplatin and a fluoropyrimidine.

¹²In combination with interferon alpha 2a.

¹³In combination with capecitibine.

¹⁴Second-line treatment.

¹⁵Patients who have received at least two prior therapies.

¹⁶First-line therapy for postmenopausal women with locally advanced or metastatic breast cancer.

¹⁷Advanced breast cancer in postmenopausal women whose disease has progressed following therapy with tamoxifen.

¹⁸Second- or third-line therapy.

As the first example of a modern, targeted molecular cancer therapeutic, the humanized monoclonal antibody trastuzumab showed substantial therapeutic activity in patients with breast cancers that overexpress the ERBB2/HER2 oncoprotein as a result of DNA amplification [12]. This population represents about 30% of patients with node-positive breast cancer, and they benefited from a 50% decrease in disease recurrence over a 20-month period. Since its first approval in 1998, trastuzumab has become part of the standard of care in treating metastatic ERBB2/HER2-positive breast cancer and exemplifies the potential of individualized treatment based on a molecular biomarker to stratify the patient population [13].

The first successful small-molecule targeted cancer therapeutic was the tyrosine kinase inhibitor (TKI) imatinib (Gleevec; see Fig. 1.1 for the chemical structures of this and other small molecules discussed in this chapter). Imatinib is often considered a prototype for drugs targeting oncogenic signal transduction proteins [14], although the extent to which it is atypical and represents a significant outlier rather than a poster child has been debated [15,16]. The primary target of imatinib is the Abelson tyrosine kinase (ABL), which is activated by a chromosomal translocation that occurs in chronic myeloid leukemia (CML), creating the BCR–ABL fusion protein. The molecular abnormality generates a unique dependence on the ABL kinase. This explains why imatinib is so impressively effective in chronic-phase CML. The median overall survival was 88% at 30 months [17]. Equally important, ABL does not seem to be very critical for normal tissues, which probably explains why the drug is so well tolerated, even during chronic treatment. Imatinib has become the first-line therapy for CML, and has been followed by multiple second-generation BCR–ABL inhibitors such as dasatinib (Table 1.1) [18]. In part, the development of new BCR–ABL inhibitors has been driven by the need to address the development of imatinib resistance in patients (discussed in this chapter).

FIGURE 1.1 Chemical structures of selected small molecules described in the text.

The first small-molecule inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, gefitinib (Iressa) and erlotinib (Tarceva), showed activity in patients with non-small-cell lung cancer (NSCLC). A survival advantage was seen with patients receiving erlotinib plus chemotherapy compared to those receiving chemotherapy alone [19]. Gefitinib showed activity in only a small subset of NSCLC patients in initial trials, subsequently identified as carrying activating mutations in EGFR [20]. While the links between EGFR mutations and sensitivity to the TKIs were initially disputed, mainly due to the low frequency of the mutations in the early trial populations and a low accrual of tumor tissue samples, multiple prospective trials in NSCLC since 2005 have confirmed the effectiveness of the agents in EGFR-mutant NSCLC [21]. More recent EGFR inhibitors in development include irreversible agents such as afatinib, which binds covalently to both the wild-type EGFR and the T790M kinase gatekeeper mutant that shows reduced binding of the first-generation inhibitors [22].

Two other recently approved signal transduction kinase inhibitors illustrate the progress that has been made in the discovery of targeted therapeutics. In 2002, mutation of the serine/threonine-protein kinase B-Raf (BRAF), particularly the V600E variant, was identified as an oncogenic driver of many different cancers, with especially high prevalence (ca. 50%) in malignant melanoma [23]. The inhibitor vemurafenib (Zelboraf) showed exceptional activity against V600E BRAF melanoma in early clinical trials [24], and was approved as the first targeted agent for the treatment of metastatic melanoma in 2011 [25]. As with imatinib in BCR–ABL-dependent CML, vemurafenib targets an essential driver oncogene in the V600E BRAF patient population, and dramatic effects on tumor response and patient survival were achieved. Importantly, a companion diagnostic using RT-PCR to detect the mutant V600E BRAF allele in patients' tumors prior to treatment was developed in parallel with the clinical development, which fortuitously also detected the V600D and V600K oncogenic variants, allowing for a truly targeted treatment strategy.

The approval of crizotinib (Xalkori) in 2011 for the treatment of EML4–ALK-positive NSCLC further exemplifies the practice-changing potential of targeted therapeutics when the appropriate combinations of molecular target, inhibitor, and companion diagnostic are brought together [26]. Approximately 5% of NSCLC result from the formation of a fusion protein between the receptor tyrosine kinase ALK and the EML4 protein, resulting in constitutive ALK activation. The identification of the rearranged oncogene in 2007 [27] was followed by the first reports of dramatic clinical activity in advanced NSCLC only three years later [28]. This rapid development was made possible in part by the repurposing of crizotinib, which was already in development as an inhibitor of hepatocyte growth factor receptor tyrosine kinase (MET) and was found to inhibit ALK as well [29]. The development of crizotinib also shows how previous experience with molecularly targeted therapy for receptor tyrosine kinases, such as gefitinib and erlotinib, can be brought to bear to accelerate the development of new agents of this type. As with vemurafenib, a companion diagnostic was developed, this time using fluorescent in situ hybridization technology to identify the ALK rearrangement [30]. The test was applied in the pivotal clinical trials of crizotinib and was approved by the FDA in parallel to the drug.

Despite certain caveats, the clinical activity with the agents discussed here is consistent with the concept of oncogene addiction, whereby cancers develop dependence upon, or become addicted to, activated oncogenes [31–33]. This idea reconciles the observation that despite the activation of several oncogenes being necessary to initiate and sustain the hallmark properties of malignant cells, inhibition of the action of just one may have significant therapeutic effect. The agents discussed in this section and in Table 1.1 also provide proof of the concept that clinically useful therapeutic activity and patient benefit can be achieved with drugs that inhibit oncogenic signal transduction.

In addition to targeting oncogenic signaling pathways, the approach of mechanism-based inhibition of angiogenesis has also been validated by both small-molecule kinase inhibitors and antibodies. New blood vessel formation is required to support the growth of solid tumors, and this process of angiogenesis is therefore a logical target for therapeutic modulation. Bevacizumab (Avastin) is a monoclonal antibody that binds vascular endothelial growth factor (VEGF), which is an important driver of the proliferation and functions of the endothelial cells responsible for angiogenesis. Activity has been seen in various solid cancers [34]. When used in combination with cytotoxic agents, bevacizumab offers an extension of survival by a few months in advanced disease, as in the case of colorectal cancer [35]. The activity of the small-molecule kinase inhibitors sunitinib (Sutent) and sorafenib (Nexovar) in renal cell cancer may well be due, at least in part, to inhibition of the tyrosine kinase activity associated with the membrane receptors for VEGF and another angiogenic growth factor, platelet-derived growth factor (PDGF). However, these drugs inhibit a range of kinases and are members of a growing class of multitargeted kinase inhibitors [36]. The attribution of action through any one molecular target is difficult for such agents, and the polypharmacology may be essential for maximizing their effects.

The approval of vorinostat (Zolinza) in 2006 for the treatment of cutaneous T-cell lymphoma represented the first entry of an agent targeting chromatin-modifying enzymes into clinical use [37]. Histone acetyl transferases (HATs) and deacetylases (HDACs) contribute to the regulation of gene expression through acetylation of residues in the histone proteins that act as the core scaffolds for DNA in nucleosomes. In turn, the conformational changes associated with the histone functionalization determine the accessibility of the DNA toward transcription factors. Mutation, overexpression, translocation, and amplification of genes encoding HATs have been observed in various cancers. HDACs cooperate with oncogenic protein products to determine their effect, acting as posttranslational modifiers of the oncogene function. The mechanism of action of vorinostat likely involves both altering gene expression through changing histone and transcription factor acetylation, and also altering the function of cell cycle proteins through inhibiting their deacetylation [38]. In a pivotal phase IIb trial in cutaneous T-cell lymphoma, vorinostat showed an overall response rate of 30% in heavily pretreated patients with persistent, progressive, or refractory disease [39].

The challenges encountered with molecular targeted therapies

There is no doubt that the agents discussed in this section are quite distinct from those from the cytotoxic era in that they target the precise molecular mechanisms that are responsible for the initiation and progression of cancer, often to the level of a single, specific protein target. They clearly have sufficient therapeutic activity to warrant regulatory approval. On the other hand, a range of complications and limitations have emerged [4,40]. These are particularly concerned with uncertainties over the clinically relevant mechanism of action, unexpected toxicities, and, most importantly, the development of resistance to the new agents [41]. As an example of the first point, it is likely that at least part of the activity of trastuzumab is due to antibody-directed cellular cytotoxicity, and the precise role of the effects on receptor signaling is still unclear. In addition, the combination of trastuzumab with anthracycline-based chemotherapy causes an increase in cardiac toxicity.

In the case of imatinib, responses in accelerated-phase and blast-crisis CML are less dramatic than those in the chronic phase, potentially due to the involvement of additional oncogenic drivers in the more advanced forms of the disease. Resistance to imatinib is common and is associated with mutations that lead to an impairment of binding to the adenosine triphosphate (ATP) site of the kinase [42], or with the activation of BCR–ABL-independent pathways such as signaling through SRC-family of non-receptor tyrosine kinases [43]. On the other hand, sensitivity of many of the mutant forms can be maintained to nilotinib (Tasigna), dasatinib (Sprycel), and bosutinib (Bosulif), which also inhibit SRC-family kinases [43,44]. Although these agents effectively tackle a spectrum of imatinib-resistant BCR–ABL mutants with modifications to the kinase domain P-loop, the T315I mutation remains intractable with currently approved agents [45].

Kinase domain mutations are likewise responsible for secondary resistance to gefitinib and erlotinib in NSCLC, particularly the T790M mutation on exon 20 of EGFR. Thus, responses to gefitinib and erlotinib have often proved to be of limited duration, and acquired resistance to current TKI therapy appears unavoidable in NSCLC [46]. Once again, alternative inhibitors are being developed to overcome this, with the irreversible EGFR, ERBB2, and ERBB4 kinase inhibitor afatinib one of the most advanced agents that has potent activity against mutant as well as wild-type EGFR [47]. Regardless of EGFR mutational status, amplification of the MET gene and/or activation of MET signaling has been observed in up to 20% of tumor samples following gefitinib or erlotinib treatment and provides another mechanism of resistance to the drugs [48], in this case treatable with crizotinib. Here, combination therapy with MET inhibitors is one means to counter the resistance. Other tractable resistance mechanisms to EGFR inhibitors have been described [49]. There are also indications that a so-called vertical blockade of EGFR signaling by combining an intracellular receptor tyrosine kinase inhibitor with an extracellular antibody therapy to the same EGFR target may offer increased prospects for disease control [50]. Kinase domain mutations also underlie resistance to crizotinib [51].

In the case of vemurafenib, resistance does not appear to result from further mutations in V600E BRAF, although splice variants of V600E BRAF that are resistant to the drug are known [52]. Instead, mechanisms that lead to retention of mitogen-activated protein kinase (MAPK) pathway signaling in a V600E BRAF-independent manner, including activation of EGFR and potentially other receptor tyrosine kinases, upregulation of RAF proto-oncogene serine/threonine-protein kinase (CRAF) expression, a pathway switch to serine/threonine-protein kinase A-Raf (ARAF) or CRAF signaling, and oncogenic activation of neuroblastoma RAS viral oncogene homolog (NRAS), are important [25,53]. Another problem is that while ATP-competitive inhibitors such as vemurafenib inhibit extracellular-signal-regulated kinase (ERK) signaling in cells with mutant V600E BRAF, paradoxical activation of signaling is seen in cells with wild-type BRAF. Several mechanistic rationales have been advanced based on the formation of homodimers and heterodimers for transactivation of wild-type BRAF and related proteins such as CRAF, ARAF, or kinase suppressor of Ras 1 (KSR), whereby binding of the kinase inhibitor to one component of the dimer inhibits that enzyme but stabilizes the dimer and promotes activation of the other component [54]. In contrast, mutant V600E BRAF is active as a monomer. A consequence of this paradoxical transactivation of wild-type BRAF signaling is a distinctive toxicity of V600E BRAF inhibitors, with the emergence of cutaneous squamous cell carcinomas and benign keratoacanthomas associated with activating RAS mutations in wild-type BRAF cells in patients treated with vemurafenib [55]. Although the superficial keratoacanthomas can be removed surgically, there are concerns about the potential for malignant progression of cells with mutant RAS in deep-seated organs such as the lung and colon [56]. Combination treatments to tackle the activation of these bypass signaling pathways have been proposed, particularly the combination of BRAF and MAPK kinase (MEK) inhibitors.

Turning to multitargeted inhibitors like sorafenib and sunitinib, understanding their mechanism of action is somewhat confounded by their multitargeted nature, and reasons for differences in the therapeutic activity of the various small molecules and antibodies targeted to the VEGF–VEGF receptor axis are unclear [57]. While bevacizumab was approved by the FDA in 2008 for treating metastatic breast cancer, approval for this indication was withdrawn in the United States in 2011. Although the agent was shown to slow disease progression, there was insufficient evidence of extension of life or improvement in quality of life to support continued approval when balanced against the adverse effects of treatment [58]. The clinical experience with bevacizumab in breast cancer exemplifies two limitations now recognized with molecularly targeted agents. First, molecularly targeted agents do not always lack significant side effects, despite the targeting of cancer-specific processes, and the side effects of each drug or antibody treatment must still be considered relative to the benefit of the treatment and the severity of the disease [59]. Of note in this regard has been the potential for cardiotoxicity observed with some receptor tyrosine kinase inhibitors, which may be mechanism based or due to off-target kinase inhibition depending on the agent [60,61].

Another limitation of molecularly targeted agents illustrated by bevacizumab in breast cancer is that the targeted mechanism may not translate across cancer tissue types as generally as originally expected. A further example of this is provided by the V600E BRAF inhibitor vemurafenib, which did not show efficacy as a single agent in the treatment of V600E BRAF colorectal cancer despite the presence of the specific mutation in the disease [62]. The lack of sensitivity was associated with feedback activation of EGFR in colon cancer cells, whereas melanoma cells express low levels of EGFR and remain sensitive to V600E BRAF inhibition. Thus, the biological context in which an oncogenic mutation occurs is important in determining the response to therapeutic interventions targeting the abnormality. On the other hand, the data in Table 1.1 very clearly show the extension of use of a number of approved molecularly targeted agents across multiple tumor tissue types, emphasizing that the targeting of underlying molecular lesions common to different tumor types remains a valid concept, exploitable for increased patient benefit.

Rising to the Current Challenges of Oncology Drug Discovery and Development

The preceding section shows that overall the results with the new molecular therapeutics have been mixed. With respect to small-molecule drug development, it is notable that many of the agents are directed to the same small set of molecular targets. While this repetition reflects the established importance of certain targets in cancer, and a choice between several drugs for a given target can be desirable in clinical practice, there are notable absences in the current selection of approved molecularly targeted agents. For example, despite significant and sustained preclinical research, there is as yet no approved small molecule directed toward a cell cycle or mitotic kinase [63]. While upstream receptor tyrosine kinases are extensively drugged, inhibitors acting at downstream nodes in mitogenic signaling pathways remain restricted to BRAF and mammalian target of rapamycin (mTOR) inhibitors. However, given current late-stage clinical trial activities, it would be expected that inhibitors of phosphatidylinositide 3-kinase (PI3K), protein Kinase B (PKB, otherwise known as AKT), MEK, and checkpoint kinase 1 (CHK1), among others, may appear in the near future to address these gaps [4].

Assessments of the overall success rates for oncology drug development illustrate how challenging an activity it is [15,64,65]. A frequently cited analysis showed that failure rates for cancer drugs in clinical trials during the period from 1990 to 2000 were worse than for most other therapeutic areas [64]. Only 5% of oncology drugs entering the clinic went on to gain regulatory approval for marketing, while 95% failed. This is compared with an 11% success rate—more than double—for other diseases. A subsequent study looking at the extended period of 1990–2006 showed a similarly low US approval success rate (8%) for oncology drugs [65]. An analysis of the economics of new drug development found that oncology drugs benefit from a disproportionally high share of FDA priority review ratings, orphan drug designations at approval, and inclusion in the FDA's expedited access programs [66]. Those authors also found that clinical approval rates were in fact similar for oncology versus other drugs, but that a greater proportion of cancer drug candidates were abandoned in advanced-stage clinical evaluation, where failures are very expensive. This study also showed that clinical oncology drug development timelines were longer than for other therapeutic areas, probably due to evaluation in a greater number of indications. In a more recent analysis, the mean development time for oncology drugs over the period 2000–2009 was determined as 6.9 years, with only central nervous system drugs taking longer on average [67]. However, the authors also noted that the numbers of approved new anticancer drugs increased steadily and significantly each decade from the 1980s through the decade 2000–2009. Data on contemporary kinase inhibitor development suggest that they have markedly better prospects (odds of one in two) for successfully completing clinical development from phase I trial initiation to new drug approval than oncology drugs as a whole [68]. However, consistent with the earlier analysis of Kola and Landis, the greatest attrition was seen at the transition from phase II to phase III, suggesting that the attainment of sufficient therapeutic activity remains the major challenge in cancer drug development. Such late-stage failures contribute in a major way to the estimated fully capitalized cost of US$1000 million or more per approved new drug [69,70].

Clearly, we would like to see the success rates for cancer drugs to be higher, development times shortened, and failing drugs to be identified earlier. It is very instructive to understand the reasons for the attrition of oncology drugs in the clinic, since this allows us to focus attention on the areas that are most problematic. Metrics show that the reasons for failure have changed with time [64]. In the early 1990s, poor pharmacokinetics and limited bioavailability were the major problems. Recognition of this led to the use of predictive assays for absorption, distribution, metabolism, and excretion (ADME) properties [71]. Implementation of these assays to weed out compounds with ADME liabilities led to a fall in the clinical failure rate due to this cause from 40% to 10% by the year 2000 [64]. As a result, the principal causes of attrition in clinical development became insufficient therapeutic efficacy (30%) and unacceptable toxicity (30%). A more recent analysis across multiple disease indications showed that lack of efficacy (51%) and toxicity (19%) continue to be the dominant reasons for failure in phase II development [72]. Particular attention therefore needs to be paid to reducing failure due to these factors.

The risk of failure because of inadequate therapeutic activity could be reduced by better selection of molecular targets [4,15,73]. For target selection and validation, perspectives on the best targets have changed as a result of clinical experiences with the first generation of molecularly targeted agents [40]. First, there is a greater understanding of the inherent differences between genetic target validation technologies, such as RNA interference (RNAi) methods, and the use of pharmacological inhibitors to probe target biology [74,75], and of the limitations of both techniques. As a result, there is increased emphasis on the validation of new targets using multiple approaches. This has led to a more explicit focus on the qualities of preclinical small molecules that will make them fit for purpose chemical probes for target validation [76]. The integrated approach to the discovery and refinement of drug candidates (discussed later in this chapter) is invaluable to the discovery and credentialing of chemical probes for specific biologies. In parallel to the better use of target validation technologies, there is also an increased understanding of what comprises a validated target and, importantly, the accompanying genetic or epigenetic backgrounds that may be required for modulation of a given target to achieve a therapeutic outcome [77]. Increased use of functional genomics technologies, such as RNAi and compound screens in panels of genetically well-characterized cancer cells, is allowing a more systematic approach to defining the right combinations of targets and the accompanying contexts in which they function [78]. The range of potential targets has also expanded to include survival pathways that may remain critical for cancer cell survival in response to the stress caused by multiple underlying genetic or epigenetic changes, and thus may operate over wider genetic contexts [79]. These include protein quality control mechanisms and DNA damage detection and repair processes [80,81].

Of note is that while the genetic understanding of many cancers and the discovery of cancer genes and potential molecular targets have increased dramatically in the era of genomic sequencing [82], the selection of targets has remained largely ad hoc. Patel et al. [83] have recently published a systematic and objective, multifaceted computational approach to assessing and prioritizing potential targets, including an estimation of the druggability of a given target based on prior structural biology and pharmacological data. A point to stress is the importance of the reproducibility of the preclinical data, whether biochemical, cellular, or in animal models, on which preclinical target validation assessments are made and drug discovery projects are launched [84].

The success rate of clinical development can also be improved by identifying animal models of human cancer that have better predictive power [85–88]. Molecularly characterized human tumor xenografts in immunosuppressed mice remain useful for drug discovery, with increasing emphasis placed on orthotopic implantation and early-passage patient-derived xenografts [89]. The use of genetically engineered mouse models (GEMMs) in the validation of proposed cancer target biology offers one approach to achieving better animal models [90,91]. For example, mice that conditionally express endogenous mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) and p53 alleles in their pancreatic cells develop pancreatic tumors with pathophysiological and molecular features resembling those of human pancreatic ductal adenocarcinoma. This model has been used to understand the marked resistance to chemotherapy of pancreatic cancer, and to validate new targets for intervention [92,93].

The risk of attrition due to unacceptable side effects can be mitigated by developing improved methods for predicting on-target and off-target toxicity [94]. First-in-class drugs acting on previously unprecedented molecular targets carry a higher level of risk compared to those that work on targets that are well precedented in the clinic [95]—but, at the same time, these high-risk drugs also have more potential to be truly innovative. An analysis by DiMasi and Grabowski [66] indicated that oncology drugs had the highest rate of first-in-class introductions. In addition to judicious target validation and selection and the use of more predictive models for efficacy and toxicity, late-stage failure can also be minimized by the careful use of biomarkers to identify the most responsive patients and to provide proof of concept for the proposed molecular mechanism, especially in phase I and II clinical trials [14,96–98]. The success of this approach has been demonstrated in the development of vemurafenib and crizotinib and their accompanying diagnostic tests as detailed in this chapter. Later in this chapter, we will advocate the use of patient selection biomarkers together with pharmacokinetic–pharmacodynamic endpoints as part of the pharmacological audit trail concept designed to aid decision making in clinical development [97,99–102].

The often rapid emergence of resistance mechanisms to molecularly targeted agents compromises the effectiveness of drugs that have been successfully developed. To address this, research into possible resistance mechanisms is being brought forward into the preclinical discovery phase of new molecularly targeted agents [41,78]. New molecular targets can be considered along with companion targets that may modulate resistance mechanisms, and may need to be inhibited in combination to maximize the effectiveness of a new targeted drug. The genetic backgrounds where a specific targeted agent will have maximal effect can also be defined using synthetic lethality approaches [103,104]. The need for combination therapies of targeted agents has encouraged the realization of commercial mechanisms for first-in-human clinical trials of drugs in combination, which might otherwise be developed separately due to distinct proprietary interests [105]. There is also renewed interest in intrinsic multitargeted small-molecule drugs, sometimes referred to as selective nonselective inhibitors [36], where a controlled polypharmacology is engineered into a single chemical agent. However, combinations of multiple specific inhibitors may give more flexibility for longitudinal adaptive therapy to target the changing genotypes' underlying resistance.

Recent sequencing studies have shown the extent of genetic heterogeneity within individual patients' tumors [106]. These indicate that patients presenting with multiple sites of disease are likely to harbor tumors with multiple, different molecular lesions, and therefore different sensitivities to treatments. For example, in a study of renal clear cell carcinoma, the somatic mutations present differed between regions of the anatomically defined primary tumor, as well as between the primary site and distant metastases [107]. In a recent study of BRAF-inhibitor-resistant melanoma, molecular heterogeneity was observed at the intralesional level, with two distinct subclones populating a single metastasis site [108]. The concept of personalized cancer medicine is changing to tackle the issues of resistance to targeted agents and tumor heterogeneity. The likely presence of multiple genotypes within an individual patient's tumor burden is reinforcing research into combining new molecular-targeted agents to increase treatment effectiveness [41,78]. Observations that the clonal constitution of tumors also changes in response to the selection pressure of treatment, as exemplified in recent longitudinal case studies in multiple myeloma [109,110], suggests that individualized iterative diagnostic and treatment sequences will be necessary to mitigate the evolution of tumor genetics in response to therapies.

Integrated Small-Molecule Drug Discovery and Development

The successful discovery and development of small-molecule cancer drugs are highly dependent upon the creative interplay between many disciplines: these include genetics, genomics, and bioinformatics; cell and molecular biology; structural biology; tumor biology; pharmacology; pharmacokinetics and metabolism; medicinal chemistry; and experimental medicine. The application of a wide range of powerful technologies has had a major impact. Examples include high-throughput genomic approaches for discovering new targets and identifying molecular biomarkers [78,111], and the use of biochemical or cell-based high-throughput screening (HTS) to discover chemical starting points for drug discovery [112,113]. Structure-based drug design using X-ray crystallography has had a profound influence [114], not least in the application of fragment-based technologies for the discovery of chemical starting points [115]. Bioinformatic and chemoinformatic approaches have become indispensable for analyzing the large amounts of data generated by the high-throughput genomic and chemical screening approaches [116,117], while in silico chemistry methods are widely and routinely applied in the generation and optimization of chemical leads [4].

Preclinical small-molecule drug development is commonly portrayed as a linear process, progressing from molecular target, to early chemical hits and leads, to highly optimized lead compounds, to preclinical development candidates, and finally to drug candidates for clinical evaluation. Although this is a useful and not wholly inaccurate depiction, the more holistic view illustrated in Fig. 1.2 captures the integrated and iterative way in which modern drug discovery often occurs [3]. According to this model, structural biology and the various approaches collectively referred to as chemical biology play central roles in accelerating the path to the clinic and in linking the multiple elements of the drug discovery endeavor. For example, small-molecule chemical probes can be used as tools for target validation and to help determine the best biological models for guiding drug development, to anticipate potential pharmacological outcomes, and to identify possible biomarkers [76]. They can also act as pathfinders to help define potential hurdles and ways to overcome them later in the drug discovery project. The visualization of the process as a circle rather than the usual straight line is particularly useful in emphasizing how the different elements can be closely connected, with opportunities for feed-forward and feedback between the various stages. As an example, observations that are made preclinically in basic and translational research can often now have an immediate impact on clinical development. Equally, feedback of information on disease response, resistance mechanisms, and biomarker changes from the clinic to the laboratory can often lead rapidly to innovation in the selection of targets or the refinement of inhibitors. Thus, the lab–clinic interaction in drug discovery and development, as in other areas of contemporary translational research, is a two-way street.

FIGURE 1.2 The integrated and nonlinear way in which modern drug discovery often occurs. Structural biology and the various approaches collectively referred to as chemical biology link together the multiple elements of the drug discovery process. (This figure is reproduced in color in the color plate section.) Reproduced with permission from [3].

While many individual approaches and technologies can have a profound influence on a particular drug discovery project, it is the integrated application of these that is particularly important in enhancing the quality and robustness of the innovative cancer drugs that enter the clinic—and also in shortening the time and reducing the cost to progress from a new molecular target to an approved drug.

New Molecular Targets: The Druggable Cancer Genome and Epigenome

The selection of the best possible molecular targets is clearly crucial to the success of a drug discovery and development project [4,15,73]. A number of factors influence the choice of target, including, in particular, (1) the involvement of the target in the initiation and progression of cancer, and (2) the technical feasibility or druggability of the target. With the mapping of the human genome sequence, the concept of the druggable genome has become popular and useful [83,118,119]. Since cancer is above all else a disease of aberrant genetics and epigenetics, and particularly with our burgeoning understanding of the differences between the genomes of cancer versus normal cells, the notion of drugging the cancer genome has been used to embrace the contemporary approach [1–3,6,120].

There are various ways in which identifying and then validating new targets can be considered. Ultimate validation can be achieved in the clinic only with the provision of evidence of therapeutic activity via the intended mechanism of action. However, projects aimed at drugging novel targets inevitably have to be initiated with less secure credentials. Figure 1.3(A) depicts the various classes of genes that are involved in malignancy and illustrates how targets can be selected so as to modulate the multiple biochemical pathways that are hijacked by cancer genes and also to act upon the resulting multiple hallmark traits of cancer, as articulated by Hanahan and Weinberg [121,122]. These include increased proliferation, inappropriate survival and decreased apoptosis, immortalization, invasion, angiogenesis, and the metastasis or spreading to distant sites around the body that is the usual cause of death from solid tumors. The introduction of reprogramming of energy metabolism pathways and evasion of immune surveillance, as additional hallmark features of cancer, has expanded the opportunities for new targeted treatments [122]. The approval of the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibody ipilimumab (Yervoy) for the treatment of metastatic melanoma demonstrates the potential of targeting immune activation in cancer therapy [123].

FIGURE 1.3 Schematic representations of the genes and biological mechanisms involved in cancer and their exploitation in the development of new treatments. (A) The classes of genes that are involved in cancer and are potential targets for drug discovery. (B) A portfolio of new drug discovery projects can be built by selecting targets in the different categories that affect different biochemical effects and phenotypic traits of cancer. Due attention should also be paid to druggability (see text of this chapter). (C) The translation of new cancer genes into drugs and biomarkers. The integrated use of biomarkers is essential for traditional drug development leading to personalized medicine. (For color version of this figure, the reader is referred to the online version of this book.)

Targeting different types of genes, pathways, and hallmark traits provides the basis with which to attack cancer in multiple ways and at distinct levels, either with single agents or, more likely, with combinations of agents to achieve greater clinical effectiveness. From the point of view of commercial pharmaceutical research, selection of different targets from the various classes provides a means to spread risk rather than have all the drug discovery eggs in one basket. The importance of druggability is also highlighted (see Fig. 1.3(B) and later in this chapter). Figure 1.3(C) emphasizes the considerable value of discovering and developing molecular biomarkers alongside molecular cancer therapeutics so that the two can be used together in a progressive move toward personalized or individualized cancer medicine. It is hard to understand why a modern drug discovery project would be initiated without a plan to produce one or more biomarkers for patient selection and target engagement (see Ref. [97] and later in this chapter). This is particularly important given our current understanding of the inherent high genetic heterogeneity of many tumors and the envisioned need to regularly reassess the matching of targeted treatment to tumor genetics during a treatment sequence [78].

At the heart of the contemporary approach to cancer drug discovery is the identification of the aberrant genes or epigenetic abnormalities that are responsible for various cancers by hijacking cellular signaling networks [124,125], and hence lead to the characteristic hallmark traits or phenotypic features of cancer. The molecular comprehension of malignancy can be traced back to the study of cancer-causing animal viruses in the 1960s and 1970s, the discovery of oncogenes and tumor suppressor genes during the 1970s and 1980s, and the understanding of how cancer genes subvert cellular processes in the 1990s [126,127]. Although our increasing understanding of cancer as a disease of abnormal genes and signaling pathways is not new, it is only over the past 15 years or so that molecular oncology has been embraced by the drug development community as a rich source of targets for cancer drug discovery. This has required cultural evolution as well as technological and scientific advances.

It seems obvious that the best targets for the development of cancer drugs with minimal side effects will be those that are responsible for major differences between cancer and healthy cells. In retrospect, the antiproliferative toxicities associated with cytotoxic agents are unsurprising, since many important normal tissues also contain rapidly proliferating cells and are affected by agents targeting the rapid DNA synthesis and cell division of tumor cells. The development of molecular cancer therapeutics seeks to avoid the more damaging toxicities associated with cytotoxic drugs through targeting processes that are more specific to cancer cells. Although significant side effects are perhaps inevitably seen with drugs that interfere with biochemical pathways and biological processes that play a key role in normal cells, the potential to achieve a therapeutic window is clear.

Kamb et al. [15] have suggested that there is a fundamental distinction between those cancer drug targets that have an essential function in at least one normal cell type in the body and those that have nonessential functions in normal cells. They proposed, not unreasonably, that drugs acting on essential functions would have a narrower therapeutic index than those that interfere with nonessential functions in normal cells. Imatinib (discussed in this chapter) is an excellent example of a drug acting on a target that does not appear to be essential in normal cells (i.e., the ABL kinase). Although Kamb et al. [15] did not rule out the development of drugs acting on targets with essential functions in normal cells, the narrower therapeutic index likely to be seen with such agents led them to describe such agents as neocytotoxics.

Referring again to Fig. 1.3(A), cancer genes—and potential drug targets—can be categorized as (1) activated oncogenes (e.g. RAS, RAF, and PIK3CA); (2) deactivated tumor suppressor genes (e.g., p53 and PTEN); (3) genes that, when inactivated, lead to DNA repair defects (e.g., BRCA1 and BRCA2); (4) genes that support oncogenic pathways, for example those encoding the molecular chaperone heat shock protein 90 (HSP90) and the histone deacetylases, which are involved in posttranslational modification of proteins, chromatin modification, and the control of gene expression; and (5) genes controlling the tumor microenvironment, including cancer–host interactions.

Another way of considering various cancer gene targets is a classification based on four different categories of dependency [73]. The first category, genetic dependency, relates to the concept of oncogene addiction outlined in this chapter. Examples cited by the authors include the use of imatinib in leukemias driven by the BRC–ABL translocation and of MEK 1/2 inhibitors in BRAF-mutated melanoma models [128]. The second category, synergy dependency, is founded on the notion of synthetic lethality, in which genetic loss of a particular function predisposes the cancer cell to respond to pharmacological modulation of a second function [103], and is exemplified by the preferential killing of BRCA-defective breast cancer cells by poly(ADP-ribose) polymerase (PARP) inhibitors [129]. The third category, lineage dependency, refers to cancers that originate from a certain tissue or cell and have multiple features in common, some of which can constitute an addiction based on the cell lineage. This is exemplified by antihormonal drugs that target the sex hormone dependency of breast and prostate cancers, which is shared with the normal tissue of origin. The identification of the differentiation regulator MITF (micropthalemia-associated transcription factor) as an amplified oncogene in melanoma [130] and the dependence of certain lung adenocarcinomas on the developmental regulator TTF1 (thyroid transcription factor-1) [131] are further examples. The final category made by Benson et al. [73] is host dependency. This is based on the recognition that physiological factors involved in the tumor microenvironment, including tumor–host cell interactions, are vitally important for malignant progression. Examples of drugs acting on such targets are the antibodies (e.g., bevacizumab) and small molecules (e.g., sorafenib and sunitinib) that inhibit the VEGF–VEGF receptor axis. Drugs blocking the functions of hypoxia-inducible factor (HIF), which is upregulated in tumor hypoxia—as well as following loss of the VHL tumor suppressor—would also fall into this category, as would drugs acting on invasion and metastasis.

Maintaining the constantly replicative malignant state is highly stressful to the cell. Pathways that are activated in response to cope with this stress have emerged as an important set of targets for therapeutic intervention. These are mechanisms that are present and useful in nonmalignant cells in response to short-term stress, but that are chronically activated in cancer cells and have become essential for survival [79]. Two key examples that are the focus of current drug discovery efforts are the proteostasis functions of molecular chaperones such as heat shock protein 90 (HSP90) [81], and the DNA damage response pathways that monitor and direct repair of damaged DNA [80]. Another example would be the switch from mitochondrial oxidative phosphorylation to aerobic glycolysis for the production of ATP that takes place in cancer cells, first noted by Warburg over 80 years ago [132]. These examples of non-oncogene addiction are of increasing interest since they may represent points of convergence for the effects of multiple underlying oncogenic or epigenetic events. Agents targeted to the critical elements in these pathways may operate over wider genetic contexts than agents targeted at the specific oncogenes.

In a matter of a few years, we have progressed from a situation in which there was a perceived lack of targets for the development of new cancer drugs to one in which there is a considerable excess. The ongoing survey from the Wellcome Trust Cancer Genome Project (http://www.sanger.ac.uk/genetics/CGP/Census) tallies the human genes that are causally implicated in cancer via mutation, with more than 450 implicated by somatic mutation alone [83]. Moreover, new cancer genes continue to be identified. Whereas cancer gene discovery previously arose from painstaking hypothesis-driven cell and molecular biology research, it is now increasingly driven by genome-wide high-throughput systematic screening technologies, including gene copy number analysis, gene expression profiling, gene resequencing, and profiling of epigenetic markers [82]. Array-based DNA copy number and gene expression profiling can be used to identify amplified and overexpressed genes and provide complementary approaches to high-throughput mutation analysis. The application of high-throughput RNAi technology is also used extensively for gene and target discovery [133].

The discovery of BRAF as a bona fide oncogene in melanoma and other cancers was the first example of the power of high-throughput cancer genome mutation detection analysis [23]. Since then, the number of oncogenes identified through high-throughput genome sequencing for mutations has burgeoned rapidly and includes EFGR in lung cancer, JAK2 in myeloproliferative disorders, FGFR2 in endometrial cancer, and ALK in neuroblastoma [134]. High-resolution copy number profiling has identified, among many others, MITF as an oncogene in melanoma [130] and CDK8 as an oncogene in colorectal cancer [135]. The improvements in throughput and the reduced cost of second-generation DNA-sequencing technologies, where individual DNA molecules are amplified on solid-phase arrays or beads before massively parallel sequence generation, have enabled the complete sequencing of entire genomes. As a result, the amount of cancer genetic information will continue to increase hugely in the future, supported by multinational efforts such as the International Cancer Genome Consortium (ICGC) [136].

The studies outlined here show that a large number of cancer genes are involved in human malignancies. Moreover, the involvement of any one of these genes across human cancers is commonly very low (e.g., compared to BRAF), and many cancers harbor a large number of potential oncogenes. This reinforces the value of high-throughput cancer genome resequencing for the detection of cancer genes. However, the findings suggest challenges for drug development with respect to the potentially small number of patients being suitable for a given drug targeted to a specific driver mutation, and also to the choice of which target to go after in a cancer with many mutations. However, there is the possibility that many mutations may occur in genes that lie on a particular pathway, which could be drugged at a common downstream locus, or that the mutations lead to a reliance on common survival mechanisms through non-oncogene dependence [79].

While all of the high-throughput, genome-wide technologies described in this chapter are invaluable for gene discovery, they do not particularly help us to validate or prioritize a potentially new target for cancer drug discovery, particularly when the number of candidate targets is very high. A prioritization has to be made. How can we do this? There is no checklist for cancer target validation and prioritization, but some rules of thumb have emerged from more than a decade of work on targeted molecular cancer therapeutics, including proposals for a systematic approach [83]. A combination of human genetics and genomics with functional analysis involving overexpression, mutation, and knockdown by RNAi, together with the use of genetically modified mouse cancer models or other model organisms, has proved effective [4,73].

Taking the example of an oncogene, high priority is likely to be given to a gene that is mutated in human cancers (ideally at a high frequency); that lies on a pathway in which other genetic or epigenetic abnormalities are found; that, when overexpressed or mutated, recapitulates the relevant cancer phenotype; that, when knocked down, leads to the reciprocal loss of the cancer phenotype; and for which the oncogenic activity can be recapitulated in an animal model. The extent of the unmet medical need will often be influential, particularly to pharmaceutical and biotechnology companies for which the potential market size is an inevitable consideration. This is, however, notoriously difficult to predict. It is well-known in the field that the imatinib development project was nearly dropped because of concerns about the size of commercial revenues, yet imatinib became a highly effective, multibillion US dollar drug. Academic and other not-for-profit drug discovery groups can be less constrained by commercial considerations, allowing potential therapies for rare cancers, such as pediatric malignancies, to be explored. Furthermore, the direction of personalized cancer medicine is to better and more narrowly define patient populations for treatment with agents targeting specific mechanisms. This fragmentation of the cancer patient population for well-targeted drugs reduces the size of potential markets. It has been suggested that new economic models for drug discovery and development will be essential to accommodate the personalized medicine paradigm [137]. This is likely to include a greater emphasis on partnerships between academic, commercial, and not-for-profit drug discovery research scientists, to gain the benefits of a more collaborative, open-source approach to target validation [138].

In addition to the factors described here that give credence to the genetic validation of a target, higher priority will usually be given to more druggable targets for which the technical feasibility of finding a drug is more likely [118]. Receptors for small endogenous molecules, enzymes with well-defined active sites (e.g., kinases), and protein–protein interactions involving small domains are all accepted as druggable with the technology that is currently available to us. On the other hand, large-domain protein–protein interactions remain very difficult, although the experience with inhibitors of the interaction of the tumour suppressor p53 and its regulator HDM2, or between the apoptosis regulators BCL and BAK shows that the boundaries of druggability are expanding [139]. Phosphatases are also challenging targets. Despite progress in identifying potent inhibitors of several phosphatase enzymes, including the progression of compounds to early-phase trials, achieving cell permeability and selectivity is still a challenge [140]. Many other potentially important targets remain stubbornly undruggable. For example, no drugs have yet emerged that are able to directly inhibit the mutant RAS G protein or the myelocytomatosis viral oncogene homolog (MYC) oncoprotein, or to reactivate mutant p53.

In situations where a particular target of interest is not druggable, knowledge of the biochemical pathway may allow a downstream target to be selected. As an example, although RAS itself cannot be inhibited, the downstream MEK 1/2 kinases proved tractable with small-molecule inhibitors, interestingly of an allosteric nature [128]. Recent clinical data show a benefit to progression-free survival of combining MEK inhibition with V600E BRAF kinase inhibition, to counter the reactivation of the MAPK signaling pathway associated with selective V600E BRAF inhibition [141]. Inhibition of RAS prenylation, which blocks the essential membrane localization of the oncoprotein, has proved technically feasible, although the clinical significance of RAS prenylation inhibitors is still unclear [142].

While one of the most classically druggable family of targets, the G protein-coupled receptors (GPCRs), did not feature significantly as cancer genes in an early census [143], there is growing evidence of links between GPCRs and the initiation and progression of cancer [144]. As well as overexpression of GPCRs in certain cancers, the extensive cross-talk between GPCRs and growth factor receptors also argues for a role for GPCRs in the development of aberrant cancer-signaling networks. The manipulation of a GPCR-like protein to treat cancer has been proven with the approval of the smoothened (SMO) receptor ligand vismodegib (Erivedge) for the treatment of basal cell carcinoma [145]. Vismodegib binds to SMO, a seven-transmembrane-helix receptor, and prevents activation of Hedgehog (Hh) pathway signaling through the receptor, inhibiting the action of the Hh transcription factors Gli1 and Gli2. Interestingly, vismodegib was discovered by a phenotypic pathway screen in murine fibroblasts, using a luciferase reporter under the control of Gli transcription factor binding to identify inhibitors of signaling elicited by sonic hedgehog (Shh) ligand [146].

The examples given here illustrate how a good target must pass the dual test of relevance to disease mechanisms and potential for druggability. At the same time it is important to not be overly conservative, but to seek creative solutions to expand the druggable cancer genome. It is not very long ago that kinases were regarded as high-risk targets, yet they now lie second in frequency only to GPCRs in the druggable genome [118]. A target's druggability is often estimated by placing it within known gene families that have been shown by past precedent to be technically feasible. Publicly available databases such as canSAR (http://cansar.icr.ac.uk), developed in our drug discovery unit, can be very helpful in assessing the protein sequence or structural homology of a new target to known cancer drug targets, and also in identifying published chemical compounds that modulate targets in a given class [117]. De novo prediction of protein druggability based on structure or structural homology is commonly attempted using a variety of computational tools that in essence seek out and enumerate potential pockets for small-molecule binding on the surface of the protein