Applying Genomics To Drug Discovery

Typically, new drug discovery begins with the selection of promising candidate chemical entities having biological activities that correlate with relevant treatment targets. Following the identification of the better candidates as leads, the process proceeds through preclinical and clinical phases to validate the safety and efficacy of the better leads.3 Candidates should consistently lead to phenotypic changes that are in harmony with the desired therapeutic effects, effects should be dose dependent, the desired phenotypic change should be inducible or mimicked in one or more relevant animal or experimental models, and the mechanism by which the target molecule brings about a particular phenotype should be known or determined. Until recently, studying functional changes of single genes in appropriate animals and in human cells were the primary approaches to validate the target. Though such approaches are powerful and time tested, they are also slow, labor intensive, and expensive.

By knowing the full complement of human genes, however, scientists have at their disposal a much broader range of targets at which to aim potential therapeutic interventions. They may also take advantage of high-throughput technologies, global gene expression analysis, and genome-wide functional analyses. With the aid of gene expression profiling, investigators can readily analyze the effects of hundreds or thousands of genes on toxicity and the efficacy of drug candidates, conducting thousands of tests in parallel instead of sequentially, thus streamlining the drug discovery process while enhancing prospects for better ther-apies.15 Thus, the real challenge of gene-based approaches to drug discovery is to define a consistently effective process, or processes, to translate gene-sequence data into drugs that provide defined physiological and clinical endpoints, or, to put it more simply, to find efficient ways to move from gene sequence data (or the protein the sequence encodes) to drug entity.

Some incremental advances have already been achieved in this endeavor as shown by the following descriptions of several successful or promising applications of genomics/proteomics to the discovery or improvement of small molecule drugs, recombinant DNA products, and monoclonal antibodies suitable for human therapy.

SMALL MOLECULE DRUG DISCOVERY Targeting Enzymatic Drug Inactivation Mechanisms

Once an enzyme has been identified as an attractive target, drug designers have tended to frame their approach in terms of designing enzyme inhibitors that bind to the active conformation of the enzyme. For tyrosine kinases, work by Schindler and colleagues16 on the antileukemic drug STI-571 suggested that the inactive conformation of the enzyme might make a better drug target than the active conformation. STI-571, the most potent of a series of 2-phenylaminopyridines, had been exceptionally successful as a therapeutic agent in clinical trials on patients with chronic myeloid leukemia. It blocked the oncogenic form of ABL, a tyrosine kinase whose activation by the acquisition of a phosphate group is linked to the proliferation of leukemic cells in affected patients, but it did not alter the activity of some 50 other closely related kinases. Studies of the crystallographic structure of the ABL-drug complex that were undertaken to explain the specificity of STI-571 for the ABL kinase revealed that the drug bound to the inactive conformation of ABL, locking the enzyme in an inactive conformation and preventing the acquisition of the activating phosphate by the drug-enzyme complex. While the active kinases looked very similar structurally to each other, the inactive ki-nases looked very different, which explained why STI-571 was selective for ABL, and why this drug did not exhibit selectivity for other kinases.

The discovery and clinical development of STI-571, now called imatinib (Gleevec®), is an excellent example of how knowledge of a specific enzyme in-activation mechanism was used to design a therapeutic agent. Imatinib mesylate has specific indications for treating chronic myelogenous leukemia, a clonal he-matological disorder that is characterized by a reciprocal translocation between chromosomes 9 and 22. As a consequence of the creation of the BCR-ABL fusion gene, proliferation of leukemic cells occurs in affected patients. Binding of im-atinib to the inactive conformation of ABL prevents proliferation of the cancer cells. The first patient was treated with imatinib in 1998, and within 3 years three large clinical trials showed the drug to be a safe and effective treatment for all stages of chronic myelogenous leukemia. In 2001, imatinib received FDA approval for treating this disorder, and in 2002, it received approval for the treatment of gastrointestinal stromal tumors.17

Imatinib mesylate (Gleevec®) is a highly effective treatment for chronic my-elogenous leukemia and causes remarkably few side effects. Unfortunately, some patients relapse and die because of Gleevec resistance.18 However, additional inhibitors of BCR-ABL appear to be active against several kinase mutations found in patients who develop Gleevec resistance.19 Recently, investigators have tested such a compound as a prototype of a new generation of anti-BCR-ABL compounds that appears to be more effective than Gleevec.20 In 2006, two second-generation tyrosine kinase inhibitors, Sutent (Sunitinib malate, Pfizer) and Sprycel (Dasatinib, Bristol-Myers Squibb), both received FDA approval for the treatment of chronic myelogenous leukemia.4

The success of imatinib suggests that strategies that target distinctive inactiva-tion mechanisms can provide a compelling approach to rational drug discovery.

Targeting Inhibitor Chemical Switches

The development of small molecule inhibitors of tyrosine kinases such as imatinib was an important advance in drug design, but the study of individual kinases has also presented a formidable problem because they are so numerous—more then 500 have been identified in the human genome21—and because they have such similar active sites. A new method of small molecule target identification, termed ''chemical genetics,'' has recently been reported.22 The chemical genetic approach devised by Shokat and colleagues23 provides a strategy, among a number of other strategies that have been proposed,24 to engineer proteins with specificity and sensitivity to small molecule, cell-permeable protein kinase inhibitors. Submitting this method to the kinase family of enzymes provides a rigorous test of this strategy because protein kinase signaling pathways are highly conserved, central mediators of many different cell signaling events, and because highly selective kinase inhibitors for dissection of these pathways have proven difficult to obtain.

In Schokat's approach, a functionally silent mutation is inserted into a kinase (e.g., v-src protein kinase) by replacing a bulky amino acid in the active site (such as threonine) by glycine (or alanine). This modification has little effect on the ability of the src kinase to transfer the activating phosphate efficiently in cells and animals, and the modified kinase retains its ability to confer unrestrained growth on cells in culture equivalent to that of the unmodified gene. However, the modified kinase can be distinguished from all other cellular kinases by an inhibitor especially synthesized for this purpose. And when the gene that encodes the modified enzyme is inserted into cells and/or living animals, its activity can be switched off by administering (feeding) them the inhibitor.

The generality of the strategy was demonstrated by repeating the procedure in several protein kinases from five distinct families, again by substituting a bulky amino acid with a glycine.23

The researchers say that the chemical genetic strategy has key advantages over strategies to study the function of closely related proteins that involve mutating or knocking out the genes that encode them, or by studying temperature-sensitive mutants.23 They point out that mutations or knockouts may disrupt embryonic development, or be lethal, and that studies of temperature-sensitive mutants can be hard to interpret because the temperature change may affect cellular processes of direct interest as well as processes other than those directly altered by the target protein.

Targeting G-Protein-Coupled Receptors

Analysis of the human genome reveals the existence of 735-802 GPCR open reading frames, of which ~375 are neither olfactory nor taste receptors.25,26 Based on sequence homology and pharmacological similarities, human GPCRs are divisible into five families: A (rhodopsin), B (secretin), C (glutamate), and adhesion and Frizzled/Smoothened/Taste2.26 Family A is the largest and its members recognize ligands including odorants, biogenic amines, neuropeptides and peptidic hormones, lipids, nucleotides, proteases, and photons. Family B recognizes hormones and peptides and family C recognizes amino acids, ions, and tastants. Adhesion receptors are believed to interact with extracellular matrix or membrane-bound proteins, while Frizzled and Taste2 receptors are activated by Wnt proteins and tastants, respectively.

The GPCR family represents one of the most attractive therapeutic targets in the human genome. According to Drews,11 cell membrane receptors constitute the largest subgroup with 45% of all therapeutic targets. In 2001, 50-60% of all therapeutics in use targeted GPCRs. The GPCR family, represented by more than 600 genes, is one of the largest gene families in the genome.10 These receptors conduct, or participate in, a wide variety of disorders associated with dysfunctional central and peripheral neurotransmission, as well as with impaired auto-crine, hormonal, and paracrine systems. Traditionally, therapeutic agents intended to modulate GPCR function act as agonists or antagonists. GPCRs consist of membrane-bound molecules that are usually classified according to the drug ligand they bind; the major GPCR classes are those that bind bioaminergic, peptide, opioid, protease, chemokine, or fMLP (chemotactic) ligands or substrates. GPCRs that have been extensively studied, or are currently of considerable interest, include the p2-adrenoceptor, the dopamine receptor, the V2 vasopressin receptor, the chemokine receptor, and the protease receptor.

A significant degree of individual variability is associated with therapeutic responses to GPCR agonists and antagonists. But pharmacogenomic studies on GPCRs are relatively scarce, and the therapeutic relevance of GPCRs is often not well defined.27 Attempts to assess the relevance of specific and variant forms of GPCR alleles are often thwarted by a variety of factors including the cross reaction of a single drug with multiple receptors, poorly defined ligand-binding receptor pockets that can accommodate drugs in different orientations and at alternative domains, the possibility of multiple receptor conformations with distinct functions, and multiple signaling pathways engaged by a single receptor.

Nevertheless, insights into the nature and significance of GPCR variability that are of interest to drug discovery are advancing. Characterization of GPCR genetic variability reveals that as much as 60% of this variability is attributed to genetic causes, and functional polymorphisms occur in multiple, potentially critical, genomic regions of GPCR genes. Studies of the p2-adrenoceptor, for example, suggest that such variability might have evolved through expression, ligand binding, G-protein coupling, or regulation, but the data indicate that variability developed most commonly in the transmembrane-spanning domains that are typical of ligand binding.28 It was suggested that there is a need to be cognizant of ligand-binding single-nucleotide polymorphisms (SNPs) and to seek to delineate such variability early in the discovery process. Also, false nonsynonymous polymorphisms of GPCR genes are frequently reported (68%), and caution is advised against exclusive reliance on databases for the selection of candidate GPCR polymorphisms for pharmacogenetic studies or disease associations.29

Targeting RGS Proteins and GPCR Regulation

The regulator of G-protein signaling (RGS) family is a recently identified protein family that plays a major role in regulating GPCR signaling pathways by modulating the activity of G-proteins.30,31 Despite advances in the understanding of GPCR signaling pathways, none of the known regulatory mechanisms satisfactorily explained the rapid turnoff of G-protein signaling. Generally speaking,

G Protein Cycle

G-proteins are fairly stable and the levels of G-protein expression do not vary significantly under different physiological conditions. In contrast, several RGS mRNAs do vary under these circumstances, suggesting that RGS proteins might provide an important mechanism for regulating G-protein signaling (Figure 10.3).

Such a rapid turnoff mechanism generally applicable to G-proteins emerged only a few years ago with the discovery of the RGS protein family.30,31 This highly diverse protein family was discovered when a conserved sequence of 120 amino acids was observed across yeast and worm proteins. Shortly thereafter, several investigators showed that RGS proteins were GTPase accelerating proteins. The GTPase activating activity of the RGS domain of the RGS proteins accounts for the rapid suppression of G-protein signaling by accelerating GTP hydrolysis of the GTP bound to the a-subunit of the G-protein. The RGS family of proteins exerts a number of additional effects on cell signaling, but their GTPase activity explains the paradox that some signals, visual responses, and cardiac potassium channels turn off much faster than expected given the slow hydrolysis of GTP by purified a-subunits.

RGS proteins are believed to act selectively by localizing to specific cell types and distinct intracellular sites, by the timing of their expression, and by the presence of domains other than the RGS domain that link these proteins to diverse signaling pathways. The gene structure, tissue-specific expression, chromosomal location, and regulation of RGS proteins in mammalian systems are as yet at an early stage of investigation, and the evidence for the physiological roles of RGS proteins as they are currently perceived comes mainly from model studies in Saccharomyces cerevisiae and Caenorhabditis elegans. However, multiple RGS mRNAs have been found in every mammalian tissue and cell type examined so far. Alternatively spliced RGS mRNAs may explain the multiplicity of RGS proteins, but many of these isoforms are incompletely characterized. Further characterization of the molecular characteristics and transcriptional regulation should be helpful in elucidating the potential involvement of RGS proteins in disorders of phamacogenetic interest.

As regulators of GPCRs, RGS proteins present numerous opportunities for new drug development. The fact that levels of RGS proteins have an important effect on GPCR signaling pathways suggests that RGS dysregulation might lead to pathological states. Because of a defect in transcriptional regulation such that RGS proteins are not expressed, or are mistargeted, G-protein signaling would be prolonged. On the other hand, should such a defect lead to RGS overexpression, they might represent potential targets for drug discovery. In their recent review of RGS proteins as novel multifunctional drug targets, Neubig and Siderovski speculate on ways that RGS inhibitors could be used, either alone or in combination with other drugs, for a variety of clinical indications.32 For example, an inhibitor that targeted RGS proteins in brain regions involved in pain control might serve as an analgesic or an analgesic potentiator. As another example, an RGS inhibitor might serve to modify and/or increase the specificity of an administered agonist. Thus a drug that decreased RhoGEF activity, an oncogene that has a role in stimulating leukemic cell proliferation and possesses an RGS homology region, could be useful as an anticancer agent. Additional possibilities for RGS proteins as attractive drug targets are also discussed.32

The structural and biochemical properties of the RGS proteins enable them to interact with a growing list of proteins with diverse cellular functions. Their ability to suppress or silence GPCR signaling pathways provides a compelling reason to investigate their role in human variations in response to drugs and other exogenous chemicals. Information gained will not only make possible a broader understanding of the physiological roles of RGS proteins, but may also open new avenues to the discovery of more effective drugs and individualized drug therapy.

RNA Aptamers as Therapeutic Agents

Finding that cells contain a bevy of RNA snippets was another genomics discovery with surprising therapeutic implications.33 These short segments of RNA, called aptamers, can bind to ligands with high affinity and specificity. This property, plus their low molecular weight, stability, and ease and low cost of preparation, suggests therapeutic aptamers may ultimately be superior to small molecule-based and monoclonal antibody therapeutics.34

Aptamers typically bind to their targets with dissociation constants in the high picomolar to low nanomolar range. They achieve their tight binding and specificity by adapting precise three-dimensional structures on binding and becoming encapsulated by the ligand. This property distinguishes them from antisense oligonucleotides and ribozymes that are linear and act to disrupt protein expression at the mRNA level via traditional hydrogen bond interactions. Studies in cell culture and of animals have shown that functionally, aptamers can be potent biological antagonists.

In addition to their high affinity and specificity, RNA aptamers can be modified relatively easily to improve their stability and bioavailability. They are nontoxic and are of low immunogenicity or are nonimmunogenic. While the first generation of aptamers was limited by nuclease sensitivity, poor availability, fast renal clearance, and limited uptake, these limitations have been largely overcome. Another significant advance in aptamer technology has shown that ''antidotes,'' which are short complementary sequences (antisense) to the aptamers, can reverse the inhibitory properties of aptamers.

At the present time, Macugen® (Pegaptanib sodium) is the only FDA-approved aptamer drug. It improves vision in some patients afflicted with age-related wet macular degeneration, the leading cause of blindness in the elderly.35 Age-related wet macular degeneration is characterized by blood vessel growth in the back of the eye or leakage of blood vessels that results in total loss of vision. The PEGylated, nuclease-stabilized aptamer is a selective antagonist of the vascular endothelial growth factor (VEGF) that plays a crucial role in angiogenesis. Macugen retards loss of vision and is currently in trial for diabetic macular edema and retinal vein occlusion.

Several additional RNA aptamers are currently in clinical development. Included among these are aptamers that target transcription factor decoys (for E2F, which plays a role in cardiovascular disease, and for nuclear factor NF-Kp), thrombin, several coagulation factors including Factors IXa, VIIa, and XII, and nucleolin.34 Aptamers designed to target extracellular as well as intracellular sites present additional prospects for antiviral therapy, including HIV therapy. The anti-HIV aptamer, RNA aptamer B4, targets the extracellular, envelop protein gp120, which controls viral entry through its interaction with chemokine receptors. This aptamer was found to be highly effective in neutralizing HIV infectivity in human blood mononuclear cells by more than 1000-fold. Other viral infections being targeted for aptamer intervention include hepatitis C virus (HCV), Rous sarcoma virus, and cyto-megalovirus.

PROTEIN THERAPEUTICS Recombinant Proteins

The synthesis of recombinant therapeutic proteins traces back to two studies performed in the early 1970s. In 1972, Paul Berg and colleagues first described the biochemical technique for joining two DNA molecules from different sources to produce a ''recombinant plasmid.''36 Following this lead, Stanley Cohen and Herbert Boyer used such a construct to transform Escherichia coli cells and replicate the recombinant protein encoded in the DNA of the construct. The perfection and commercialization of this technology by the 1980s, complemented by the polymerase chain reaction invented in 1986, provided a rapid, reliable means for producing unique recombinant proteins in quantities sufficient for therapeutic purposes.

This technology was used initially to produce recombinant versions of biological proteins heretofore available only from extracts derived from natural sources such as insulin from pigs and cows and growth hormone and anti-hemophilia factor from human tissues. The first human recombinant protein, recombinant insulin (Humulin), was approved for marketing in 1982, the second recombinate product in 1985 was the human growth hormone, somatotropin, while the third, antihemophilia factor, was not produced until 1992 (Table 10.1). From 1982 through 2002, 54 rDNA products (Table 10.1) received FDA approval for therapeutic use. During this 20-year period, the number of approved products expanded rapidly with 6 approved between 1985 and 1989, 11 between 1990 and 1994, 19 between 1995 and 1999, and 17 between 2000 and 2002. Approximately half of these were approved between 1997 and 2002. Recombinant DNA technology permits the synthesis of rDNA products that are slightly altered, or completely different from naturally occurring proteins, and hence may have superior safety and efficacy profiles. Today, the rDNA product industry numbers more than 100 companies, over 70 marketed products, and more than 100 new products in clinical development.

To be efficacious and safe, a successful recombinant protein must satisfy several criteria. Optimally, its efficacy should closely match that of the native protein, and it should have high affinity and specificity, and good stability. It must also have properties favorable to expression in cell culture, fold and secrete correctly, be subject to the appropriate posttranslational modifications, and be soluble at concentrations apropos of those used under therapeutic conditions. Delivery of protein drugs presents another problem. As oral administration results in their denaturation by the acid in the stomach, protein drugs must be delivered parenterally. Still further, as the half-life of protein products in the body is usually very short, it is advantageous if its clearance can be slowed. Drug formulation relies on a strategy known as PEGylation for this purpose.37

As genetic engineering technology improved, different cell lines were employed for production of rDNA products. Between 1980 and 1983, most (86%) therapeutic products were produced in E. coli while only one (14%) was produced in a mammalian cell line, whereas between 1984 and 1987 the percentage produced in E. coli dropped to 58% with 25% produced in mammalian cell lines and 17% in yeast. Between 1988 and 1991, the preferred cell line for production was mammalian (60%, Chinese hamster ovary and baby hamster kidney cells). But bacterial, mammalian, and yeast cell lines differ in the abilities regarding protein glycosylation, glycosylation pattern, and posttranslational modification, and mammalian cell lines were not necessarily better and did not supplant older cell lines. In 2002,46% of approved products were produced in E. coli, with 38% produced in mammalian cell lines and 15% produced in yeast.38

Some interesting trends are revealed by the time intervals required for completion of the clinical development and FDA-approval phases of rDNA thera-peutics.38 When the data were stratified according to the therapeutic indication (Table 10.2A), it is apparent that the mean clinical phase varied greatly and depended on the indication. Products with immunological and antiinfective

Recombinant proteins Monoclonal antibodies (mAbs) Breakthrough targets

FDA Trade name Protein FDA

approval (generic name) type Generic name Description Application Drug Target approval

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