Estrogen receptor

Serotonin 5-HT1D

receptor INF-b-1a INF-b-1b Serotonin 5-HT1

receptor Granulocyte colony-stimulating factor


Farensyl diphosphate synthase, probably

ß-Tubulin m-Opioid receptor Luteinizing hormone

Dopamine, serotonin, and histamine receptors

Factor X

Zestril, Lotensin, Tritace, Accupril Cozaar Diovan

Norvasc, Adalat Ambien, Stilnox Rituxan

Premarin, Evista, Noladex (breast)









Muscarinic M3 receptor Detrol

Duragesic, Ultram Lupron, Leuplin, Zoladex

Zyprexa, Risperidal, Seroquel


Low blood pressure

Low blood pressure Low blood pressure Not available

Hyperactive, hyperresponsive Depletion of a subpopulation of B cells Reproductive defects, reduced bone mineral density Not available

Not available Not available Not available

Deficiency in the total bone marrow cells, granulocytes and monocyte precursors in the bone marrow PLRP2, decreased fat absorption, carboxyl ester lipase reduced dietary cholesterol ester absorption Embryonic lethal;

heterozygous males have increased bone mineral density Not available

Increased urine retention in males Increased sensitivity to pain Luteinizing hormone receptor: releasing hormone hypogonadism and reduced steroidogenesis Multiple targets; related KOs display behavioral phenotypes (movement, activity, and anxiety) Neonatal death due to massive bleeding

DRUG DISCOVERY Table 10.5 (continued)




Mouse phenotype

Transplant Calcineurin, thymocytes, Sandimmun rejection defective leukocytes

KO of calcineurin Ab exhibits reduced T cells in periphery, reduced activation, and impaired allograft rejection

Transplant Inosine monophosphate rejection dehydrogenase


Embryonic lethal; heterozygotes show significant impairment of T cell activation and function

Source: Modified from Zambrowicz and Sands6 (Table 8).

These two studies indicate that KO mice can be highly informative in the discovery of efficacious drug targets. They suggest that the prospective application of ''reverse genetics'' to KO mice is likely to afford a productive source of new targets for future drug discovery.

Simple Eukayotic Organisms in Drug Discovery

The use of simple eukaryotic organisms as models for the study of human disease (see p. 292) can be extended to the discovery of better therapeutics.51-53 For instance, Giaever and colleagues53 have recently explored the possibility of the genomic profiling of yeast S. cerevisiae for drug sensitivities via induced hap-loinsufficiency. This approach is based on the idea that lowering the dosage of a single gene from two copies to one in diploid yeast yields a heterozygote that is sensitized to any drug that acts on the product of this gene. The ''haploinsuffi-cient'' phenotype serves to identify the gene product of the heterozygous locus as the drug target.

This observation was exploited in a genomic approach to drug-target identification as follows. First, a set of heterozygous yeast strains was constructed carrying deletions in genes encoding drug targets. Next, each strain was grown in the presence of sublethal concentrations of the drug that directly targets the protein encoded by the heterozygous locus and was analyzed for drug sensitivity (e.g., a reduced growth rate in the presence of the drug) on high-density oligonucleotide microarrays. A feasibility study on individual heterozygous strains verified six known targets. In each case, the result was highly specific as no sensitivity was exhibited when these strains were tested with other drugs. Additionally, parallel analysis of a mixed culture of 233 strains in the presence of the drug tunicamycin (a well-characterized glycosylation inhibitor) identified the known target and two unknown hypersensitive loci.

Today, drug discovery is driven, in part, by combinatorial chemistry followed by high-throughput screening of agents against a preselected target. Giaever's method does not require any prior knowledge of the target, and will identify only those targets that affect the fitness of the organism. The discovery that both drug target and hypersensitive loci exhibit drug-induced haploinsufficiency may help elucidate mechanisms underlying heterozygous disease phenotypes as well as variable drug toxicities.

Yeast, Flies, Worms, and Zebrafish in Drug Discovery

In the search for the genetic basis of a disease, it is not uncommon to discover a new protein whose normal function is unknown. Information about a new protein may be obtained by studying its distribution in normal tissues and subcellular compartments, or alternatively by examining the consequences of overexpression of the protein in cultured cells, or inactivation of the corresponding gene in knockout mice, as noted above.

In their article on yeast, flies, worms, and zebrafish, Hariharan and Haber54 discuss the usefulness of these simple model organisms for the identification of genes with direct relevance to human disease, a topic of great interest to new drug discoveries, noting that the use of simple organisms in medical research is based on two premises. The first is that most of the important biological processes that occur in simple organisms have remained unchanged throughout evolution and are conserved in humans. The second is that these processes are easier to dissect in simple organisms than in humans. Their short generation times accelerate genetic studies of these organisms, mutant strains can be generated efficiently, and the effects of gene inactivation or overexpression on phenotype can be identified rapidly. Finally, the genetic approaches used to study each of these organisms have been extensively described.

Apart from the study of disease-causing genes, the potential usefulness of yeast, flies, worms, and zebrafish to pharmacological and toxicological studies has recently gained momentum. Their usefulness is dictated in part by their suitability for investigating particular cellular pathways. Yeast cells are particularly suited for studying the effects of mutations on cell division while fly embryos are well suited for studying mutations that disturb tissue organization and cell differentiation, Genetic studies in worms, in which the developmental fate of individual cells can be tracked, are especially suited for understanding programmed cell death (apoptosis). Vertebrate zebrafish, which have a counterpart for almost every disease-causing gene in humans, have been used mainly in the fields of molecular genetics and developmental biology of vertebrates. But since their amenability to large-scale forward genetic screens, a technique previously limited to yeast, flies, and worms, was demonstrated in 1996, zebrafish have been explored as a model to accelerate drug discovery.55,56 They are amenable to high-throughput chemical screens because of their small size, and are more permeable than invertebrates because of the absence of a cuticle. The genes of zebrafish show an overall identity of about 70% with human orthologs at the amino acid level, but the similarity is much higher in functional domains. In substrate-binding regions, for instance, identity approaches 100%, which explains why many drugs elicit responses in this organism comparable to those in humans, and why zebrafish are suitable for modeling human pharmacological, toxico-

logical, and behavioral research.57 Because zebrafish combine a way to study the physiological complexity in the whole organism with high-throughput scale of in vitro screens, useful in drug target identification, lead discovery, and toxicology, this model offers an alternative opportunity to decrease the time and cost, simplify the performance, and enhance the prospects for new drug discovery.

A Perspective on Epigenetic Therapy

Methylation of CpG islands and conformational changes in chromatin involving histone acetylation are reversible, interacting processes that are associated with transcriptional silencing of gene expression (Figure 6.6). Disruption of either or both of these processes can lead to inappropriate gene expression, resulting in epigenetic disease including cancer (Table 6.2). Encouraged by the possibility that reversal of these processes and upregulation of genes could be important in preventing or reversing the disease phenotype, these processes have become therapeutic targets in the treatment of cancer and other epigenetic disorders.

Numerous preclinical and clinical trials have resorted to the treatment of various hemoglobinopathies (b-thalessemia, sickle cell anemia), myelodysplastic and leukemic syndromes, and the fragile-X syndrome with demethylating agents, histone deacetylase (HDAC) inhibitors, or the combined manipulation of cyto-sine methylation and histone acetylation, as described earlier (see p. 172). Agents used in these trials included older (5-azacytidine, 2-deoxy-5-azacytidine, or decitabine) and newer (MG98, an antisense DNMT1 inhibitor) demethylating agents, older (sodium butyrate, sodium phenyl butyrate), and newer (trichostatin, suberoylanilide hydroxamic acid, and depsipeptide) HDAC inhibitors. The results of most of these trials, though limited in scope, were encouraging, but the risks of such therapy at present would appear to be largely unknown. First, it is clear that hypomethylation is observed in malignant cells in vivo at doses of demethylating agents such as decitabine that overlap with clinical responses. Second, it should be noted that multiple genes may be methylated, or undergo histone modification in epigenetic diseases, and there is the possibility of hitting many targets with one drug. The evidence for decitabine, for example, favors DNMT inhibition as the critical event in demethylation, but other possible events might include reactivation of hypermethylated tumor suppressor genes and activation of retroposons through hypomethylation. Furthermore, the extent to which other downstream events such as apoptosis and differentiation might occur is unclear. And finally, because methylation increases with age, demethylation might contribute to senescence and the development of age-related chronic diseases, including cancer, in the elderly.58-60

In 2004, 5-azacytidine was the first agent to receive FDA approval for the treatment of several myelodysplastic syndromes. In 2006, one demethylating agent, Decitabine® (5-aza-2'-deoxycytidine), received FDA approval for the treatment of the myelodysplastic syndrome, and one HDAC inhibitor, Zolinza® (vorinostat), received FDA approval for the treatment of cutaneous T cell lym-phoma.4 Several other epigenetic drugs are capable of altering DNA methylation patterns or of the modification of histones. Those targeting methylation include

FCDR (5-fluoro-2'-deoxycytidine), EGCG (epigalocatechin-3-gallate), zebular-ine, procainamide, SAHA (suberoylanilide hydroxamic acid, vorinostat, Zolinza), Psammaplin A, and antisense oligomers. Those targeting HDACs include phe-nylbutyric acid, SAHA, depsipeptide, and valproic acid. Several of these agents are in clinical trial.59,61

We are just beginning to understand the contributions of epigenetics to human disease, and how to optimize its management. As we learn more about the proteins that are targeted by therapeutic agents, and the molecular interactions that they perturb, rationally designed drugs and individualized therapy may become reasonable goals.58-60 Various HDAC inhibitors seem to enhance the tumor response to ionizing radiation and thereby may protect normal tissues from radiation damage, and combinations of demethylating agents with HDAC inhibitors are also being studied with great interest.61

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