Combinatorial Chemistry & High
Throughput Screening, Vol. 7, No. 7, 2004
Contents
Reverse
Pharmacology and the Pharmaceutical Industry
Guest
Editor: George G. Harrigan
Quality, not Quantity:
The Role of Natural Products and Chemical Proteomics in Modern Drug Discovery Pp.607-630
[Abstract]
Current Progress in
Natural Product-like Libraries for Discovery Screening Pp.631-643
[Abstract]
Forward Chemical
Genetics: Library Scaffold Design Pp.645-652
[Abstract]
Synthetic Molecules
that Modulate Transcription and Differentiation: Hints for Future Drug
Discovery Pp.653-659
[Abstract]
Identification and
Characterization of Sir2 Inhibitors Through Phenotypic Assays in Yeast Pp.661-668
[Abstract]
Mapping Chemical Space
Using Molecular Descriptors and Chemical Genetics: Deacetylase Inhibitors Pp.669-676
[Abstract]
Target Identification
Strategies in Chemical Genetics Pp.677-688
[Abstract]
Targeted Degradation
of Proteins by Small Molecules: A Novel Tool for Functional Proteomics† Pp.689-697
[Abstract]
Photo-Affinity Labeling
Strategies in Identifying the Protein Ligands of Bioactive Small Molecules:
Examples of Targeted Synthesis of Drug Analog Photoprobes Pp.699-704
[Abstract]
Abstracts
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Quality, not Quantity: The Role of Natural Products and Chemical Proteomics
in Modern Drug Discovery
Chemical genetics and reverse chemical genetics parallel classical genetics but target genes at the protein level and have proven useful in recent years for screening combinatorial libraries for compounds of biological interest. However, the performance of combinatorial chemistry in filling pharmaceutical pipelines has been lower than anticipated and the tide may be turning back to Nature in the search for new drug candidates. Even though diversity oriented synthesis is now producing molecules that are natural product-like in terms of size and complexity, these molecules have not evolved to interact with biomolecules. Natural products, on the other hand, have evolved to interact with biomolecules, which is why so many can be found in pharmacopoeias. However, the cellular targets and modes of action of these fascinating compounds are seldom known, hindering the drug development process. This review focuses on the emergence of chemical proteomics and reverse chemical proteomics as tools for the discovery of cellular receptors for natural products, thereby generating protein/ligand pairs that will prove useful in identifying new drug targets and new biologically active small molecule scaffolds. Such a system-wide approach to identifying new drugable targets and their small molecule ligands will help unblock the pharmaceutical product pipelines by speeding the process of target and lead identification.
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Current Progress in Natural Product-like Libraries for Discovery Screening
Natural product-like libraries represent an effort to combine the attractive features of natural products and combinatorial libraries for high-throughput screening. Three approaches to natural product-like library design are discussed: (1) Libraries based on core scaffolds from individual natural products, (2) libraries of diverse structures with general structural characteristics of natural products, and (3) libraries of diverse structures based on specific structural motifs from classes of natural products. Examples of successful applications in discovery screening are described for each category. These studies highlight the exciting potential of natural product-like libraries in both chemical biology and drug discovery.
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Forward Chemical Genetics: Library Scaffold Design
With the unraveling of the entire human genome, it has become imperative to understand the function of the gene products, proteins. Within the past several years, chemical genetics has gained recognition as a powerful approach to study protein function by using small molecules as gene knock-out or knock-in mimics. Forward chemical genetics is a three-step process; the design and synthesis of a small molecule library represents the first step followed secondly by the search for novel phenotypes and then by isolation and identification of target protein(s). This review will focus on the first step, the design of the scaffold for small molecule libraries. It will also examine the connection between the choice of a scaffold and the propensity of that library to demonstrate enhanced biological activity when tested in certain cellular systems.
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Synthetic Molecules that Modulate Transcription and Differentiation: Hints
for Future Drug Discovery
Regulation of gene transcription and cell differentiation often induces drastic phenotypes in living organisms. External, precise control over these processes through small organic molecules represents a challenge in chemistry and biology. Our laboratory has been discovering small organic molecules that modulate transcription or differentiation and using them as a tool to understand biological phenomena. This personal perspective summarizes our contributions to chemical biology and chemical genetics in the fields of gene transcription and cell differentiation. Our case studies may foreshadow the promise and the challenge of future drug discovery.
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Identification and Characterization of Sir2 Inhibitors Through Phenotypic
Assays in Yeast
Yeast Sir2 is a defining member of a large family of protein deacetylases found in organisms ranging from bacteria to humans. SIR2 was discovered as a gene required for mating in S. cerevisiae 25 years ago, but it was only recently that Sir2's activity as an NAD-dependent protein deacetylase was established. However, years of extensive research did generate a large body of knowledge about the cellular roles of Sir2 in yeast long before its biochemical function was discovered. In addition to Sir2, yeast have four additional NAD-dependent histone deacetylases Hst1-4 (for homologue of Sir2), with distinct cellular roles. Detailed knowledge of the phenotypes of SIR2 and HST loss of function mutants has allowed design of a series of cell based screens that yielded the first inhibitors of NAD-dependent protein deacetylases. These phenotypic assays, amenable to high throughput screening, and coupled with transcript array analysis for evaluation of compound specificity, allowed the identification and detailed characterization of a series of Sir2 inhibitors, entirely bypassing traditional biochemical approaches.
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Mapping Chemical Space Using Molecular Descriptors and Chemical Genetics:
Deacetylase Inhibitors
An objective of chemical genetics is to understand the relationships between the structures of small molecules and their phenotypic effects in intact living systems. We present here the results of a global analysis of a molecular descriptor space constructed using structural descriptors of an aryl 1, 3-dioxane-based diversity-oriented synthesis-derived library containing structural biasing elements directed at inhibiting protein deacetylases. Using principal component analysis and three-dimensional visualization, we generated metric space maps with morphological features contributed by different diversity positions within the library. Filtering these maps using phenotypic descriptors derived from measurements of small-molecule activities in an array of cell-based assays revealed different densities of biological activity within specific subspaces. These results provide evidence that certain structural features may be important for conferring potency and selectivity on deacetylase inhibitors with respect to tubulin and histone acetylation. Moreover, these results highlight an example of the importance of using functional measures to assess molecular diversity. Similar analyses of other chemical spaces and activity classes promise to facilitate the development of chemical genetics.
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Target Identification Strategies in Chemical Genetics
Chemical inhibitors have had a profound impact on many diverse fields of biology. The goal of chemical genetics is to use small molecules to perturb biological systems in a manner conceptually similar to traditional genetics. Key to the advancement of the chemical genetic paradigm is the further development of tools and approaches for the identification of the protein targets of active compounds identified in chemical genetic screens. This review will address historic examples in which forward chemical genetics yielded new insight into a biological problem through successful identification of the target of an active molecule. The approaches covered have been grouped into two broad classes: target identification by affinity-based methods and target identification by deduction. Strengths and shortcomings of each approach as it pertains to their application to modern chemical genetics will be discussed. Finally, a series of new genomic and proteomicbased techniques for target identification will be described. Although a truly general approach to target identification has yet to be developed, these examples illustrate that there are many effective strategies for successfully elucidating the biological targets of active small molecules.
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Targeted Degradation of Proteins by Small Molecules: A Novel Tool for Functional
Proteomics†
A novel strategy that targets protein for degradation has recently been developed by exploiting a protein-targeting chimeric molecule (‘Protac’). Typically, the chimeric Protac is composed of a small-molecule ligand (‘bait’) on one end and a synthetic octapeptide on the other. This octapeptide is recognized by E3 ubiquitin ligase pVHL (von Hippel Lindau tumor suppressor protein), thereby recruiting a small moleculebound protein (‘prey’) to pVHL for ubiquitination and degradation. Since selective degradation of a cellular protein generates a “loss of function” mutation, this protein knock-out strategy may be useful to study the function of a given protein or to evaluate whether a cellular protein is a potential target for drug intervention, in a manner reminiscent of gene knock-out or siRNA approaches. Herein, we show that a synthetic pentapeptide is sufficient to interact with pVHL E3 ligase, and that the pentapeptide-based Protac efficiently induces ubiquitination and degradation of target protein. Our results also demonstrate that the pentapeptide-based Protac can enter cells efficiently to exerts its biological activity effectively. These results suggest that the synthetic pentapeptide can be used either directly in the preparation of cell-permeable Protacs or as a template to develop peptidomimetic or non-peptide Protacs.
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Photo-Affinity Labeling Strategies in Identifying the Protein Ligands of Bioactive
Small Molecules: Examples of Targeted Synthesis of Drug Analog Photoprobes
The discovery of many new targets by chemical genetics has frequently exploited the fact that their biologically active chemical ligands were reactive and thus could covalently bind to their protein target(s). When experimental compounds or therapeutic agents with unidentified mechanisms of action do not contain reactive groups that can covalently label the putative site of molecular action, it may be possible to create a reactive photo-affinity probe if there is sufficient knowledge of the structure-activity relationship of the chemical series. Two specific examples are presented. These include the use of photo-affinity probes in the identification of the mechanism of action of synthetic oxazolidinones, a class of novel acting antibiotics and in the identification of a novel target for the insulin-sensitizing thiazolidinediones. Developments in photoaffinity labeling and combinatorial library design now imply that the parallel incorporation of photo-probes into screening library design could, at least in principle, greatly facilitate reverse pharmacological and chemical genetics approaches to protein target discovery.