The Chemical Genetic Approach: The Interrogation of Biological Mechanisms with Small Molecule Probes

MARTIN FISHER AND ADAM NELSON

1.1 Introduction to Chemical Genetics

The chemical genetic approach exploits small molecule probes to modulate the function of biological macromolecules, and hence to reveal insights into biological mechanisms. The term "chemical genetics" stems from the analogy of the approach to classical genetics. Chemical genetics exploits small molecule probes to modulate protein function; in contrast, classical genetics modulates protein function indirectly through mutation of the corresponding gene. The features of the chemical genetics and classical genetics are rather different, making the two approaches broadly complementary.

The use of small molecule probes can confer a number of advantages over conventional genetic manipulations. First, the effects of small molecules on the function of a target protein are conditional and, in addition, are usually rapid (usually diffusion-controlled) and reversible. Second, by varying the concentration of a small molecule probe, it may be possible to tune the activity of a target protein in a refined way. Third, the chemical genetic approach can sometimes allow multiple functions of an individual protein to be independently modulated. Fourth, by using more than one small molecular probe in combination, the function of multiple proteins may be independently modulated. Finally, the approach is useful when classical genetics is difficult to employ: for example, in organisms (especially mammals) with diploid genomes and a slow reproductive rate. The chemical genetic approach is particularly useful for investigating tightly coordinated, dynamic biological mechanisms, especially when classical genetics may render an organism inviable.

One of the obvious features of classical genetics is its incredible specificity: mutation of a specific gene can allow a single protein (from a family of closely related proteins) to be targeted. In addition, classical genetics is highly general, and can be used to study the function of many proteins. However, classical genetics does not generally allow the conditional modulation of a specific protein (unless, for example, a temperature-sensitive mutant is used). RNA interference (RNAi) is a popular alternative to classical genetics which works by inhibiting the synthesis of a specific protein by targeting a specific RNA transcript. However, RNAi is also poorly suited to time-sensitive studies because the modulation is made at the mRNA level: degradation of the protein target needs to occur before the effects of modulation are observed.

The chapter is not an exhaustive review of chemical genetics: the field, and its impact on our understanding of fundamental biological mechanisms, has already been comprehensively reviewed elsewhere. Rather, the chapter will emphasise the approaches that may be used to discover small molecule probes, the solutions that address the challenges raised by chemical genetics, and the insights into fundamental biological mechanisms that may be revealed.

1.1.1 Forward and Reverse Chemical Genetics

The chemical genetic approach comes in two broad guises that are contrasted in Figure 1.1: forward chemical genetics and reverse chemical genetics. In the forward chemical genetic approach, the target of the small molecule modulator is not known at the outset of the investigation. The active ligand is, therefore, identified on the basis of the observation of a specific phenotypic change that it induces. A major challenge of the forward chemical genetic approach is the subsequent identification of the protein that the active ligand targets. The interrogation of biological mechanisms using the forward chemical genetic approach (and the associated challenges associated with target identification) are described in Section 1.3.2. The forward chemical genetic and the forward genetic approaches are broadly analogous in that a phenotypic change to a biological system is induced in both cases: through modulation using either small molecule probes (sometimes known as "perturbogens") (forward chemical genetics) or random mutagenesis (forward genetics).

In contrast, the reverse chemical genetic approach begins with the discovery of a small molecule modulator of a specific target protein. Most usually, the small molecule is identified on the basis of the modulation of the activity of the purified protein; however, assays of the cellular activity of a specific protein are also possible. Both reverse chemical genetics and reverse genetics modulate the function of a specific protein: either directly by the binding of a ligand (reverse chemical genetics), or indirectly through targeted mutagenesis of the corresponding gene. The discovery of ligands for use in reverse chemical genetics, and some of the insights into biological mechanism that have thereby been revealed, are described in Section 1.3.1.

1.1.2 Screening Small Molecule Libraries: Some General Considerations

The forward and reverse chemical genetic approaches both require the identification of an appropriate small molecule modulator. Most usually, active ligands are initially discovered using a high-throughput primary screen. In such cases, it is, of course, imperative that active molecules can be reliably distinguished from inactive molecules, and the performance of an assay must be assessed using appropriate statistical parameters (such as plate-based Z factors). In addition, it is necessary to correct screening results for systematic error by normalisation to controls (for example, to controls on each screening plate). Although guidelines for reporting data from high-throughput screening of small molecule libraries have been suggested, appropriate standards have not yet been formalised.

Having identified a "positive" in a primary screen, further investigation is necessary to confirm its validity. Greater confidence is gained if an active compound is related structurally to other active compounds identified in the assay (allowing preliminary structure-activity relationships to be formulated). It is important to confirm that the active compound did not interfere with the primary screen: possible problems can include inhibition of a coupling enzyme in an assay (rather than the target protein), or the interference of a fluorescent compound with a fluorescence-based readout. The chemical structure of the active compound must be verified, and then, ideally, resynthesised, repurified and retested. Ideally, the activity of the compound should be confirmed using one or more independent assays.

1.2 Synthetic Strategies for Exploring Biologically Relevant Chemical Space

A challenge in chemical biology is to design small molecule libraries that span large tracts of biologically relevant chemical space. The chemical space defined by small molecules is vast: it has been estimated that there are >1060 compounds with molecular weight less than 500 Da (only a tiny fraction of which have been prepared by chemical synthesis). How then, can the biologically relevant sub-fractions of chemical space be identified and targeted?

One approach is to design libraries around known scaffolds that have been biologically validated such as those found in drug molecules or natural products. In biology-oriented synthesis (BIOS), for example, natural product scaffolds are regarded as pre-validated starting points for ligand design since they have been selected through evolution to interact with protein binding sites. In a related approach, libraries may be designed around scaffolds that are closely related to those that are already known to be biologically relevant.

Alternatively, a fragment-based approach may be used in ligand design. Essentially, a fragment-based approach dramatically reduces the chemical space that needs to be searched. Emphasis is placed on ligands with high "ligand efficiency" (binding energy per heavy atom) rather than high absolute affinity. The approach allows a much higher proportion of the relevant chemical space to be explored, whilst leaving ample scope (and providing a better starting point) for ligand optimisation.

The diversity of chemical libraries may derive from the high substitutional, stereochemical and scaffold diversity of its members. Combinatorial chemistry is focused on the synthesis of chemical libraries with only high substitutional diversity. In general, combinatorial syntheses exploit a rather limited palette of chemical transformations to vary the substitution of library members. The synthesis of compound libraries with high substitutional diversity is described in Section 1.2.1.

Varying the configuration of library members is more challenging, but has been hugely facilitated by the many reliable asymmetric transformations that have been developed in the past 30 years. In many cases, the high enantioselectivity of these transformations can dominate over substrate-based diastereoselectivity, and allow the configuration of compounds to be varied systematically. The synthesis of a compound library with both high substitutional and stereochemical diversity is described in Section 1.2.2.

Charting chemical space systematically, however, does require the preparation of libraries with high scaffold diversity. Historically, chemists' exploration of chemical space has been highly uneven and unsystematic, and much emphasis has been placed on a small number of molecular scaffolds. (Note that the ~30 million cyclic organic compounds in the CAS registry are overwhelmingly dominated by a remarkably small number of molecular scaffolds: thirty (of the 2.5 million!) molecular scaffolds are found in 17% of the compounds, and 0.25% of the scaffolds are found in 50% of the compounds.) A key goal of diversity-oriented synthesis (DOS) is to address this problem through the preparation of libraries of small molecules that populate broad tracts of chemical space. In Section 1.2.3, the progress that has been made towards developing synthetic strategies that allow the systematic variation of scaffolds of ligands will be described.

1.2.1 Synthesis of Compound Libraries with High Substitutional Diversity

The combinatorial chemistry approach has been extended to the diversification of complex scaffolds, including natural product and natural product-like scaffolds. For example, a library of conformationally restricted 1,3-dioxanes 4 with high substitutional diversity has been prepared (Scheme 1.1). A split-pool approach was used to diversify three substituents in the final compounds 4. The supported epoxides 1 were opened with either a thiol or an amine nucleophile to yield 1,3-diols 2, which were converted into Fmoc-substituted 1,3-dioxanes. Some of the nucleophiles used in the first step were hydroxy-substituted, leading to the formation of mixed acetals in the second step: these acyclic acetals were, therefore, hydrolysed by treatment of the resins with PPTS in THF-MeOH. Following removal of the Fmoc group (-> 3), the resulting free amines were converted into amides, ureas, thioureas or sulfonamides (-> 4), and the final compounds cleaved from the beads. Using material cleaved from a single bead, and with knowledge of the substituent added into the final step, it was possible to use mass spectrometry to identify possible combinations of substituents added in the first two diversification steps. The library has been exploited in the discovery of small molecule tools: uretupamine (see Section 1.3.1.1), which targets Ure2p, a repressor of metabolic genes, and tubacin, a class II histone deacetylase inhibitor. In addition, derivatisation of the scaffold of the alkaloid, galanthamine, yielded secramine, a potent inhibitor of protein trafficking from the Golgi apparatus to the plasma membrane; crucially, although secramine's structure was inspired by galanthamine, it had an entirely distinct biological function.

1.2.2 Synthesis of Compound Libraries with High Stereochemical Diversity

The synthesis of stereochemically diverse compound libraries has been facilitated by the development of a wide range of reliable asymmetric transformations. For example, an asymmetric hetero-Diels-Alder reaction was used to prepare a library of dihydropyrancarboxamides in which both substitution and configuration was varied (structures 7 and their enantiomers) (Scheme 1.2). The library of 4320 structures was encoded using a binary encoding protocol which involved the attachment of tags to individual macrobeads. The asymmetric step (5 -> 6) served a number of purposes: it generated the molecular scaffolds; it introduced some stereochemical diversity; and it incorporated the variable substituent, R1. Deprotection of the ester, amide formation, and release from solid support gave the final compounds. The library enabled the discovery of haptamide A, a ligand which targeted a subunit of the transcription factor, Hap3p.

1.2.3 Synthesis of Compound Libraries with High Scaffold Diversity

The development of robust and reliable synthetic methods that yield a range of diverse small molecule scaffolds has proved extremely demanding. In this section, we describe the progress that has been made towards developing synthetic strategies that allow the scaffolds of small molecules to be systematically varied: in particular, the development of "folding" pathways (Section 1.2.3.1), "branching" pathways (Section 1.2.3.2) and oligomer-based approaches (Section 1.2.3.3).

1.2.3.1 Use of "Folding Pathways" to Introduce Scaffold Diversity

The "folding pathway" approach uses common reaction conditions to transform a range of substrates into products with alternative molecular scaffolds. The substrates are encoded to "fold" into the alternative scaffolds through strategically placed appending groups (sometimes known as σ-elements). This strategy leads to scaffold diversification under substrate control.

Schreiber has developed a folding pathway which exploits the Achmatowicz reaction (Scheme 1.3). The fate of oxidation of the furan substrates 10 depends on the functionalisation of groups elsewhere in the molecule (the σ-elements). Hence, oxidation of the furan 10c, which does not bear any free hydroxy groups, simply generated the ene-dione 11c. With suitably positioned nucleophilic groups in the starting material, however, a cis-enedione intermediate could be intercepted. Hence, upon oxidation, the furyl alcohol 10b folded, and eliminated water, to yield the alkylidene-pyran-3-one 11b. In contrast, with two free hydroxyl groups, 10a folded to yield the bicyclic ketal 11a. The combination of solid phase chemistry and common reaction conditions made the strategy amenable to split-pool synthesis, which significantly increased the efficiency of library generation.

Schreiber used substrate-based control to create alternative indole alkaloidlike scaffolds (Scheme 1.4). An α-diazo-β-keto-carbonyl group and an indole ring were strategically placed at different positions on the scaffolds of the starting materials (12). The densely functionalised starting materials were assembled either using an Ugi four-component coupling reaction or by alkylation of a common scaffold; subsequent conversion into a diazo compound yielded the cyclisation precursors. The substrates, 12, were treated with a catalytic amount of rhodium(II) octanoate dimer in benzene (80 °C). Presumably, generation of a carbonyl ylid was followed by intramolecular 1,3-dipolar cycloaddition with the indole ring to give the alternative polycyclic scaffolds, 13.

The "folding" pathway strategy has been shown to be useful in the preparation of small molecule libraries based on a few diverse scaffolds. In addition to the examples highlighted here, radical chemistry has been harnessed to "fold" precursors into a range of fused and bridged amine scaffolds. The scope of the "folding" pathway strategy has, however, been enormously expanded by using an oligomer-based approach to prepare precursors for a "folding" step (see Section 1.2.3.3).

1.2.3.2 Use of "Branching Pathways" to Introduce Scaffold Diversity

The "branching pathway" strategy involves the conversion of common precursors into a range of distinct molecular scaffolds through careful choice of the reaction conditions. Ideally, a range of flexible precursors would be designed which could participate in complementary reactions leading to alternative scaffolds.

An outstanding example of a "branching pathway" exploited complementary cyclisation reactions to yield alternative molecular scaffolds (Scheme 1.5). A four-component Petasis condensation reaction was used to assemble flexible cyclisation precursors (e.g. 14). Alternative cyclisation reactions were then used to yield products with distinct molecular scaffolds: Pd-catalysed cyclisation (-> 15a); enyne metathesis (-> 15b); Ru-catalysed cycloheptatriene formation (-> 15c); Au-catalysed cyclisation of the alcohol onto the alkyne (15d); base-induced cyclisation (-> 15e); Pauson–Khand reaction (-> 15f); and Miesenheimer [ 2,3]-sigmatropic rearrangement (not shown). Four of these cyclisation reactions could be used again to convert the enyne 15e into molecules with four further scaffolds (17a–d). In addition, Diels–Alder reactions with 4-methyl-1,2,4-triazoline-3,5-dione converted the dienes 15b, 17a and 17d into the corresponding polycyclic products (e.g. 16; other products not shown). The key to this powerful synthetic approach lay in the design of precursors (e.g. 14) which were effective substrates in a wide range of efficient and diastereoselective cyclisation reactions.