Dissection of melanogenesis with small molecules identifies prohibitin as a regulator.

Bioactive compounds can be used to selectively modulate gene function. We utilized a chemical genetic approach to dissect the mammalian pigmentation pathway and identify protein regulators. We screened a tagged library of 1170 small molecules in a cell-based assay and discovered a class of pigment-enhancing chemicals. From this class we characterized the small molecule melanogenin. Using melanogenin bound to an affinity matrix and amino acid sequencing, we identified the mitochondrial protein, prohibitin, as an intracellular binding target. Studies employing siRNA demonstrate that prohibitin is required for melanogenin to exert its propigmentary effects and reveal an unsuspected functional role for this protein in melanin induction. This represents a mechanism by which propigmentary signals are transduced and ultimately provides a potential target for the treatment of pigmentary disorders.

Identification of compounds that bind mitochondrial F1F0 ATPase by screening a triazine library for correction of albinism.

A triazine-based combinatorial library of small molecules was screened in albino murine melanocytes to identify compounds that induce pigmentation. Six compounds (of 1536 screened) produced at least 3-fold increases in pigmentation. Immunohistochemical studies demonstrated that the compounds conferred correct routing of the mistrafficked enzyme tyrosinase, which is critical to normal melanogenesis. Affinity matrices of the immobilized compounds allowed the cellular target to be identified as the mitochondrial F1F0-ATP synthase. Oligomycin and aurovertin B, small molecules known to inhibit the mitochondrial ATP synthase, were shown to compete with the triazine-based compounds for their cellular target in albino melanocytes and confer similar effects on pigmentation and tyrosinase rerouting. This is the first demonstration of the mitochondrial ATP synthase as a potential therapeutic target for restoring pigmentation in albino melanocytes.

Identification of the F1F0 mitochondrial ATPase as a target for modulating skin pigmentation by screening a tagged triazine library in zebrafish.

A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a dramatic increase in the pigmentation of early stage zebrafish embryos. This compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. Oligomycin, a small molecule known to inhibit the mitochondrial ATPase, competed with PPA for its cellular target in melanocytes. In addition, PPA was shown to alter the membrane potential of mitochondria, consistent with inhibition of the mitochondrial ATPase. Thus, PPA has been successfully used as a chemical probe in a forward chemical genetic approach to establish a link between the phenotype and the protein. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells.

Recent advances in glycomics and glycogenetics.

As the human genome sequence is nearly deciphered, it is important to turn the attention to the physiological functions of the genes. Thus, the study of the gene products, the proteins, is the next big challenge. The proteins, however, are not the final gene products in many cases. It has been shown that carbohydrates participate in post-translational modifications and in many other functional regulations, hence the study of the glycome, the entire collection of carbohydrates is essential in order to determine the functions of all genes, and will greatly enhance the field of chemical genetics. Known biological function / targets of carbohydrates and combinatorial synthesis & structural analysis of natural / non-natural carbohydrates are surveyed in this review. Methods to search for new biological targets that include carbohydrate mimetics and carbohydrate scaffolds along with chip technology, are also presented.

Novel orthogonal strategy toward solid-phase synthesis of 1,3,5-substituted triazines.

[reaction: see text] To improve upon the previous orthogonal method for synthesis of a triazine library, an alternative strategy has been developed via oxidation-activation of the thioether to the sulfone. Through a comparison between these two methods, the sulfone strategy was demonstrated as an enhanced method in the generation of highly pure triazine library compounds.

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.

(+/-)-1,2:5,6-Di-O-isopropylidene-myo-inositol and (+/-)-6-O-benzoyl-1,2:4,5-di-O-isopropylidene-myo-inositol: a practical preparation of key intermediates for myo-inositol phosphates.

A simple and practical synthetic procedure for the versatile intermediates, (+/-)-1,2:5,6-di-O-isopropylidene-myo-inositol and (+/-)-6-O-benzoyl-1,2:4,5-di-O-isopropylidene-myo-inositol, is described.

Forward chemical genetic approach identifies new role for GAPDH in insulin signaling.

Insulin and insulin-like growth factor have an essential role in growth, development and the maintenance of metabolic homeostasis, including glucose uptake from the bloodstream. Researchers have identified mutations in insulin receptors that cause severe insulin resistance, and a temperature-sensitive daf-2 (a gene encoding an insulin receptor-like protein) mutant in Caenorhabditis elegans has served as an insulin resistance model. Here we report a forward chemical genetic approach with a tagged library that we used to identify a small molecule, GAPDH segregator (GAPDS), that suppresses the dauer formation induced by the daf-2 mutant. Like insulin, GAPDS increased both glucose uptake and the concentration of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in mammalian preadipocytes. Using affinity matrices and RNA interference, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a GAPDS target. We discovered that GAPDH stimulates phosphatase activity against not only PtdIns(3,4,5)P(3) but also PtdIns(4,5)P(2). These results suggest that GAPDH is both an active regulator in the phosphoinositide-mediated signaling pathway and a potential new target for insulin resistance treatment.

Identification of a new class of prostaglandin transporter inhibitors and characterization of their biological effects on prostaglandin E2 transport.

Prostaglandins (PGs) are involved in several major signaling pathways. Their effects are terminated when they are transported across cell membranes and oxidized intracellularly. The transport step of PG metabolism is carried out by the prostaglandin transporter (PGT). Inhibition of PGT would therefore be expected to change local or circulating concentrations of prostaglandins, and thus their biological effects. To develop PGT-specific inhibitors with high affinity, we designed a library of triazine compounds and screened 1842 small molecules by using Madin-Darby canine kidney cells stably expressing rat PGT. We found several effective PGT inhibitors. Among them, the most potent inhibitor had a Ki of 3.7 +/- 0.2 microM. These inhibitors allowed us to isolate the efflux process of PGE2 and to demonstrate that PGT does not transport PGE2 outwardly under physiological conditions.

Triazine-based tyrosinase inhibitors identified by chemical genetic screening.

As most of the available depigmenting agents exhibit only modest activity and some exhibit toxicities that lead to adverse side effects after long-term usage, there remains a need for novel depigmenting agents. Chemical genetic screening was performed on cultured melanocytes to identify novel depigmenting compounds. By screening a tagged-triazine library, we identified four compounds, TGH11, TGD10, TGD39 and TGJ29, as potent pigmentation inhibitors with IC50 values in the range of 10 microM. These newly identified depigmenting compounds were found to function as reversible inhibitors of tyrosinase, the key enzyme involved in melanin synthesis. Tyrosinase was further confirmed as the cellular target of these compounds by affinity chromatography. Kinetic data suggest that all four compounds act as competitive inhibitors of tyrosinase, most likely competing with L-3,4-dihydroxyphenylalanine (L-DOPA) for binding to the DOPA-binding site of the enzyme. No effect on levels of tyrosinase protein, processing or trafficking was observed upon treatment of melanocytes with these compounds. Cytotoxicity was not observed with these compounds at concentrations up to 20 muM. Our data suggest that TGH11, TGD10, TGD39 and TGJ29 are novel potent tyrosinase inhibitors with potential beneficial effects in the treatment of cutaneous hyperpigmentation.

Safety-catch approach to orthogonal synthesis of a triazine library.

A novel safety-catch method for orthogonal synthesis of highly pure trisubstituted triazines was developed. Since the polymer-support used in this method is not acid-labile, this strategy can be uniquely applied to the synthesis of acid-sensitive triazine library compounds. This method will dramatically increase the diversity of triazine and other related heterocyclic library compounds.

System dynamics of subcellular transport.

In pharmacokinetic experiments, interpretations often hinge on treating cells as a "black box": a single, lumped compartment or boundary. Here, a combinatorial library of fluorescent small molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution. Most probes accumulate in cytoplasmic vesicles and probe kinetics conform to a nested, two-compartment dynamical system. At steady state, probes preferentially partition from the extracellular medium to the cytosol, and from the cytosol to cytoplasmic vesicles, with hydrophobic molecules favoring sequestration. Altogether, these results point to a general organizing principle underlying the system dynamics of subcellular, small molecule transport. In addition to plasma membrane permeability, subcellular transport phenomena can determine the active concentration of small molecules in the cytosol and the efflux of small molecules from cells. Fundamentally, direct observation of intracellular probe distribution challenges the simple boundary model of classical pharmacokinetics, which considers cells as static permeability barriers.

Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG.

A novel and highly diverse tagged triazine library incorporating a triethylene glycol-based linker was synthesized using an orthogonal combinatorial approach on the solid phase and covalently immobilized on a glass substrate as a small molecule microarray (SMM). The SMM was screened with a fluorophore-conjugated human IgG, and 4 novel binders from a library of 2688 compounds were identified from the fully spatially addressable array without the need for compound decoding. Using surface plasmon resonance (SPR) analysis, binding seen on the array was confirmed, and a binding constant as low as Kd = 2.02 x10(-6) M was measured.

Facilitated forward chemical genetics using a tagged triazine library and zebrafish embryo screening.

An improved forward chemical genetics approach was successfully demonstrated using a tagged library concept. A small-molecule triazine library with linkers was used to screen for brain/eye developmental phenotypes in a zebrafish embryo system. This approach enabled the rapid isolation of the target proteins by facile affinity matrix preparation and elucidated the first small-molecule inhibitors for several ribosomal accessory proteins or their complex as the target.

A novel microtubule destabilizing entity from orthogonal synthesis of triazine library and zebrafish embryo screening.

The first orthogonal combinatorial synthesis of a high-purity triazine library was demonstrated. Novel triazine-based microtubule inhibitors were discovered by an efficient zebrafish embryo screening and in vitro microtubule polymerization assay.