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Synthetic molecules that modulate and probe biological events are critical tools in chemical biology. Utilizing combinatorial and diversity-oriented synthetic strategies, access to large numbers of small molecules is becoming more and more feasible, and research groups in this field can take advantage of the power of chemical diversity. Since the majority of early studies were focused on the discovery of compounds that perturb protein functions, diversity-based approaches are often considered as therapeutic lead discovery tactics. However, the diversity-oriented approach can also be applied to advance distinct aims, such as target protein identification, or the development of imaging probes and sensors. This review provides a personal perspective of the chemical-diversity-based approach and how this principle can be adapted to various chemical biology studies.

Pancreatic Langerhans islets are mainly composed of insulinsecreting beta cells and glucagon-secreting alpha cells, along with other minor cell types, and play a central role in the regulation of blood glucose levels. Because of this, imaging of viable pancreatic islets is an important component in research on diabetes both in clinical and experimental medicine. The conventional imaging technique for pancreatic islets is antibody-based immunostaining directly on pancreatic sections, or using transgenic mice with luminescent reporter genes linked to islet-specific promoters. Among small molecule probes, Newport Green and dithiazone (DTZ) have been used for ex vivo fluorescent staining of pancreatic islets, based on their Zn ion binding affinity, which are abundant in beta cells in complex with insulin. For in situ application, fluorescently labeled exendin-4 (a GLP1R binding peptide: M.W. is about 5 kDa) has been recently introduced for the measurement of the mass of pancreatic islet beta cells. However, small molecule probes for selective staining of beta cells in pancreatic islets of live animals have not yet been reported. We predicted that a diversity-oriented fluorescence library approach (DOFLA), an expedited bioimaging probe discovery method using high throughput synthesis and high contents screening, would be a powerful method to achieve this goal. Using a similar approach, we have previously elucidated probes for pluripotent stem cells (CDy1), muscle cells (CDy2), neuronal stem cells (CDr3), and pancreatic alpha cells (GY= glucagon yellow). Although the glucagon-targeting probe GY selectively stains alpha cells in isolated cell culture, it did not clearly mark mouse pancreatic islets in tissue, partially owing to the small population of alpha cells (around 15–20% in mouse islets). We expected that a fluorescent probe for pancreatic beta cells (with a larger population of 75–80% in mouse islets) would be more effective for visualizing pancreatic islets. As a first step, we synthesized fluorescent smallmolecule libraries composed of 1200 compounds, and screened them against beta TC-6 cells in comparison to alpha TC-1 cells and acinar cells (exocrine cells in the pancreas) as controls. The three cell types were compared in 384-well plates and incubated with the library compounds (1 mm) at incubation times ranging from one to 48 hours. The fluorescence live-cell images were acquired by an automated imaging microscope system, ImageXpress Micro. One compound from the BDNCA series (Scheme 1), BDNCA-325 (labs/lem= 558/585 nm, extinction cooefficient e= 58,000m 1 cm , quantum yield F= 0.06) was chosen as the most selective for the beta TC-6 cells (Figure 1a) in comparison to the two control cell types in terms of relative fluorescence intensity. The BDNCA library was prepared from a BODIPY-aniline (BDN) series by chloroacetylation. While BDN has very low fluorescence emission (less than 1% quantum yield) owing to photoinduced electron transfer (PET), by converting the amine to an amide, the fluorescence of BDNCA was moderately increased (F= 5–10%). Therefore, this amide motif is a modulator of the fluorescence intensity of the BDNCA series through interaction with the surrounding environment or binding partner. However, when we injected BDNCA-325 intravenously into a mouse, the pancreatic islets were not selectively stained at various incubation times and concentrations (data not shown). Because BDNCA-325 contains a chemically reactive chloroacetyl group, we hypothesized that the compound might have reacted with other tissues in the animal before reaching the pancreatic islets. Thus, BDNCA-325 was modified by removing the reactive alpha-chloride and also by [*] Dr. N.-Y. Kang, Dr. S.-C. Lee, Dr. S.-J. Park, Dr. S.-W. Yun, Dr. E. Kostromina, Dr. N. Gustavsson, Dr. Y. Ali, Y. Chandran, Dr. W. Han, Dr. G. K. Radda, Prof. Y.-T. Chang Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR) 138667, Singapore (Singapore) E-mail: chmcyt@nus.edu.sg Homepage: http://ytchang.science.nus.edu.sg

Genetic and biochemical studies suggest that Alzheimer's disease (AD) is caused by a series of events initiated by the production and subsequent aggregation of the Alzheimer's amyloid beta peptide (Abeta), the so-called amyloid cascade hypothesis. Thus, a logical approach to treating AD is the development of small molecule inhibitors that either block the proteases that generate Abeta from its precursor (beta- and gamma-secretases) or interrupt and/or reverse Abeta aggregation. To identify potent inhibitors of Abeta aggregation, we have developed a high-throughput screen based on an earlier selection that effectively paired the folding quality control feature of the Escherichia coli Tat protein export system with aggregation of the 42-residue AD pathogenesis effecter Abeta42. Specifically, a tripartite fusion between the Tat-dependent export signal ssTorA, the Abeta42 peptide and the beta-lactamase (Bla) reporter enzyme was found to be export incompetent due to aggregation of the Abeta42 moiety. Here, we reasoned that small, cell-permeable molecules that inhibited Abeta42 aggregation would render the ssTorA-Abeta42-Bla chimera competent for Tat export to the periplasm where Bla is active against beta-lactam antibiotics such as ampicillin. Using a fluorescence-based version of our assay, we screened a library of triazine derivatives and isolated four nontoxic, cell-permeable compounds that promoted efficient Tat-dependent export of ssTorA-Abeta42-Bla. Each of these was subsequently shown to be a bona fide inhibitor of Abeta42 aggregation using a standard thioflavin T fibrillization assay, thereby highlighting the utility of our bacterial assay as a useful screen for antiaggregation factors under physiological conditions.