Multicomponent therapies, originating through deliberate mixing of drugs in a clinical setting, through happenstance, and through rational design, have a successful history in a number of areas of medicine, including cancer, infectious diseases, and CNS disorders. We have developed a high-throughput screening method for identifying effective combinations of therapeutic compounds. We report here that systematic screening of combinations of small molecules reveals unexpected interactions between compounds, presumably due to interactions between the pathways on which they act. Through systematic screening of Ϸ120,000 different two-component combinations of reference-listed drugs, we identified potential multicomponent therapeutics, including (i) fungistatic and analgesic agents that together generate fungicidal activity in drug-resistant Candida albicans, yet do not significantly affect human cells, (ii) glucocorticoid and antiplatelet agents that together suppress the production of tumor necrosis factor-␣ in human primary peripheral blood mononuclear cells, and (iii) antipsychotic and antiprotozoal agents that do not exhibit significant antitumor activity alone, yet together prevent the growth of tumors in mice. Systematic combination screening may ultimately be useful for exploring the connectivity of biological pathways and, when performed with reference-listed drugs, may result in the discovery of new combination drug regimens.M odern biological research and much of drug discovery is often driven by the search for new molecularly targeted therapeutics (1-3). In this approach, a specific protein is studied in vitro, in cells and in whole organisms, and evaluated as a drug target for a specific therapeutic indication (3, 4). The refinement of this approach has resulted in the ability to discover compounds with great selectivity for a chosen protein target. The recent success of Gleevec (imatinib mesylate), an inhibitor of the breakpoint cluster regionabelson (BCR-ABL) kinase, and of selective cyclooxygenase-2 (COX-2) inhibitors Vioxx (rofecoxib) and Celebrex (celecoxib) are evidence that the target-based approach can be successful (5, 6).Systems biology, however, has revealed that human cells and tissues are composed of complex, networked systems with redundant, convergent and divergent signaling pathways (7-10). For example, the redundant function of proteins involved in cell-cycle regulation (11) has inspired efforts to intervene simultaneously at multiple points in these signaling pathways (12). A drug discovery approach consonant with this systems biology framework, and complementary to the target-based approach, entails identification of combinations of small molecules that perturb cellular signaling networks in a desired fashion.Recognition of the potential for multipoint intervention in biology and medicine has a long history. As early as 1928, Loewe (13) observed and quantified effects of combinations of compounds that were different from, and not predicted by, the activities of the constituents. The conc...

The retinoic acid-inducible transcription factor AP-2 is expressed in epithelial and neural crest cell lineages during murine development. AP-2 can regulate neural and epithelial gene transcription, and is associated with overexpression of c-erbB-2 in human breast-cancer cell lines. To ascertain the importance of AP-2 for normal development, we have derived mice containing a homozygous disruption of the AP-2 gene. These AP-2-null mice have multiple congenital defects and die at birth. In particular, the AP-2 knockout mice exhibit anencephaly, craniofacial defects and thoraco-abdominoschisis. Skeletal defects occur in the head and trunk region, where many bones are deformed or absent. Analysis of these mice earlier in embryogenesis indicates a failure of cranial neural-tube closure and defects in cranial ganglia development. We have shown that AP-2 is a fundamental regulator of mammalian craniofacial development.

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According to a model based on embryological studies in amphibia, dorsoventral patterning is regulated by the antagonizing function of ventralizing bone morphogenetic proteins (BMPs) and dorsalizing signals generated by Spemann's organizer. Large-scale mutant screens in the zebrafish, Danio rerio, have led to the isolation of two classes of recessive lethal mutations affecting early dorsoventral pattern formation, dino mutant embryos are ventralized, whereas swirl mutants are dorsalized. We show that at early gastrula stages, dino and swirl mutants display an expanded or reduced Bmp4 expression, respectively. The dino and swirl mutant phenotypes both can be phenocopied and rescued by the modulation of BMP signaling in wild-type and mutant embryos. By suppressing BMP signaling in dino mutants, adult fertile dino -/-fish were generated. These findings, together with results from the analysis of dino-swirl double mutants, indicate that dino fulfills its dorsalizing activity via a suppression of swirl-dependent, BMP-like ventralizing activities. Finally, cell transplantation experiments show that dino is required on the dorsal side of early gastrula embryos and acts in a non-cell-autonomous fashion. Together, these results provide genetic evidence in support of a mechanism of early dorsoventral patterning that is conserved among vertebrate and invertebrate embryos.[Key Words: Dorsoventral pattern formation; dino; swirl; BMP4; noggin; Spemann's organizer; zebrafish; Danio rerio]Received June 10, 1996; revised version accepted July 22, 1996.Our understanding of how the axes of the vertebrate embryo are patterned is dominated by studies of amphibia. Patterning of the anterior-posterior and dorsal-ventral axes of the amphibian embryo is thought to depend on discretely localized signals (for review, see Sive 1993). Maternal signals emanating from vegetal cells induce ventral mesoderm in most of the marginal zone and the dorsal mesoderm of Spemann's organizer in a rather small dorsal region at the site where the formation of the blastopore is initiated. Later, zygotic signals generated by the ventral and dorsal mesoderm themselves regulate the refinement of dorsoventral pattern within the mesoderm and the induction of neuroectoderm in the dorsal animal zone of the early gastrula embryo.Recent evidence, principally based on studies in Xenopus laevis, supports a simple model of opposing dorsal and ventral activities. Two members of the family of bone morphogenetic proteins (BMPs), members of the TGF[3 superfamily, BMP2 and BMP4, are expressed strongly on the ventral side of early gastrula and have strong ventralizing properties. Overexpression of either 1Corresponding author. Present address:

Angiogenesis is necessary for tumor growth, making inhibition of vessel formation an excellent target for cancer therapy. Current assays for angiogenesis, however, are too complex to be practical for drug screening. Here, we demonstrate that the zebrafish is a viable whole animal model for screening small molecules that affect blood vessel formation. Blood vessel patterning is highly characteristic in the developing zebrafish embryo and the subintestinal vessels (SIVs) can be stained and visualized microscopically as a primary screen for compounds that affect angiogenesis. Small molecules added directly to the fish culture media diffuse into the embryo and induce observable, dose-dependent effects. To evaluate the zebrafish as a model, we used two angiogenesis inhibitors, SU5416 and TNP470, both of which have been tested in mammalian systems. Both compounds caused a reduction in vessel formation when introduced to zebrafish embryos prior to the onset of angiogenesis. Short duration (1 h) exposure of SU5416 was sufficient to block new angiogenic and vasculogenic vessel formation. In contrast, TNP470 required continuous exposure to block SIV formation and had no apparent effect on vasculogenic vessel formation. To ascertain whether blood vessels in the zebrafish embryo respond to angiogenic compounds, we introduced human VEGF into embryos. Injection of VEGF caused an observable increase in SIV formation.