Virtual Screening (VS) is designed to prospectively help identifying potential hits, i.e., compounds capable of interacting with a given target and potentially modulate its activity, out of large compound collections. Among the variety of methodologies, it is crucial to select the protocol that is the most adapted to the query/target system under study and that yields the most reliable output. To this aim, the performance of VS methods is commonly evaluated and compared by computing their ability to retrieve active compounds in benchmarking datasets. The benchmarking datasets contain a subset of known active compounds together with a subset of decoys, i.e., assumed non-active molecules. The composition of both the active and the decoy compounds subsets is critical to limit the biases in the evaluation of the VS methods. In this review, we focus on the selection of decoy compounds that has considerably changed over the years, from randomly selected compounds to highly customized or experimentally validated negative compounds. We first outline the evolution of decoys selection in benchmarking databases as well as current benchmarking databases that tend to minimize the introduction of biases, and secondly, we propose recommendations for the selection and the design of benchmarking datasets.

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417 IntroductionSeveral new targets have recently been identified in neoplastic mast cells (MCs), and various targeted drugs have been examined for their effectiveness in malignant MC disorders. However, most drugs, including new KIT D816V-blocking agents, such as midostaurin (PKC412), 1 and various BCR-ABL1 inhibitors known to block KIT-activity, such as imatinib or dasatinib, 2 have failed to achieve long-lasting remissions in aggressive systemic mastocytosis (ASM). There is a similar problem in Ph + CML, where imatinib-resistant patients do not reach molecular remission even when secondor third-line BCR-ABL1 tyrosine kinase inhibitors (TKIs) are applied, particularly in patients exhibiting the BCR-ABL1 T315I mutant.3 During disease progression, additional signal transduction pathways become activated in neoplastic cells. Among these, AKT and STAT5 are critical downstream signaling molecules constitutively phosphorylated imatinibresistant chronic myeloid leukemia (CML) and KIT D816V + SM. 4,5 This has been demonstrated in vitro using KIT D816V + and BCR-ABL1 + imatinib-resistant cell lines, where inhibition of phosphorylation of these targets has shown their crucial role in cell proliferation. 6,7 It has also been reported that STAT5 and AKT remained activated in neoplastic myeloid cells, even after inhibition of BCR-ABL1 by TKIs. 8 Moreover, during disease progression, the levels of STAT5 mRNA and protein increase, and STAT5 production and activation is triggered not only by BCR-ABL1 or mutant KIT, but also by other pro-oncogenic pathways relevant to disease progression and resistance. 9 Taken together, these observations strongly suggest that STAT5 and AKT are key drivers in drug-resistant CML and SM, and thus represent potential therapeutic targets. Small molecules targeting STAT5 or AKT may indeed be effective in these malignancies, especially in TKIs-resistant patients. However, inhibitors available today, such as pimozide or BP-1-108 for STAT5, 10,11 or perifosine for AKT,12 are neither specific nor potent enough to be applicable in clinical practice. Therefore, it seems important to develop compounds that specifically and potently target STAT5 and AKT. Mast cells and mastocytosis KIT and cytokine signaling in normal mast cellsMCs originate from bone marrow (BM)-derived hematopoietic stem cells (HSCs) that enter the peripheral tissues via the bloodstream and undergo maturation under the Co-operating STAT5 and AKT signaling pathways in chronic myeloid leukemia and mastocytosis: possible new targets of therapy open-access paper. doi:10.3324/haematol.2013.098442 Manuscript received on October 8, 2013. Manuscript accepted on December 20, 2013 Chronic myeloid leukemia and systemic mastocytosis are myeloid neoplasms sharing a number of pathogenetic and clinical features. In both conditions, an aberrantly activated oncoprotein with tyrosine kinase activity, namely BCR-ABL1 in chronic myeloid leukemia, and mutant KIT, mostly KIT D816V, in systemic mastocytosis, is key to disease evolution. The appreciation of...