Search

In cattle, parasitic flies such as the horn fly, Haematobia irritans, or the stable fly, Stomoxys calcitrans, are a serious nuisance for the animals and, in high populations, can cause substantial reduction in milk production, as well as significantly defer weight gain in calves. In 2012 annual losses associated with such pests were estimated to be up to 2200 million USD in the United States alone, and new effective treatments were therefore deemed highly valuable. The dinitriles were identified as a very promising class for combatting these pests, with additional potential for flea and tick control in companion animals. The molecules act as highly potent GABA-gated chloride channel blockers with IC50 values down to the single-digit nanomolar range. In addition, they possess fast knockdown activities close to state-of-the-art pyrethroids. In this chapter, the research efforts from early hit discovery to initial field testing will be described in further detail.

SummaryDefining the molecular targets of insecticides is crucial for assessing their selectivity and potential impact on environment and health. Two commercial insecticides are now shown to target a transient receptor potential (TRP) ion channel complex that is unique to insect stretch receptor cells. Pymetrozine and pyrifluquinazon disturbed Drosophila coordination and hearing by acting on chordotonal stretch receptor neurons. This action required the two TRPs Nanchung (Nan) and Inactive (Iav), which co-occur exclusively within these cells. Nan and Iav together sufficed to confer cellular insecticide responses in vivo and in vitro, and the two insecticides were identified as specific agonists of Nan-Iav complexes that, by promoting cellular calcium influx, silence the stretch receptor cells. This establishes TRPs as insecticide targets and defines specific agonists of insect TRPs. It also shows that TRPs can render insecticides cell-type selective and puts forward TRP targets to reduce side effects on non-target species.

The protein kinase family represents an enormous opportunity for drug development. However, the current limitation in structural diversity of kinase inhibitors has complicated efforts to identify effective treatments of diseases that involve protein kinase signaling pathways. We have identified a new structural class of protein serine/threonine kinase inhibitors comprising an aminoimidazo[1,2-a]pyridine nucleus. In this report, we describe the first successful use of this class of aza-heterocycles to generate potent inhibitors of cyclin-dependent kinases that compete with ATP for binding to a catalytic subunit of the protein. Co-crystal structures of CDK2 in complex with lead compounds reveal a unique mode of binding. Using this knowledge, a structure-based design approach directed this chemical scaffold toward generating potent and selective CDK2 inhibitors, which selectively inhibited the CDK2-dependent phosphorylation of Rb and induced caspase-3-dependent apoptosis in HCT 116 tumor cells. The discovery of this new class of ATP-site-directed protein kinase inhibitors, aminoimidazo[1,2-a]pyridines, provides the basis for a new medicinal chemistry tool to be used in the search for effective treatments of cancer and other diseases that involve protein kinase signaling pathways.

G protein-coupled receptors (GPCRs) are the target of approximately 40% of all approved drugs and continue to represent a significant portion of drug discovery portfolios across the pharmaceutical industry. As a result, GPCRs are the focus of many high-throughput screening (HTS) campaigns. Historically, ligand-binding assays were used to identify compounds that targeted GPCRs. Current GPCR drug discovery efforts have moved toward the utilization of functional cell-based assays for HTS. Many of these assays monitor the accumulation of a second messenger such as cAMP or calcium in response to GPCR activation. Calcium stores are released from the endoplasmic reticulum when Galphaq-coupled GPCRs are activated. Although Galphai- and Galphas-coupled receptors do not normally result in this mobilization of intracellular calcium, they can often be engineered to do so by expressing a promiscuous or a chimeric Galphaprotein, which couples to the calcium pathway. Thus calcium mobilization is a readout that can theoretically be used to assess activation of all GPCRs. The fluorometric imaging plate reader (FLIPR) has facilitated the ability to monitor calcium mobilization in the HTS setting. This assay format allows one to monitor activation and inhibition of a GPCR in a single assay and has been one of the most heavily utilized formats for screening GPCRs.

G protein-coupled receptors (GPCRs) are the target of approximately 40% of all approved drugs and continue to represent a significant portion of drug discovery portfolios across the pharmaceutical industry. As a result, GPCRs are the focus of many high-throughput screening (HTS) campaigns. Historically, ligand-binding assays were used to identify compounds that targeted GPCRs. Current GPCR drug discovery efforts have moved toward the utilization of functional cell-based assays for HTS. Many of these assays monitor the accumulation of a second messenger such as cAMP or calcium in response to GPCR activation. Calcium stores are released from the endoplasmic reticulum when Galphaq-coupled GPCRs are activated. Although Galphai- and Galphas-coupled receptors do not normally result in this mobilization of intracellular calcium, they can often be engineered to do so by expressing a promiscuous or a chimeric Galphaprotein, which couples to the calcium pathway. Thus calcium mobilization is a readout that can theoretically be used to assess activation of all GPCRs. The fluorometric imaging plate reader (FLIPR) has facilitated the ability to monitor calcium mobilization in the HTS setting. This assay format allows one to monitor activation and inhibition of a GPCR in a single assay and has been one of the most heavily utilized formats for screening GPCRs.

Publisher Summary Baculovirus infected insect cells represent an effective system for the overexpression of heterologous proteins. The level of soluble K-ras produced by these cells is quite high as seen in the chapter. Kirsten-ras is the most frequently activated oncogene in human tumors. Activated K-ras is bound to GTP and possesses intrinsic GTPase activity that leads to its inactivation. Determination of the crystal structure of K-ras is likely to aid in the rational design of anticancer therapeutics. Truncated K-ras is expressed in baculovirus infected insect cells that has been suggested as an appropriate system for its production. K-ras is expressed in abundance from these cells and is purified to apparent homogeneity evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Analytical Diethylaminoethyl (DEAE) chromatography and electrospray-ionization mass spectrometry (ESI-MS) of the purified protein indicated substantial heterogeneity. The different proteins have been characterized by tryptic mapping utilizing LC-MS. This analysis indicated the presence of at least four different N-terminal variants of K-ras and additional heterogeneity because of dissociation of bound nucleotide, indicating that unwanted cellular processing of proteins may occur in baculovirus infected cells. The study described in this chapter includes discussion on the utility of analytical DEAE coupled with ESI-MS in the determination of bound nucleotide. This technique may be useful in evaluation of K-ras protein that has been loaded with GTP analogues to activate it.

Two cDNA clones coding for the human protein kinase C-delta (PKC-delta) were fortuitously isolated during the process of screening a human library for a cDNA clone of an unrelated protein, the nucleolar protein fibrillarin. The two human homologues have about 88% nucleotide sequence identity to the rat and mouse PKC-delta cDNA clones. A comparison of the predicted amino acid sequences of the two human PKC-delta clones with the rat and mouse homologues indicated a greater degree of sequence divergence (89-90% homology) compared to the high degree of sequence conservation observed with other human PKC family members and their mammalian counterparts. Expression of the clones in the baculovirus insect-cell expression system indicated that both proteins exhibited phorbol ester binding activity, and were dependent upon phosphatidylserine and diacylglycerol for maximal activation. Further characterization of the properties of the human PKC-delta revealed substrate and lipid dependencies distinct from other members of the protein kinase C family; including PKC-deltas isolated from other species. The dissimilarities in the predicted amino acid sequences between the human and other mammalian species could account in part for some of these observed biochemical differences.