Discovery and Characterization of a Potent and Selective Inhibitor of Aedes aegypti Inward Rectifier Potassium Channels

Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the world''s population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes aegypti Kir1 (AeKir1) channels heterologously expressed in HEK293 cells. Of 283 confirmed screening ‘hits’, the small-molecule inhibitor VU625 was selected for lead optimization and in vivo studies based on its potency and selectivity toward AeKir1, and tractability for medicinal chemistry. In patch clamp electrophysiology experiments of HEK293 cells, VU625 inhibits AeKir1 with an IC₅₀ value of 96.8 nM, making VU625 the most potent inhibitor of AeKir1 described to date. Furthermore, electrophysiology experiments in Xenopus oocytes revealed that VU625 is a weak inhibitor of AeKir2B. Surprisingly, injection of VU625 failed to elicit significant effects on mosquito behavior, urine excretion, or survival. However, when co-injected with probenecid, VU625 inhibited the excretory capacity of mosquitoes and was toxic, suggesting that the compound is a substrate of organic anion and/or ATP-binding cassette (ABC) transporters. The dose-toxicity relationship of VU625 (when co-injected with probenecid) is biphasic, which is consistent with the molecule inhibiting both AeKir1 and AeKir2B with different potencies. This study demonstrates proof-of-concept that potent and highly selective inhibitors of mosquito Kir channels can be developed using conventional drug discovery approaches. Furthermore, it reinforces the notion that the physical and chemical properties that determine a compound''s bioavailability in vivo will be critical in determining the efficacy of Kir channel inhibitors as insecticides.     

Potent small molecule inhibitors of spleen tyrosine kinase (Syk)

A series of oxindoles demonstrating inhibition of the phosphorylation of biotinylated substrates of Syk and IgE/Fc epsilon RI triggered basophil cell degranulation has been identified. A study of the SAR around sulfonamide 31 (IC(50)=5 nM, EC(50)=1400 nM) is discussed. The modest cellular activity representative of the sulfonamide series was overcome when the Polar Surface Area was lowered to <110 A(2), leading to the identification of amide 32 (IC(50)=145 nM, EC(50)=100 nM).     

Incorporating uncertainty and social values in managing invasive alien species: a deliberative multi-criteria evaluation approach

The management of Invasive Alien Species (IAS) is stymied by complex social values and severe levels of uncertainty. However, these two challenges are often hidden in the conventional model of management by “value-free” analyses and probability-based estimates of risk. As a result, diverse social values and wide margins of error in risk assessment carry zero weights in the decision-making process, leaving IAS risk decisions to be made in the wake of political pressure and the crisis atmosphere of incursion. We propose to use a Deliberative Multi-Criteria Evaluation (DMCE) to incorporate multiple social values and profound uncertainty into decision-making processes. The DMCE process combines the advantages of conventional multi-criteria decision analysis methods with the benefits of stakeholder participation to provide an analytical structure to assess complex multi-dimensional objectives. It, therefore, offers an opportunity for diverse views to enter the decision-making process, and for the negotiation of consensus positions. The DMCE process can also function as a platform for risk communication in which scientists, stakeholders, and decision-makers can interact and discuss the uncertainty associated with biological invasions. We examine two case studies that demonstrate how DMCE provides scientific rigor and transparency in the decision-making process of invasion risk management. The first case regards pre-border priority ranking for potential invasive species and the second relates to selecting the most desirable policy option for managing a post-border invader.     

Detection of low prevalence somatic mutations in solid tumors with ultra-deep targeted sequencing

Ultra-deep targeted sequencing (UDT-Seq) can identify subclonal somatic mutations in tumor samples. Early assays' limited breadth and depth restrict their clinical utility. Here, we target 71 kb of mutational hotspots in 42 cancer genes. We present novel methods enhancing both laboratory workflow and mutation detection. We evaluate UDT-Seq true sensitivity and specificity (> 94% and > 99%, respectively) for low prevalence mutations in a mixing experiment and demonstrate its utility using six tumor samples. With an improved performance when run on the Illumina Miseq, the UDT-Seq assay is well suited for clinical applications to guide therapy and study clonal selection in heterogeneous samples.