An AKAP-Lbc-RhoA interaction inhibitor promotes the translocation of aquaporin-2 to the plasma membrane of renal collecting duct principal cells

Stimulation of renal collecting duct principal cells with antidiuretic hormone (arginine-vasopressin, AVP) results in inhibition of the small GTPase RhoA and the enrichment of the water channel aquaporin-2 (AQP2) in the plasma membrane. The membrane insertion facilitates water reabsorption from primary urine and fine-tuning of body water homeostasis. Rho guanine nucleotide exchange factors (GEFs) interact with RhoA, catalyze the exchange of GDP for GTP and thereby activate the GTPase. However, GEFs involved in the control of AQP2 in renal principal cells are unknown. The A-kinase anchoring protein, AKAP-Lbc, possesses GEF activity, specifically activates RhoA, and is expressed in primary renal inner medullary collecting duct principal (IMCD) cells. Through screening of 18,431 small molecules and synthesis of a focused library around one of the hits, we identified an inhibitor of the interaction of AKAP-Lbc and RhoA. This molecule, Scaff10-8, bound to RhoA, inhibited the AKAP-Lbc-mediated RhoA activation but did not interfere with RhoA activation through other GEFs or activities of other members of the Rho family of small GTPases, Rac1 and Cdc42. Scaff10-8 promoted the redistribution of AQP2 from intracellular vesicles to the periphery of IMCD cells. Thus, our data demonstrate an involvement of AKAP-Lbc-mediated RhoA activation in the control of AQP2 trafficking

Comprehensive characterization of the Published Kinase Inhibitor Set

Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome

The crystal structure of the RhoA-AKAP-Lbc DH-PH domain complex

The RhoGEF domain of AKAP-Lbc (AKAP13) catalyses nucleotide exchange on RhoA and is involved in development of cardiac hypertrophy. The RhoGEF activity of AKAP-Lbc has also been implicated in cancer. We have determined the X-ray crystal structure of the complex between RhoA:GDP and the AKAP-Lbc RhoGEF (DH-PH) domain to 2.1 {Angstrom} resolution. The structure reveals important differences compared to related RhoGEF proteins such as Leukemia-associated RhoGEF. Nucleotide exchange assays comparing the activity of the DH-PH domain to the DH domain alone showed no role for the PH domain in nucleotide exchange, which is explained by the RhoA:AKAP-Lbc structure. Comparison to a structure of the isolated AKAP-Lbc DH domain revealed a change in conformation of the N-terminal 'GEF switch' region upon binding to RhoA. Isothermal titration calorimetry showed that AKAP-Lbc has only micromolar affinity for RhoA which combined with the presence of potential binding pockets for small molecules on AKAP-Lbc raises the possibility of targeting AKAP-Lbc with guanine nucleotide exchange factor inhibitors

What Controls the Flushing Efficiency and Particle Transport Pathways in a Tropical Estuary? Cochin Estuary, Southwest Coast of India

Estuaries with poor flushing and longer residence time retain effluents and pollutants, ultimately resulting in eutrophication, a decline in biodiversity and, finally, deterioration of water quality. Cochin Estuary (CE), southwest coast of India, is under the threat of nutrient enrichment by the anthropogenic interventions and terrestrial inputs through land runoff. The present study used the FVCOM hydrodynamic model coupled with the Lagrangian particle module (passive) to estimate the residence time and to delineate site-specific transport pathways in the CE. The back and forth movements and residence time of particles was elucidated by using metrics such as path length, net displacement and tortuosity. Spatio-temporal patterns of the particle distribution in the CE showed a similar trend during monsoon and post-monsoon with an average residence time of 25 and 30 days, respectively. During the low river discharge period (pre-monsoon), flood-ebb velocities resulted in a minimum net transport of the water and longer residence time of 90 days compared to that of the high discharge period (monsoon). During the pre-monsoon, particle released at the southern upstream (station 15) traversed a path length of 350 km in 90 days before being flushed out through the Fortkochi inlet, where the axial distance was only 35 km. This indicates that the retention capacity of pollutants within the system is very high and can adversely affect the water quality of the ecosystem. However, path length (120 km) and residence time (7.5 days) of CE were considerably reduced during the high discharge period. Thus the reduced path length and the lower residence time can effectively transport the pollutants reaching the system, which will ultimately restore the healthy ecosystem. This is a pioneer attempt to estimate the flushing characteristics and residence time of the CE by integrating the hydrodynamics and Lagrangian particle tracking module of FVCOM. This information is vital for the sustainable management of sensitive ecosystems