State-Dependent Allosteric Inhibition of the Human SLC13A5 Citrate Transporter by Hydroxysuccinic Acids, PF-06649298 and PF-06761281 s

ABSTRACT In the liver, citrate is a key metabolic intermediate involved in the regulation of glycolysis and lipid synthesis and reduced expression of the hepatic citrate SLC13A5 transporter has been shown to improve metabolic outcomes in various animal models. Although inhibition of hepatic extracellular citrate uptake through SLC13A5 has been suggested as a potential therapeutic approach for Type-2 diabetes and/or fatty liver disease, so far, only a few SLC13A5 inhibitors have been identified. Moreover, their mechanism of action still remains unclear, potentially limiting their utility for in vivo proof-of-concept studies. In this study, we characterized the pharmacology of the recently identified hydroxysuccinic acid SLC13A5 inhibitors, PF-06649298 and PF-06761281, using a combination of 14 C-citrate uptake, a membrane potential assay and electrophysiology. In contrast to their previously proposed mechanism of action, our data suggest that both PF-06649298 and PF-06761281 are allosteric, state-dependent SLC13A5 inhibitors, with low-affinity substrate activity in the absence of citrate. As allosteric state-dependent modulators, the inhibitory potency of both compounds is highly dependent on the ambient citrate concentration and our detailed mechanism of action studies therefore, may be of value in interpreting the in vivo effects of these compounds

Cardiac-Specific Elevations in Thyroid Hormone Enhance Contractility and Prevent Pressure Overload-Induced Cardiac Dysfunction

Thyroid hormone (TH) is critical for cardiac development and heart function. In heart disease, TH metabolism is abnormal, and many biochemical and functional alterations mirror hypothyroidism. Although TH therapy has been advocated for treating heart disease, a clear benefit of TH has yet to be established, possibly because of peripheral actions of TH. To assess the potential efficacy of TH in treating heart disease, type 2 deiodinase (D2), which converts the prohormone thyroxine to active triiodothyronine (T3), was expressed transiently in mouse hearts by using the tetracycline transactivator system. Increased cardiac D2 activity led to elevated cardiac T3 levels and to enhanced myocardial contractility, accompanied by increased Ca(2+) transients and sarcoplasmic reticulum (SR) Ca(2+) uptake. These phenotypic changes were associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) 2a expression as well as decreased Na(+)/Ca(2+) exchanger, beta-myosin heavy chain, and sarcolipin (SLN) expression. In pressure overload, targeted increases in D2 activity could not block hypertrophy but could completely prevent impaired contractility and SR Ca(2+) cycling as well as altered expression patterns of SERCA2a, SLN, and other markers of pathological hypertrophy. Our results establish that elevated D2 activity in the heart increases T3 levels and enhances cardiac contractile function while preventing deterioration of cardiac function and altered gene expression after pressure overload

Characterization of KCNQ5/Q3 potassium channels expressed in mammalian cells

Heteromeric KCNQ5/Q3 channels were stably expressed in Chinese Hamster ovary cells and characterized using the whole cell voltage-clamp technique. KCNQ5/Q3 channels were activated by the novel anticonvulsant, retigabine (EC(50) 1.4 μM) by a mechanism that involved drug-induced, leftward shifts in the voltage-dependence of channel activation (−31.8 mV by 30 μM retigabine). KCNQ5/Q3 channels were inhibited by linopirdine (IC(50) 7.7 μM) and barium (IC(50) 0.46 mM), at concentrations similar to those required to inhibit native M-currents. These findings identify KCNQ5/Q3 channels as a molecular target for retigabine and raise the possibility that activation of KCNQ5/Q3 channels may be responsible for some of the anti-convulsant activity of this agent. Furthermore, the sensitivity of KCNQ5/Q3 channels to linopirdine supports the possibility that potassium channels comprised of KCNQ5 and KCNQ3 may make a contribution to native M-currents