Development of Drugs Acting at Ion Channels

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Linked publications & trials

Abstract

The P2X ion channels mediate a number of potent and possibly important biological effects in the cardiovascular, inflammatory, and central nervous systems. Activators of ATP-gated ion channels (P2X receptors) are being synthesized and investigated for cardioprotection in collaboration with Dr. Bruce Liang (University of Connecticut). Previous studies have shown that extracellular ATP can cause an ionic current in murine, rat and guinea pig cardiac ventricular myocytes. The receptor that mediates this current appears to be a P2X receptor, of which the P2X4 receptor is an important subunit. Activation of P2X receptors leads to the opening of a nonselective cation channel permeable to sodium, potassium, and calcium ions. The current is inward at negative membrane potentials, reverses near 0 mV, and becomes outward at positive potentials. The continuous activation of this receptor channel by endogenous extracellular ATP may assume an important biological function. This constant activation under the resting or negative membrane potentials would produce an inward current, whereas its activation during depolarized portions of the action potential should lead to an outward current. These currents represent a possible ionic mechanism by which the cardiac P2X channel achieves its biological effects. A potential biologically important role of the cardiac P2X receptor was suggested by the finding that cardiac myocyte-specific overexpression of the P2X4 receptor can rescue the hypertrophic and heart failure phenotype of the calsequestrin (CSQ) model of cardiomyopathy. However, little is known regarding regulation of the cardiac P2X receptor in cardiac hypertrophy or failure. Furthermore, it is not clear whether an increased activation of the endogenous P2X receptor channel is beneficial or harmful in the progression of heart failure. The regulation of the P2X receptor-mediated ionic current and its potential role in heart failure was investigated using several novel nucleotide agonists. MRS2339, synthesized in our lab, is a nucleotide activator of a P2X4R ion channel present in the cardiac muscle cells. We have explored the structure activity relationships of this nucleotide. Certain phosphonate derivatives are more stable to hydrolysis than the phosphate derivative MRS2339 and are being explored in vivo. MRS2339 is currently being licensed by private industry for the treatment of heart failure. Chronic administration of a novel nucleotidase-resistant P2 receptor agonist MRS2339, which was capable of inducing this ionic current and was devoid of any vasodilator action, reduced cardiac hypertrophy and increased lifespan. The data suggests that an important biological function of the cardiac P2X current is to favorably modulate the progression of cardiac hypertrophy and failure. Recently we identified uncharged carbocyclic nucleotide analogues (including nonhydrolyzable phosphonates) related structurally to MRS2339, that represent potential candidates for the treatment of heart failure, suggesting this as a viable and structurally broad approach. We also found a beneficial therapeutic effect of 2-cyclohexylthio-adenosine 5-monophosphate in mice with heart failure (HF). We discovered MRS2978 as a nucleotide agonist of the cardiac P2X4 receptor that is not charged and has oral biovailability. Like MRS2339, it is cardioprotective in heart failure models, but it can be administered by oral gavage in experimental animals. We have studied the pharmacokinetics of this compound and found it to be drug-like. We are synthesizing and exploring P2X4 receptor antagonists for treatment of stroke. In a mouse stroke model, treatment with a known P2X4 receptor antagonist 5-BDBD decreased the infiltration of peripheral myeloid cells, which contributed to recovery by reducing P2X4R activation in both infiltrated monocytes/macrophages. Reduced activation of resident microglia specifically in the perilesional cortex upon blocking P2X4 suggests that acute inhibition of this receptor in microglia is also beneficial for stroke recovery. We have discovered the first allosteric modulators of the dopamine transporter (DAT) in collaboration with Aaron Janowsky (University of Oregon). These rigidified nucleoside derivatives inhibited dopamine uptake, but they enhanced the binding of a tropane radioligand to the transporter. The most potent analogue displayed an EC50 value of 35 nM in the enhancement of radioligand binding. In some cases modulation of the norepinephrine transporter (NET) was also observed. The behavioral effects of these agents are being probed. These allosteric ligands stabilize the human form of DAT such that it can be purified to homogeneity for the first time (with Eric Gouaux, University of Oregon). We have studied the interactions of adenosine derivatives that act as potent A3 adenosine receptor agonists with pumps, including P-glycoprotein (P-gp). This is an undesired activity when the nucleosides are administered for treatment of pain or inflammation. However, we have identified novel modified analogues that contain a rigid methanocarba ring in place of ribose (considered potency-enhancing at the A3 adenosine receptor) that also inhibit transport in P-gp. These analogues are being used to study the binding site of P-gp.