Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains function of numerous intracellular signaling nodes utilized by cancer cells for proliferation and survival. Hsp90 is also detected on the plasma membrane of tumor cells and its expression has been suggested to correlate with metastatic potential. Given the abundance and diverse functions of the intracellular pool of this protein, the precise contribution of cell surface Hsp90 to cell motility and tumor metastasis remains to be determined. In this study we utilized the small molecule DMAG-N-oxide, a novel cell-impermeable Hsp90 inhibitor, to specifically examine the role of cell surface Hsp90 in cell motility. We observed that, while not affecting intracellular Hsp90 function, DMAG-N-oxide significantly retarded tumor cell migration and integrin/extracellular matrix-dependent cytoskeletal reorganization. Concomitant with these findings, targeting cell surface Hsp90 significantly inhibited tumor cell motility and invasion in vitro, and had a dramatic impact on melanoma cell lung colonization in vivo. These data indicate that cell surface Hsp90 plays an important role in modulating cancer cell migration that is independent of the function of the intracellular Hsp90 pool, and that small molecule inhibitors of surface Hsp90 may provide a new approach to targeting the metastatic phenotype.

The management of sodium intake is clinically important in many disease states including heart failure, kidney disease, and hypertension. Tenapanor is an inhibitor of the sodium-proton (Na(+)/H(+)) exchanger NHE3, which plays a prominent role in sodium handling in the gastrointestinal tract and kidney. When administered orally to rats, tenapanor acted exclusively in the gastrointestinal tract to inhibit sodium uptake. We showed that the systemic availability of tenapanor was negligible through plasma pharmacokinetic studies, as well as autoradiography and mass balance studies performed with (14)C-tenapanor. In humans, tenapanor reduced urinary sodium excretion by 20 to 50 mmol/day and led to an increase of similar magnitude in stool sodium. In salt-fed nephrectomized rats exhibiting hypervolemia, cardiac hypertrophy, and arterial stiffening, tenapanor reduced extracellular fluid volume, left ventricular hypertrophy, albuminuria, and blood pressure in a dose-dependent fashion. We observed these effects whether tenapanor was administered prophylactically or after disease was established. In addition, the combination of tenapanor and the blood pressure medication enalapril improved cardiac diastolic dysfunction and arterial pulse wave velocity relative to enalapril monotherapy in this animal model. Tenapanor prevented increases in glomerular area and urinary KIM-1, a marker of renal injury. The results suggest that therapeutic alteration of sodium transport in the gastrointestinal tract instead of the kidney--the target of current drugs--could lead to improved sodium management in renal disease.

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A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase. When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha2/beta2 structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.

In CKD, phosphate retention arising from diminished GFR is a key early step in a pathologic cascade leading to hyperthyroidism, metabolic bone disease, vascular calcification, and cardiovascular mortality. Tenapanor, a minimally systemically available inhibitor of the intestinal sodium-hydrogen exchanger 3, is being evaluated in clinical trials for its potential to (1) lower gastrointestinal sodium absorption, (2) improve fluid overload-related symptoms, such as hypertension and proteinuria, in patients with CKD, and (3) reduce interdialytic weight gain and intradialytic hypotension in ESRD. Here, we report the effects of tenapanor on dietary phosphorous absorption. Oral administration of tenapanor or other intestinal sodiumhydrogen exchanger 3 inhibitors increased fecal phosphorus, decreased urine phosphorus excretion, and reduced [ 33 P]orthophosphate uptake in rats. In a rat model of CKD and vascular calcification, tenapanor reduced sodium and phosphorus absorption and significantly decreased ectopic calcification, serum creatinine and serum phosphorus levels, circulating phosphaturic hormone fibroblast growth factor-23 levels, and heart mass. These results indicate that tenapanor is an effective inhibitor of dietary phosphorus absorption and suggest a new approach to phosphate management in renal disease and associated mineral disorders.

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Takeda G protein-coupled receptor 5 (TGR5) agonists induce systemic release of glucagon-like peptides (GLPs) from intestinal L cells, a potentially therapeutic action against metabolic diseases such as nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and Type 2 diabetes. Historically, TGR5 agonist use has been hindered by side effects, including inhibition of gallbladder emptying. Here, we characterize RDX8940, a novel, orally administered TGR5 agonist designed to have minimal systemic effects and investigate its activity in mice fed a Western diet, a model of NAFLD and mild insulin resistance. Agonist activity, binding selectivity, toxicity, solubility, and permeability of RDX8940 were characterized in standard in vitro models. RDX8940 pharmacokinetics and effects on GLP secretion, insulin sensitivity, and liver steatosis were assessed in C57BL/6 mice fed normal or Western diet chow and given single or repeated doses of RDX8940 or vehicle, with or without dipeptidyl peptidase-4 (DPP4) inhibitors. Gallbladder effects were assessed in CD-1 mice fed normal chow and given RDX8940 or a systemic TGR5 agonist or vehicle. Our results showed that RDX8940 is minimally systemic, potent, and selective, and induces incretin (GLP-1, GLP-2, and peptide YY) secretion. RDX8940-induced increases in plasma active GLP-1 (aGLP-1) levels were enhanced by repeated dosing and by coadministration of DPP4 inhibitors. RDX8940 increased hepatic exposure to aGLP-1 without requiring coadministration of a DPP4 inhibitor. In mice fed a Western diet, RDX8940 improved liver steatosis and insulin sensitivity. Unlike systemic TGR5 agonists, RDX8940 did not inhibit gallbladder emptying. These results indicate that RDX8940 may have therapeutic potential in patients with NAFLD/NASH. NEW & NOTEWORTHY Takeda G protein-coupled receptor 5 (TGR5) agonists have potential as a treatment for nonalcoholic steatohepatitis and nonalcoholic fatty liver disease (NAFLD) but have until now been associated with undesirable side effects associated with systemic TGR5 agonism, including blockade of gallbladder emptying. We demonstrate that RDX8940, a potent, selective, minimally systemic oral TGR5 agonist, improves liver steatosis and insulin sensitivity in a mouse model of NAFLD and does not inhibit gallbladder emptying in mice.