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The oxycyclohexyl acid BMS-986278 (33) is a potent lysophosphatidic acid receptor 1 (LPA1) antagonist, with a human LPA1 Kb of 6.9 nM. The structure-activity relationship (SAR) studies starting from the LPA1 antagonist clinical compound BMS-986020 (1), which culminated in the discovery of 33, are discussed. The detailed in vitro and in vivo preclinical pharmacology profiles of 33, as well as its pharmacokinetics/metabolism profile, are described. On the basis of its in vivo efficacy in rodent chronic lung fibrosis models and excellent overall ADME (absorption, distribution, metabolism, excretion) properties in multiple preclinical species, 33 was advanced into clinical trials, including an ongoing Phase 2 clinical trial in patients with lung fibrosis (NCT04308681).

The oxycyclohexyl acid BMS-986278 (

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal fibrosing lung disease with limited approved treatment options. The bioactive lysophospholipid lysophosphatidic acid (LPA) and the LPA1 receptor have been implicated in the etiology and pathogenesis of IPF, positioning LPA1 as a promising therapeutic target for IPF and other fibrotic diseases. The LPA1 antagonist BMS-986020 600 mg BID was efficacious in a 6-month placebo-controlled Phase 2 clinical trial in IPF patients by slowing lung function decline as measured by rate of decline of forced vital capacity (FVC) (CHEST, 154, p1061–69, 2018). BMS-986278 is a novel next generation LPA1 antagonist currently in Phase I clinical trials. BMS-986278 is a potent and complete antagonist of LPA action at LPA1-mediated Gi, Gq, G12, and β-arrestin signaling pathways in both cells heterologously expressing human LPA1 and in primary human lung fibroblasts. In vivo, BMS-986278: 1) inhibits LPA-stimulated histamine release in mice; 2) demonstrates antifibrotic activity, as shown by decreases in picrosirius red staining area of the lung in the chronic rodent bleomycin model. BMS-986278 demonstrates excellent pharmacokinetics in preclinical species: 1) oral bioavailability = 70% (mouse); 100% (rat); 79% (monkey) and 2) clearance (mL/min/kg) = 37 (mouse); 15 (rat); 2 (monkey). BMS-986278 has negligible activity at bile acid and other clinically relevant drug transporters (BSEP, MDR3 IC50 = >100 μM; OATP1B1 IC50 = 35.5 μM). BMS-986278 represents a novel promising LPA1 antagonist for the treatment of multiple fibrotic diseases.

Rationale and Aim: Studies linking the lysophosphatidic acid 1 receptor (LPA1) to the pathogenesis of lung and kidney fibrosis have identified LPA1 as a potential clinical target for idiopathic pulmonary fibrosis (IPF). LPA1 antagonism has shown efficacy in a Phase 2 clinical IPF trial. Here we report the evaluation of BMS-986327 as a PET ligand to detect LPA1 expression and assess engagement and occupancy of LPA1-targeted drugs. Methods: BMS-986327 binding was measured in competition with LPA1 antagonists in a whole-cell binding assay. PET imaging was performed in wild-type (WT) and pulmonary fibrosis (PF) rat models to determine target expression and drug target engagement. Similar PET studies were carried out in healthy non-human primates (NHPs) as a prelude to clinical studies. Results: In vitro studies showed tracer binding to LPA1 and concentration-dependent inhibition with LPA1 antagonists. In vivo, preferential tracer accumulation was measured in the lungs of PF rats compared to WT controls. Drug competition studies showed a correlation between tracer signal in the lungs of PF rats and the plasma concentration of LPA1 antagonist. PET studies in NHPs confirmed lung tracer localization and dose-dependent blockade with LPA1 antagonist, with test-retest variability within typical limits of PET. Conclusions: In vitro and in vivo studies demonstrated specific binding of BMS-986327 to LPA1. Increased lung tracer accumulation in PF compared to WT animals and displacement with LPA1 antagonists showed the ability of BMS-986327 to assess LPA1 expression and drug target engagement in vivo in both rat and NHP models. Minimal test-retest variability supports the translatability of this tracer to clinical study.

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.

Squalene synthase catalyzes the reductive condensation of two identical substrate molecules, farnesyl diphosphate, to the hydrocarbon squalene via an obligatory intermediate, presqualene pyrophosphate. Since the kinetic mechanism of the transformation is sequential, two substrate binding pockets that recognize the same molecule must exist in the enzyme active site. This raises the possibility of a choice of binding pockets for inhibitors that are designed as substrate or reaction intermediate analogs and thus may provide some information on the mechanism of differentiation of the two identical molecules. In this report, we have investigated the mechanism of inhibition of a series of farnesyl diphosphate analog inhibitors. The inhibitors fall into two categories. One class of compounds binds to free enzyme as well as the enzyme substrate complex, and the binding is refractory to the concentration of the substrate. The second class binds only to the free enzyme, and its binding is significantly modulated by the substrate concentration. Very modest structural changes in the compounds appear to dictate which class of inhibitor any compound may fall into. The significance of these observations with respect to the mechanism of the enzyme are discussed.

Glucokinase (GK) plays a central role in the sensing of glucose in pancreatic β-cells and parenchymal cells of the liver. Glucokinase regulatory protein is a physiological inhibitor of GK in the liver. To understand the role of the interaction of these two proteins in glucose sensing, we carried out a series of experiments to localize the protein in the liver cell. The regulatory protein was found to be present mainly in the nucleus of the cell under a variety of conditions that mimicked the glucose status of the fed and fasted state. GK was localized in the nucleus when the cells were exposed to low glucose concentrations. At higher glucose concentrations or in the presence of low concentrations of fructose, GK translocated to the cytoplasm. The effect of fructose was more robust and rapid than the effect of high glucose concentrations. Furthermore, the effect of fructose and high glucose on the translocation of GK from the nucleus could be partially reversed by glucagon. This unusual localization and behavior suggests a role for GK and its regulatory protein in hepatic energy metabolism that may be broader than glucose phosphorylation.

Glucokinase is the predominant hexokinase in pancreatic β-cells and liver parenchymal cells and functions as a critical component of the glucose-sensing apparatus in these glucose-responsive cell types. In the β-cells, the sensing leads to insulin secretion, while the role in hepatocytes is thought to be in hepatic glucose uptake. To determine the physiological response to an increase in hepatic glucokinase expression, transgenic mice expressing the human hepatic glucokinase gene under the control of a liver-specific human apolipoprotein A-I gene enhancer were generated. Transgenic mice had twofold higher total fasting hepatic glucokinase mRNA, which resulted in a modest 20% increase in fasting glucokinase activity. These animals showed lower fasting plasma glucose, insulin, and lactate levels and improved tolerance to glucose. In addition, glucokinase transgenic animals weighed less and had lower BMI than nontransgenic animals. Thus, glucokinase transgenic animals demonstrate that a modest change in hepatic glucokinase activity enhances the metabolism of glucose.

Glucokinase is a critical component of the physiological glucose sensor found in cell types that are responsive to changes in plasma glucose levels. The acute regulation of glucokinase activity has been shown to occur via a regulatory protein found in liver parenchymal cells (Van Schaftingen E, Detheux M, Da Cunha MV. Faseb J 8:414–419, 1994). The action of this protein is modulated by phosphate esters of fructose. In the presence of fructose-6-phosphate, the protein inhibits glucokinase in an allosteric competitive manner, while fructose-1-phosphate reverses this inhibition. A cDNA potentially encoding the rat liver regulatory protein has been cloned, but its identity is uncertain because of the small amounts of soluble protein obtained by expression in bacteria. We report the heterologous expression of the regulatory protein in Escherichia coli and its purification to homogeneity and high specific activity in a single chromatographic step. The properties of this recombinant protein are very similar to those of the liver protein. Direct demonstration of the binding of the recombinant protein to glucokinase has been obtained in vitro using coprecipitation experiments and in vivo, using the yeast two-hybrid system. These studies establish that the protein encoded by the cDNA is identical to the glucokinase regulatory protein and also validate tools with which to carry out structure-function studies on the interaction of the regulatory protein with glucokinase.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme that regulates cholesterol synthesis, lower serum cholesterol by increasing the activity of low density lipoprotein (LDL) receptors in the liver. In rat liver slices, the dose-response curves for inhibition of [14C]acetate incorporation into cholesterol were similar for the active acid forms of lovastatin, simvastatin, and pravastatin. The calculated IC50 values were approximately 20-50 nM for all three drugs. Interest in possible extrahepatic effects of reductase inhibitors is based on recent findings that some inhibitors of HMG-CoA reductase, lovastatin and simvastatin, can cause cataracts in dogs at high doses. To evaluate the effects of these drugs on cholesterol synthesis in the lens, we developed a facile, reproducible ex vivo assay using lenses from weanling rats explanted to tissue culture medium. [14C]Acetate incorporation into cholesterol was proportional to time and to the number of lenses in the incubation and was completely eliminated by high concentrations of inhibitors of HMG-CoA reductase. At the same time, incorporation into free fatty acids was not inhibited. In marked contrast to the liver, the dose-response curve for pravastatin in lens was shifted two orders of magnitude to the right of the curves for lovastatin acid and simvastatin acid. The calculated IC50 values were 4.5 +/- 0.7 nM, 5.2 +/- 1.5 nM, and 469 +/- 42 nM for lovastatin acid, simvastatin acid, and pravastatin, respectively. Thus, while equally active in the liver, pravastatin was 100-fold less inhibitory in the lens compared to lovastatin and simvastatin. Similar selectivity was observed with rabbit lens. Following oral dosing, ex vivo inhibition of [14C]acetate incorporation into cholesterol in rat liver was similar for lovastatin and pravastatin, but cholesterol synthesis in lens was inhibited by lovastatin by as much as 70%. This inhibition was dose-dependent and no inhibition in lens was observed with pravastatin even at very high doses. This tissue-selective inhibition of sterol synthesis by pravastatin was likely due to the inability of pravastatin to enter the intact lens since pravastatin and lovastatin acid were equally effective inhibitors of HMG-CoA reductase enzyme activity in whole lens homogenates. We conclude that pravastatin is tissue-selective with respect to lens and liver in its ability to inhibit cholesterol synthesis.