P. Douglas Boatman

Niacin Lipid Efficacy Is Independent of Both the Niacin Receptor GPR109A and Free Fatty Acid Suppression

GPR109A is not the target mediating niacin’s lipid efficacy and the free fatty acid hypothesis does not explain niacin’s mechanism of action. Breaking Free of the “FFA Hypothesis” Free fatty acids (FFAs) appear in the blood plasma after a meal. Niacin—a vitamin that helps to regulate lipid levels in the body—is given to patients to reduce the amount of FFAs. It also works to raise “good” cholesterol [high-density lipoprotein (HDL)] and lower both “bad” cholesterol [low-density lipoprotein (LDL)] and triglycerides. The “FFA hypothesis” suggests that niacin works to exert these beneficial lipid effects by limiting the amount of FFAs available to synthesize triglycerides. Lauring, Taggart, and colleagues now challenge this long-standing theory. In studies in mice and humans, the authors debunk the hypothesis, showing that the effect on HDL, LDL, and triglycerides is not directly linked to FFAs. To study the lipid-modifying effects of niacin (nicotinic acid), Lauring et al. used a genetic, humanized mouse model lacking the LDL receptor. In these animals, niacin increased HDL cholesterol levels, as expected. Lack of GPR109A in these animals blocked the anti-lipolytic effect of nicotinic acid on FFAs but had no effect on drug-related changes in plasma HDL and LDL cholesterol or triglyceride levels. Treatment of the mice with a GPR109A agonist, MK-1903, also caused an anti-lipolytic effect but did not affect levels of triglyceride or LDL and HDL cholesterol. Together, these in vivo preclinical studies suggest that niacin works to lower FFAs through GPR109A but has an independent mechanism of action on other lipids. The authors addressed the role of GPR109A in humans by testing the effects of MK-1903 and of another synthetic GPR109A agonist in clinical trials. Both agonists affected FFA lipolysis but had only minor effects on HDL cholesterol and triglyceride levels in patients, thus mirroring results seen in animals and showing that niacin works independently of GPR109A to modify dyslipidemia. The studies by Lauring et al. point to a new, yet-uncovered mechanism of action for niacin’s beneficial effects on lipids in the blood. Despite overturning the FFA hypothesis and potentially redirecting drug development away from GPR109A agonists, niacin could still be useful for treating other diseases in patients, including atherosclerosis and inflammation, where GPR109A plays a major role in cell signaling. Nicotinic acid (niacin) induces beneficial changes in serum lipoproteins and has been associated with beneficial cardiovascular effects. Niacin reduces low-density lipoprotein, increases high-density lipoprotein, and decreases triglycerides. It is well established that activation of the seven-transmembrane Gi-coupled receptor GPR109A on Langerhans cells results in release of prostaglandin D2, which mediates the well-known flushing side effect of niacin. Niacin activation of GPR109A on adipocytes also mediates the transient reduction of plasma free fatty acid (FFA) levels characteristic of niacin, which has been long hypothesized to be the mechanism underlying the changes in the serum lipid profile. We tested this “FFA hypothesis” and the hypothesis that niacin lipid efficacy is mediated via GPR109A by dosing mice lacking GPR109A with niacin and testing two novel, full GPR109A agonists, MK-1903 and SCH900271, in three human clinical trials. In mice, the absence of GPR109A had no effect on niacin’s lipid efficacy despite complete abrogation of the anti-lipolytic effect. Both MK-1903 and SCH900271 lowered FFAs acutely in humans; however, neither had the expected effects on serum lipids. Chronic FFA suppression was not sustainable via GPR109A agonism with niacin, MK-1903, or SCH900271. We conclude that the GPR109A receptor does not mediate niacin’s lipid efficacy, challenging the long-standing FFA hypothesis.

3-(1H-Tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (MK-0354): A Partial Agonist of the Nicotinic Acid Receptor, G-Protein Coupled Receptor 109a, with Antilipolytic but No Vasodilatory Activity in Mice

The discovery and profiling of 3-(1H-tetrazol-5-yl)-1,4,5,6-tetrahydro-cyclopentapyrazole (5a, MK-0354), a partial agonist of GPR109a, is described. Compound 5a retained the plasma free fatty acid lowering effects in mice associated with GPR109a agonism, but did not induce vasodilation at the maximum feasible dose. Moreover, preadministration of 5a blocked the flushing effect induced by nicotinic acid but not that induced by PGD2. This profile made 5a a suitable candidate for further study for the treatment of dyslipidemia.

Highly efficient and versatile synthesis of libraries of constrained β-strand mimetics

The general approach of using a bicyclic template to produce inhibitors of the protease superfamily of enzymes has been investigated. The Diels-Alder cycloaddition reaction on solid support has been found to be highly efficient for the synthesis of libraries of compounds that mimic the β-strand secondary structure of proteins. Several potent and selective inhibitors of proteases have been discovered.

(1aR,5aR)1a,3,5,5a-Tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalene-4-carboxylic acid (MK-1903): a potent GPR109a agonist that lowers free fatty acids in humans.

G-protein coupled receptor (GPCR) GPR109a is a molecular target for nicotinic acid and is expressed in adipocytes, spleen, and immune cells. Nicotinic acid has long been used for the treatment of dyslipidemia due to its capacity to positively affect serum lipids to a greater extent than other currently marketed drugs. We report a series of tricyclic pyrazole carboxylic acids that are potent and selective agonists of GPR109a. Compound R,R-19a (MK-1903) was advanced through preclinical studies, was well tolerated, and presented no apparent safety concerns. Compound R,R-19a was advanced into a phase 1 clinical trial and produced a robust decrease in plasma free fatty acids. On the basis of these results, R,R-19a was evaluated in a phase 2 study in humans. Because R,R-19a produced only a weak effect on serum lipids as compared with niacin, we conclude that the beneficial effects of niacin are most likely the result of an undefined GPR109a independent pathway.

Inhibition of Mas G-protein signaling improves coronary blood flow, reduces myocardial infarct size, and provides long-term cardioprotection

The Mas receptor is a class I G-protein-coupled receptor that is expressed in brain, testis, heart, and kidney. The intracellular signaling pathways activated downstream of Mas are still largely unknown. In the present study, we examined the expression pattern and signaling of Mas in the heart and assessed the participation of Mas in cardiac ischemia-reperfusion injury. Mas mRNA and protein were present in all chambers of human hearts, with cardiomyocytes and coronary arteries being sites of enriched expression. Expression of Mas in either HEK293 cells or cardiac myocytes resulted in constitutive coupling to the G(q) protein, which in turn activated phospholipase C and caused inositol phosphate accumulation. To generate chemical tools for use in probing the function of Mas, we performed a library screen and chemistry optimization program to identify potent and selective nonpeptide agonists and inverse agonists. Mas agonists activated G(q) signaling in a dose-dependent manner and reduced coronary blood flow in isolated mouse and rat hearts. Conversely, treatment of isolated rat hearts with Mas inverse agonists improved coronary flow, reduced arrhythmias, and provided cardioprotection from ischemia-reperfusion injury, an effect that was due, at least in part, to decreased cardiomyocyte apoptosis. Participation of Mas in ischemia-reperfusion injury was confirmed in Mas knockout mice, which had reduced infarct size relative to mice with normal Mas expression. These results suggest that activation of Mas during myocardial infarction contributes to ischemia-reperfusion injury and further suggest that inhibition of Mas-G(q) signaling may provide a new therapeutic strategy directed at cardioprotection.

High-throughput synthesis and optimization of thrombin inhibitors via urazole α-addition and Michael addition

A novel alpha-addition of propiolates to urazoles followed by Michael addition of a variety of nucleophiles has been developed for rapid production and optimization of peptidomimetic drug leads. This technology has produced a number of highly potent and selective inhibitors of the serine protease, thrombin.

Potent tricyclic pyrazole tetrazole agonists of the nicotinic acid receptor (GPR109a)

Tricyclic pyrazole tetrazoles which are potent partial agonists of the high affinity niacin receptor, GPR109a, have been discovered and optimized. One of these compounds has proven to be effective at lowering free fatty acids in vitro and in vivo.

Discovery of a new series of potent prostacyclin receptor agonists with in vivo activity in rat

The design and synthesis of two closely related series of prostacyclin receptor agonist compounds that showed excellent human IP receptor potency and efficacy is described. Compounds from this series showed in vivo activity after SC dosing in the monocrotaline model of PAH in rat.

Discovery of 2-(((1r,4r)-4-(((4-Chlorophenyl)(phenyl)carbamoyl)oxy)methyl)cyclohexyl)methoxy)acetate (Ralinepag): An Orally Active Prostacyclin Receptor Agonist for the Treatment of Pulmonary Arterial Hypertension

The design and synthesis of a new series of potent non-prostanoid IP receptor agonists that showed oral efficacy in the rat monocrotaline model of pulmonary arterial hypertension (PAH) are described. Detailed profiling of a number of analogues resulted in the identification of 5c (ralinepag) that has good selectivity in both binding and functional assays with respect to most members of the prostanoid receptor family and a more modest 30- to 50-fold selectivity over the EP3 receptor. In our hands, its potency and efficacy are comparable or superior to MRE269 (the active metabolite of the clinical compound NS-304) with respect to in vitro IP receptor dependent cAMP accumulation assays. 5c had an excellent PK profile across species. Enterohepatic recirculation most probably contributes to a concentration-time profile after oral administration in the cynomolgus monkey that showed a very low peak-to-trough ratio. Following the identification of an acceptable solid form, 5c was selected for further development for the treatment of PAH.

Synthesis of dipeptide secondary structure mimetics

Masakatsu Eguchi, Hwa-Ok Kim, Benjamin S. Gardner, P. Douglas Boatman, Min S. Lee, Hiroshi Nakanishi and Michael Kahn Molecumetics Ltd., 2023 120th Ave. N.E., Suite 400, Bellevue, WA 98005, USA and Department of Pathobiology, University of Washington, Seattle, WA, 98195, USA Secondary structures in proteins and peptides play an important role in biological recognition systems. In principle, conformationally restricted mimetics of such bioactive local structures can enhance the activity of bioactive molecules. Our previous studies [1] showed that a 3-benzyl 6,5-bicyclic dipeptide mimetic for DPhe-Pro coupled with an Arg moiety (MOL-098) afforded effective thrombin inhibitory activity X-ray co-crystal structural analysis of MOL-098 with human revealed that the potent inhibitory activity was due to the extended strand structure of MOL-098 interacting with the backbone structure of the thrombin active site, and the benzyl group at the P3 position of the inhibitor interacting favorably through a hydrophobic aromatic interaction with of thrombin. In this study, we have designed a conformationally constrained analogue of the 3-benzyl 6,5-bicyclic dipeptide, (3R and 3S, 6S, 9S)-3-(t-butyoxycarbony)amino-3-benzyl-l-aza[4.3.0]nonan-2-one-9-carboxylic acid (1, 2), and developed efficient syntheses of these templates.