We assessed the effects of melatonin, N 1 -acetyl-N 2 -formyl-5-methoxykynuramine (AFMK) and N 1 -acetyl-5-methoxykynuramine (AMK) on neuronal nitric oxide synthase (nNOS) activity in vitro and in rat striatum in vivo. Melatonin and AMK (10)10 )3 M), but not AFMK, inhibited nNOS activity in vitro in a dose-response manner. The IC 50 value for AMK (70 lM) was significantly lower than for melatonin (>1 mM). A 20% nNOS inhibition was reached with either 10 )9 M melatonin or 10 )11 M AMK. AMK inhibits nNOS by a non-competitive mechanism through its binding to Ca 2+-calmodulin (CaCaM). The inhibition of nNOS elicited by melatonin, but not by AMK, was blocked with 0.05 mM norharmane, an indoleamine-2,3-dioxygenase inhibitor.In vivo, the potency of AMK to inhibit nNOS activity was higher than that of melatonin, as a 25% reduction in rat striatal nNOS activity was found after the administration of either 10 mg/kg of AMK or 20 mg/kg of melatonin. Also, in vivo, the administration of norharmane blocked the inhibition of nNOS produced by melatonin administration, but not the inhibition produced by AMK. These data reveal that AMK rather than melatonin is the active metabolite against nNOS, which may be inhibited by physiological levels of AMK in the rat striatum.

In the present study we have investigated whether pharmacophore models may account for the activity and selectivity of the known cyclooxygenase-2 (COX-2) selective inhibitors of the phenylsulfonyl tricyclic series, i.e., Celecoxib (1) and Rofecoxib (3), and whether transferring this structural information onto the frame of a nonsteroidal antiinflammatory drug (NSAID), known to tightly bind the enzyme active site, may be useful for designing novel COX-2 selective inhibitors. With this aim we have developed a pharmacophore based on the geometric disposition of chemical features in the most favorable conformation of the COX-2 selective inhibitors SC-558 (2; analogue of Celecoxib (1)) and Rofecoxib (3) and the more restrained compounds 4 (DFU) and 5. The pharmacophore model contains a sulfonyl S atom, an aromatic ring (ring plane A) with a fixed position of the normal to the plane, and an additional aromatic ring (ring plane B), both rings forming a dihedral angle of 290 degrees +/- 10 degrees. The final disposition of the pharmacophoric groups parallels the geometry of the ligand SC-558 (2) in the known crystal structure of the COX-2 complex. Moreover, the nonconserved residue 523 is known to be important for COX-2 selective inhibition; thus, the crystallographic information was used to position an excluded volume in the pharmacophore, accounting for the space limits imposed by this nonconserved residue. The geometry of the final five-feature pharmacophore was found to be consistent with the crystal structure of the nonselective NSAID indomethacin (6) in the COX-2 complex. This result was used to design indomethacin analogues 8 and 9 that exhibited consistent structure-activity relationships leading to the potent and selective COX-2 inhibitor 8a. Compound 8a (LM-1685) was selected as a promising candidate for further pharmacological evaluation.

The farnesoid X receptor (FXR) is activated by endogenous bile acids (BAs) and plays a variety of physiological roles related to modulation of gene transcription. In particular, FXR positively regulates the cholesterol catabolism while feedback inhibits the BA synthesis by repressing the expression of the CYP7A and CYP8B genes. We have previously shown that 6alpha-ethyl-CDCA (6ECDCA) is a potent and selective FXR agonist. In this paper we report an extensive structure-activity relationship for a series of synthetic bile acids. Our results indicate that the 6alpha position plays a fundamental role in determining affinity and that the side chain of BA is amenable to a variety of chemical modification. Although none of the new derivatives is more potent than 6ECDCA, we show here that a wide variability in efficacy, from full agonists to partial antagonists, can be obtained.

Melatonin prevents mitochondrial failure in models of sepsis through its ability to inhibit the expression and activity of both cytosolic (iNOS) and mitochondrial (i-mtNOS) inducible nitric oxide synthases. Because Parkinson's disease (PD), like sepsis, is associated with iNOS induction, we assessed the existence of changes in iNOS/i-mtNOS and their relation with mitochondrial dysfunction in the MPTP model of PD, which also displays increased iNOS expression. We also evaluated the role of melatonin (aMT) and its brain metabolite, N(1)-acetyl-5-methoxykynuramine (AMK), in preventing i-mtNOS induction and mitochondrial failure in this model of PD. Mitochondria from substantia nigra (SN) and, to a lesser extent, from striatum (ST) showed a significant increase in i-mtNOS activity, nitrite levels, oxidative stress, and complex I inhibition after MPTP treatment. MPTP-induced i-mtNOS was probably related to mitochondrial failure, because its prevention by aMT and AMK reduced oxidative/nitrosative stress and restored complex I activity. These findings represent the first experimental evidence of a potential role for i-mtNOS in the mitochondrial failure of PD and support a novel mechanism in the neuroprotective effects of aMT and AMK.

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