Reevaluation of a Bicyclic Pyrazoline as a Selective 15-Lipoxygenase V-Type Activator Possessing Fatty Acid Specificity

Abstract

Regulation of lipoxygenase (LOX) activity is of great interest due to the involvement of the various LOX isoforms in the inflammatory process and hence many diseases. The bulk of investigations have centered around the discovery and design of inhibitors. However, the emerging understanding of the role of h15-LOX-1 in the resolution of inflammation provides a rationale for the development of activators as well. Bicyclic pyrazolines are known bioactive molecules that have been shown to display antibiotic and anti-inflammatory activities. In the current work, we reevaluated a previously discovered bicyclic pyrazoline h15-LOX-1 activator, PKUMDL_MH_1001 (written as 1 for this publication), and determined that it is inactive against other human LOX isozymes, h5-LOX, h12-LOX, and h15-LOX-2. Analytical characterization of 1 obtained in the final synthesis step identified it as a mixture of cis- and trans-diastereomers: cis-1 (12%) and trans-1 (88%); and kinetic analysis indicated similar potency between the two. Using compound 1 as the cis-trans mixture, h15-LOX-1 catalysis with arachidonic acid (AA) (AC₅₀ = 7.8 +/- 1 μM, A _max = 240%) and linoleic acid (AC₅₀ = 5.3 +/- 0.7 μM, A _max = 98%) was activated, but not with docosahexaenoic acid (DHA) or mono-oxylipins. Steady-state kinetics demonstrate V-type activation for 1, with a β value of 2.2 +/- 0.4 and an K _x of 16 +/- 1 μM. Finally, it is demonstrated that the mechanism of activation for 1 is likely not due to decreasing substrate inhibition, as was postulated previously. 1 also did not affect the activity of the h15-LOX-1 selective inhibitor, ML351, nor did 1 affect the activity of allosteric effectors, such as 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) and 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA). These data confirm that 1 binds to a distinct activation binding site, as previously postulated. Future work should be aimed at the development of selective activators that are capable of activating h15-LOX-1 catalysis with DHA, thus enhancing the production of DHA-derived pro-resolution biomolecules.

Figure 1

An overview of indazole core structures with potential as tools for anti-inflammatory pharmacology.

Figure 2

Indazole-containing compounds possessing anti-inflammatory potential or other relevant bioactivity properties. (A) 2H-indazole, COX-2 selective (IC₅₀ = 409 nM); (B) tetrahydro-2H-indazole, best of 18 analogues with COX-2 selectivity; (C) hexahydro-2H-indazole, two best of 19 analogues with solid tumor cytotoxicity and mild antibiotic activity.

Figure 3

Molecular structures of anti-inflammatory tools and pharmaceutics. (A) ML351, h15-LOX-1 selective (IC₅₀ = 200 nM); B) (±) ketoprofen, human NSAID therapeutic; (C) (±) phenylbutazone, animal NSAID therapeutic; and (D) (±) zileuton, human 5-LOX therapeutic.

Scheme 1. Preparation of 1 (aka PKUMDL_MH_1001) as a Mixture of Isomers

Figure 4

Trans-1 (aka PKUMDL_MH_1001).

Figure 5

Diagnostic ³J_HH and dihedral angle (DH) values between H12–H14 to track trans/cis orientations in bicyclic pyrazolines; 1, 2, and 3.

Figure 6

LC–MS/MS data for cis-1 and trans-1. (A) LC–MS TIC trace showing minor peak (cis-1) at 9.83 min (12%) and major peak (trans-1) at 11.38 min (88%). (B) trans-1, Parent ion of MS TIC peak at 11.38 min. (C) cis-1, Parent ion of MS TIC peak at 9.83 min. (D) trans-1, MS² fragments for 11.38 min after the 429.3 mass filter was applied. (E) cis-1, MS² fragments for 9.83 min after the 429.3 mass filter was applied.

Figure 7

Proposed (+ ion) MS/MS fragmentation of 1 (trans or cis) from CID at 429.3.

Figure 8

The saturation curves are shown of compound 1 with various substrates. The calculated AC₅₀ values (μM) were determined with LA (squares, AC₅₀ = 5.3 +/– 0.7 μM), AA (circles, AC₅₀ = 7.8 +/– 1 μM), and DHA (triangles, AC₅₀ > 100 μM).

Figure 9

Lag phase activity plots of compound 1 with AA and h15-LOX-1 (left) and DHA and h15-LOX-1 (right). Time is in seconds and error is shown as bars for each measurement.

Figure 10

The k_cat apparent is plotted against increasing concentrations of 1 (μM) and fit to eq 4 from Scheme 2, resulting in β and K_x being 2.2 +/– 0.4 and 16 +/– 1 μM, respectively (α was set to 1).

Scheme 2. Kinetic Scheme of Hyperbolic Activation with Concomitant Equations