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    • Some of the earliest LOX inhibitors were redox

    2023-01-14

    Some of the earliest 12-LOX inhibitors were redox inhibitors, including nordihydroguaiaretic AS-605240 (NDGA), BW 755C, and baicalein 48, 49, 50. Redox inhibitors block the oxidation of the nonheme iron at the cataylytic site, preventing its conversion from the inactive (Fe2+) to the active (Fe3+) state [15]. All LOX isoforms require the activation of nonheme iron; therefore, redox inhibitors are pan-lipoxygenase antagonists. Redox inhibitors have been instrumental in demonstrating the potentiating effects of 12-LOX on platelet activation in vitro because 12-LOX is the only LOX isoform to be identified in human platelets. Unfortunately, their lack of selectivity has limited their utility as an in vivo approach for limiting platelet reactivity (Figure 4). While higher concentrations of redox inhibitors have been shown to inhibit COX, researchers have demonstrated that NDGA inhibits platelet activation in the presence of aspirin, suggesting the antiplatelet effects of NDGA are not entirely due to its effects on COX [51]. Another subset of LOX inhibitors are the fatty acid analogs, such as 5,8,11,14-eicosatetraynoic acid (ETYA), which have similar antiplatelet effects as redox inhibitors in vitro but also lack selectivity, and thus have not been further developed as potential antiplatelet therapies 25, 52. 12-LOX must translocate to the glycerophospholipid membrane to produce 12-HETE, therefore, 12-LOX translocation inhibitors block 12-HETE formation without directly inhibiting enzymatic activity. Two such 12-LOX translocation inhibitors, OPC-29030 and L-655,238, have been shown to prevent 12-HETE formation in platelets 24, 53. In response to U46619 stimulation in vitro, OPC-29030-treated platelets have a decrease in Ca2+ mobilization, granule secretion, αIIbβ3 activation, and aggregation compared to control-treated platelets. The antiplatelet activity of OPC-29030 resulted in a decrease in thrombus formation in canine models of thrombosis [54]. As with previous 12-LOX inhibitors, however, OPC-29030 lacks selectivity compared to other LOX isoforms, and therefore, cannot be considered for in vivo treatment of thrombosis without the potential of significant off-target effects both within and beyond the vessel [24].
    Development of Potent and Selective 12-LOX Inhibitors The antiplatelet and antithrombotic effects of nonselective 12-LOX inhibitors have helped identify 12-LOX as a promising therapeutic target for the prevention of thrombosis. However, initial attempts to develop a potent and selective 12-LOX inhibitor using natural products and traditional medicine chemistry approaches were unsuccessful, largely due to poor selectively over other LOX and COX isoforms [55]. The successful development of the selective 5-LOX inhibitor, zileuton, for the treatment of asthma has helped to renewed interest in the development of a potent and selective inhibitor of 12-LOX. These efforts were realized recently, when a quantitative high-throughput screen of the NIH Molecular Libraries Probe Production Center Network (MLPCN), which is comprised of 153 607 unique compounds, identified two chemotypes, 8-hydroxyquinoline-based scaffold and 4-((2-hydroxy-3-methoxybenzyl)amino)-benzenesulfonamide-based scaffold that had nanomolar potency against purified 12-LOX and >50-fold selectivity over other LOX isozymes and COX and low micromolar potency in the human platelets for selectively targeting 12-LOX 5, 56. Structure–activity relationship (SAR) refinement strategies with the 8-hydroxyquinoline-based scaffold produced two lead compounds, NCTT-956 (N-((8-hydroxy-5-nitroquinolin-7-yl)(thiophen-2-yl)methyl)propionamide) and ML127 (N-((5-bromo-8-hydroxy-quinolin-7-yl)(thiophen-2-yl)methyl)acetamide) that were potent noncompetitive, nonreductive inhibitors of 12-LOX that exhibited excellent selectivity (>50-fold) over other LOX isozymes. While NCTT-956 inhibited intracellular Ca2+, αIIbβ3 activation, and platelet aggregation mediated by thrombin or collagen, further biological characterization of NCTT-956 determined that it was cytotoxic [50]. Refinement of NCTT-956 to decrease its cytotoxicity severely reduced its potency; therefore, development of NCTT-956 was stopped. The initial characterization of ML127 demonstrated that it inhibits 12-HETE production in platelets stimulated with PAR1-AP; however, further studies are required to determine how it effects platelet activation and thrombosis. The development of ML127 as a potential antithrombotic remains an area of active investigation and is being developed in parallel with benzenesulfonamide-based 12-LOX inhibitors to help determine which of these chemotypes will eventually be best-in-man approaches for limiting 12-LOX activity and platelet function while maintaining enzyme selectivity.