Contribution of a Low-Barrier Hydrogen Bond to Catalysis Is Not Significant in Ketosteroid Isomerase

Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ5-3-ketosteroid to its conjugated Δ4-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1–11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7–2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI

Identification of a New Morpholine Scaffold as a P2Y12 Receptor Antagonist

The P2Y12 receptor is critical for platelet activation and is an attractive drug target for the prevention of atherothrombotic events. Despite the proven antithrombotic efficacy of P2Y12 inhibitors, these thienopyridine scaffolds are prodrugs that lack important features of the ideal antithrombotic agent. For this reason, ticagrelor—a new chemical class of P2Y12 receptor antagonist—was developed, but it can cause shortness of breath and various types of bleeding. Moreover, ticagrelor is a cytochrome P450 3A4 substrate/inhibitor and, therefore, caution should be exercised when it is used concomitantly with strong CYP3A4 inducers/inhibitors. There is a need for novel P2Y12 receptor antagonist scaffolds that are reversible and have high efficacy without associated side effects. Here, we describe a novel antagonist containing a morpholine moiety that was identified by screening libraries of commercially available compounds. The molecule, Compound E, acted on P2Y12, but not P2Y1 and P2Y13, and exhibited pharmacological characteristics that were distinct from those of ticagrelor, acting instead on P2Y12 via an allosteric mechanism. These results provide a basis for the development/optimization of a new class of P2Y12 antagonists

Identification of a New Morpholine Scaffold as a P2Y12 Receptor Antagonist

The P2Y12 receptor is critical for platelet activation and is an attractive drug target for the prevention of atherothrombotic events. Despite the proven antithrombotic efficacy of P2Y12 inhibitors, these thienopyridine scaffolds are prodrugs that lack important features of the ideal antithrombotic agent. For this reason, ticagrelor—a new chemical class of P2Y12 receptor antagonist—was developed, but it can cause shortness of breath and various types of bleeding. Moreover, ticagrelor is a cytochrome P450 3A4 substrate/inhibitor and, therefore, caution should be exercised when it is used concomitantly with strong CYP3A4 inducers/inhibitors. There is a need for novel P2Y12 receptor antagonist scaffolds that are reversible and have high efficacy without associated side effects. Here, we describe a novel antagonist containing a morpholine moiety that was identified by screening libraries of commercially available compounds. The molecule, Compound E, acted on P2Y12, but not P2Y1 and P2Y13, and exhibited pharmacological characteristics that were distinct from those of ticagrelor, acting instead on P2Y12 via an allosteric mechanism. These results provide a basis for the development/optimization of a new class of P2Y12 antagonists

Inhibition of c-Kit signaling by diosmetin isolated from Chrysanthemum morifolium

The interaction of stem cell factor (SCF) with its cognate receptor c-Kit is closely associated with the survival and maturation of melanocytes. To investigate novel depigmentation agents, we screened 2,000 plant extracts for c-Kit inhibitors to identify active small molecules by using time-resolved fluorescence enzyme assays. For the active extracts identified as inhibitors of c-Kit enzyme, we evaluated the effects of the active extracts and isolated flavonoids on c-Kit phosphorylation in MO7e/melanocytes. Anti-melanogenic activity was also examined in melanocytes and melanoderm model. The flavonoids such as diosmetin, apigenin, acacetin and luteolin isolated from Chrysanthemum morifolium were found to be active in inhibiting c-Kit both at enzyme and cellular levels. In addition, these flavonoids attenuated SCF-induced proliferation of human primary melanocytes without toxicity and suppressed ultraviolet (UV) B irradiation-mediated melanin synthesis significantly. Among the active flavonoids, diosmetin was found to inhibit SCF-induced melanogenesis in a human melanoderm model. These results strongly suggest that C. morifolium extract and diosmetin have potential to suppress SCF-/UVB-induced melanogenesis, and could be developed as anti-pigmentation agents